MOLECULAR CHARACTERISATION OF SOME MULTI-DRUG RESISTANT SALMONELLA ENTERICA OF HUMAN ORIGIN IN SOUTHEAST NIGERIA BY NATHANIEL EJIKEME ONYENWE B.Sc. (IMSU), M. Sc. Pharmaceutical Microbiology (Ibadan) A Thesis in the Department of Pharmaceutical Microbiology Submitted to the Faculty of Pharmacy in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN JANUARY, 2016. i CERTIFICATION This is to certify that this research study was carried out by Onyenwe Nathaniel Ejikeme under my supervision, in the Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ibadan, Nigeria. …………………………………………………………………………… Supervisor O. E. Adeleke (Ph. D) Reader ii DEDICATION This work is dedicated to the almighty God, my beloved Children, Miracle (Ada), Chizitere (Precious), Godswill (Osinachi), Ifeanyi (Francis), and wife Oluchi (Norah). iii ACKNOWLEDGEMENT I acknowledge the support of the Almighty God who ignites his wisdom and understanding in me through the period of my research work in the University of Ibadan. Special thanks go to my supervisor and former Ag. Head of Department, Dr. O. E. Adeleke who through his fatherly love, painstakingly read through my work and made necessary corrections, I say may God bless you abundantly. My appreciation also goes to Professor (Mrs.) B.O. Adeniyi for all her effort and encouragement so far, may God equally bless you accordingly. I want to specially thank the departmental Co-ordinator, Dr. Idowu, P. A. for his brotherly advice and effort in making sure that this thesis becomes a reality. I also want to appreciate the dean, faculty of Pharmacy, Prof. Chinedum P. Babalola for her effort in making this thesis a success. My humble regards goes to my Parents, Mr. and Mrs. Anthony Umelo Onyenwe, who prayed and made sure that this journey of mine was fruitful and successful. My brothers, Mr. and Mrs. Richard Onyenwe, Kelechi Onyenwe, Nnamdi Onyenwe, Mr. and Mrs.Oliver Onyenwe , Mr. and Mrs. Alphonsus Onyenwe , Mr. and Mrs. Kenneth Onyenwe, Mr. Ferguson Onyenwe, Mr. and Mrs. Chidi Onyenwe, for their encouragement and moral supports. My sincere appreciation to my class mate and friend, Pharm. Alabi Muyiwa who took it upon himself seeing that this work becomes a reality. My appreciation also goes to my other class mates and friends like Christy, Odumosu Dele and those who have one way or the other contributed to this work. I wish to show my gratitude to my loving wife, Mrs. Norah O. Onyenwe and my children, who through their encouragement have also helped immensely in the completion of this research. My special thanks go to the management and staff of Nigeria Institute of Medical Research (NIMR) especially the Microbiology / Molecular biotechnology department, Dr. Mrs. Smith, S.I., Mrs. (Alhaja) Fowora Muinah, I., Miss Magrete and others including the I.T students for their contributions to the success of this work. Also, my thanks go to the staff of the Federal Medical Centres (FMC) and Teaching hospital, especially the staff of FMC Owerri Dr. Amah, Oga Edi, Sister Christy, Chigbu and the H.O.D. Microbiology Department for making their laboratory and facilities available throughout my stay there, may almighty God bless and keep you all. My regards to my in-laws, Mr. and Mrs. Zephaniah Iroegbu, Mrs. Stella Amadi and the rest, for their prayers, financial and moral support. My friends, Pastor Ifeanyi, roommates Obinna, iv Johnpaul and all my charismatic brethren everywhere for their assistance and prayers. May His Majesty, the King of kings, Jesus Christ and His most precious blood that comes out of His Sacred Head, Divine Temple of wisdom, Terbanacle of Divine Knowledge and Sunshine of Heaven and Earth cover us now and protect us forever, Amen. v ABSTRACT There has been an increase in the occurrence of antibiotic resistance among Salmonella enterica, one of the commonest causative agents of Salmonella infections. Fluoroquinolones and third generation cephalosporins are usually the drugs of choice in the management of Salmonella infections. Previous reports have indicated common occurrence of multi-drug resistance (MDR) including resistance to β-lactams and fluoroquinolones among clinical Gram-negative organisms. However, there is paucity of information on the genetic determinants of resistance to β-lactam and fluoroquinolones from S. enterica in Southeast Nigeria. This study screened for the presence of Extended-Spectrum Beta-Lactamases (ESBL) and mutations in gyrA and parC genes of S. enterica from human origin in the Southeast Nigeria. Twenty-five S. enterica isolates from stool of patients suspected to have Salmonella infection were collected from each of four hospitals (one teaching hospital and three Federal Medical Centres) in Southeast Nigeria between July and September, 2010. The isolates were confirmed ® using Microbact identification kit . Antibiogram for the isolates was determined by disc- diffusion based on Clinical and Laboratory Standards Institute breakpoints. Five commonly used antibiotics (amoxicillin-clavulanic acid, cefotaxime, ceftriaxone, ciprofloxacin and levofloxacin) in the treatment of Salmonella infections were selected for determination of Minimum Inhibitory Concentrations (MIC) against the isolates using broth-dilution method. Isolates resistant to two or more different classes of antibiotics were classified as MDR. Isolates resistant to fluoroquinolones and cephalosporins were exposed to mutagens for R-plasmid curing, and ESBL were detected phenotypically using Double-Disk Synergy Test. Genomic and plasmid DNA of mutagen treated and untreated isolates were extracted by boiling and alkaline lysis, respectively. Polymerase chain reaction was used to amplify blaTEM, blaSHV and blaCTX-M among the ESBL positive isolates, and Quinolone Resistance Determining Regions (QRDR) among fluoroquinolone resistant isolates, followed by sequencing of the QRDRs. Antibiogram data were analysed using ANOVA at p = 0.05. The 100 clinical isolates collected were confirmed to be S. enterica. Percentage resistance obtained was: amoxicillin-clavulanic acid (87%), chloramphenicol (80%), amoxicillin (80%), co- trimoxazole (78%), sparfloxacin (78%), streptomycin (77%), gentamicin (51%), ceftazidime (44%), perfloxacin (29%), ciprofloxacin (29%), ofloxacin (28%), cefotaxime (27%), ceftriaxone vi (22%) and levofloxacin (22%). Eighty of the 100 isolates were MDR and the ranges of MICs of the selected antibiotics were: amoxicillin-clavulanic acid (≥ 50 µg/mL), cefotaxime (6.25 - 25 µg/mL), ceftriaxone (6.25 – 12.5 µg/mL), ciprofloxacin (6.25 – 12.5 µg/mL) and levofloxacin (12.5 - 25 µg/mL). Of the 100 isolates, nine MDR isolates carrying R-plasmid were cured. Thirty six of the MDR isolates produced ESBL phenotypically, of which 13 were blaCTX-M positive. DNA sequencing revealed single point mutations in gyrA at amino acid positions Asp-87-Gly, Asp-87-Asn and Ser-83-Tyr in 55 (68.8%), and double mutation in parC at positions Asp-87-Gly in 14 (17.5%). There was significant difference in the activity of the individual antibiotics against the isolates. The occurrence of mutations in gyrA and parC genes, and chromosomal blaCTX-M were responsible for fluoroquinolones and cephalosporins resistance, respectively in some of the Salmonella enterica from Southeast Nigeria. Hence, alleviating the fear of easy spreading of quinolone and cephalosporin resistant isolates. Keywords: Multi-drug resistant Salmonella enterica, R-plasmids, quinolone-resistance determining region, blaCTX-M, fluoroquinolones Word count: 499. vii LIST OF MAIN ABBREVIATIONS Abbreviation Full Meaning MDR Multidrug Resistance BLAST Basic Local Alignment Search Tool DNA Deoxyribonucleic acid bla Beta-Lactamase bla CTX-M Cefotaximase bla SHV Beta-Sulfhydryl variable bla TEM Beta-Temoniera ESBLs Extended-Specrum Beta-Lactamases MIC Minimum Inhibitory Concentration CLSI Clinical Laboratory Standards Institute PCR Polymerase Chain Reaction µg/mL Microgram per millilitres HindIII Haemophilus influenzae III Qnr Quinolone Resistant Protein gyrA DNA gyrase enzyme parC Topoisomerase IV enzyme Qnr-B Quinolone Resistant Protein Class B viii TABLE OF CONTENTS Pages Title page i Certification ii Dedication iii Acknowledgement iv Abstract vi List of main abbreviations viii Table of contents ix List of Tables xiii List of Plates xiv List of Figures xv Chapter One 1 Introduction 1 1.1. Background 1 1.1.1 Background Description of Salmonella enterica 1 1.2. Justification for this study 3 1.3. Overall aims for the study 3 1.4. Specific objectives for the study 4 Chapter Two 5 Literature review 5 2.1. Characteristics of Salmonella enterica 5 2.2. Salmonella enterica infections in man 10 2.2.1 Antigenic structure of S. enterica 11 2.2.2 Pathogenesis of S. enterica 12 2.2.3 The enteric fever 12 2.2.4 Salmonella septicaemias 13 2.2.5 Gastroenteritis 13 ix 2.2.6 Carriers 14 2.2.7 Laboratory diagnosis of S. enterica infections 14 2.2.8 Immunity of S. enterica infection 14 2.2.9 Treatment of S. enterica infection 14 2.2.10 Prevention of S. enterica infection 15 2.3. Salmonella infections associated with animal 16 2.4. Salmonella and the quinolones 18 2.5. Adverse effects of quinolone administration 22 2.6. Characteristic of Extended-Spectrum Beta-Lactamase (ESBL) 23 2.7. Overview of the Quinolones 27 2.8. Levofloxacin 31 2.9. Ciprofloxacin 35 2.10. Cephalosporins 38 2.11. Cefotaxime 40 2.12. Ceftriaxone 41 2.13. Ethidium bromide 43 2.14. Acridine 45 Chapter Three 47 Materials and methods 47 3.1. Materials 47 3.1.1. Equipment and glassware 47 3.1.2. Chemicals and culture media used 48 3.1.3. The antibiotics and primers used 48 3.1.4. Test microorganism 49 3.2. Methods 49 3.2.1. Maintainance of pure isolates 49 3.2.2. Collection of bacterial isolates 49 3.2.3. Culturing and identification of the S. enterica 50 3.2.4. Identification of Salmonella species using Microbact® kit GNB12E 50 3.3. Antibiogram screening of the Salmonella 51 x 3.4. Determination of the Minimum Inhibitory Concentration (MIC) 51 3.5. β-lactamase production test using nitrocefin Sticks 52 3.6. Plasmid DNA isolation 53 3.7. Curing of antibiotic resistance in salmonella Strains 53 3.8. Detection of ESBL’s using Double Disc Synergy Test (DDST) 54 3.9. Preparation of purified chromosomal DNA for PCR analysis using boiling method 55 3.10. Agarose gel electrophoresis of DNA products 55 3.11. Spectrophotometric quantification and purity test of chromosomal DNA 55 3.12. PCR analysis and DNA sequencing 56 Chapter Four 58 Results 58 4.1. Authentication of Salmonella enterica Serovar.Typhi 58 4.2. Antibiogram screening and resistant pattern of the S.enterica 58 4.3. Determination of the Minimum Inhibitory Concentration (MIC) using tube dilution method on five selected antibiotics 65 4.4. Prevalence rate of S. enterica in various departmental units and hospitals in the Southeast region of Nigeria 70 4.5. Beta-lactamase production and plasmid profiling of S. enterica isolates in relation to gender distribution 72 4.6. Distribution of resistant determinants of S. enterica in relation to patient’s age from the Southeast region of Nigeria. 84 4.7. Antibiotics susceptibility of S. enterica in relation to their resistant determinants in the four (a-d) hospitals. 94 4.8. Percentage antibiotic susceptibility pattern of S. enterica in relation to the resistant determinants 99 4.9. Phenotypic antibiotic resistance patterns of S. enterica isolates from the various hospitals in relation to β- lactamase and ESBL’s production 102 4.10. Phenotypic antibiotic resistance pattern of the isolates of S. enterica against five (5) selected antibiotics in relation to resistance genes 108 xi 4.11. Prevalence of β-lactamase linked phenotypic resistance grouping against the 5 (five) selected antibiotic in relation to enzymatic productionby S. enterica isolates. 113 4.12. Curing of antibiotic resistance in Salmonella enterica strains with ethidium bromide and acridine orange 115 4.13. Analysis of variance (ANOVA) to determine the treatment effect of the antibiotic drugs on the S. enterica isolates 118 4.14: BLAST analysis of the gene sequencing and alignment of S. enterica from Southeast Nigeria 119 Chapter Five 125 Discussion 125 5.1. Discussion 125 Chapter Six Conclusion and Recommendations 140 6.1. Conclusion 140 6.2. Recommendations 141 References 143 Appendix1: Laboratory equipment, primers, Reagent and media used 159 Appendix II: Spectroctrophotometric, Antibiogram, Summary of MIC and Percentage prevalence formulae 168 Appendix III: Analysis of variance (ANOVA) for S. enterica; two-way grouping (symbolic) and some Curing Analysis 173 Appendix IV: The Fast Minimum Evolution and Neighboring joining Taxonomic tree of sequenced isolates SO3, SO14, SU33, SA96, and SA98. 181 xii LIST OF TABLES 4.1. Biochemical test authentication of Salmonella enterica Serovar.Typhi 60 4.2. Antibiogram screening and resistant pattern of the S. enterica 61 4.3. The MICs and MBCs (µg/l) of five selected antibiotics against 25 isolates of S. enterica 66 4.4. Prevalence rate of S. enterica in various departmental units and hospitals in the Southeast region of Nigeria 71 4.5. Beta-lactamase production and plasmid profiling of S. enterica isolates in relation to gender distribution 75 4.6. Distribution of resistant determinants of S. enterica in relation to patient’s age from the Southeast region of Nigeria. 86 4.7. Antibiotic susceptibility of S. enterica in relation to their resistant determinants in the various hospital (a-d) 95 4.8. Percentage antibiotic susceptibility pattern of S. enterica in relation to the resistant determinants 100 4.9. Phenotypic antibiotic resistance patterns of S. enterica isolates from the various hospitals in relation to β- lactamase and ESBL’s production 103 4.10. Phenotypic antibiotic resistance pattern of the isolates of S. enterica against five (5) selected antibiotics in relation to resistant genes 109 4.11. Prevalence of β-lactamase linked phenotypic resistance grouping against the 5 (five) selected antibiotics in relation to enzymatic production by S. enterica isolates. 114 4.12. Analysis of variance (ANOVA) to determine the treatment effect of the antibiotics drugs on the S. enterica isolates 120 4.13. BLAST analysis of the gene sequencing and alignment of S. enterica from Southeast Nigeria. 121 xiii LIST OF PLATES Plate 4.1: Agarose gel electrophoresis pattern showing single PCR amplification products of BlaCTX-M genes from S. enterica isolates from FMC Owerri 79 Plate 4.2: Agarose gel electrophoresis pattern showing single PCR amplification products of BlaCTX-M genes from S. enterica isolates from FMC Umuahia and UNTH Enugu 80 Plate 4.3: Agarose gel electrophoresis pattern showing single PCR amplification products of BlaCTX-M genes from S. enterica isolates from FMC Owerri and Umuahia. 81 Plate 4.4: Agarose gel electrophoresis pattern showing single PCR amplification Products of BlaCTX-M genes from S. enterica isolates from FMC Umuahia and UNTH, Enugu. 82 Plate 4.5: Agarose gel electrophoresis Pattern showing plasmid of S. enterica serovar. before treatment with dyes. 83 Plate 4.6: Agarose gel electrophoresis pattern showing single PCR amplification products of GyrA genes from S. enterica. isolates from FMC Owerri 87 Plate 4.7: Agarose gel electrophoresis pattern showing single PCR amplification products of GyrA genes from S. enterica isolates from FMC Umuahia 88 Plate 4.8: Agarose gel electrophoresis pattern showing single PCR amplification products of GyrA genes from S. enterica. isolates from UNTH Enugu 89 Plate 4.9: Agarose gel electrophoresis pattern showing single PCR amplification products of GyrA genes from S. enterica. isolates from FMC Abakaliki 90 Plate 4.10: Agarose gel electrophoresis pattern showing single PCR amplification products of ParC genes from S. enterica. isolates from FMC Owerri, Umuahia and UNTH Enugu 91 Plate 4.11: Agarose gel electrophoresis pattern showing single PCR amplification products of ParC genes from S.enterica. isolates from UNTH Enugu and FMC Abakaliki 92 Plate 4.12: Agarose gel electrophoresis pattern showing cured plasmids of S.enterica after exposure to Ethidium bromide (dye) 116 xiv Plate 4.13: Agarose gel electrophoresis pattern showing cured plasmids of S.enterica after exposure to Acridine orange ( dye) 117 xv LIST OF FIGURES Figure 2.1: Essential structure of all quinolone antibiotics 31 Figure 2.2: Structure of Nalidixic acid 31 Figure 2.3: Structure of Levofloxacin 35 Figure 2.4: Structure of ciprofloxacin 38 Figure 2.5: Core structure of the cephalosporins 39 Figure 2.6: Structure of Cefotaxime 41 Figure 2.7: Ethidium bromide 45 Figure 2.8: Acridine orange 46 Figure 4.1: The incidence rate of qnrB, gyrA, parC, blaSHV, blaTEM, blaCTX-M and plasmids on patients infected with S. enterica in Southeast region of Nigeria. 93 Figure 4.2: Graphical representation of the genetic constituents, the number of antibiotics and resistant pattern of isolates that produced Plasmids or a type of mutation in a gene detected using PCR amplification 101 Figure 4.3: Graphical representation of the genetic constituents on the isolates from different hospitals in the Southeast Region and the level of Distribution 107 Figure 4.4: Isolate SO3, Taxonomic Neighboring joining tree 123 Figure 4.5: Isolate SO14, Taxonomic Fast Minimum Evolution tree 124 xvi CHAPTER ONE INTRODUCTION 1.1. Background 1.1.1. Description of Salmonella enterica Typhoid fever is a systemic infection caused by Salmonella enterica serotype typhi. This is a highly adapted human specific pathogen and possesses remarkable mechanism for persistence in host (Mushtaq, 2006). Most of the disease burden occurs in developing countries due to poor sanitary conditions (Parry et al., 2002; Parry and Beeching, 2009). In the late 1980s, some S. typhi and S. paratyphi strains (multidrug resistant) developed plasmid-mediated resistance simultaneously to all of the first line antibacterial agents, which are ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole (TMP-SMZ) (Parry and Beeching, 2009; Brusch et al., 2010). The use of antimicrobial agents in any environment creates selection pressure that favours the survival of antibiotic-resistance pathogens (White et al., 2007). According to the infectious disease report that was released by the World Health Organisations in 2000, such organisms have become increasingly prevalent world wide (White et al., 2003). Multi-drug resistant Salmonella typhi (MDRST) is defined as Salmonella typhi resistant to all first line antibiotics i.e. chloramphenicol, ampicillin, and trimethoprim- sulphamethoxazole (Mushtaq, 2006). Multiple outbreaks of infections with these resistant strains occurred in India, Pakistan, Bangladesh, Vietnam, Middle East and Africa (Thong, et al., 2000; Connerton et al., 2000; Mirza et al., 2000). These strains were also found to be resistant to sulphonamide, tetracycline and streptomycin. Amoxicillin and trimethoprim-sulphamethoxazole were effective alternatives till the end of 1990s when strains resistant to all the first line anti-salmonella drugs used at that time, were reported (Mushtaq, 2006). This resistance rarely develops during course of treatment but instead results from clonal dissemination of individual multi-drug resistant S. typhi or from transfer of R-plasmid. However, there are reports from some areas, of strains, fully susceptible to all first line drugs (Mushtaq, 2006). 1 Fluoroquinolones are now recommended by most authorities for the treatment of typhoid fever. They are highly effective against susceptible organisms, yielding a better cure rate than cephalosporins. Unfortunately, resistance to first-generation fluoroquinolones is widespread in many parts of Asia (Brusch et al., 2010). In recent years, third-generation cephalosporins have been used in regions with high fluoroquinolone resistance rates, particularly in South Asia and Vietnam. Unfortunately, sporadic resistance has been reported, so it is expected that these will become less useful over time (Brusch et al., 2010). Threlfall and Ward (2001) have reported that S. typhi with decreased sensitivity to ciprofloxacin is endemic in several Asian countries, and incidence of such strains has increased in travelers from the Indian subcontinent. They suggested 3rd generation cephalosporins such as ceftriaxone or cefotaxime as possible alternatives, and in their study, it was assured that all strains were sensitive to these drugs. Reduced susceptibility to fluoroquinolones has become a major problem mostly in Asia (Hawkey, 2003; Mushtaq, 2006). Outbreak with such strains affected eight thousand people and killed 150 people in Tajikistan in 1997. Isolates responsible for this outbreak of fluoroquinolone resistance had their MIC to be ten times of those fully susceptible to the drug. This decreased susceptibility is resulting in treatment failure (Mushtaq, 2006). At the moment the emergence of resistant strains to two major second line drugs like ciprofloxacin and ceftriaxone is posing a major problem (Ackers et al., 2000). Antibiotic resistance is a moving target and reports are quickly outdated, thus recommendation regarding antibiotic treatment must not be taken with a grain of salt (Brusch et al., 2010), if the origin of the infection is unknown, the combination of a first-generation fluoroquinolone and a third-generation cephalosporin should be used (Brusch et al., 2010). Based on these facts, this research was aimed to carry out molecular characterisation of some multidrug resistant clinical isolates of Salmonella enterica from human origin from Southeast part of Nigeria and to detect the resistance determinants, mutations in the gyrA and parC genes and possible variations of resistant gene involved. 2 1.2 Justification for this study: There has been an increase in the multidrug-resistance of Salmonella enterica, the causative agent of typhoid fever in Nigeria (Yah, 2007). This organism also causes other diseases such as enteric fever, salmonella septicaemia and gastroenteritis, thereby indicating the organism as an important human pathogen of these nosocomial infections (Cheesbrough, 2006). The increasing clinical importance of these infections has led to the search for information on the prevalence of multidrug resistance pathogens such as Salmonella enterica in Africa including Nigeria (Yah, 2010). Notably, high level of antimicrobial resistance and multidrug resistant Salmonella enterica isolates have not been reported in the Southeast region of Nigeria, thereby leading to scarcity of published or documented data in this region. Amongst the drugs implicated in Nigeria and other parts of the world were chloramphenicol and co- trimoxazole which have been the fronline drugs for the treatment of the infections caused by S. enterica, and resistance to these drugs has become a real challenge especially in the developing world (Brusch et al., 2010). Currently, the generations of antibiotics implicated were the quinolones (fluoroquinolone) and cephalosporins (Mustaq, 2006; Yah, 2007). Hence, the need to determine and characterize the genetic determinants associated with the resistance to the currently implicated drugs using phenotypic and molecular techniques in the Southeast Nigeria. 1.3 Overall aim for this study The overall aim of this study is to characterize the resistance determinants of the multidrug resistant clinical isolates of Salmonella enterica of human origin, in other to identify the types of genes involved in their acquisition and their possible locations in the organism from the Southeast part of Nigeria. 3 1.4 Specific objectives for the study 1. To determine the antibiogram and Minimum Inhibitory Concentrations (MIC) of antibiotics mostly used against Salmonella enterica infections in Southeast Nigerian hospitals. 2. To determine and characterize the Salmonella isolates producing Extended Spectrum Beta-lactamase (ESBL) enzymes such as bla-CTX-M, bla-SHV, bla-TEM-1 and plasmid-mediated quinolone resistant gene such as QnrB gene type. 3. To determine the Quinolone Resistance Determining Regions (QRDRs) in gyrA and ParC of Salmonella enterica. 4. To determine the resistant plasmid and genetic location of the different resistance determinants harbored by the resistant Salmonella enterica. 4 CHAPTER TWO LITERATURE REVIEW 2.1 Characteristics of Salmonella enterica Salmonellae are Gram-negative, motile aerobic rods that characteristically ferment glucose and manose but fail to ferment lactose or sucrose, they are pathogenic for humans or animals by the oral route (Cheesbrough, 2006). Salmonellae grow readily on simple media. The selective media for the organisms from faces contain brilliant green, cholate, selenite, tetra chlorate, or citrate to suppress the growth of coliforms. Formerly classified as separate species, DNA hybridization studies have now shown that all pathogenic Salmonellae belong to a single species, Salmonella enterica which is subdivided into 7 subspecies (subsp). S. enterica subsp. enterica has over 2000 serovars which can cause disease in humans (Cheesbrough, 2006). For convenience the Serovars (first letter in capital) are written in an abbreviated form, e.g, the accepted abbreviation for S.enterica subsp. Enteric Serovar Typhi is S. typhi (italics is not used for the serovar) (Cheesbrough, 2006). They usually use citrate as their sole carbon source for growth and produce hydrogen sulphide. Multidrug-resistant Salmonella typhi has become a problem in a developing country like Nigeria and in developed countries like the United States and United Kingdom. This chronic infection appears to be peculiar to isolates from temperate climate zones. Strains native to Africa do not give rise to chronic infection. The commonest group of carriers are women in their 50's with gallstones (Brusch et al., 2010). Typhoid fever appears to be a disease that has been associated with man since close to the first appearance of hominids and it may have first infected human ancestors anywhere from 200,000 to two million years ago. The bacterium can survive in contaminated water, but does not have any host other than man. There are over 1,000 different strains of the bacterium of which only a few cause typhoid (Mushtaq, 2006). Salmonella typhi, a potentially lethal organism was successfully managed with the introduction of chloramphenicol. Since then emergence of resistant strains began and now Multidrug resistant Salmonella typhi (MDRST) has become a real challenge especially in the developing world. There have been reports from different parts of the world about resistance pattern. Most reports from 5 developing countries are showing MDRST strain (Ackers et al., 2000). The original clinical indication of chloramphenicol was in the treatment of typhoid, but with the universal presence of multi-drug resistant Salmonella typhi it is seldom used for this indication except where the organism is known to be sensitive (Mandal et al., 2010). Chloramphenicol susceptibility test following disk diffusion is not enough for its re- selection in the treatment of typhoid fever, (Gautam et al., 2002) and therefore, it is imperative to compare the Minimum Inhibitory Concentration values (MICs) of chloramphenicol for the sensitive isolates with chloramphenicol MICs for the resistant isolates. The genes for antibiotic resistance in S. typhi and S. paratyphi are acquired from Escherichia coli and other Gram-negative bacteria via plasmids (Brusch et al., 2010). The plasmids contain cassettes of resistance genes that are incorporated into a region of the Salmonella genome called an integron. Some plasmids carry multiple cassettes and immediately confer resistance to multiple classes of antibiotics. This explains the sudden appearance of MDR strains of S. typhi and S. paratyphi, often without intermediate strains that have less-extensive resistance (Brusch et al., 2010). The primary target of the organism is the intestine. It attacks through tissues that are a part of the immune system called Peyer's patches. These tissues on the inner surface of the intestine are normally the first line of defense against food and water-borne infection, but the typhoid bacterium subverts them (Cheesbrough, 2006). It does not have the cell surface features that normally trigger a defensive response but it can fool the cells of the patches to take it in without attacking it. The infected tissues become inflamed and the intestine begins to lose function, leading to diarrhoea or constipation. There may be bleeding of the intestine leading to bloody stools. In severe cases, the disease may punch holes in the intestine leading to peritonitis (infection of the abdominal cavity) and death (Cheesbrough, 2006). Even if the patient recovers from the infection, it may leave residual damage, such as the formation of attachments of the damaged areas of the intestine to the abdominal wall, or the development of a chronic infection that leads to the patient becoming a carrier (Cheesbrough, 2006). A closely related bacterium Salmonella typhimurium causes a similar disease in the mouse, but does not affect man and the combination of the pathogen and the mouse is used as a model to study the human disease. Resistance to fluoroquinolones is evolving in an ominous direction. 6 Fluoroquinolones target DNA gyrase and topoisomerase IV, bacterial enzymes that are part of a complex that uncoils and recoils bacterial DNA for transcription (Brusch et al., 2010). Patients in the United Kingdom and United States detected strains resistant to all three first-line drugs (ampicillin, trimethoprim-sulphamethoxazole and chloramphenicol) were reported to be infected with Multi-drug Resistant Salmonella typhi (MDRST) (Brusch et al., 2010), while ciprofloxacin and ceftriaxone resistant isolates were reported as a result of traveling to the developing world especially Southeast Asia (Ackers et al., 2000). In high-prevalence areas outside the areas mentioned above, the rate of intermediate sensitivity or resistance to fluoroquinolones is 3.7% in the Americas (P =.132), 4.7% (P =.144) in sub-Saharan Africa, and 10.8% (P =.706) in the Middle East. Therefore, for strains that originate outside of south or Southeast Asia, the WHO recommendations may still be valid—that uncomplicated disease should be treated empirically with oral ciprofloxacin and complicated typhoid fever from these regions should be treated with intravenous ciprofloxacin (Brusch et al., 2010). A single-point mutation gyrA confers partial resistance. If a second gyrA point mutation is added, the resistance increases somewhat. However, a mutation in parC added to a single gyrA mutation confers full in vitro resistance to first-generation fluoroquinolones. Clinically, these resistant strains show a 36% failure rate when treated with a first-generation fluoroquinolone such as ciprofloxacin. The risk of relapse after bacterial clearance is higher in both partially and fully resistant strains than in fully susceptible strains (Brusch et al., 2010). According to Mushtaq (2006), susceptibility testing on 350 isolates reported that 16% of the isolates were MDRST. No resistance was reported on ciprofloxacin, ceftriaxone, gentamicin and kanamicin (Ackers et al., 2000). Nadeem and colleagues reported from Quetta, Pakistan that 69% of isolates were found to be MDRST (Nadeem et al., 2002). In yet another report from Bahawalpur, Pakistan: 53.8% of isolates were found MDRST and all strains were sensitive to fluoroquinolones and third-generation cephalosporins (Munir et al., 2001). In their study, 28 isolates were checked against all three first-line anti-salmonella drugs of which 18 (65%) isolates turned out to be MDRST (Munir et al., 2001). The third-generation fluoroquinolone, gatifloxacin, appears to be highly effective against all known clinical strains of S. typhi both in vitro and in vivo. 7 However, any two of a number of gyrA mutations, when added to the parC mutation, confer full in-vitro resistance. Although such a combination is yet to be discovered in- vivo, all of these mutations exist in clinical (Brusch et al., 2010). Studies by Ackers et al. (2000) and Nadeem et al. (2002) demonstrated all the isolates including MDRST were fully susceptible to ciprofloxacin and ceftriaxone, while data in Mushtaq (2006) reported that out of 18 MDRST four of the isolates were found resistant to ciprofloxacin, four resistant to ceftriaxone and two of them were found resistant to both of these drugs. The overall resistance to ciprofloxacin and ceftriaxone was found to be 19.2% and 17.9% respectively (Mushtaq, 2006). Threlfall and Ward (2001) reported decreased sensitivity to ciprofloxacin and suggested possible alternatives as ceftriaxone and cefotaxime, and reassured that organisms were fully susceptible to these drugs. According to Mushtaq (2006) resistance to ceftriaxone was 21.1% out of 76 isolates that were found resistant to cefotaxime as well. Cases of Salmonella typhi resistant to ciprofloxacin and ceftriaxone have been reported from Bangladesh (Mushtaq, 2006). Pattern of S. typhi resistance is changing rapidly. MDRST and strains resistant to ciprofloxacin and ceftriaxone are a major threat in developing world (Mushtaq, 2006). In some areas strains fully susceptible to all first-line anti-salmonella drugs (ampicillin, trimethoprim-sulphamethoxazole and chloremphenicol) have re-emerged (Mushtaq, 2006). Mushtaq (2006) reported that 65 isolates were tested against two or more first- line anti-salmonella drugs and found that they were sensitive to at least two first-line drugs, and only one isolate was found sensitive to all three first-line drugs. The author also reported that resistance to trimethoprim/sulphamethoxazole and chloramphenicol was found to be 94.2% and 65.3% respectively. Similarly, resistance to cefuroxime was 53.1%, cefaclor 49.2% and amoxiclavulanic acid 42.5% of the isolates (Mushtaq, 2006). Cui et al. (2008) in their study reported a high incidence of quinonlone-resistant S. typhimurium isolates might have been affected by several factors. First, patients infected by antimicrobial drug-resistant S. typhimurium strains had higher rates of hospitalization than the patients infected by susceptible strains (Martin et al., 2004 ; Varma et al., 2005), and the isolates in their study were from a university-affiliated medical centre that usually treats patients with severe illness (Cui et al., 2008). In China, inappropriate prescriptions 8 might be even more common because antimicrobial drug prescriptions in hospitals were a source of profit (Cui et al., 2008). Cui et al., (2008), stated that though they do not have the patient antimicrobial drug–use information, but the easy access to antimicrobial drugs raises the possibility that before the collection of stool specimens, the outpatients might have taken fluoroquinolones after the onset of the illness. Secondly, because livestock products are a common source of salmonellosis (Cui et al., 2008), the dissemination of ciprofloxacin-resistant S. typhimurium might have been facilitated by the use of fluoroquinolones in livestock production. Lastly, use of other antimicrobial drugs, such as ampicillin, gentamicin, or streptomycin, may also contribute to the spreading of fluoroquinolone-resistant S. typhimurium because all the ciprofloxacin-resistant isolates were also resistant to 8-11 additional antimicrobial drugs, they used in their study (Cui et al., 2008). Hakanen et al. (2001) stated that when looking for reasons for the rapidly increased quinolone resistance in travelers' Salmonella isolates, three issues must be considered, such as; transferable resistance, mutational resistance, and clonal spread. However, transferable fluoroquinolone resistance appears to be rare in bacteria in vivo (Hakanen et al., 2001). Thus, either clonal spread or resistance due to mutations in chromosomal genes remains the potential mechanism accounting for the high level of reduced fluoroquinolone susceptibility in Southeast Asia (Hakanen et al., 2001). The emergence of mutation-based resistance may be fostered by selection pressure caused by the use of antimicrobial agents in either human medicine or agriculture (Hakanen et al., 2001). In Europe, none of the fluoroquinolones licensed for humans is approved for animal use, although many other quinolone preparations are allowed for the treatment of livestock, poultry, and fish. The policy is stricter in the United States, where the only quinolone licensed for food animals is enrofloxacin (Hakanen et al., 2001). According to Hakanen et al. (2001), no conclusions can be drawn on a potential link between the reduced fluoroquinolone susceptibility of salmonellae and the use of quinolones in animal husbandry in the area studied. In direct contrast, treatment with first-generation quinolones (e.g., nalidixic acid) is known to cause rapid emergence of resistance in the family of Enterobacteriaceae (Hakanen et al., 2001). Another topic of major interest involves the potential influence of antimicrobial use in travelers on infections with 9 quinolone-resistant Salmonella strains (Hakanen et al., 2001). However, strains with reduced fluoroquinolone susceptibility are currently not identified in any microbiological laboratory worldwide according to current CLSI recommendations, with MIC 4 µg/mL of ciprofloxacin as a breakpoint for resistance (CLSI, 2007). These breakpoint values are considered adequate, as the clinical importance of the reduced fluoroquinolone susceptibility of salmonellae remains unproven. Nevertheless, it is suggested that laboratories worldwide aim at recognizing these less susceptible strains, to reveal their eventual clinical impact and that laboratories use the nalidixic acid screening test (CLSI, 2007) or the E-test to aid identification (Hakanen et al., 2001). The CLSI (2014), reported in 2013, that the ciprofloxacin, levofloxacin, and ofloxacin MIC interpretive criteria (breakpoints) was revised for Salmonella and the disk diffusion breakpoints for ciprofloxacin, in part to better detect these fluoroquinolone resistance mechanisms in Salmonella spp. Though, at present, no levofloxacin or ofloxacin disk diffusion breakpoints for Salmonella have been established by the CLSI (CLSI, 2014; Deak et al., 2015). The recent professional guideline for the treatment of typhoid fever in South Asia was issued by the Indian Association of Pediatrics (IAP) in October, 2006 (Brusch et al., 2010). Although these guidelines were published for pediatric typhoid fever, the authors felt that they are also applicable to adult cases. For empiric treatment of uncomplicated typhoid fever, the IAP recommends cefixime and, as a second-line agent, azithromycin. For complicated typhoid fever, cefriaxone was recommended. Aztreonam and imipenem are second-line agents for complicated cases (Brusch et al., 2010). The authors believe that the IAP recommendations have more validity than the WHO recommendations for empiric treatments of typhoid fever in both adults and children (Brusch et al., 2010). 2.2 Salmonella enterica infections in man Salmonella infections represent a major health problem worldwide, particularly in the developing countries where they are recognized as the most frequent cause of morbidity and mortality (Yah, 2010). Mortality is highly associated with infants under one year of age (South Australia Department of Health, 2008). The impact of lives lost, together with 10 the high costs to local public health care systems, makes prevention and control a priority (Yah, 2010). Antibiotic resistant Salmonella are of global concern because they affect both developed and developing countries due to increased international travel (Yah, 2010). Antimicrobial drug resistance has become increasingly common in S. enterica, which can complicate therapy (Ajibade et al., 2010). These concerns have been further reinforced in recent years by the emergence of antimicrobial resistance among the major groups of the enteric pathogens. The presence of antibiotic- resistant bacteria from hospitalized patients has been documented (Yah, 2010). Reports have shown that the resistance of gastroenteric Salmonella strains to these antimicrobial agents is in large part due to the production of extended-spectrum β- lactamases (ESBLs) encoded on plasmids, as well as on the chromosome (David and Frank, 2000); Yujuan et al. (2006) and Yah (2010). According to Momtaz et al. (2002), the development of S. typhi strains that are resistant to antibiotics historically used to treat S. typhi infection has forced physicians to prescribe fluoroquinolones or third-generation cephalosporins. So far, data from Momtaz et al. (2002) study showed that neither quinolone resistance nor third-generation cephalosporin resistance has emerged in Egypt (Momtaz et al., 2002). However, extrapolation from data in the literature suggests that quinolone resistance is likely to develop unless use of this drug class is restricted (Momtaz et al., 2002). Also Momtaz et al. (2002) stated that resurgence in chloramphenicol-susceptible S. typhi strains has been reported in recent years. They further stated that eighty percent of Indian strains isolated in 1991-1993 were susceptible to chloramphenicol, compared with only 33.3% of such strains isolated in 1990-1991 (P < .001). This observation was attributed to the restricted use of chloramphenicol for 2.5 years (Momtaz et al., 2002). 2.2.1 Antigenic structure of S. enterica While Salmonellae are initially detected by their biochemical characteristics, groups and species must be identified by antigenic analysis. Like other enterobacteriace, Salmonellae possess several O antigens and different H antigens (Adeleke et al., 2006). Some Salmonellae have capsular antigens, referred to as Vi, which may be associated with 11 virulence (Cheesbrough, 2006). Organisms may possess H antigens and become immotile and loss of O antigen is associated with change from smooth to rough. Colony which forms Vi antigen may be lost partially or completely (Cheesbrough, 2006; Adeleke et al., 2006). 2.2.2 Pathogenesis of S. enterica Salmonella typhi and perhaps S. paratyphi A and S. schottmulleri (formerly S. paratyphi B) are primarily infective for humans, and infection with these organisms implies acquisition from a human source (Cheesbrough, 2006). The rest majority of salmonellae, however, are chiefly pathogenic in animals (e.g poultry, pigs, rodents, cattle, pets and in many others) that constitute the reservoirs for human infections (Cheesbrough, 2006). The organisms are acquired through the oral route, usually with contaminated food and 5 8 drinks. The mean infective dose for humans is 10 - 10 Salmonellae to produce clinical and subclinical infection. Among the host factors that contribute to resistance to Salmonellae infection are gastric acidity, normal intestinal microbial flora and local intestinal immunity (IgA). In humans, salmonellae produce 3 main types of disease (enteric fever, Salmonellae septicaemia and gastroenteritis) but mixed forms are also frequent (Cheesbrough, 2006). 2.2.3 The enteric fever Enteric fever including typhoid and paratyphoid fever is caused by S. enterica serovar. typhi. The disease usually begins insidiously after an incubation period of 7 - 14 days, with malaise, anorexia and headache, followed by the onset of fever. The ingested organisms multiply in the gastrointestinal tract, and some enter the intestinal lymphatics, from which they are disseminated throughout the body by the blood stream and are excreted in urine (Cheesbrough, 2006). The bile is a good culture medium for S. typhi, and so luxuriant growth occurs in the biliary tract and provides a continued flow of organisms into the small bowel (flat patches of lymphoid tissue situated in the small intestine but mainly in the ileum, seat of infection in typhoid fever) where they tend to localize in the payer’s patches. 12 Their ability to persist in the biliary tract may result in a chronic carrier state, with continued excretion in the faeces (Cheesbrough, 2006). Fever often increases in a step- like manner and is accompanied by relative bradycardia (slow rate of heart contraction resulting in slow pulse rate). Diarrhoea is usually absent. Cough and signs of bronchitis may be present, “rose spots”, which last for only a few days, may appear in the truck, and sphenomegaly and leucopenia are common (Cheesbrough, 2006). After the third week the fever usually subsides. In fatal case the most prominent (excessive formation of cells.) lesion found at autopsy is lymphoid hyperplasia, ulcerations in the payer’s patches may lead to intestinal haemorhages or perforation of the bowel (Cheesbrough, 2006). In active lesions, bacilli are often detectable in the phagocytic monocular cells, where they can multiply intracellular bacilli seen to be protected from the bacteriolytic action of specific Antibody, which appears in the blood long before the disease subsides. The paratyphoid fever caused by S. parartyphi A and B is usually milder and have shorter incubating period (1 - 10 days). Bacterial infection occurs early; fever usually lasts for 1- 3 weeks and rose spots are rare (Cheesbrough, 2006). 2.2.4 Salmonella septicaemia It is characterized by high intermitent fever, and focal suppurative lesions may develop almost anywhere in the body including the brainy tract, kidneys, heart, spleen, meninges, joints and lungs. Prolonged septicaemia of this type is most commonly caused by S. choleraesuis (Cheesbrough, 2006). 2.2.5 Gastroenteritis This is most commonly the kind of Salmonellae infection, primary confined to the gastrointestinal tract. The most frequent cause in the U.S is S. typhimurium (Cheesbrough, 2006). Symptoms begin 8 - 48hour after the consumption of contaminated food, with diarrhea, coughing from mild to fulminate (developing quickly with rapid termination) from the sudden and or let or set (food poisoning). Headache, chills and abdominal pain are followed by nausea, vommitting, and diarrhoea, accompanied by fever lasting from 1 - 4 days. Blood cultures are rarely positive, but the organisms can usually be cultured from faeces (Cheesbrough, 2006). 13 2.2.6 Carriers Following active salmonellosis, the organisms occasionally become established in the 6 9 host, who then continues indefinitely to excrete as many as 10 - 10 S. typhi per gram of faeces. The source is usually a chronic superlative focus in the bilary tract. When treated with broad – spectrum antibiotics carriers may come down with active Salmonellae disease (Cheesbrough, 2006). 2.2.7 Laboratory diagnosis of Salmonella infection A diagnosis of salmonella’s infection is made by isolation of this organism. Isolation from blood or urine establishes the diagnosis, but a salmonellae organism isolated from the faeces is not necessarily the cause of the individual’s illnesses. Blood culture is often positive in the first week of the disease. Bone marrow cultures may be useful, urine cultures may be positive after the second week. Stool specimens must be taken repeatedly, in enteric fevers, the stools are positive from the second and third weeks or, in gastroenteritis, during the first week (Cheesbrough, 2006). 2.2.8 Immunity to Salmonella infection Infection with S. typhi, S. Paratypli and S. chottmulleri usually confers certain dose of immunity. However, relapses may occur in 2 - 3 weeks after recovery in spite of antibodies (IgA) which may prevent attachment of salmonellae to intestinal epithelium (Cheesbrough, 2006). 2.2.9 Treatment of Salmonella infection Definitive treatment of typhoid fever (enteric fever) is based on susceptibility. As a general principle of antimicrobial treatment, intermediate susceptibility should be regarded as equivalent to resistance. Between 1999 and 2006, 13% of S. typhi isolates collected in the United States were multidrug resistant (Multidrug-resistant S. typhi is, by definition, resistance to the original first-line agents, ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole.) as stated by Brusch et al. (2010). 14 Until susceptibilities are determined, antibiotic prescription should be empiric, for which there are various recommendations (Brusch et al., 2010). For typhoid fever, and salmonellae septicemia, chloramphenicol has long been the drug of choice, but chloramphenicol resistant strains have appeared (Galanis et al., 2006). Based on antibiotic sensitivity testing parenteral ampicillin and trimethoprime-sulfamethaxazole are usually effective alternatives. Plasmid mediated resistance to many antimicrobials has been observed (Helms et al., 2002). Response to therapy is usually rapid, but because the organisms tend to survive within phagocytic cell, relapses frequently occur unless the patient is treated for at least 2 weeks (Cheesbrough, 2006). Antibiotics are not indicated in salmonellae gastroenteritis (except in the very young and those over 60) since the disease is brief but limited to the gastroenteritinal tract. In addition, the unnecessary use of antibiotics prolongs salmonellae excretion, promotes the incidence of the carrier state, and favours the acquisition of resistance by the infection strain (Cheesbrough, 2006). In schistomiasis endemic areas there is a high indence of chronic S. typhi and S. paratypi A. infections and carriers (Cheesbrough, 2006). The salmomellae colonize adult schistosome flukes (protected from antibiotics). An immume complex disorder of the kidneys can occur in those with urinary schistomiasis (nephrotyphoid), characterized by fever, oedema, marked albuminuria and haematuria. Infection with S. typhi can also cause osteomyelitis and typhoid arthritis particularly in those with sickle cell disease and thalassaemia (Cheesbrough, 2006). This can be biochemically differentiated from other Salmonellae by being citrate negative, not producing gas and forming only small amount of H2S. Isolates of S. typhi can be identified serologically (Cheesbrough, 2006). In eliminating the carrier state the bactericidal ampicillin is much more effective than the bacteriostatic chloramphenicol. Prolonged treatment with large doses of ampicillin is effective in 60% - 80% of cases. In carriers who relapse after one or more course of therapy cholecytectomy truncates the carrier state in 98 out of 100 cases (Galanis et al., 2006). 2.2.10 Prevention of Salmonella infection Most important in preventing the man to man transmissions of typhoid fever have been; i) Proper sewage disposal and Pasteurization of milk 15 ii) Maintenance of unpolluted water supplies Exclusion of chronic carriers as food handlers S. typhi infects only humans and its control is relatively feasible. The incidence of typhoid fever in them has been declining steadily (eg from 5593 cases in 1942 to 398 in 1977). Eradication of human disease due to salmonellae that infect animals as well as man is difficult and also elimination of the animal reservoir is impossible (Zhao et al., 2006). Domestic fouls probably constitutes the largest reservoir of almonellae having been isolated from 4% of apparently healthy chickens or turkeys and from many other domestic and wild animals including; turtles meat and pooled preparations of dived eggs. Practical ways for controlling salmonellae in animals only recently started and improved techniques of food processing should lower the incidence of human infections (Gautam et al., 2002). 2.3 Salmonella infections associated with animal Salmonella is recognized as one of the major food-borne pathogens in the United States, causing an estimated 1.4 million cases of illness, approximately 20,000 hospitalizations, and more than 500 deaths annually (Paveen et al., 2007). Salmonellae are a common cause of foodborne disease worldwide (Galanis et al., 2006). Although a growing number of human salmonellosis cases are associated with contaminated fruits and vegetables, traditionally, illness has been linked with consumption of contaminated food of animal origin, especially poultry and poultry products (Roy et al., 2002). More problematic is the fact that antimicrobial resistance, in particular multidrug resistance (MDR), is being increasingly identified among numerous Salmonella serotypes recovered from animals and humans worldwide (Zhao et al., 2006). The levels and degree of resistance vary globally and are influenced by antimicrobial use practices in humans and animals and geographical variations in the epidemiology of Salmonella infections (Zhao et al., 2006). Salmonella isolates showing resistance to clinically important antibiotics have been reported since the early 1960s, when most of the reported resistance was limited to a single antibiotic (Paveen et al., 2007). However, since the mid-1970s, there has been an increasing trend of Salmonella isolates exhibiting MDR phenotypes worldwide. The recovery of antimicrobial-resistant Salmonella in foods of animal origin has raised 16 concerns that the treatment of human salmonellosis may be compromised because antimicrobial-resistant strains appear to be more often associated with severe disease than are susceptible isolates (Helms et al., 2002); of significant concern is the isolation of Salmonella exhibiting decreased susceptibility to fluoroquinolones (e.g., ciprofloxacin) and extended-spectrum cephalosporins (e.g., ceftiofur and ceftriaxone) because these two antimicrobial agents are important in treating Salmonella infections in adults and children, respectively (Gupta et al., 2003). The majority of these antimicrobial-resistant phenotypes in Salmonella and other pathogens are gained from extrachromosomal genes that may impart resistance to an entire antimicrobial class. In recent years, a number of these resistance genes have been associated with large transferable plasmids on which may be other DNA mobile elements, such as transposons and integrons. Recent data indicate that different resistance determinants can amass in linked clusters, such that antimicrobials of a different class or substances such as disinfectants or heavy metals may select for MDR in bacteria (Harbottle et al., 2006). Although resistance, in particular MDR, appears to be most serious in certain serotypes, this situation may be shifting. Thus, there is a continuing need for increased surveillance of antimicrobial- resistant phenotypes in Salmonella isolates of animal and human origin on a global basis. The role of meat and poultry products in the dissemination of antimicrobial-resistant zoonotic bacterial pathogens is well documented (Larkin et al., 2004), though, the hygienic standards for meat production was quites high (Paveen et al., 2007). In most developed countries faecal contamination of meat products cannot be completely prevented. Procedures such as handling during processing also may contribute to cross- contamination among carcasses (Paveen et al., 2007). Recently, several investigators suggested that processing conditions may play a significant role in promoting and influencing the selection of pathogens, including antimicrobial-resistant variants (Logue et al., 2003). However, the factors that contribute to this selection have yet to be fully evaluated. Several studies have been conducted on the prevalence and antimicrobial resistance of Salmonella in processed poultry, poultry products, and poultry processing plants (Jam and Chen, 2006; Paveen et al., 2007). 17 2.4 Salmonella and the quinolones The quinolone class of antibiotics comprises a relatively large and expanding group of synthetic compounds, and has since their discovery in the early 1960s evolved to become important and effective agents in the treatment of a wide range of bacterial infections (Haugum et al., 2006). The quinolones inhibit the bacterial enzymes, DNA gyrase and DNA topoisomerase, both of which are essential for bacterial DNA replication (Corbett . et al., 2004). Bacterial type II DNA topoisomerases are A2B2 hetero tetramers (Corbett et al., 2004). DNA gyrase is composed of two GyrA and two GyrB subunits, encoded by gyrA and gyrB (Wang, 2002; Haugum et al., 2006) and topoisomerase by two ParC and two ParE subunits, encoded by parC and parE Type. DNA topoisomerases catalyse the ATP-dependent transport of one intact DNA double helix which give several topological transformations, including relaxation of positively or negatively supercoiled DNA and decatenation unlinking of double strand DNA rings (Wang, 2002). Quinolones act by binding to complexes that form between DNA and gyrase or topoisomerase and formation of this quinolone-enzyme-DNA complex that contains broken DNA inhibits DNA synthesis. Important mechanisms for quinolone resistance are mutations accumulating in the genes encoding DNA gyrase and topoisomerase gyrA, gyrB, parC and parE (Haugum et al., 2006; Hawkey, 2003). In Salmonella and Escherichia coli, the majority of mutations in DNA gyrase are found between residues 67 and 106 in gyrA, in a region called the quinolone resistance-determining region (QRDR). While some mutations in parC in salmonellae have been found (Eaves et al., 2004), between residues 57 and 84, they may only be required to achieve high-level resistance (Hopkins et al., 2005). Mutations in gyrB and parE are considered rare in salmonellae (Hopkins et al., 2005; Haugum et al., 2006). Mutations in gyrB in salmonellae are found between residues 420 and 464, and mutations in parE in salmonellae are found between residues 453 and 512 (Hopkins et al., 2005; Haugum et al., 2006). Other reported mechanisms for quinolone resistance are active efflux mechanisms, and decreased outer membrane permeability. In E. coli, the AcrAB-TolC system is an efflux system involved in multi- drug resistance (Cloeckaert and Chaslus-Dancla, 2001; Hopkins et al., 2005; Haugum et al., 2006). Over-expression of the AcrAB efflux pump has also been shown in strains of Salmonella enterica serovar Typhimurium S. typhimurium with reduced susceptibility to 18 fluoroquinolones (Cloeckaert and Chaslus-Dancla, 2001). There have been some reports on plasmid-mediated quinolone resistance, but only in Klebsiella and E. coli (Hopkins et al., 2005; Haugum et al., 2006). Among salmonellae, and especially Salmonella enterica serovars Enteritidis, Hadar, typhimurium and Virchow, there have been reports of increase in quinolone resistance (Threlfall, 2002). In a previous study it was discovered that a geographically dependent distribution of gyrA mutation at codon 83 and 87 in S. hadar (Lindstedt et al., 2004). Earlier studies have observed that in salmonellae, the relative frequency of different mutations in gyrA was dependent on the quinolone antibiotic used for selection (Levy and Marshall, 2004; Haugum et al., 2006), and that the position and type of amino acid substitution in gyrA varied with the serovar (Haugum et al., 2006). Among S. typhi isolates obtained in the United States between 1999 and 2006, 43% were resistant to at least one antibiotic. Nearly half of S. typhi isolates found in the United States now come from travelers to the Indian subcontinent, where fluoroquinolone resistance is endemic (Brusch et al., 2010). The rate of fluoroquinolone resistance in the South and Southeast Asia and, to some extent, in East Asia is generally high and rising (Brusch et al., 2010). Susceptibility to chloramphenicol, TMP-SMZ, and ampicillin in these areas is rebounding. In Southeast Asia, MDR strains remain predominant, and some acquired resistance to fluoroquinolones by the early 2000s. Fluoroquinolones have become the first-line drugs for the treatment of typhoid fever (Hirose et al., 2002). Fluoroquinolones are active drugs against isolates of the Salmonella species (Hakanen et al., 2001). There are several reports, however, of treatment failures when these antimicrobials have been used to treat Salmonella infections caused by strains with reduced fluoroquinolone susceptibility (Hakanen et al., 2001). However, some Salmonella enterica serovar typhi strains with decreased susceptibilities to fluoroquinolones have been already reported (Hirose et al., 2001; Deak et al., 2015). The emergence and spread of these organisms have been reported in developing countries (Hirose et al., 2002). There is evidence that the incidence of strains that are resistant to nalidixic acid with decreased susceptibilities to the most recent fluoroquinolones used for the treatment of typhoid fever is on the increase. In most strains, the acquired fluoroquinolone resistance was attributed to mutations in the genes encoding DNA gyrase 19 (gyrA, gyrB) (Hirose et al., 2002) or DNA topoisomerase IV (parC, parE) (Hirose et al., 2002). According to Hirose et al. (2002), the mutations responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar typhi and serovar Paratyphi A were investigated and the sequences of the quinolone resistance- determining region (QRDRs) of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes. According to Hirose et al. (2002), these findings indicate that gyrA mutations are of principal importance for the fluoroquinolone resistance of serovars typhi and Paratyphi A. Double mutations at positions 83 and 87 of the gyrA amino acid sequence were also reported in clinical isolates of serovar Schwarzengrund, which caused nosocomial infections in the United States and which exhibited ciprofloxacin resistance (Hirose et al., 2002). Although strains with high-level fluoroquinolone resistance due to double mutations at codons 83 and 87 in the gyrA amino acid sequence have not been found in clinical isolates of serovars typhi and Paratyphi A, several cases of the failure of treatment for typhoid fever due to strains with decreased susceptibilities to fluoroquinolones have been reported (Hirose et al., 2002). The difference in fluoroquinolone resistance between two closely related species may be explained by differences in outer membrane permeabilities for fluoroquinolones and differences in active efflux activities (Hirose et al., 2002). Fluoroquinolone resistance in Salmonella enterica is of clinical importance because ciprofloxacin is the drug of choice for treating invasive human salmonellosis (Eaves et al., 2004). Fluoroquinolone resistance in S. enterica is usually mediated by at least one mutation in a DNA topoisomerase gene gyrA resulting in elevated ciprofloxacin and levofloxacin MICs (0.12 to 0.5 µg/ml) according to Deak et al. (2015). However, in clinical human and veterinary isolates of Salmonella spp., mutations are usually confined to gyrA. Whilst a single mutation in gyrA on its own is not sufficient for clinical resistance to fluoroquinolones, a gyrA mutation is a good marker indicating that fluoroquinolones should not be chosen for treating the respective infection (Randall et al., 2005). According to Gaind et al. (2006) analysis revealed that in S. typhi and paratyphi A, a single gyrA mutation (Ser-83-->Phe or Ser-83-->Tyr) was associated with reduced 20 susceptibility to ciprofloxacin (MICs 0.125-1 mg/L); an additional mutation in parC (Ser- 80-->Ile, Ser-80-->Arg, Asp-69-->Glu or Gly-78-->Asp) was accompanied by an increase in ciprofloxacin MIC (> or = 0.5 mg/L) (Gaind et al., 2006). They further stated that three mutations conferred ciprofloxacin resistance: two in gyrA (Ser-83-->Phe and Asp-87-->Asn or Asp-87-->Gly) and one in parC. This is the first report of parC mutations in S. typhi. Ciprofloxacin-resistant S. typhi and S. paratyphi A, differed in their MICs and mutations in gyrA and parC. Moreover S. typhi harboured a 50 kb transferable plasmid carrying a class 1 integron (dfrA15/aadA1) that confers resistance to co- trimoxazole and tetracycline but not to ciprofloxacin (Gaind et al., 2006). Pulse field gel electrophoresis (PFGE) revealed undistinguishable XbaI fragment patterns in ciprofloxacin-resistant S. typhi as well as in S. paratyphi A isolates and showed that ciprofloxacin-resistant S. typhi have emerged from a clonally related isolate with reduced susceptibility to ciprofloxacin after sequential acquisition of a second mutation in gyrA (Gaind et al., 2006). The presence of a plasmid-borne integron in ciprofloxacin-resistant S. typhi may lead to a situation of untreatable enteric fever (Gaind et.al., 2006). According to Haugum et al. (2006), reporting their study on the effect of quinolone antibiotics and chemicals on mutation types in Salmonella enterica serovars Enteritidis, confirmed gyrA codon 83 and codon 87 as the main targets for mutations in S. enteritidis. According to Hakanen et al. (2001), based on their preliminary report on fluoroquinolone, stated an increasing trend in quinolone resistance among Salmonella isolates classified as being of foreign origin in Finland. Among all 1,210 Salmonella isolates, 78 (6.4%) exhibited reduced susceptibility to ciprofloxacin (MIC greater than 0.125µg/ml) (Hakanen et al., 2001). Hakanen et al. (2001) conclusively reported that their analysis clearly showed that in the era of frequent international connections, microbes may be easily transmitted from one place to another. Correspondingly, factors furthering the emergence and spread of antimicrobial resistance in any country may soon have an impact on resistance of bacterial pathogens, or even of normal human flora, in faraway regions, even different continents. On this basis, the emergence of antimicrobial resistance in any part of the world may have a global bearing and thus deserves universal attention (Hakanen et al., 2001). 21 2.5 Effects of quinolone administration All quinolones cause erosion of cartilage in weight-bearing joints. They may cause convulsions, increased intracranial pressure, toxic psychosis, CNS stimulation (i.e.nervousness, lightheadedness, confusion, hallucinations) and should not be used by anyone with seizure disorders, or cerebral arteriosclerosis (Haugum et al., 2006). There have been deaths due to anaphylactic shock, and cardiovascular collapse. Also occurring are tingling, itching, facial swelling, and difficult breathing. At the first sign of a rash anyone taking ciprofloxacine and having diarrhoea should immediately check with his prescribing physician (Haugum et al., 2006). Antibacterial drugs may kill off normal intestinal flora, resulting in an overgrowth of clostridia. It produces a toxin that is a primary cause of "antibiotic-associated- colitis". Achilles and other tendon ruptures requiring surgical repair, resulting in prolonged disability can occur from quinolone use. Hence, it is adviced to discontinue ciprofloxacin and consult your physician, if you experience pain, inflammation, or tendon rupture (Hirose et al., 2002; Haugum et al., 2006). Crystaluria (particles out of solution in urine) may occur, particularly if the urine is alkaline. While taking ciprofloxacin, hydration should be monitored (8 - 8oz glasses of water daily minimum) and drink orange or cranberry juice, or apple cider vinegar (2 teaspoon with 1 teaspoon honey in 8 oz water) to maintain acidity of the urine. Photosensitivity (sunburn) is reported to occur easily (Haugum et al., 2006). Although more than 2,500 serotypes have been reported, Salmonella enterica serotype Typhimurium is 1 of the leading serotypes causing salmonellosis worldwide (Galanis et al., 2006). Fluoroquinolones such as ciprofloxacin are strongly recommended for treatment of severe S. typhimurium infections in adults (Guerrant et al., 2001; Cui et al., 2008). 22 2.6 Antibiotic resistance associated with extended-spectrum beta-lactamase (ESBL) production Among Gram-negative bacteria, the emergence of resistance to expanded-spectrum cephalosporins has been a major concern. It appeared initially in a limited number of bacterial species that could mutate to hyperproduce their chromosomal class C β- lactamase. A few years later, resistance appeared in bacterial species not naturally- producing AmpC enzymes (K. pneumoniae, Salmonella spp., P. mirabilis) due to the production of TEM- or SHV-type ESBLs. Characteristically, such resistance has included oxyimino- (for example ceftizoxime, cefotaxime , ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam), but not 7-alpha-methoxy-cephalosporins (cephamycins) (Paterson et al., 2003 ;Woodford et al., 2006). Plasmid-mediated AmpC β-lactamases represent a new threat, since they confer resistance to 7-alpha-methoxy- cephalosporins (cephamycins) such as cefoxitin or cefotetan are not affected by commercially-available β-lactamase inhibitors, and can, in strains with loss of outer membrane porins, provide resistance to carbapenems (Woodford et al., 2006). Studies have also shown that resistance to broad-spectrum β-lactams is highly mediated by extended-spectrum β-lactamase (ESBL) enzymes, increasing the world health problem in clinical settings (Yujuan & Ling, 2006; Valverde et al., 2008; Yah, 2010). According to Yah (2010), the plasmid-borne β-lactamases are also competent enough to hydrolyze β- lactam antibiotics, as well as the mechanism of resistance to β-lactam agents among gram-negative bacteria (Yah, 2010). Members of the family Enterobacteriaceae commonly express plasmid-encoded β- lactamases (e.g., TEM-1, TEM-2, and SHV-1) which confer resistance to penicillins but not to expanded-spectrum cephalosporins. In the mid-1980s, a new group of enzymes, the extended-spectrum b-lactamases (ESBLs), was detected (First detected in Germany in 1983) (Philippon et al., 2002; George et al., 2005). ESBLs are beta-lactamases that hydrolyze extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino-monobactam aztreonam. Thus ESBLs confer resistance to these antibiotics and related oxyimino-beta lactams (Philippon et al., 2002). In general, an isolate is suspected 23 to be an ESBL producer when it shows in vitro susceptibility to the second-generation cephalosporins (cefoxitin, cefotetan) but resistance to the third-generation cephalosporins and to aztreonam (Woodford et al., 2006). Moreover, one should suspect these strains when treatment with these agents for Gram-negative infections fails despite reported in vitro susceptibility. Once an ESBL-producing strain is detected, the laboratory should report it as "resistant" to all penicillins, cephalosporins, and aztreonam, even if it is tested (in vitro) as susceptible (Woodford et al., 2006). Associated resistance to aminoglycosides and trimethoprim-sulfamethoxazole, as well as high frequency of co- existence of fluoroquinolone resistance, creates problems. Beta-lactamase inhibitors such as clavulanate, sulbactam, and tazobactam in vitro inhibit most ESBLs, but the clinical effectiveness of beta-lactam/beta-lactamase inhibitor combinations cannot be relied on consistently for therapy (Woodford et al., 2006). Cephamycins (cefoxitin and cefotetan) are not hydrolyzed by majority of ESBLs, but are hydrolyzed by associated AmpC-type β-lactamase (AmpC type β-lactamases are commonly isolated from extended-spectrum cephalosporin-resistant Gram-negative bacteria. The characteristics of β- lactamases is based on the nucleotide and amino acid sequences in these enzymes (Paterson et al., 2003). AmpC β-lactamases (also termed class C or group 1 are typically encoded on the chromosome of many Gram-negative bacteria including Citrobacter, Serratia and Enterobacter species where its expression is usually inducible (Philippon et al., 2002). Also, β-lactam/β-lactamase inhibitor combinations may not be effective against organisms that produce AmpC-type β-lactamase (Lee et al., 2004). Sometimes these strains decrease the expression of outer membrane proteins, rendering them resistant to cephamycins. In vivo studies have yielded mixed results against ESBL-producing K. pneumoniae. (Cefepime, a fourth-generation cephalosporin, which has demonstrated an in-vitro stability in the presence of many ESBL/AmpC strains). In typical circumstances, genes from TEM-1, TEM-2, or SHV-1 are altered in their amino acid configuration around the active site by mutations (Kim et al., 2006). This extends the spectrum of β-lactam antibiotics susceptible to hydrolysis by these enzymes. The ESBLs are frequently plasmid encoded. Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes (for example, aminoglycosides). Therefore, antibiotic options in the treatment of ESBL-producing 24 organisms are extremely limited (Kim et al., 2006). Carbapenems are the treatment of choice for serious infections due to ESBL-producing organisms, yet carbapenem-resistant isolates have recently been reported (Kim et al., 2006). ESBL-producing organisms may appear susceptible to some extended-spectrum cephalosporins. However, treatment with such antibiotics has been associated with high failure rates (Philippon et al., 2002; George et al., 2005). According to Woodford et al. (2006) CTX-M beta-lactamases (class A) enzymes were named for their greater activity against cefotaxime than other oxyimino-beta-lactam substrates (e.g., ceftazidime, ceftriaxone, or cefepime). Rather than arising by mutation, they represent examples of plasmid acquisition of beta-lactamase genes normally found on the chromosome of Kluyvera species, a group of rarely pathogenic commensal organisms. These enzymes are not very closely related to TEM or SHV beta-lactamases in that they show only approximately 40% identity with these two commonly isolated beta-lactamases (Woodford et al., 2006). More than 80 CTX-M enzymes are currently known. Despite their name, a few are more active on ceftazidime than cefotaxime (Woodford et al., 2006). They have mainly been found in strains of Salmonella enterica serovar Typhimurium and E. coli, but have also been described in other species of Enterobacteriaceae and are the predominant ESBL type in parts of South America. (They are also seen in Eastern Europe) CTX-M-14, CTX-M-3, and CTX-M-2 are the most widespread. CTX-M-15 is currently (Woodford et al., 2006) the most widespread type in E. coli the UK and is widely prevalent in the community (Woodford et al., 2006). Although the inhibitor-resistant β-lactamases are not ESBLs, they are often discussed with ESBLs because they are also derivatives of the classical TEM- or SHV-type enzymes (Bradford, 2001). These enzymes were at first given the designation IRT for inhibitor-resistant TEM β-lactamase; however, all have subsequently been renamed with numerical TEM designations. There are at least 19 distinct inhibitor-resistant TEM β- lactamases. Inhibitor-resistant TEM β-lactamases have been found mainly in clinical isolates of E. coli, but also some strains of K. pneumoniae, Klebsiella oxytoca, P. mirabilis, and Citrobacter freundii (Bradford, 2001). Although the inhibitor-resistant TEM variants are resistant to inhibition by clavulanic acid and sulbactam, thereby showing clinical resistance to the beta-lactam/ lactamase inhibitor combinations of 25 amoxicillin-clavulanate (co-amoxaclav), ticarcillin-clavulanate, and ampicillin /sulbactam, they normally remain susceptible to inhibition by tazobactam and subsequently the combination of piperacillin / tazobactam, although resistance has been described. To date, these beta-lactamases have primarily been detected in France and a few other locations within Europe (Bradford, 2001). Also, according to Bradford (2001), Paterson et al. (2003), George et al. (2005), TEM-1 is the most commonly-encountered Beta-lactamase in Gram-negative bacteria which belong to the TEM beta-lactamases (class A). Up to 90% of ampicillin resistance in E. coli is due to the production of TEM- 1. Also responsible for the ampicillin and penicillin resistance seen in H. influenzae and N. gonorrhoeae in increasing numbers. Although TEM-type beta-lactamases are most often found in E. coli and K. pneumoniae, they are also found in other species of Gram- negative bacteria with increasing frequency. The amino acid substitutions responsible for the ESBL phenotype cluster around the active site of the enzyme changed its configuration, allowing access to oxyimino-beta- lactam substrates (Bradford, 2001; Paterson et al., 2003; George et al., 2005). Opening the active site to beta-lactam substrates also typically enhances the susceptibility of the enzyme to b-lactamase inhibitors, such as clavulanic acid. Single amino acid substitutions at positions 104, 164, 238, and 240 produce the ESBL phenotype, but ESBLs with the broadest spectrum usually have more than a single amino acid substitution. Based upon different combinations of changes, currently 140 TEM-type enzymes have been described. TEM-10, TEM-12, and TEM-26 are among the most common in the United States (Bradford, 2001; Paterson et al., 2003; George et al., 2005), while SHV-1 which is in the SHV beta-lactamases (class A) shares 68 percent of its amino acids with TEM-1 and has a similar overall structure, the SHV-1 beta-lactamase is responsible for up to 20% of the plasmid-mediated ampicillin resistance in this species of E. coli and K. pneumoniae. SHV-1, the original member of the SHV β-lactamase family, is present in most strains of Klebsiella pneumoniae and may be either chromosomally or plasmid mediated (Bradford, 1999). ESBLs in this family also have amino acid changes around the active site, most commonly at positions 238 or 238 and 240. More than 60 SHV varieties are known. They are the predominant ESBL type in Europe and the United States and are found worldwide. SHV-5 and SHV-12 are among the most common 26 (Paterson et al., 2003). While ESBL-producing organisms were previously associated with hospitals and institutional care, these organisms are now increasingly found in the community. CTX-M-15-positive E.coli are a cause of community-acquired urinary infections in the UK, (Woodford et al., 2006) and tend to be resistant to all oral β-lactam antibiotics, as well as quinolones and sulfonamides (Woodford et al., 2006). 2.7 Overview of the quinolones The quinolones are a family of synthetic broad-spectrum antibiotics. The term quinolones (s) refers to potent synthetic chemotherapeutic antibacterials (Nelson et al., 2007; Ivanov, and Budanov, 2006). The first generation of the quinolones begins with the introduction of nalidixic acid in 1962 for treatment of urinary tract infection in humans (Sanofi- Aventis, 2008). Quinolones and fluoroquinolone are chemotherapeutic bactericidal drugs, eradicating bacteria by interfering with DNA replication. The other antibiotics used today, (e.g., tetracyclines, lincomycin, erythromycin, and chloramphenicol) do not interact with components of eukaryotic ribosomal particles and, thus, have not been shown to be toxic to eukaryotes, (Murray et al., 2006) as opposed to the fluoroquinolone class of drugs. Other drugs used to treat bacterial infections, such as penicillins and cephalosporins, inhibit cell wall biosynthesis, thereby causing bacterial cell death, as opposed to the interference with DNA replication as seen within the fluoroquinolone class of drugs (Ambrose and Owens, 2000). Quinolones inhibit the bacterial DNA gyrase or the topoisomerase II enzyme, thereby inhibiting DNA replication and transcription (Ambrose and Owens, 2000). Quinolone-induced DNA damage was first reported in 1986 (Lamb, 2008; Lardizabal, 2009). There continues to be debate as to whether or not this DNA damage is to be considered one of the mechanisms of action concerning the severe and non-abating adverse reactions experienced by some patients following fluoroquinolone therapy (Sissi and Palumbo, 2003; Lardizabal, 2009). Recent evidence has shown that topoisomerase II is also a target for a variety of quinolone-based drugs. Quinolones can enter cells easily via porins and, therefore, are often used to treat intracellular pathogens such as Legionella pneumophila and Mycoplasma pneumoniae. For many Gram-negative bacteria, DNA gyrase is the target, 27 whereas topoisomerase IV is the target for many Gram-positive bacteria. It is believed that eukaryotic cells do not contain DNA gyrase or topoisomerase IV. However, there is debate concerning whether the quinolones still have such an adverse effect on the DNA of healthy cells, in the manner described above, hence contributing to their adverse safety profile. This class has been shown to damage mitochondrial DNA (Kaplowitz, 2005). Resistance to quinolones can evolve rapidly, even during a course of treatment. Numerous pathogens, including Staphylococcus aureus, enterococci, and Streptococcus pyogenes now exhibit resistance worldwide (Jacobs, 2005). Widespread veterinary usage of quinolones, in particular in Europe, has been implicated (Nelson et al., 2007). There are three known mechanisms of resistance (Robicsek et al., 2006). Some types of efflux pumps can act to decrease intracellular quinolone concentration (Ambrose and Owens, 2000). In Gram-negative bacteria, plasmid-mediated resistance genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally, mutations at key sites in DNA gyrase or topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drugs' effectiveness. They prevent bacterial DNA from unwinding and duplicating (Hooper, 2001). Quinolones in comparison to other antibiotic classes have the highest risk of causing colonization with MRSA and Clostridium difficile. Finally, mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to quinolones, decreasing the drug's effectiveness (Liu and Mulholland, 2005). A general avoidance of fluoroquinolone is recommended based on the available evidence and clinical guidelines (Muto et al., 2003; Tacconelli et al., 2008; Vonberg, 2009). Though it is generally accepted that nalidixic acid is to be considered the first quinolone drug, this has been disputed over the years by a few researchers that believed that chloroquine, from which nalidixic acid is derived, should be considered the first quinolone drug, rather than nalidixic acid. Since the introduction of nalidixic acid in 1962, more than 10,000 analogs have been synthesized but only a handful have found their way into clinical practice (Stacy and Childs, 2000). Floroquinolones are not recommended as first-line antibiotics for acute sinusitis, as this condition is usually self-limiting, and the risks outweigh the benefits in comparison to other antibiotic classes (Karageorgopoulos et al., 2008; Le Saux, 2008). The basic pharmacophore, or active structure, of the fluoroquinolone class is based upon the 28 quinoline ring system (Schaumann, and Rodloff, 2007). The addition of the fluorine atom at C6 is what distinguishes the successive-generation floroquinolones from the first- generation quinolones. It has since been demonstrated that the addition of the C6 fluorine atom is not a necessary requirement for the antibacterial activity (Ambrose and Owens, 2000). The first generation is rarely used today. A number of the second-, third-, and fourth-generation drugs have been removed from clinical practice due to severe toxicity issues or discontinued by their manufacturers. The drugs most frequently prescribed today consist of Avelox (moxifloxacin), Cipro (ciprofloxacin), Levaquin (levofloxacin), and, to some extent, their generic equivalents. First-generation quinolone; - cinoxacin (Cinobac) (Removed from clinical use, according to Oliphant and Green (2002), - flumequin (Flubactin) (Genotoxic carcinogen)(Veterinary use) [ - nalidixic (NegGam, Wintomylon) (Genotoxic carcinogen) - oxolic acid (Uroxin) (Currently unavailable in the United States) - piromidic acid (Panacid) (Currently unavailable in the United States) - pipemidic acid (Dolcol) (Currently unavailable in the United States) - rosoxacin (Eradacil) (Restricted use, currently unavailable in the United States) (Oliphant and Green, 2002). Second-generation quinolone; The second-generation class is sometimes subdivided into "Class 1" and "Class 2" (Oliphant, and Green, 2002) -ciprofloxacin (Zoxan, Ciprobay, Cipro, Ciproxin) - enoxacin (Enroxil, Penetrex) (Removed from clinical use) -fleroxacin (Megalone, Roquinol) (Removed from clinical use) -lomefloxacin (Maxaquin) (Discontinued in the United States) - nadifloxacin (Acuatim, Nadoxin, Nadixa) (Currently unavailable in the United States) - norfloxacin (Lexinor, Noroxin, Quinabic, Janacin) (restricted use) - ofloxacin (Floxin, Oxaldin, Tarivid) (Only as ophthalmic in the United States) - pefloxacin (Peflacine) (Currently unavailable in the United States) 29 -rufloxacin (Uroflox) (Currently unavailable in the United States) (Oliphant and Green, 2002). Third-generation quinolone; Unlike the first- and second-generations, the third-generation is active against Streptococcus (Ambrose and Owens, 2000) -balofloxacin (Baloxin) (Currently unavailable in the United States), - grepafloxacin (Raxar) (Removed from clinical use), - levofloxacin (Cravit, Levaquin) , -pazufloxacin (Pasil, Pazucross) (Currently unavailable in the United States) -sparfloxacin (Zagam) (Currently unavailable in the United States), - temafloxacin (Omniflox) (Removed from clinical use), -tosufloxacin (Ozex, Tosacin) (Currently unavailable in the United States) (Oliphant and Green, 2002; Ambrose and Owens, 2000). Fourth-generation quinolone; Fourth generation fluoroquinolones act at DNA gyrase and topoisomerase IV (Gupta, 2009). This dual action slows development of resistance. -clinafloxacin (Currently unavailable in the United States (Ambrose and Owens, 2000) -gatifloxacin (Zigat, Tequin) (Zymar -opth.) (Tequin removed from clinical use) -gemifloxacin (Factive)(Currently unavailable in the United States) - moxifloxacin (Avelox,Vigamox) (restricted use). -sitafloxacin (Gracevit) (Currently unavailable in the United States) -trovafloxacin (Trovan) (Removed from clinical use) Oliphant and Green, 2002) - prulifloxacin (Quisnon) (Currently unavailable in the United States) in development (Ambrose and Owens, 2000) - garenoxacin (Geninax)(Application withdrawn due to toxicity issues) - delafloxacin The quinolones have been widely used in agriculture, and several agents that have veterinary but not human use exist such as; danofloxacin (Advocin, Advocid) difloxacin (Dicural, Vetequinon), enrofloxacin (Baytril) Ibaflin), marbofloxacin (Marbocyl, Zenequin), orbifloxacin (Orbax, Victas), sarafloxacin (Floxasol, Saraflox, Sarafin) (Ambrose and Owens, 2000). 30 Figure 2.1: Essential structure of all quinolone antibiotics (Sanofi-Aventis, 2008): The blue drawn remainder of R is usually piperazine; if the connection contains fluorine (red), it is a fluoroquinolone. Figure 2.2: Structure of Nalidixic acid (Sanofi – Aventis, 2008). 2.8 Levofloxacin Levofloxacin, a fluoroquinolone is a synthetic chemotherapeutic antibiotic of the quinolone class (Nelson et al., 2007) and is used to treat severe or life-threatening bacterial infections or bacterial infections that have failed to respond to other antibiotic classes (Liu and Mulholland, 2005; MacDougall et al., 2005). It is sold under various brand names, such as Levaquin and Tavanic, the most common. In form of ophthalmic solutions, it is known as Oftaquix, Quixin, Edolev, L-flox and Iquix etc. The Systematic 31 (IUPAC) name is (S)-7-fluoro-6-(4-methylpiperazin-1-yl) -10-oxo-4-thia-1- 5,13 azatricyclo[7.3.1.0 ] trideca-5(13),6,8,11-tetraene-11-carboxylic acid with Molecular formulae C18H20FN3O4 and molar mass of 361.368g/mol. (Nelson et al., 2009; Corbett and Berger, 2004). Levofloxacin is a chiral fluorinated carboxyquinolone. Investigation of ofloxacin, an older drug that is the racemic mixture, found that the l form [the (-)-(S) enantiomer] is more active. This specific component is levofloxacin (Janssen Pharmaceuticals, 2008). Levofloxacin interacts with a number of other drugs, as well as a number of herbal and natural supplements. Such interactions increase the risk of cardiotoxicity and arrhythmias, anticoagulation, the formation of non-absorbable complexes, as well as increasing the risk of toxicity (DrugBank, 2009). Levofloxacin is associated with a number of serious and life-threatening adverse reactions as well as spontaneous tendon ruptures and irreversible peripheral neuropathy. Such reactions may manifest long after therapy had been completed and in severe cases may result in life-long disabilities. Hepatoxicity has also been reported with the use of levofloxacin. (Albrecht, 2007; Albrecht, 2008). Levofloxacin is a broad-spectrum, third-generation fluoroquinolone antibiotic and optically active L-isomer of ofloxacin with antibacterial activity. Levofloxacin diffuses through the bacterial cell wall and acts by inhibiting DNA gyrase (bacterial topoisomerase II), an enzyme required for DNA replication, RNA transcription, and repair of bacterial DNA. Inhibition of DNA gyrase activity leads to blockage of bacterial cell growth (Liu, and Mulholland, 2005) Levofloxacin was first patented in 1987 (Levofloxacin European patent – Daiichi Pharmaceutical Co., Ltd.) and was approved by the United States Food and Drug Administration on December 20, 1996 (Nelson et al., 2007) for use in the United States to treat severe and life-threatening bacterial infections. Within a significant number of medical publications and books, levofloxacin was described as a second generation fluoroquinolone (Lamb, 2008) whereas within a number of medical web sites it has been described as a third-generation fluoroquinolone (Lamb, 2008). Levofloxacin is considered to be same as ofloxacin by the U.S. Food and Drug Administration (FDA), with the exception of the potency shown in vitro against mycobacteria. In vitro, it is, in 32 general, twice as potent as ofloxacin, whereas d-ofloxacin is less active against mycobacteria (Nelson et al., 2007). Levofloxacin is limited to the treatment of proven serious and life-threatening bacterial infections such as urinary tract infections, community-acquired pneumonia, skin and skin structure infections, nosocomial pneumonia, chronic bacterial prostatitis (Albrecht, 2007; Albrecht, 2008). Levofloxacin has shown moderate activity against anaerobes, and about twice as potent as ofloxacin against Mycobacterium tuberculosis and other mycobacteria, including Mycobacterium avium complex (Nelson et al., 2007; Albrecht, 2007; Albrecht, 2008). Although claimed to be effective, levofloxacin is not to be considered a first line agent for inhalational anthrax in the pediatric population due to severe adverse reactions involving the musculoskeletal system and other serious adverse reactions, including fatalities (Nelson et al., 2007; Albrecht, 2007; Albrecht, 2008). The Center For Disease Control (CDC) revoked its recommendation regarding the use of fluoroquinolones (ciprofloxacin) as a first line agent in treating anthrax (in part) due to the risk of adverse reactions documented within the Antimicrobial Postexposure Prophylaxis for Anthrax study (aka Cipro 60-day study), (Dolui et al., 2007). However, the fluoroquinolones are licensed to treat lower respiratory infections in children with cystic fibrosis in the UK (Dolui et al., 2007; Johnson & Johnson, 2009). Caution should be exercised in prescribing to patients with liver disease. Levofloxacin is also considered to be contraindicated in patients with epilepsy or other seizure disorders (Albrecht, 2008; Janssen Pharmaceuticals, 2008). Research indicates that the fluoroquinolones can rapidly cross the blood-placenta and blood-milk barriers, and are extensively distributed into the fetal tissues (Cahill et al., 2005). Peak concentration in human breast milk is similar to levels attained in plasma. Breast-feeding mothers that take levofloxacin may expose their infants to severe adverse reactions, and pregnant women are at risk of spontaneous abortions and birth defects (Nardiello et al., 2002; Cahill et al., 2005). For this reason, the prescription of levofloxacin is contraindicated during pregnancy. Other fluoroquinolones have also been reported to be present in the mother’s milk and are passed on to the nursing child (Cahill et al., 2005; Shin et al., 2003). Oral and I.V. Levofloxacin is not licensed for use in the pediatric population, except as noted above, 33 due to the risk of serious, life-threatening and permanent injury to the pediatric patient (Nardiello et al., 2002). Within one study, a pediatric patient has a 3.8% chance of experiencing a serious musculoskeletal adverse event (Noel et al., 2007). However, the two most recent pediatric studies involving the use of levofloxacin indicated paediatric patient has a greater than 50% chance of experiencing one or more adverse reactions (Noel et al., 2007). Within the first paediatric study, it is stated that 275 (52%) levofloxacin-treated subjects experienced one or more adverse event, out of the total 712 subjects tested. Serious adverse events were reported in 33 (6%) levofloxacin- treated subjects. Two serious adverse events in levofloxacin-treated subjects resulted in fatal outcomes. Within the second pediatric study, 122 experienced one or more adverse events, out of the 204 subjectes tested. Twelve subjects (6%) discontinued the drug study due to an adverse event. Seven subjects (3%) experienced 8 serious adverse events (Albrecht, 2008). Levofloxacin pharmacokinetics is linear and predictable after single and multiple oral or I.V dosing regimens. Levofloxacin is rapidly and, in essence, completely absorbed after oral administration. Peak plasma concentrations are usually attained one to two hours after oral dosing. The plasma concentration profile of Levofloxacin after I.V administration was similar and comparable in extent of exposure (AUC) to that observed for levaquin tablets when equal doses (mg/mg) are administered (Janssen Pharmaceuticals, 2008; Noel et al., 2007). Levofloxacin is excreted largely unchanged in the urine. Its mean terminal plasma elimination half-life ranges from approximately 6 to 8 hours following single or multiple doses of Levofloxacin given orally or intravenously (Janssen Pharmaceuticals, 2008; Noel et al., 2007). Levofloxacin is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-negative bacteria. Levofloxacin should be used only to treat or prevent infections that are proven or strongly suspected to be caused by susceptible bacteria (Albrecht, 2008). Normally, levofloxacin should only be used in patients who have failed at least one prior therapy. It is reserved for use in patients who are seriously ill that require immediate hospitalization (Johnson and Johnson, 2004; Janssen Pharmaceuticals, 2008; Shin et al., 2003). Though, it was 34 considered to be a very important and necessary drug required to treat severe life threatening bacterial infections. The associated prescription abuse of levofloxacin remains unchecked, which has contributed to the problem of bacterial resistance. The overuse of antibiotics, such as what happens with children suffering from otitis media, has given rise to super-bacteria that are resistant to antibiotics entirely (Fraunfelder and Fraunfelder, 2009; Shin et al., 2003). Fluoroquinolones, including levofloxacin, had become the most commonly prescribed class of antibiotics to adults in 2002. Nearly half (42%) of these prescriptions were for conditions not approved by the FDA, such as acute bronchitis, otitis media, and acute upper respiratory tract infection, according to a study that was supported in part by the Agency for Healthcare Research and Quality (Linder et al., 2005; Grépinet et al., 2008; Shin et al., 2003) In addition, they are commonly prescribed for medical conditions that are not even bacterial to beging with such as viral infections, or those to which no proven benefit exists. Figure 2.3: Structure of Levofloxacin (Sanofi-Aventis, 2008) 2.9 Ciprofloxacin The Systematic (IUPAC) name of ciprofloxacin is 1-cyclopropyl- 6-fluoro- 4-oxo- 7- piperazin- 1-yl- quinoline- 3-carboxylic acid. The molecular formula is C17H18FN3O3 with the molar mass 331.346 (Nelson et al., 2007). Ciprofloxacin hydrochloride (USP) is 35 the monohydrochloride monohydrate salt of ciprofloxacin. Ciprofloxacin is a synthetic chemotherapeutic antibiotic of the fluoroquinolone class of drug (Nelson et al., 2007) and was was first patented in 1983 by Bayer A.G. and subsequently approved by the United States Food and Drug Administration (FDA) in 1987. Ciprofloxacin has 12 FDA- approved human uses and other veterinary uses, but it is often used for non-approved uses (off-label) (Bayer Corporation, 2001; Nelson et al., 2007). Ciprofloxacin interacts with other drugs, herbal and natural supplements, and thyroid medications (Cooper et al., 2005). It is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-negative bacteria and functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV (Nelson et al., 2007) enzymes necessary to separate bacterial DNA, thereby inhibiting cell division (Jam and Chen, 2006) This mechanism can also affect mammalian cell replication. In particular, some congeners of this drug family (for example those that contain the C-8 fluorine) display high activity not only against bacterial topoisomerases but also against eukaryotic topoisomerases and are toxic to cultured mammalian cells and in vivo tumor models. Although quinolones are highly toxic to mammalian cells in culture, its mechanism of cytotoxic action is not known. Recent studies have demonstrated a correlation between mammalian cell cytotoxicity of the quinolones and the induction of micronuclei (Jam and Chen, 2006) As such some fluoroquinolones may cause injury to the chromosome of eukaryotic cells (Jam and Chen, 2006). Oral and intravenous fluoroquinolones are not licensed by the U.S. FDA for use in children due to the risk of permanent injury to the musculoskeletal system, with two exceptions as outlined below (Connerton et al., 2000; Galanis et al., 2006). Within the studies submitted in response to a Pediatric Written Request, the rate of arthropathy was reported to be 9.3% and 13.6% in one month and year respectively (Connerton et al., 2000), as such the pediatric use of ciprofloxacin is restricted to proven complicated urinary tract infections and pyelonephritis due to E. coli and inhalation anthax (Bayer Corporation, 2001). Although claimed to be effective, ciprofloxacin is not to be considered a first line agent for inhalation anthrax in the pediatric population (Threlfall et al., 2001; Galanis et al., 2006). The CDC revoked its recommendation regarding the use 36 of ciprofloxacin as a first line agent in treating anthrax due to the unacceptable risk documented within the Antimicrobial Postexposure Prophylaxis for Anthrax study (aka Cipro 60 day study) (Dolui et al., 2007). However, the fluoroquinolones are licensed to treat lower respiratory infections in children with cystic fibrosis in the U.K (Bayer Corporation, 2001). Current recommendations by the American Academy of Pediatrics noted that the systemic use of ciprofloxacin in children should be restricted to infections caused by multidrug resistant pathogens or when no safe or effective alternatives are available (Robicsek et al., 2006). Ciprofloxacin is not recommended to treat CAP (community acquired pneumonia) as a stand-alone first-line agent. The current guidelines (Infectious Diseases Society of America) (Dolui et al., 2007) stated that, in very limited circumstances, ciprofloxacin or levofloxacin should be combined with other drugs such as a ßeta-lactam drug to treat specific CAP infections, but neither drug was recommended to be used separately as a stand-alone first-line agent (Nelson et al., 2007). In addition, the current guidelines stated that: “Data exist, suggesting that resistance to macrolides and older fluoroquinolones (ciprofloxacin and levofloxacin) resulted in clinical failure (Nelson et al., 2007). Other studies have shown that repeated use of fluoroquinolones predicted an increased risk of infection with fluoroquinolone-resistant pneumococci (Hooper, 2001). As noted above, under licensed use, ciprofloxacin was also considered to be contraindicated for the treatment of certain sexually transmitted diseases by some experts due to bacterial resistance (Hooper, 2001). Due to growing prevalence of antibiotic resistance to the fluoroquinolones in the Southeast Asia, the use of ciprofloxacin in patients have been discouraged (Nelson et al., 2007; Brusch et al., 2010). Ciprofloxacin is also considered contraindicated within the pediatric population (except for the indications outlined under licensed use above), pregnancy, nursing mothers, and in patients with epilepsy or other seizure disorders (Brusch et al., 2010). The fluoroquinolones have also been reported as being present in the mother’s milk and are passed on to the child, which may increase the risk of the child suffering from this syndrome as well, even though the child had never been prescribed with or taken any of the drugs found within this class (Brusch et al., 2010). Overdose of ciprofloxacin may 37 result in reversible renal toxicity. Treatment of overdose included emptying of the stomach via induced vomiting or by gastric lavage. Careful monitoring and supportive treatment, monitoring of renal function and maintaining adequate hydration are recommended by the manufacturer (Brusch et al., 2010). Ciprofloxacin is commonly used for urinary tract and intestinal infections (traveler's diarrhoea) and was once considered a powerful antibiotic of last resort (Jacobs, 2005), used to treat especially persistent infections. Not all physicians agreed with this assessment, as evidenced by its wide-spread use to treat minor infections as well as non-approved uses. As a result, in recent years, many bacteria have developed resistance to this drug, leaving it significantly less effective than it would have been otherwise (Jacobs, 2005) Resistance to ciprofloxacin and other fluoroquinolones may evolve rapidly, even during a course of treatment. Numerous pathogens, including Staphylococcus aureus, enterococci, and Streptococcus pyogenes now exhibit resistance worldwide (Tacconelli et al., 2008). Widespread veterinary usage of the fluoroquinolones, particularly in Europe, has been implicated (Nelson et al., 2007). Figure 2.4: Structure of ciprofloxacin (Sanofi-Aventis, 2008). 2.10 Cephalosporins The Cephalosporins are a class of β-lactam antibiotics originally derived from the fungus Acremonium, which was previously known as Cephalosporium (Stork, 2006; Pegler and Healy, 2007). Cephalosporins are indicated for the prophylaxis and treatment of 38 infections caused by susceptible bacteria. First-generation cephalosporins are active predominantly against Gram-positive bacteria, and successive generations have increased activity against Gram-negative bacteria (albeit often with reduced activity against Gram- positive organisms) (Richard and Ronald, 2009; Kollef, 2009). The cephalosporin nucleus can be modified to gain different properties. Cephalosporins are sometimes grouped into "generations" by their antimicrobial properties (Stork, 2006). The first generation cephalosporins were designated first-generation cephalosporins, whereas, later, more extended-spectrum cephalosporins were classified as second- generation cephalosporins. Each newer generation of cephalosporins has significantly greater Gram-negative antimicrobial properties than the preceding generation, in most cases with decreased activity against Gram-positive organisms. Fourth-generation cephalosporins, however, have true broad-spectrum activity (Pichichero, 2006). Cephalosporins are bactericidal and have the same mode of action as other ß-lacatam antibiotics (such as penicillins) but are less susceptible to penicillinases. Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidases known as penicillin- binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala at the end of peptidoglycan precursors to crosslink the peptidoglycan. Beta-lactam antibiotics mimic the D-Ala-D- Ala site, thereby competitively inhibiting PBP crosslinking of peptidoglycan (Kollef, 2009). Figure 2.5: Core structure of the cephalosporins (Sanofi-Aventis, 2008). 39 2.11 Cefotaxime The systematic IUPAC name of cefotaxime is (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2- aminothiazol-4-yl)-2-(methoxyimino)acetamido)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2- ene-2-carboxylic acid. Its chemical formular is C16H17N5O7S2, while the molecular weight is 455.47g/mol. Cefotaxime is a third-generation cephalosporin antibiotic (Bonnet, 2004). Like other third-generation cephalosporins, it has broad spectrum activity against Gram positive and Gram negative bacteria. In most cases, it is considered to be equivalent to ceftriaxone in terms of safety and efficacy (Bonnet, 2004). The syn-configuration of the methoxyimino moiety confers stability to β-lactamase enzymes produced by many Gram-negative bacteria. Such stability to β-lactamases increases the activity of cefotaxime against otherwise resistant Gram-negative organisms. Cefotaxime inhibits bacterial cell wall synthesis by binding to one or more of the penicillin-binding proteins (PBPs) which in turn inhibits the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls, thus inhibiting cell wall biosynthesis(Onyenwe et al., 2012). Bacteria eventually lyse due cell wall autolytic enzymes (autolysins and murein hydrolases) while cell wall assembly is arrested. Cefotaxime is used for infections of the respiratory tract, skin, bones, joints, urogenital system, meningitis, and septicemia. It generally has good coverage against most Gram- negative bacteria, with the notable exception of Pseudomonas (Bonnet, 2004). It is also effective against most Gram-positive cocci except Enterococcus. It is active against penicillin-resistant strains of Streptococcus pneumoniae. It has modest activity against the anaerobic Bacteroides fragilis. Cefotaxime crosses blood-brain barrier better than Cefuroxime in meningitis. Cefotaximases (CTX-M) are class - A β-lactamases that in general present higher levels of hydrolytic activity against cefotaxime than against ceftazidime. Ceftazidime MICs for organisms producing these enzymes are sometimes in the susceptible range (Bonnet, 2004). Many laboratories use ceftazidime resistance alone as an indicator of extended- spectrum β-lactamase production. For this reason, CTX-M-producing isolates may be missed by routine susceptibility testing performed by clinical microbiology laboratories (Bonnet, 2004). CTX-M enzymes comprise a rapidly growing family distributed both 40 over wide geographic areas and among a wide range of bacteria of clinical significance (Bonnet, 2004; Batchelor et al., 2005). Figure 2.6: Structure of Cefotaxime (Sanofi- Aventis, 2008). 2.12 Ceftriaxone The systemic IUPAC name of ceftriaxone is (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4- yl)->2- (methoxyimino)acetyl]amino}-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4- triazin-3-yl) thio]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid. The formular is C18H18N8O7S3, while the molar mass is 554.58 g/mol. Ceftriaxone is a third-generation cephalosporin antibiotic. Like other third-generation cephalosporins, it has broad spectrum activity against Gram-positive and Gram-negative bacteria (Bradley et al., 2009). In most cases, it is considered to be equivalent to cefotaxime in terms of safety and efficacy. Resistance to ceftriaxone, the drug of choice for invasive salmonella disease, is a public health concern, especially with respect to children, since fluoroquinolones, which can also be used to treat this disease, are not approved for use in children (Fey et al., 2000). Ceftriaxone is often used (in combination, but not direct, with macrolide and/or aminoglycoside antibiotics) for the treatment of community-acquired or mild to moderate health care-associated pneumonia. It is also a choice drug for treatment of bacterial 41 meningitis. In pediatrics, it is commonly used in febrile infants between 4 and 8 weeks of age who are admitted to the hospital to exclude sepsis. The dosage for acute ear infection in the very young is 50mg/kg I.M, one dose only) (Bradley et al., 2009). It has also been used in the treatment of Lyme disease, typhoid fever and gonorrhea. Intravenous dosages may be adjusted for body mass in younger patients and is administered every 12–24 hours, at a dose that depended on the type and severity of the infection (Bradley et al., 2009). Treatment for chlamydia infection was also recommended (usually with azithromycin) unless it is specifically ruled out (Barclay, 2007). It must not be mixed or administered simultaneously (within 48 hours) with calcium-containing solutions or products for patients younger than 28 days old (Bradley et al., 2009), even via different infusion lines (rare fatal cases of calcium- ceftriaxone precipitates in lung and kidneys in neonates have been described (Bradley et al., 2009). To reduce the pain of intramuscular injection, ceftriaxone may be reconstituted with 1% lidocaine (Bradley et al., 2009). Ceftriaxone has also been investigated for efficacy in preventing relapse to cocaine addiction (Fey et al., 2000). Ceftriaxone is a white crystalline powder readily soluble in water, sparingly soluble in methanol and very slightly soluble in ethanol. The pH of a 1% aqueous solution is approximately 6.7 (Barclay, 2007). The syn-configuration of the methoxyimino moiety confers stability to β-lactamase enzymes produced by many Gram-negative bacteria. Such stability to β-lactamases increases the activity of ceftriaxone against otherwise resistant Gram-negative bacteria. In place of the easily hydrolysed acetyl group of cefotaxime, ceftriaxone has a metabolically stable thiotriazinedione moiety (Barclay, 2007; Bradley et al., 2009). The emergence of resistance to antimicrobial agents within the salmonellae is a worldwide problem that has been associated with the use of antibiotics in livestock. Resistance to ceftriaxone and the fluoroquinolones, which are used to treat invasive Salmonella infections, was rare in the United States (Fey et al., 2000). According to Fey et al. (2000), they analyzed the molecular characteristics of a ceftriaxone-resistant strain of Salmonella enterica serotype typhimurium isolated from a 12-year-old boy with fever, abdominal pain, and diarrhoea. The ceftriaxone-resistant isolate from the child was indistinguishable from one of the isolates from cattle, which was also resistant to ceftriaxone. Both ceftriaxone-resistant isolates were resistant to 13 42 antimicrobial agents; all but one of the resistance determinants were on a conjugative plasmid of 160 kpb that encoded the functional group 1 (Beta-lactamase CMY-2). Both ceftriaxone-resistant isolates were closely related to the three other salmonella isolates obtained from cattle, all of which were susceptible to ceftriaxone (Fey et al., 2000). Also their study provides additional evidence that antibiotic-resistant strains of Salmonella in the United States evolve primarily in livestock (Fey et al., 2000). 2.13 Ethidium bromide The IUPAC name is 3, 8-Diamino-5-ethyl-6-phenylphenanthridinium bromide with the Molecular formular is C21H20BrN3. The molar mass of the compound is 394.294 g/mol with the appearance of Purple-red solid, and a melting point of 260 - 262 °C. Its solubility in water is approximately 40 g/l (Diaz et al., 2002; Von Wurmb-Schwark et al., 2006). Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis (NTP, 2005). It is commonly abbreviated as "EtBr", which is also an abbreviation for bromoethane (Huang and Fu, 2005). When exposed to ultraviolet light, it fluoresces with an orange colour, intensifying almost 20-fold after binding to DNA (NTP, 2005). Ethidium bromide may be a mutagen, carcinogen or teratogen although this depends on the organism and the conditions (Huang and Fu, 2005; NTP, 2005). Ethidium bromide is commonly used to detect nucleic acids in molecular biology laboratories when viewed under ultra violet light (Huang and Fu, 2005; Von Wurmb-Schwark et al., 2006). Where direct viewing is needed, the viewer's eyes and exposed skin should be protected. In the laboratory the intercalating properties have long been utilized to minimize chromosomal condensation when a culture is exposed to mitotic arresting agents during harvest. The resulting slide preparations permit a higher degree of resolution, and thus more confidence in determining structural integrity of chromosomes upon microscopic analysis (Von Wurmb-Schwark et al., 2006). Ethidium bromide has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells (Diaz et al., 2002). Ethidium bromide is thought to act as a mutagen because it intercalates double 43 stranded DNA, thereby deforming the molecule (Von Wurmb-Schwark et al., 2006). This can affect DNA biological processes, like DNA replication and transcription. Ethidium bromide has been shown to be mutagenic to bacteria via the Ames test (with liver homogenate) (Von Wurmb-Schwark et al., 2006). The requirement of S9 liver homogenate indicates that Ethidium bromide isn't mutagenic, but its metabolites are. The identities of these mutagenic metabolites are unknown. The National Toxicology Program states it is nonmutagenic in rats and mice (NTP, 2005). Ethidium bromide (Homidium) use in animals to treat trypanosome infection suggests that toxicity and mutagenicity are not high. Studies have been conducted in animals to evaluate EtBr as a potential antitumorigenic chemotherapeutic agent (NTP, 2005). Its chemotherapeutic use is due to its toxicity to mitochondria (Von Wurmb-Schwark et al., 2006). The above studies do not support the commonly held idea that Ethidium bromide is a potent mutagen in humans, but they do indicate that it can be toxic at high concentrations. Most use of Ethidium bromide in the laboratory (0.25 - 1 microgram/ml) is below the level required for toxicity (NTP, 2005). The level is high enough that exposure may interfere with replication of mitochondrial DNA in some human cell lines, although the implications of that are not clear. Testing in humans and longer studies in any mammalian system are required (NTP, 2005). R-plasmids may be classified as transferrable or non transferable. Transferable R-plasmid is detected by conjugation where as-non transferrable R-plasmids are detected by their loss from host cell spontaneously due to some errors in replication or segregation. Ethidium bromide is not technically hazardous waste at low concentrations (NTP, 2005; Von Wurmb-Schwark et al., 2006), but is treated as hazardous waste by many organizations. Waste should be treated in accordance with federal, state and local guidelines (NTP, 2005). Ethidium bromide can be degraded chemically, or collected and incinerated. It is common for ethidium bromide waste below a mandated concentration to be disposed of normally. A common practice is to treat ethidium bromide with sodium hypochlorite (bleach) before disposal (NTP, 2005). Chemical degradation using bleach yields compounds which are mutagenic by the Ames test (NTP, 2005; Von Wurmb-Schwark et al., 2006). Data is lacking on the mutagenic effects of degradation products. EtBr can be removed from 44 solutions with activated charcoal or amberlite ion exchange resin. Various commercial products are available for this use (NTP, 2005). Figure 2.7: Ethidium bromide (Sanofi-aventis, 2008). 2.14 Acridine −1 The molecular formula of acridine is C13H9N with Molar mass of 179.22 g mol . Its melting point is 107 °C and boiling point is 346 °C , Acidity (pKa) is 5.60. Acridine is an organic compound and a nitrogen heterocycle. Acridine is also used to describe compounds containing the C13N tricycle. Acridine, a colorless solid, was first isolated from coal tar. It is separated from coal tar by extracting with dilute sulfuric acid; addition of potassium dichromate to this solution precipitates acridine bichromate. It is a raw material used for the production of dyes and some valuable drugs. Many acridines, such as proflavine, also have antiseptic properties. Acridine and related derivatives bind to DNA and RNA due to their abilities to intercalate. Many synthetic processes are known for the production of acridine and its derivatives. Bernthsen condensed diphenylamine with carboxylic acids, in the presence of zinc chloride in the Bernthsen acridine. With formic acid as the carboxylic acid the reaction yields acridine itself, and with the higher homologues the derivatives substituted at the meso carbon atom are generated (Moloney et al., 2001). A classic method for the synthesis of acridones is the Lehmstedt-Tanasescu reaction (Moloney et al., 2001). Acridine orange (3, 6-dimethylaminoacridine) is a nucleic acid-selective metachromatic stain useful for cell cycle determination. Acridarsine is formally derived from Acridine 45 by replacing the nitrogen atom with one of arsenic, and acridophosphine by replacing it with one of phosphorus. Acridine is a known human carcinogen. It causes mutations in incorporating into the DNA, and doing so creating an additional base on the opposite strand. If that mutation occurs in a coding sequence, it almost always leads to inactivation of the protein it encoded. Increased tolerance of disinfecting agents can be caused by energy – dependent efflux pumps located in the cell membrane (Surolia and Surolia, 2001). The use of acridine dyes such as acridine orange for curing and recognizing resistant plasmid in resistant S. aureus had been reported by Adeleke et al. (2002). Apart from acridine, other agents used for curing were ethidium bromide and mepacrine (Onyenwe et al., 2011). The genetic effects of acriflavine (an example of acridine dyes) have been studied on two different strains of Fusarium with regard to photodynamic inactivation and reversion of genetic markers (Surolia and Surolia, 2001). In the presence of acriflavine photodynamic inactivation was observed and did not result in a change in the shapes of the survival curves, but induces reversion of the genetic markers in each strain of organisms used (Surolia and Surolia, 2001). Since the report of elimination of resistance cytoplasmic factors of yeast with acriflavine and acridine orange was made by Surolia and Surolia (2001) and Adeleke et al. (2002) respectively, loss of antibiotic resistance after exposure to a curing agent has been widely accepted as an evidence of Plasmid mediated resistance to some extent. Figure 2.8: Acridine orange (Sanofi-aventis, 2008). 46 CHAPTER THREE MATERIALS AND METHODS 3.1 Materials 3.1.1 Equipment and glassware The items used in this study were: Dry-block heater (Model FDB03AP-Techne, Barloworld scientific Ltd. USA): It was used for the boiling of microbial cells during DNA extraction. Vortex mixer (Denville scientific Inc.England): It was used for mixing and homogenising cell pellets, DNA and Primers before centrifuging. Centrifuge (Spectrafuge-7M. Labnet international Edison,USA.): Separation of cells and their products was done using this equipment Hot-air oven (gallenkamp England): It was used for the sterilization of all equipment o made of glassware at 160 C for 1hour. Incubator (Gallenkamp England and Uniscope-SM9082, Surgifield medicals England): It o was used for the purpose of incubating culture media used at 37 C. Gel tank (Bioneer Agarose-power TM, Germany): It was used for the separation of the plasmid DNA extracts and PCR products after amplification. Autoclave (Gallenkamp, England): It was used for the sterilization of all the relevant o culture media used at 121 C for 15 mins. Transilluminator (an ultraviolet lightbox, CBS.Scientific Company Inc., Germany): Ethidium bromide-stained DNA in gels was visualized on this equipment. Weighing balance (Mattler PC 400 and Bartonus BS2005, England): It was used for the purpose of weighing appropriate antibiotic powder samples and culture media and other relevant material. Eppendorf pipette (Uniscope, Labnet and Oxford research pipette, Germany): It was used for incubating microbial cells, centrifuging of cell pellets and during PCR analysis. Water bath (Electrothermal, England): It was used for dissolving powdered culture media to suspension before sterilization and also for heating some powdered reagents mixed with water to solution. 47 PCR Machine (Models; Eppendorf vapo-protect, Master cycler gradient and Eppendorf thermomixer comfort, Germany): They were used for DNA amplification Spectrophotometer (Nano drop ND1000, Germany): It was used for testing DNA purification level and quantification. Glassware (Conical flask, measuring cylinder, Petri dishes, glass beakers, pipettes, and test tubes) (Pyrex, England): They were used for measuring solutions. Agarose voltameter or Electrophoretic machine (model EPS-300,11v, size 1-34 well,CBS Scientific company Inc. Germany): The meter was used to convey electric current to the agarose gel during electrophoresis. Hot plate (Jenway 1000-Burboword scientific Ltd., England): It was used in heating or boiling liquid substances. Sanger sequencing machine (ABI 3730 x l of Applied Biosystems, Germany): It was used for DNA sequencing). 3.1.2 Chemicals and culture media The lists and compositions of the culture media and chemicals used are given in the Appendix 1. 3.1.3 Antibiotics and primers The five selected antibiotic referenced standards used for MIC determination were; Cefotaxime powder (Aventis Pharma) Ceftriaxone powder (Aventis Pharma) Amoxicillin-clavulanic acid powder (Merck Sharpe and Dohme Ltd) Ciprofloxacin powder (Merck Sharpe and Dohme Ltd) Levofloxacin (Merck- Sharpe and Dohme, Ltd.). The other antibiotic impregnated discs used for the antibiogram were from Oxoid, England and are as follows; Septrin® (co-trimoxazole) (30µg), chloramphenicol (30µg), sparfloxacin (10µg), amoxicillin (30µg), gentamycin (10µg), pefloxacin (30µg), ofloxacin (10µg), streptomycin (30µg), amoxicillin/clavulanic acid (30µg), cefotaxime (30µg), ceftazidime (30µg), ciprofloxacine (10µg), ceftriaxone (30µg), and levofloxacin (5µg). 48 The primers used were as shown in appendix I. 3.1.4 Test microorganism Clinical human isolates of the Salmonella enterica from stool were obtained from the Routine Section of the Medical Microbiology Laboratory in the South-eastern part of Nigeria, namely; Federal Medical Centre Owerri, Imo State; University of Nigeria Teaching Hospital Enugu, Enugu State; Federal Medical Centre Umuahia, Abia State; Federal Medical Centre Abakiliki, Ebonyi State. Thus the codes were designated as ‘O’ for isolates from Owerri, ‘U’ (Umuahia), ‘E’ (Enugu) and ‘A’ (Abakaliki) accordingly. 3.2 Methods 3.2.1 Maintainance of pure isolates All purified isolates were maintained as stock cultures on nutrient agar slants and stored o at 4 C. Sub culturing was carried out fortnightly into Salmonella Shigella Agar (SSA) to ensure viability of the isolates. 3.2.2 Collection of bacteria isolates The bacteria isolates from stool samples of patients diagnosed with typhoid fever were obtained from one teaching hospital and three federal medical centres in South-east Nigeria between July, 2009 and September, 2010. Out of which a total of one hundred (100) bacteria isolate of S. enterica from the different hospitals (both the teaching hospitals and federal medical centres) in the different States of the South east Nigeria namely; Federal Medical Centre Owerri, University of Nigeria Teaching Hospital Enugu, Federal Medical Centre Umuahia, and the Federal Medical Centre Abakaliki. A total of twenty- five isolates each were collected from the hospitals. The isolates were recovered from different Departments such as General out Patients Department (GOPD), In pataients Unit (IPU), National Health Insurance Scheme (NHIS), Children Out Patience (CHOP), and Emergency Patient Unit (EPU) as stated in their record ledger in each Microbiology unit visited. Each isolate was transferred into a sterile media slant for further confirmatory test and biochemical characterization. They were then further authenticated as S. enterica using Microbact® 12E kit. 49 3.2.3 Culturing and identification of the Salmonella enterica All the isolates were appropriately labeled for the purpose of clearity and identification. The isolates from FMC Owerri were designated SO1-SO25, FMC Umuahia SU26-SU50 and UNTH Enugu SE51-SE75, while FMC Abakaliki SA76-SA100 accordingly. All the colonies of Salmonella enterica previously isolated from these hospitals were subcultured into a selective medium such as Salmonella shigella agar (SSA), to further verify their H2S production through exhibiting tiny black spots on the culture plate. They were later transferred into Mueller Hinton agar (MHA) for Grams staining and biochemical characterization using microbact identification kit (oxoid-England) as specified by the manufacturer, to be certain of the right isolates as described by Cheesbrough (2006). 3.2.4. Identification of Salmonella species using Microbact® kit GNB12E Procedures: For the test, a single colony from an 18-24 hour plate culture was inoculated aseptically into 5ml sterile normal saline solution (0.9% NaCl) and the suspension was homogenized 9 to a cell suspension with turbidity equivalent to that of 0.5 McFarland standard (1.5 × 10 CFU/mL). Using a sterile pipette, few drops of the bacterial suspension were distributed into 12 wells of the Microbact® 12E (Oxoid Ltd. Australia) and sealed after the addition of two drops of mineral oil (Oxoid reagent) into 1 - 3 of the wells and reseal the well for incubation. The microbact 12E kit consist of 12 different biochemical test specific for the identification of Enterobacteriaceae. In the absence of colour change in the wells as 0 suggested by the manufacturer. The kit was incubated at 35 ±2 C for 18-24 hours. After incubation, colour changes were read against colour chart as provided by the manufacturer. Further test were performed on the unchanged wells or tubes. Then one to two drops of the reagent were introduced in each well 8 and 10 to check for indole and vogues proskauer reaction respectively. The reaction colour change or values corresponding to each group were added to get a profile (A four digit number). Then the numbers are entered into the softwasre of Microbact® 12E for final identification. The Microbact® API 20E could also be used for this purpose. 50 The slide agglutination antigen kit (Lab-Care Diagnostics, India) was used for this analysis. A bit of discrete colony from nutrient agar culture plate was put on the grease free slide and mixed with a drop of physiological saline solution using a mixing stick. The results were observed macroscopically after one minute, for auto-agglutinating strains after rocking the slide for 30–60 seconds. This procedure was repeated simultaneously with a drop of the antiserum (Lab-care diagnostics, India). The O antigen suspensions were tested first. If they were positive, then the monovalent O antisera belonging to this group will also be tested. A positive result shows that the isolated bacterium possesses the antigen corresponding to the antiserum. The same procedures were subsequently carried out on the H antigen suspension 3.3 Antibiogram of the clinical isolate Salmonella enterica The antibiogram was carried out by the multidisc agar- diffusion method. Using sterile -2 pipette, 0.1ml of 10 dilution of 18- 24 hours culture of the test isolates was added to o 20ml molten mueller Hinton Agar cooled at 45 C, the contents were gently swirled to mix before pouring into sterile petri dishes. The seeded culture plates were allowed to set o and subsequently dried for 20mins, in the incubator at 37 C. After drying, the antibiotic discs were aseptically introduced on the surface of the medium using apair of sterile o forceps and allowed for 10-15 minutes. before incubation at 37 C for 24 hours. Thereafter, the zones of growth inhibition were interpreted using CLSI breakpoint as standard zones depending on the antibiotics used (CLSI, 2007; CLSI, 2011), as sensitive / intermediate sensitive/ enzyme inactivation / resistant. Statistics analysis by Analysis of Variance (ANOVA) was used to determine the level of significance among the zone of growth inhition produced by the five selected antibiotic against the clinical isolate of S.enterica. 3.4 Determination of the Minimum Inhibitory Concentration (MIC) of five selected antibiotics Using the tube broth-dilution method the MIC of Five selected antibiotics; ceftriaxone, cefotaxime, augmentine® (amoxicillin/ clavulanic acid), levofloxacin and ciprofloxacin) 51 against the standard and clinical strains of S. enterica was determined. Two sets of graded drug concentrations were prepared in duplicates in nutrient broth. For each test antibiotic, a stock concentration of 100 µg/ml was prepared by dissolving 1mg in 10ml of sterile water. From the stock, 100 µg/ml was prepared in duplicates each of which was diluted down serially by doubling fold dilution to 0.39 µg/ml. Using 2- fold serial dilution (by introducing 5mls of the stock into another 5mls nutrient broth and diluting serially). The -2 serially diluted test drugs were then inoculated each with 0.1ml of 10 dilutions of the 7 respective overnight broth cultures and equivalent of x 10 cell/ml. Control tubes were also set up consisting of nutrient broth incubated with the same o organism, but drug free as positive control at 37 C for 24hours, and then examined for growth. Also, 5ml from the last tube in each set of diluted concentrations was pipetted into a clean sterile test tube, was similarly incubated without inoculum as a transference negative control. Another negative control was a mixture of 0.1ml from the stock concentration and 5ml sterile broth, similarly were incubated and examined. The least drug concentration showing no visible growth was chosen as the MIC for the particular antibiotic. Such concentration and the next two higher concentrations were subsequently o plated using 0.1ml on drug- free nutrient agar plates and incubated at 37 C for 24hours- 48hours. Observation of scanty growth on the plate of MIC broth dilution confirms the chosen concentration as MIC while absence of growth indicates a bactericidal concentration; hence, the Minimum Bactericidal Concentration (MBC). A standard strain of S.enterica serovar Typhimurium (ATCC14028) was also used as control organism and included with each batch of the isolates tested. 3.5 Beta-Lactamase production test using nitrocefin sticks Nitrocefin (Oxoid) is the chromogenic cephalosporin (Yah, 2010), developed by Glaxo Research Limited coded 87/312; 3-(2,4 dinitrostyrl) –6R, 7R) –7-(2 thienylacetamido)- ceph-3-em-4 carboxylic acid, E-isomer. This compound exhibits a rapid distinctive colour change from yellow to red as the amide bond in the beta-lactam ring is hydrolysed by a Beta-lactamase. It is sensitive to hydrolysis by all known Beta-lactamases produced by Gram-positive and Gram-negative bacteria. 52 Procedure; The container was removed from the freezer and allowed to reach room temperature. Then a well separated representative colony from the primary isolation medium was selected. The colony was touched with the impregnated tip of the stick, which was rotated to pick up a small mass of cells. The inoculated tip of the stick was placed between the lid and the base of the inverted plate. The reaction requires moisture, so the inoculated tip of the stick was placed in the moisture condensate in the lid or in absence of condensate in the inverted lid; one or two drops of distilled water were added to the lid to moisten the tip of the stick. The reagent end of the stick was examined for up to five minutes or up to the end of fifteen minutes. A positive reaction was shown by the development of a pink/red colour. No colour change shows absence of Beta-lactamase. In every experiment an unused nitrocephine stick serves as a control. 3.6. Plasmid DNA isolation The alkaline Lysis method was adopted for the extraction. Buffer 1A, 200 µl, (see appendix 1) was added to the cell pellets and vortexed. Then, 400 µl of lysing solution was added and the tubes were inverted 20 times gently at room temperature bet (25-30 o C) and 300 µl ice cold buffers 2B (see appendix1) was added followed by vortexing and the mixture was placed on ice for 30 minutes. Centrifuge followed at 3,000 xg for 15 mins. To the supernatant was added 700 µl of chloroform and mixture was vortexed. Centrifugation again followed at 3,000 xg for 10 minutes. Then 500 µl of the aqueous layer was transfered into a fresh tube, and 1ml of absolute ethanol was added. The mixture was kept on ice for 1 hour. followed by centrifugation at 3,000 xg for 30minutes and then washing of pellets with 70% ethanol. By decantation, dry pellets were obtained, to which 50 µl of buffer 3C (appendix I) was added and then kept for electrophoresis on ice. The Buffer constituents are as shown in appendix I. 3.7. Curing of antibiotic resistance in Salmonella strains Twenty-five resistant clinical strains of Salmonella enterica (Human) were randomly selected from among the identified antibiotic resistant groups. The method used for this 53 test was the modified method as described by Onyenwe et al. (2011) with further modification in the use of more than one subinhibitory concentration of the curing agents. Overnight broth culture of each resistant strain and the control strain were obtained each in 10ml nutrient broth (5 test tubes per strain) containing 5, 2.5, 1.25, 0. 625, 0.3125 µg/ml, of the mutagens (acridine orange and ethidium bromide). The mixtures were o incubated at 37 C for 24hours. After the incubation, each mutagen-exposed culture was plated on Mueller Hinton agar medium and incubated. Colonies were subcultured each o into 5ml Mueller Hinton broth and incubated at 37 C for 24 hours. Then following -2 incubation, a 10 in sterile distilled water was made and after shaking, 0.1ml of the -2 dilution (10 ) was seeded into a molten Mueller Hinton agar (10-15ml), swirled to mix and then poured on a sterile culture plate and allowed to solidify. Wells of 6.5mm diameter each were later made on the set agar medium. The wells were then filled each with referenced MIC and resistant MIC (before curing) of each test antibiotic against each strain of Salmonella enterica, for cefotaxime, ceftriaxone, levofloxacin, augmentin®, and ciprofloxacin. After pre-incubation diffusion period of 2hours, plates o were incubated at 37 C for 24hours, and then examined for zones of growth inhibition measured in millimeter (mm). 3.8. Detection of ESBL’s using double disc synergy test (DDST). Procedure; 2 The test inoculum, (0.2 ml of 10 of over night broth culture) was spread evenly onto Mueller-Hinton agar (MHA-Oxoid, India) using a sterile cotton wool swab. A disc of augmentin® (20 µg amoxycillin + 10 µg Clavunalic acid) was placed on the surface of MHA; then discs of ceftriaxone CRO (30 µg), Ceftazidime CAZ (30 µg) and Cefotaxime CTX (30 µg) were kept around the disc of augumentin ® in such a way that each disc was at a distance ranging between 16 and 20 mm from the augmentin® disc (centre to centre). The plate was incubated at 37 °C 18- 24hours. The organisms were considered to be producing ESBL when the zone of growth inhibition around any of the Broad- spectrum cephalosporin discs showed a clear-cut increase towards the augmentin® (amoxycillin + clavunalic acid) disc (Yah, 2010). 54 3.9. Preparation of purified chromosomal DNA for PCR analysis using boiling method. Procedures; An overnight broth culture of 1.5ml was pipetted into an eppendorf tube and centrifuged at 5r.p.m. The supernatant was discarded and sterile water was introduced into the tube and the mixture was shaken vigorously by a vortexing machine and the supernatant was decanted. This process was repeated twice. Then the mixture was resuspended in 100µl sterile distilled water, vortexed and placed in a water block to boil for 10-20mins at o 100 C. After boiling, the mixture was then centrifuged for 10 mins, and the purified chromosomal DNA pellets were transferred into a new tube and stored on ice. 3.10. Agarose gel electrophoresis of DNA products The agarose powder was mixed with electrophoresis buffer to the concentration of 0.5µg/ml, and then heated in a microwave oven until completely melted. Then, ethidium bromide was added to the gel (0.5µg/ml concentration of agarose gel) at this point to facilitate visualization of plasmid DNA and PCR products after electrophoresis. After o cooling the solution to about 60 C, it was poured into a casting tray containing a sample comb and allowed to solidify. Then the comb was carefully removed. The gel, still in its plastic tray, was inserted horizontally into the electrophoresis chamber and covered with buffer solution. Samples containing DNA mixed with loading buffer were then pipetted into the sample wells; the lid and power leads were placed on the apparatus, and current was applied. The current was confirmed to be flowing by observing bubbles coming off the electrodes and movement of the tracking dyes. 3.11. Spectrophotometric quantification and purity test of chromosomal DNA Procedures: The spectrophotometer lens was cleaned with sterile distilled water. Then the screen of the computer to which it was connected to was adjusted, after blanking the spectrophotometer with sterile water. DNA sample of 0.2 µl was added on the lens, closed and the measure on the computer screen was clicked waiting for the readings. After each sample the lens was cleaned with sterile water and dried with dry cotton wool. 55 Note: The normal quantity required in DNA analysis is between 10 - 200 µl, while the purity level is detectable within the volume 1.5 - 2 µl. Results are shown on Appendix 3. 3.12. PCR analysis and gene sequencing The Polymerase Chain Reaction (PCR) was performed under the following conditions with the Solis biodyne 5x FIREPol Master mix Ready to load. The thermocycling o condition for Bla- SHV-1, Bla-TEM, and BlaCTX-M were 30 – 35 cycles at 95 C for o o o 30secs, 72 C for 1min.,66.2 for1 min., 72 C for 1min, 95 C for 30 sec., (PCR timing o 1.38- 2.58 hrs). The cycling condition for qnrB, GyrA and ParC were 30 cycles of 95 C o o o for 30 secs., 42 C for 1 mins, 72 Cfor 1 mins, and 95 C for 30 sec., (PCR timing 12.38 - 2.34 hrs.). The PCR primers to amplify the Bla- SHV-1, Bla-TEM, BlaCTX-M (universal primer) genes, Quinolone Resistant Regions QRDR’s of the GyrA and ParC genes and quinolone plasmid-mediating resistant qnrB type respectively were; Bla-SHV-1; F (5′-GTA TTG AAT TCA TGC GTT ATA TTC GCC TGT GTA-3′), R (5′- CAG AAT TCG GCT AGC GTT GCC AGT GCT CGA T-3’)oligonumber; 80303Y1185C02). Bla-TEM-1; F (5’-ATGAGTATTCAACATTTCCG-3’), R-(5’-ACC AAT GCT TAA TCA GTG AG -3’) (oligo number; 80303Y1185C01). BlaCTX-M ; F (5’ATG TGC AGY ACC AGT AAR GTK ATG GC-3’) ( oligo number; 90303X1185C03), R ( 5’- TGG GTR AAR TAR GTS ACC AGA AYC AGC GG- 3’) ( oligo number; 90303X1185C04) where R in the sequence is purine,Y is pyrimidine, and S is G or C. GyrA; F (5’CGT TGG TGA CGT AAT CGG- 3’) (oligo number; 00123839_3), R (5’CCG TAC CGT CAT AGT TAT- 3’) (oligo number; 00123839_4). ParC ; F (5’CTA TGC GAT GTC AGA GCT GG-3’) (oligo number;00123839_5), R(5’TAA CAG CAG CTC GGC GTA TT- 3’) (oligo number; 00123839_6), while QnrB is F-(5’-GAT CGT GAA AGC CAG AAA GG- 3’) (oligo number; 00123839_7), R- (5’-ATG AGC AAC GAT GCC TGG TA-3’) (oligo number; 00123839_8). 56 Procedure; The purified DNA template in PCR eppendorf tubes (2.0µl DNA) was mixed with the Master mix ready to load containing all the required contents as stated in the Appendix including the primers for the amplification. The mixture was vortexed to mix and then centrifuged before introducing it into the PCR machine (Eppendorf-Germany).Then the machine was adjusted to annealing temperature and switched to start. After the time for the amplification was completed, the PCR products were then taken for electrophoresis on the agarose gel as described earlier and finally viewed on the UV light for amplified image of the genes. The PCR products were now selected for gene sequencing, if the gene of interest is amplified and recovered. The DNA (amplified gene of the PCR products) was packed and sent to Gatc Biotech Laboratory, Germany, through correspondence in an Eppendorf tubes for sequencing. The gene sequencing was carried out at Gatc Biotech Germany using Sanger sequencing machine (ABI 3730 x l of Applied Biosystems, Germany) automatically edited with PhredPhrap. The Mega-5 soft ware was used for the alignment of the gene and the blasting was done using the NCBI database BLAST. 57 CHAPTER FOUR RESULTS 4.1 Authentication of Salmonella enterica Serovar.Typhi A total of 100 isolates of Salmonella enterica serovar.typhi were obtained from the Federal Medical Centres and University Teaching Hospital in the Southeast region of Nigeria between July, 2009 and September, 2010. The isolates were identified by cultural ® and biochemical characteristics using Microbact identification kit 2E. The slide agglutination test identified the antigenic property of the organism into O and H antigen, thereby identifying the isolate to be S. enterica serovar. typhi. All the isolates were Gram negative rods, oxidase negative, catalase positive, indole and Voges Proskauer (VP) negative, methyl red and Simmons citrate positive, H2S producing and urea negative. Some of these characteristics were used for the biochemical confirmation of Salmonella in this study as shown in Table 4.1. 4.2 Antibiogram screening and resistance pattern of the S.enterica Table 4.2 a- d shows the susceptibility pattern of 100 isolates out of which each twenty- five clinical strains of S. enterica serovar.Typhi obtained were from Federal Medical Centre Owerri (FMC), Federal Medical Centre (FMC) Umuahia (Table 4.2 b), University of Nigeria Teaching hospital (UNTH) Enugu (Table 4.2 c) and Federal Medical Centre (FMC) Abakaliki (Table 4.2 d). Some of the S. enterica strains were sensitive, but exhibited some forms of resistant on which 1or 2 colonies where found to be resistant (Resistant mutants- appendix II, plate 2) (SO10, 11, 13 in Table 4.2 a) in the area of zone of growth inhibition. These types of resistant were classified as resistant mutant (R.M), while some were intermediately sensitive and still exhibited the form of resistance. Some of the S.enterica strain on screening exhibited some forms of enzyme inhibition other than the conventional Beta-lactamase (SU38 in Table 4.2 b) which was responsible for their reduced susceptibility i.e Enzyme inactivation (E.I) of the antibiotics tested. Other strains though, completely showed total resistance (R) to some group of antibiotic tested 58 (SE69 and SE75 in Table 4.2 c), and isolates from SA77 and SA88 (Table 4.2 d). Similarly, analysis showed that the same trend of results (resistant pattern) was observed when the fluoroquinolone and cephalosporins groups of antibiotics were used on S. enterica serovar isolates, as stated on Tables 4.2 a-d of the various hospitals analysed. The resistant mutant and enzyme inhibition plates 1 and 2 are as shown in appendix II. 59 Table 4.1. Authentication of Salmonella enterica Serovar.Typhi Biochemical test probability rate o f C o l o u r / r e a c t ion 99.8 (%) based on the Microbact soft ware Oxidase Yellow - Lysine decarxylase Yellow + Glucose fermentation Yellowish + HS2 Black deposit + Mannose fermentation Yellowish + Catalase Effervescent bubbles + Nitrate utilization or reduction (ONPG) Red + Indole Red - Urease Orange - Simone Citrate utilization Blue-green + Voges Proskauer Pink - Trypton Deaminase Yellowish brown - Gram reactions under microscope Tiny short negative rods KEY: + = Positive reaction, - = Negative reaction 60 Table 4.2 a. Antibiogram of S. enterica from Federal medical center (FMC) Owerri, Imo state. S/NO SXT CH SP CPX AM GEN PEF OFX STR LEV CRO CTX CAZ AMC RM O1. 18 15 R 30 R 23 25 24 R 20 21 23 R R RM RM RM O2. 18 11 18 22 18 23 23 23 18 25 28 28 25 R O3. 15 12 20 23 18 17 23 23 18 30 14 23 29 R EI RM O4. 8 8 17 23 18 19 23 23 15 25 25 24 28 18 O5. R R R 32 R 18 25 17 R 22 24 25 R R O6. 19 12 18 20 16 21 23 23 18 R 14 10 10 R RM O7. 10 R 15 21 15 20 23 22 14 25 23 23 19 R O8. 10 16 18 21 15 23 23 25 15 20 29 29 R 19 O9. R R R 8 R 23 17 16 8 22 21 24 R R RM O10. R R R R R 23 16 16 10 14 21 25 20 R RM RM RM O11. R R R R R R 18 16 R 20 22 28 R R O12. R R R R R 14 15 17 10 22 21 23 R R EI RM RM RM O13. R R 10 24 R R 20 22 10 20 20 23 22 13 O14. R R R 24 R 16 20 23 10 10 R R R R O15. R R 11 23 R 21 21 23 10 26 20 23 24 R EI O16. R R 10 18 R 15 23 23 10 25 21 26 R R O17. R R R 24 R R 14 14 10 22 20 24 R R O18. R R R R R R R R R 24 21 28 20 R O19. R R R 10 R 14 8 8 R 20 21 25 27 R O20. 18 15 R 35 R 26 24 25 R R R R 9 R O21. R 11 R 28 R 20 18 15 R 25 27 25 23 R EI O22. 16 15 16 28 R 18 17 14 R 30 30 30 30 12 O23. 16 16 12 30 R 16 19 15 11 25 20 24 8 9 O24. 18 15 20 30 10 18 24 18 13 14 R 31 9 R O25 16 R 12 28 10 16 15 16 16 25 25 28 8 8 T/C. 16 R 27 25 15 18 17 16 R 28 26 28 24 12 KEY: SXT= cotrimoxazole 30µg, CH= chloramphenicol 30µg, SP = sparfloxacin 10µg, CPX= ciprofloxacin 10µg, AM= amoxicillin 30µg, GEN= gentamycin 10µg, PEF= pefloxacin 30µg, OFX= ofloxacin 10µg, STR= streptomycin 30µg, LEV= levofloxacin 5µg, CRO= ceftriaxone 30µg, CTX= cefotaxime 30µg, CAZ= ceftazidime 30µg, AMC= amoxicillin /clavulanic acid 30µg. R= Resistant, S= Sensitive, R.M = Resistant mutant, E.I=Enzyme inhibition. 61 Table 4.2 b. Antibiogram of S. enterica from Federal medical center ( FMC) Umuahia, Abia State (Continue) S/NO SXT CH SP CPX AM GEN PEF OFX STR LEV CRO CTX CAZ AMC U26. 14 R 12 28 12 16 16 17 13 24 22 23 9 R RM U27. R R R 32 R 30 28 26 11 25 25 25 20 8 RM RM RM RM U28. R R 22 36 R R R R R 23 17 28 20 15 U29. 18 15 22 28 10 14 17 17 15 23 20 29 R R U30. R R R 29 R 16 16 15 10 25 24 23 20 8 U31. 15 14 15 32 12 18 14 15 16 25 26 30 21 15 RM U32. R R R 32 R 23 26 26 10 27 26 27 25 12 U33. 10 14 12 24 12 17 R 12 17 25 29 29 10 R EI U34. 14 13 15 29 10 13 14 14 11 28 21 23 24 R U35. R R R 30 R 25 22 22 14 25 21 23 27 13 RM U36. R R R 34 15 24 19 18 17 28 29 29 22 R EI U37. 14 13 R 30 R 13 16 16 R 25 20 29 22 R EI U38. R 14 R 28 12 13 15 17 11 24 20 23 10 R U39. 14 R R 27 R R R 14 R 27 20 28 20 18 RM U40. R R R 27 12 R R R R 16 10 10 20 R U41. 10 R R 25 14 13 16 17 14 25 20 20 26 R U42. 17 R R 30 14 R 16 16 10 23 24 25 21 R RM RM RM U43. R R R 28 15 R 16 15 R 22 21 27 25 R RM U44. R R R 25 R R R R R 26 25 25 21 R U45. 16 R R 28 17 16 16 18 14 23 24 25 19 R RM RM RM U46. R R R 29 18 17 15 13 10 22 20 23 20 R U47. 18 15 10 30 13 12 16 17 R 24 21 23 25 R RM U48. R R R 25 15 16 R R R 15 21 23 15 R U49. 14 R 10 23 14 R R R 11 11 8 R R R U50 R R 11 20 10 R 18 18 R 26 28 28 27 R KEY: SXT= cotrimoxazole 30µg, CH= chloramphenicol 30µg, SP = sparfloxacin 10µg, CPX= ciprofloxacin 10µg, AM= amoxicillin 30µg, GEN= gentamycin 10µg, PEF= pefloxacin 30µg, OFX= ofloxacin 10µg, STR= streptomycin 30µg, LEV= levofloxacin 5µg, CRO= ceftriaxone 30µg, CTX= cefotaxime 30µg, CAZ= ceftazidime 30µg, AMC= amoxicillin /clavulanic acid 30µg. R= Resistant, S= Sensitive, R.M = Resistant mutant, E.I=Enzyme inhibition. 62 Table 4.2 c. Antibiogram of S. enterica from (UNTH) Enugu, Enugu State. (Continue) S/NO SXT CH SP CPX AM GEN PEF OFX STR LEV CRO CTX CAZ AMC E51. R R R 18 11 R 16 16 R 28 21 23 26 R E52. R R R 19 11 R R R R 20 25 25 15 R E53. R R R R R R 16 16 R 18 22 20 24 R E54. R R 12 22 15 12 19 19 R 25 23 25 26 R E55. R R R 24 R R 15 14 R 28 24 23 22 R E56. R R 12 20 13 R 17 16 18 23 22 23 22 R E57. R R 11 22 15 R 20 20 R 25 29 28 26 R EI E58. R 13 15 22 18 12 16 16 R 29 25 24 28 R E59. 10 14 15 25 12 12 15 16 11 15 31 31 30 R RM E60. 16 10 R 20 R 17 10 R 14 R 28 25 26 R RM E61. R R R 18 R 10 R R R 20 25 26 27 R RM RM RM RM RM E62. 10 15 15 18 R 16 17 12 R 25 28 25 22 15 E63. R 14 14 19 R 15 15 R R 11 21 26 25 R EI RM E64. 16 16 15 30 10 12 R R R 11 11 R 14 R RM RM RM RM RM E65. R R R R R 12 14 15 10 14 17 10 10 14 E66. R R R R R 15 10 10 R 11 8 8 11 R RM E67. R R R 22 R 16 10 R R 10 R R 14 R RM E68. R R R 17 R 12 10 16 R 11 R R 14 R E69. 10 8 R 15 R 16 R R R 8 8 8 8 R E70. R R R 17 R R 17 14 10 28 26 25 26 R E71. R R R R R R R 18 R 19 20 28 20 R RM RM E72. R R R R R R R R R 18 28 9 25 R RM RM RM RM RM RM RM E73. R R R 18 R R 14 14 R 20 24 R 12 15 E74. R R 13 20 R 12 14 14 R 18 14 24 20 R E75 R R R 8 R R R R R 16 8 8 R R KEY: SXT= cotrimoxazole 30µg, CH= chloramphenicol 30µg, SP = sparfloxacin 10µg, CPX= ciprofloxacin 10µg, AM= amoxicillin 30µg, GEN= gentamycin 10µg, PEF= pefloxacin 30µg, OFX= ofloxacin 10µg, STR= streptomycin 30µg, LEV= levofloxacin 5µg, CRO= ceftriaxone 30µg, CTX= cefotaxime 30µg, CAZ= ceftazidime 30µg, AMC= amoxicillin /clavulanic acid 30µg. R= Resistant, S= Sensitive, R.M = Resistant mutant, E.I=Enzyme inhibition. 63 Table 4.2 d. Antibiogram of S. enterica from (Federal Medical Center (FMC) Abakiliki, Ebonyi State (Continue) S/NO SXT CH SP CPX AM GEN PEF OFX STR LEV CRO CTX CAZ AMC RM A76. R R R 20 R R R R R 14 21 11 23 R A77. R R R R R R R 11 R 11 R R 23 R RM A78. R R R 9 R R R 18 R 10 R 10 8 R RM EI A79. R R 12 12 R R R 14 R 18 24 28 8 8 RM A80. R R R R R R R 16 R 10 22 R R R RM RM A81. R R R R R 10 14 14 R 11 R R 8 15 RM A82. R R R 14 R R 14 10 R 10 28 28 12 R A83. R 15 R R R 10 R 11 R 11 R R 26 R A84. R R R R R R 14 13 R 11 R R R R A85. R 17 R R R 16 14 15 R 13 20 10 9 R A86. R R R R R R 14 14 R 13 20 10 9 R EI A87. R R R 23 R 13 14 14 R 14 R R 13 R RM RM RM RM A88. R R R R R R 14 13 R R 25 25 R R A89. R R 14 22 R R 18 18 11 24 22 23 20 R RM EI A90. R R R 25 R R 16 15 R 16 24 13 R R A91. R R R R R R R R 14 17 22 27 12 14 A92. 16 12 16 20 15 16 20 21 18 21 25 25 22 11 A 93. R R R R R 14 R R R 20 21 24 20 14 A 94. R R R R R R R R R 18 26 25 8 R EI A 95. R 12 12 18 R 14 20 20 15 14 27 25 8 R RM RM RM A 96. R R R 16 R R 17 14 15 14 28 14 27 R RM A 97. R R R R R R 17 17 16 17 23 23 24 15 RM RM A 98. R R R R R R 16 16 R 14 20 29 23 15 A 99. R R R 22 R R 14 14 11 14 28 15 9 R A100. R R R 25 R R 15 R R 13 23 13 R R KEY: SXT= cotrimoxazole 30µg, CH= chloramphenicol 30µg, SP = sparfloxacin 10µg, CPX= ciprofloxacin 10µg, AM= amoxicillin 30µg, GEN= gentamycin 10µg, PEF= pefloxacin 30µg, OFX= ofloxacin 10µg, STR= streptomycin 30µg, LEV= levofloxacin 5µg, CRO= ceftriaxone 30µg, CTX= cefotaxime 30µg, CAZ= ceftazidime 30µg, AMC= amoxicillin /clavulanic acid 30µg. R= Resistant, S= Sensitive, R.M = Resistant mutant, E.I=Enzyme inhibition. 64 4.3. Determination of the Minimum Inhibitory Concentration (MIC) using tube dilution method on five selected antibiotics Table 4.3. shows the Minimum Inhibitory Concentration (MIC) and Minimum Bacteriocidal Concentration (MBC) of the isolates of S. enterica using the selected antibiotics as follows; levofloxacin (LEV), ciprofloxacin (CPX), amoxiclavulanic acid (AMC), cefotaxime (CTX) and ceftriaxone (CRO). From Table 4.7; having the code number SO1- SO25, levofloxacin (a fluoroquinolone) and ceftriaxone had their MIC within the range of 1.56 µg/ml - 3.125 µg/ml and 1.56 µg/ml- 6.25 µg/ml respectively, except in some cases where the isolates were very resistant wherby MIC increased to 25.0 µg/ml and 12.5 µg/ml respectively, followed by cefotaxime and ciprofloxacin having the same range (1.56 - 3.125 µg/ml) except in cases where some isolates showed high resistance, thereby increasing the MIC’s of both drugs to 6.25 µg/ml and 12.5 µg/ml respectively. The typed culture used had MIC range of 1.56 µg/ml except with the antibiotic, amoxi-clavulanic acid where it had MIC’s range of 12.5 µg/ml- 25 µg/ml. All the isolates showed increased MIC’s for amoxi-clavulanic acid up to 50 µg/ml. The same trend of MIC results was observed in the other isolates from Umuahia (SU26-SU50), but with difference from Enugu (SE51-SE75), and Abakaliki (SA76 - SA100), which showed sharp increase in the MIC’s. However, some of the antibiotics like levofloxacin, ciprofloxacin, ceftriaxone and cefotaxime showed good activity as relate to having their potency intact, but amoxi-clavulanic acid showed increased MIC in almost all the isolates of the centres as shown in Tables 4.3 a- d. 65 Table 4.3 a. The MICs and MBCs (µg/l) of five selected antibiotics against 25 isolates of S. enterica from FMC owerri S/NO SEROTYPE LEV CPX AMC CTX CRO MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC O1. S.typhi O/H 1.56 3.125 3.125 6.25 50.0 >50.0 3.125 6.25 1.56 3.125 O2. ,, 6.25 12.5 3.125 6.25 50.0 >50.0 3.125 6.25 1.56 3.125 O3. ,, 1.56 3.125 3.125 6.25 50.0 >50.0 6.25 12.5 6.25 12.5 O4. ,, 1.56 3.125 1.56 3.125 25.25 >50.0 1.56 3.125 1.56 3.125 O5. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 O6. ,, 25.0 >25.0 3.125 6.25 50.0 >50.0 6.25 12.5 6.25 12.5 O7. ,, 1.56 3.125 3.125 6.25 50.0 >50.0 3.125 6.25 3.125 6.25 O8. ,, 1.56 3.125 3.125 6.25 50.0 >50.0 3.125 6.25 1.56 3.125 O9. ,, 3.125 6.25 12.5 25.0 50.0 >50.0 3.125 6.25 1.56 3.125 O10. ,, 3.125 6.25 12.5 25.0 50.0 >50.0 6.25 12.5 1.56 3.125 O11. ,, 1.56 3.125 12.5 25.0 50.0 >50.0 1.56 3.125 1.56 3.125 O12. ,, 1.56 3.125 12.5 25.0 50.0 >50.0 1.56 3.125 1.56 3.125 O13. ,, 1.56 3.125 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 O14. ,, 25.0 >25.0 3.125 6.25 50.0 >50.0 6.25 12.5 6.25 12.5 O15. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 3.125 6.25 O16. ,, 3.125 6.25 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 O17. ,, 3.125 6.25 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 O18. ,, 1.56 3.125 12.5 25.0 50.0 >50.0 3.125 6.25 1.56 3.125 O19. ,, 1.56 3.125 12.5 25.0 50.0 >50.0 3.125 6.25 1.56 3.125 O20. ,, 25.0 >25.0 1.56 3.125 50.0 >50.0 12.5 25.0 12.5 25.0 O21. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 O22. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 O23. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 O24. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 12.5 25.0 12.5 25.0 O25 ,, 1.56 3.125 1.56 3.125 50.0 >50.0 6.25 12.5 3.125 6.25 T/C ATCC14028 1.56 3.125 1.56 3.125 25.5 50.0 1.56 3.125 1.56 3.125 KEY: CPX= ciprofloxacin 10 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg. MIC = Minimum Inhibitory Concentration, MBC = Minimum Bacteriocidal Concentration, TC= Typed culture, O1- 25= Isolates from Owerri. 66 Table 4.3 b. The MICs and MBCs (µg/l) of five selected antibiotics against 25 isolates of S. enterica from FMC Umuahia (continued) S/NO SEROTYPE LEV CPX AMC CTX CRO MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC U26. S.typhi O/H 3.125 6.25 3.125 6.25 50.0 >50.0 3.125 6.25 1.56 3.125 U27. ,, 6.25 12.5 6.25 12.5 50.0 >50.0 3.125 6.25 6.25 12.5 U28. ,, 12.5 25.5 6.25 12.5 50.0 >50.0 3.125 6.25 6.25 12.5 U29. S.typhi O 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U30. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 3.125 6.25 1.56 3.125 U31. ,, 1.56 3.125 1.56 3.125 12.5 25.0 1.56 3.125 1.56 3.125 U32. ,, 1.56 3.125 1.56 3.125 25.0 >25.0 1.56 3.125 1.56 3.125 U33. S.typhi O/H 1.56 3.125 1.56 3.125 25.0 >25.0 1.56 3.125 1.56 3.125 U34. S.typhi O/H 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U35. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 3.125 6.25 1.56 3.125 U36. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 3.125 6.25 U37. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U38. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U39. ,, 6.25 12.5 1.56 3.125 12.5 25.0 6.25 12.5 6.25 12.5 U40. ,, 6.25 12.5 1.56 3.125 50.0 >50.0 6.25 12.5 6.25 12.5 U41. ,, 1.56 3.125 1.56 3.125 25.0 >25.0 1.56 3.125 1.56 3.125 U42. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U43. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 6.25 12.5 6.25 12.5 U44. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 6.25 12.5 1.56 3.125 U45. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 3.125 6.25 1.56 3.125 U46. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U47. ,, 3.125 6.25 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 U48. ,, 6.25 12.5 6.25 12.5 50.0 >50.0 6.25 12.5 3.125 6.25 U49. ,, 12.5 25.0 1.56 3.125 50.0 >50.0 12.5 25.0 12.5 25.0 U50 ,, 1.56 3.125 1.56 3.125 50.0 >50.0 3.125 6.25 3.125 6.25 T/C ATCC14028 1.56 3.125 1.56 3.125 25.5 50.0 1.56 3.125 1.56 3.125 KEY: CPX= ciprofloxacin 10 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg. MIC = Minimum Inhibitory Concentration, MBC = Minimum Bacteriocidal Concentration, TC= Typed culture, U26- 50 = Isolates from Umuahia. 67 Table 4.3 c. The MICs and MBCs (µg/l) of five selected antibiotics against 25 isolates of S. enterica from UNTH Enugu (continued) S/NO SEROTYPE LEV CPX AMC CTX CRO MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC E51. S.typhi O/H 1.56 3.125 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 E52. ,, 3.125 6.25 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 E53. ,, 3.125 6.25 12.5 25.0 50.0 >50.0 6.25 12.5 1.56 3.125 E54. S.typhi H 1.56 3.125 3.125 6.25 50.0 >50.0 6.25 12.5 3.125 6.25 E55. S.typhi O/H 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 E56. ,, 1.56 3.125 1.56 3.125 25.0 >25.0 1.56 3.125 1.56 3.125 E57. ,, 1.56 3.125 1.56 3.125 25.0 >25.0 3.125 6.25 3.125 6.25 E58. S.typhi H 1.56 3.125 3.125 6.25 25.0 >25.0 1.56 3.125 1.56 3.125 E59. S.typhi O/H 6.25 12.5 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 E60. ,, >12.5 >25.0 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 E61. ,, 3.125 6.25 3.125 6.25 50.0 >50.0 6.25 12.5 6.25 12.5 E62. ,, 1.56 3.125 3.125 6.25 50.0 >50.0 1.56 3.125 1.56 3.125 E63. ,, 12.5 25.0 1.56 3.125 50.0 >50.0 3.125 6.25 1.56 3.125 E64. ,, 12.5 25.0 1.56 3.125 25.0 >25.0 12.5 25.0 6.25 12.5 E65. ,, 12.5 25.0 12.5 25.0 25.0 >25.0 12.5 25.0 3.125 6.25 E66. ,, 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 E67. ,, 12.5 25.0 3.125 6.25 50.0 >50.0 12.5 25.0 12.5 25.0 E68. ,, 12.5 25.0 6.25 12.5 50.0 >50.0 12.5 25.0 12.5 25.0 E69. ,, 12.5 25.0 6.25 12.5 50.0 >50.0 12.5 25.0 12.5 25.0 E70. ,, 3.125 6.25 3.125 6.25 50.0 >50.0 3.125 6.25 3.125 6.25 E71. ,, 3.125 6.25 12.5 25.0 25.0 >25.0 3.125 6.25 3.125 6.25 E72. ,, 3.125 6.25 12.5 25.0 25.0 >25.0 12.5 25.0 3.125 6.25 E73. ,, 3.125 6.25 3.125 6.25 12.5 25.0 12.5 25.0 6.25 12.5 E74. ,, 3.125 6.25 1.56 3.125 25.0 >25.0 6.25 12.5 6.25 12.5 E75 ,, 6.25 12.5 12.5 25.0 25.0 >25.0 25.0 >25.0 12.5 25.0 T/C ATCC14028 1.56 3.125 1.56 3.125 25.5 50.0 1.56 3.125 1.56 3.125 KEY: CPX= ciprofloxacin 10 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg. MIC = Minimum Inhibitory Concentration, MBC = Minimum Bacteriocidal Concentration, TC= Typed culture, E51- 75 = Isolates from Enugu. 68 Table 4.3 d. The MICs and MBCs (µg/l) of five selected antibiotics against 25 isolates of S. enterica from FMC, Abakaliki (continued). S/NO SEROTYPE LEV CPX AMC CTX CRO MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC A76. S.typhi O/H 12.5 25.0 1.56 3.125 50.0 >50.0 12.5 25.0 12.5 25.0 A77. ‘’ 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A78. ‘’ 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A79. ‘’ 3.125 6.25 12.5 25.0 50.0 >50.0 12.5 25.0 3.125 6.25 A80. S.typhi H 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A81. S.typhi O/H 12.5 25.0 12.5 25.0 25.0 >25.0 12.5 25.0 12.5 25.0 A82. ,, 12.5 25.0 6.25 12.5 25.0 >25.0 3.125 6.25 3.125 6.25 A83. ,, 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A84. ,, 12.5 25.0 12.5 25.0 25.0 >25.0 12.5 25.0 12.5 25.0 A85. ,, 12.5 25.0 12.5 25.0 25.0 >25.0 12.5 25.0 12.5 25.0 A86. ,, 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A87. ,, 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A88. ,, 12.5 25.0 12.5 25.0 50.0 >50.0 12.5 25.0 12.5 25.0 A89. ,, 1.56 3.125 1.56 3.125 50.0 >50.0 1.56 3.125 1.56 3.125 A90. S,typhi O 6.25 12.5 6.25 12.5 50.0 >50.0 6.25 12.5 6.25 12.5 A91. S.typhi O/H 3.125 6.25 12.5 25.0 25.0 >25.0 3.125 6.25 1.56 3.125 A92. ,, 1.56 3.125 1.56 3.125 25.0 >25.0 1.56 3.125 1.56 3.125 A93. ,, 1.56 3.125 12.5 25.0 25.0 >25.0 1.56 3.125 1.56 3.125 A94. ,, 3.125 6.25 12.5 25.0 50.0 >50.0 6.25 12.5 6.25 12.5 A95. ,, 12.5 25.0 3.125 6.25 50.0 >50.0 6.25 12.5 3.125 6.25 A96. ,, 12.5 25.0 3.125 6.25 50.0 >50.0 12.5 25.0 3.125 6.25 A97. ,, 3.125 6.25 12.5 25.0 50.0 >50.0 1.56 3.125 1.56 3.125 A98. ,, 12.5 25.0 12.5 25.0 50.0 >50.0 3.125 6.25 1.56 3.125 A99. ,, 12.5 25.0 12.5 25.0 25.0 >25.0 6.25 12.5 3.125 6.25 A100. ,, 12.5 25.0 6.25 12.5 25.0 >25.0 12.5 25.0 12.5 12.5 T/C ATCC14028 1.56 3.125 1.56 3.125 25.5 50.0 1.56 3.125 1.56 3.125 KEY: CPX= ciprofloxacin 10 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg. MIC = Minimum Inhibitory Concentration, MBC = Minimum Bacteriocidal Concentration, TC= Typed culture, A76 – 100 = Isolates from Abakaliki. 69 4.4. Prevalence of S. enterica in various Departmental units and hospitals in the Southeast region of Nigeria. Table 4.4 shows the percentage prevalence rate of S. enterica recovered from various units in the hospitals from the Southeast region investigated. Analysis revealed that the hospital in Owerri had a total of Twenty-five isolates of S. enterica (the same number of isolates applies to other hospitals in the region) 14 (56%) of the isolates were recovered from the general outpatient department (GOPD). Other departmental units were National Health insurance scheme (NHIS) 5 (20%), In-patient units (IPU) 4 (16%), Children out Patient units and Emergency Patient units were found to have only 1 (4%) of the isolates each. In the hospital in Umuahia, IPU had the highest number of S. enterica isolated 23 (92%) and GOPD 2 (8%). No isolates were recovered from other units from the hospital in Umuahia during the period of investigation (i.e other units like NHIS, CHOP, EPU and the SKIN CLINIC recorded 0 (0%) of isolates. From other hospitals in Enugu and Abakaliki, the percentage recovery of isolates were; NHIS 0 (0%) and 3 (12%), IPU 8 (35%) and 13 (52%), GOPD 15 (60%) and 8 (32%), CHOP 1 (4%) and 0 (0%), EPU 0 (0%) and1 (4%), while the SKIN CLINIC recorded 1 (4%) and 0 (0%) respectively. 70 Table 4.4. Prevalence of S. enterica in various Departmental unit and hospitals in the South-east region of Nigeria S/NO Unit/Department (Location) (Location) (Location) (Location) Owerri Umuahia Enugu Abia S.enterica S.enterica S.enterica S.enterica 1-25 26-50 51-75 76-100 Total 1. NHIS 5 (20%) 0 (0%) 0 (0%) 3 (12%) = 8 2. IPU 4 (16%) 23 (92%) 8 (32%) 13 (52%) = 48 3. GOPD 14 (56%) 2 (8%) 15 (60%) 8 (32% ) = 39 4. CHOP 1 (4%) 0 (0%) 1 (4%) 0 (0%) = 2 5. EPU 1 (4%) 0 (0%) 0 (0%) 1 (4%) = 2 6. SKIN CLINIC 0 (0%) 0 (0%) 1 (4%) 0 (0%) = 1 TOTAL 25 25 25 25 = 100 KEY: NHIS = National Health Insurance Scheme, IPU= In - Patient Unit , GOPD= General Out Patient Department , CHOP= Children Out Patient , EPU= Emergency Patient Unit, SKIN= Skin Clinic. 71 4.5 Beta-lactamase production and plasmid profiling of S. enterica isolates in relation to gender distribution Tables 4.5 show Beta-lactamase production and plasmid profiling of S. enterica isolates in relation to gender distribution. Results (Table 4.5 a) show that only 2 (8%) isolates out of the twenty-five isolates recovered in GOPD unit from Federal Medical Center Owerri harboured plasmids, while 20 (80%) isolates produced the conventional Beta- lactamase. It was also observed that isolates recovered from a female child of 2years in the CHOP unit produced the conventional Beta-lactamase enzyme but was resistant to only 4 (28.6%) antibiotics used in this study, while an isolate recovered in the EPU unit from a female child of 1 year old was resistant to the 4 (28.6%) of the antibiotics used but produced no conventional Beta-lactamase and harboured no plasmid. From the results obtained from the S. enterica isolates (Table 4.5b) from Federal Medical Centre (FMC) Umuahia, only 3 (12%) harboured plasmid, while 18 (72%) of the isolates recovered produced the conventional Beta-lactamase enzyme. It was also observed that isolates recovered from 2 (8%) female children in the IPU produced Beta-lactamase, though no plasmids were found on them, they were resistant to only 5 (35.7) of the 14 antibiotic used. Results from University of Nigeria Teaching Hospital (UNTH) Enugu (Table 4.5c) showed that there were no plasmid recovered from the twenty-five isolates but 20 (80%) of the isolates produced the conventional Beta-lactamase enzyme. It was also observed from this study that an isolate recovered from a child of 10years from CHOP unit produced no conventional Beta-lactamase, harboured no plasmid but was resistant to 9 (64.3%) of the antibiotics used for the study. Results on the S. enterica recovered from Federal Medical Centre (FMC) Abakaliki (Table 4.5 d) showed that 4 (16%) of the isolates harboured plasmids, with one of isolates habouring the plasmid coming from a female child of 6 years old in the NHIS unit. Analysis showed that the isolates recovered from the female child of 6 years as described above produced the conventional ßeta- lactamse and was resistant to 10 (71.4%) of the 14 antibiotics used in this study. Almost all the isolates recovered from this part of Southeast (Abakaliki) in this study, produced the conventional ßeta-lactamase. This could be as a result of the isolates been recovered 72 from IPU and GOPD unit of the hospital. It was also observed that one isolate recovered from a female patient in the IPU unit harboured no plasmid, but produced the Beta- lactamase enzyme and was resistant to only one antibiotic used in this study (Table 4.5 d). The resistance by S. enterica on the number of antibiotics tested was found in FMC Abakaliki to be the highest in the region (Southeast Nigeria). Plate 4.1 shows the Agarose gel Electrophoresis pattern revealing single PCR amplification of BlaCTX-M gene on S. enterica from Federal Medical Centre (FMC) Owerri. The PCR amplicons size of 593 bp’s were as shown on lane numbers 3 (SO3), 14 (SO14), 15 (SO15) and 24 (SO24) and were positive for BlaCTX-M gene (A class of CTX-M) type enzyme of the extended spectrum B-lactamases (ESBLs); the other blaSHV, blaTEM and quinolone resistance protein were not detected on the isolates. The marker used here was DNA molecular marker of 100 bp ladder (see Fig. 4.1, lane M), while lane T was a type culture strain (ATCC14028) negative for BlaCTX-M gene used as positive control (though no amplicon was seen) and lane N was the negative control containing sterile water. Plate 4.2 shows the agarose gel electrophoresis pattern, revealing single PCR amplification of BlaCTX-M gene in S. enterica from Umuahia and Enugu. The PCR amplicons size of 593bp’s were as shown on lanes 28 (SU28), 39 (SU39), 40 (SU40), 49 (SU49) and 50 (SU50) and were positive for BlaCTX-M gene. Lane numbers 53 (SE53) and 54 (SE54) having the same amplicons size of 593 bp’s were from UNTH Enugu. The lanes labeled M, T, and N were for DNA molecular marker, typed culture for positive control and sterile water for negative control. Plate 4.3 shows the agarose gel electrophoresis pattern, revealing single PCR o amplification of BlaCTX-M gene amplified at different annealing temperature of 62.5 C at the same cycling condition. The results of the PCR amplicons size were still seen at 593bp’s on lane number 15 (SO15) at different annealing temperature (Plate 4.1, lane 15 the PCR amplicons sizes for the BlaCTX-M gene was almost faded, while at the same annealing temperature it was prominent). Also on lane number 27 (SU27), there were no 73 amplicons, while at this temperature, the PCR amplicon size 593bp’s were amplified. Note: The primer used here is a universal primer. Plate 4.4 shows the agarose gel electrophoresis pattern of single PCR amplification of S. enterica from Umuahia and Enugu. The PCR amplicons sizes of 593 were as shown on only BlaCTX-M gene on lane number 44 (SU44). Note also, that there were no amplicons on lane number 44 (Plate 4.2). Lane M shows DNA molecular marker of 100bp’s ladder. Plate 4.5 shows the plasmid pattern of S. enterica serovar typhi on the agarose gel electrophoresis. It could be deduced that the molecular weights of the plasmid were considerably low. Lane numbers 2 and 9 (SO2 and SO9) show molecular weight of 1.39 kbs and 1.37 kbs (SO2) and 1.37 kbs (SO9). The isolates on lane number 28, 40, and 43 (SU28, SU40 and SU43) had the molecular weight of 1.37kbs respectively, just as M.wt of 1.37 kbs was obtained for each of the isolates SA76, SA81, SA83, and SA85. Thus, the plasmid profile revealed that most isolate of S. enterica screened harbored plasmids of low molecular weight. 74 Table 4.5 a. Beta-lactamase production and plasmid profiling of isolates of S. enterica from Federal Medical Center Owerri, in relation to gender distribution. Org. Unit/ Sex Age Beta Lactamase Plasmid /Mwt. Antibiotics Code Dept. Range Production Resistant S/NO (yrs) (%) O1. IPU M Adult + - 5 (35.7%) O2. GOPD F 20 + + 2 (14.3%) O3. GOPD F Adult + - 2 (14.3%) O4. GOPD F 26 - - 3 (21.4%) O5. GOPD F 22 + - 7 (50.0%) O6. GOPD F Adult + - 4 (28.6%) O7. GOPD F Adult + - 3 (21.4%) O8. GOPD F Adult + - 2 (14.3%) O9. GOPD F Adult + + 8 (57.1%) O10. GOPD M Adult + - 7 (50.0%) O11. NHIS F Adult + - 9 (64.3%) O12. GOPD M Adult + - 9 (64.3%) O13. NHIS F Adult + - 7 (50.0%) O14. GOPD M Adult + - 10 (71.4%) O15. GOPD F 50 + - 6 (42.9%) O16. GOPD F 50 + - 7 (50.0%) O17. NHIS M Adult - - 8 (57.1%) O18. GOPD F 65 - - 10 (71.4%) O19. NHIS F Adult + - 10 (71.4%) O20. IPU M Adult + - 8 (57.1%) O21. IPU M Adult + - 5 (35.7%) O22. IPU F Adult - - 3 (21.4%) O23. EPU F 1 - - 4 (28.6%) O24. NHIS M Adult + - 6 (42.9%) O25 CHOP F 2.5 + - 4 (28.6%) KEY: F= Female, M= Male , NHIS = National Health Insurance Scheme , IPU= In - Patient Unit, GOPD = General Out Patient Department , CHOP = Children Out Patient, EPU= Emergency Patient Unit. Note: Adult (includes male and female between 18 and above, whose actual age was not determined), Nil= Negative 75 Table 4.5 b. Beta-lactamase production and plasmid profiling of isolates of S. enterica from Federal Medical Center Umuahia, in relation to gender distribution (continue). Org. Unit/ Sex Age Beta- Plasmid/ Mwt. Antibiotic Code Dept. Range Lactamase Resistant S/NO (yrs) Production (%) U26. IPU F Adult - - 5 (35.7%) U27. IPU F Adult + - 6 (42.9%) U28. IPU M Adult + + 7 (50.0%) U29. IPU F Adult - - 3 (21.4%) U30. IPU F Adult - - 6 (42.9%) U31. IPU F 28 + - 1 (71.0%) U32. IPU F Adult + - 6 (42.9%) U33. IPU M Adult + - 7 (50.0%) U34. IPU F Adult - - 4 (28.6%) U35. IPU M 19 + - 5 (35.7%) U36. IPU M Adult + - 4 (28.6%) U37. IPU F 13 - - 5 (35.7%) U38. IPU F Adult - - 6 (42.9%) U39. IPU M 24 + 6 (42.9%) U40. IPU F Adult + + 11 (78.5%) U41. IPU F 3 + - 5 (35.7%) U42. IPU F Adult - - 4 (28.6%) U43. IPU M Adult + + 5 (35.7%) U44. IPU F Adult + - 9 (64.3%) U45. IPU M Adult + - 3 (21.4%) U46. IPU F 4 + - 5 (35.7%) U47. GOPD F Adult + - 4 (28.6%) U48. GOPD M 44 + - 7 (50.0%) U49. IPU F Adult + - 11 (78.5%) U50 IPU M Adult + - 7 (50.0%) KEY: F= Female , M= Male , NHIS = National Health Insurance Scheme, IPU = In - Patient Unit, GOPD = General Out Patient Department, CHOP = Children Out Patient, EPU = Emergency Patient Unit. Note: Adult (includes male and female between 18 and above, whose actual age was not determined), Nil= Negative 76 Table 4.5 c. Beta-lactamase production and plasmid profiling of isolates of S. enterica from University of Nigeria Teaching Hospital (UNTH) Enugu, in relation to gender distribution (continue). Org. Unit/ Sex Age Beta Plasmid/Mwt. Antibiotic Code Dept. Range Lactamase Resistant S/NO (yrs) Production (%) E51. IPU F 60 + - 7 (50.0%) E52. GOPD F 56 + - 9(64.3%) E53. GOPD M Adult + - 8 (57.1%) E54. IPU M Adult + - 5 (35.7%) E55. IPU F Adult + - 7 (50.0%) E56. SKIN M 80 + - 6 (42.9%) E57. GOPD F 56 + - 6 (42.9%) E58. IPU M 21 + - 5 (35.7%) E59. IPU M Adult + - 4 (28.6%) E60. IPU F Adult - - 7 (50.0%) E61. CHOP M 10 - - 9 (64.3%) E62. GOPD M Adult - - 3 (21.4%) E63. GOPD M Adult + - 6 (42.9%) E64. GOPD M Adult + - 8 (57.1%) E65. GOPD F Adult - - 8 (57.1%) E66. GOPD F 40 - - 13 (92.9%) E67. GOPD F 60 + - 11 (78.5%) E68. GOPD F 101 + - 10 (71.4%) E69. GOPD M 21 + - 12 (85.7%) E70. GOPD F 27 + - 7 (50.0%) E71. IPU F 76 + - 9 (64.3%) E72. IPU M 45 + - 11 (78.5%) E73. GOPD M 33 + - 8 (57.1%) E74. GOPD F Adult + - 6 (42.9%) E75 GOPD M 60 + - 13 (92.9%) KEY: F= Female, M= Male , NHIS = National Health Insurance Scheme , IPU = In - Patient Unit, GOPD = General Out Patient Department , CHOP = Children Out Patient, EPU = Emergency Patient Unit. Note: Adult (includes male and female between 18 and above, whose actual age was not determined), Nil= Negative 77 Table 4.5 d. Beta-lactamase production and plasmid profiling of isolates of S. enterica from Federal Medical Center (FMC) Abakiliki, in relation to gender distribution (Continue). Org. Unit/ Sex Age Beta Plasmid/Mwt. Antibiotics Code Dept. Range Lactamase Resistant (%) S/NO (yrs) Production A76. GOPD M Adult +ve +ve 12 (85.7%) A77. GOPD M 20 + - 14 (100%) A78. EPU F 59 + - 13 (92.7%) A79. IPU M 24 + - 10 (71.4%) A80. IPU M Adult + - 13 (92.7%) A81. NHIS F 6 + + 10 (71.4%) A82. GOPD M Adult + - 10 (71.4%) A83. GOPD F 58 + + 12 (85.7%) A84. IPU F 68 + - 13 (92.7%) A85. IPU F Adult + + 10 (71.4%) A86. GOPD M 34 + - 12 (85.7%) A87. GOPD F Adult + - 9 (64.3%) A88. IPU M Adult + - 11 (78.5%) A89. GOPD F 29 + - 6 (42.9%) A90. HNIS M 39 + - 9 (64.3%) A91. IPU F 39 - - 8 (57.1%) A92. IPU F 20 + - 1 (7.1%) A 93. IPU M 34 - - 9 (64.3%) A 94. GOPD F 39 + - 11 (78.5%) A 95. IPU M 25 + - 7 (50.0%) A 96. IPU F Adult + - 6 (42.9%) A 97. IPU F Adult + - 6 (42.9%) A 98. NHIS F Adult + - 8 (57.1%) A 99. IPU F Adult + - 9 (64.3%) A100. IPU F Adult - - 12 (85.7%) KEY: F= Female , M= Male , NHIS = National Health Insurance Scheme , IPU= In - Patient Unit, GOPD = General Out Patient Department, CHOP= Children Out Patient, EPU = Emergency Patient Unit. Note: Adult (includes male and female between 18 and above, whose actual age was not determined), Nil= Negative 78 M T N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 bp’s 1200 1000 800 600 400 Plate 4.1. Agarose gel electrophoresis pattern of PCR amplification products of BlaCTX-M genes from S. enterica serovar. typhi. KEY: Lanes 1- 25 show isolates from FMC Owerri. The amplicons size (59 3bp) on lane 3, 14, 15 and24 shows positive BlaCTX-M genes present in the DNA of S.enterica serovar. typhi. Lane M shows DNA molecular marker (100- bp ladder). Lane T is the typed isolate of S. typhi ATCC (negative for BlaCTX-M ). Lane N contains only sterile water for control. 79 M T N 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 bp’s 1200 1000 800 600 400 Plate 4.2. Agarose gel electrophoresis pattern of PCR amplification products of BlaCTX-M genes from S.enterica serovar. Typhi. KEY: Lines 26-50 show isolates from FMC Umuahia. The amplicon size (593bp) on lane 28,39,40,49 and 50 shows positive BlaCTX-M genes. Lanes 51-54 are from UNTH Enugu. Amplicons on lane 53 and 54 shows positive BlaCTX-M genes present in the DNA of S. enterica serovar. typhi respectively. Lane M shows DNA molecular marker (100- bp ladder). 80 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 bp’s 1400 1000 600 400 200 Plate 4.3. Agarose gel electrophoresis pattern of PCR amplification products of BlaCTX-M genes from S.enterica serovar. Typhi. KEY: Lanes 1-25 show isolates from FMC Owerri while lanes 26-33 are from FMC Umuahia. The amplicon size (593bp) on lane 15 and 27 shows positive BlaCTX-M genes present in the DNA of S. enterica serovar. typhi from Owerri and Umuahia respectively. Lane M shows DNA molecular marker (100- bp ladder). 81 M 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 bp’s 1400 1300 1000 600 400 Plate 4.4. Agarose gel electrophoresis pattern of PCR amplification products of BlaCTX-M genes from o S.enterica serovar. typhi at 62.5 C. KEY: Lanes 34-50 show isolates from FMC Umuahia while lanes 50-66 are isolates from UNTH Enugu. The amplicons size (593bp) on lane 44 shows positive BlaCTX-M genes present in the DNA of S. enterica serovar.Typhi from Umuahias. Lane M shows DNA molecular marker (100- bp ladder). 82 M 2 9 14 20 24 28 40 43 46 48 49 64 66 67 68 69 76 77 80 81 82 83 84 85 90 T kpb’s 23.13 9.42 6.54 4.36 2.32 2.03 1.39 1.37 Plate 4.5. Agarose gel electrophoresis Pattern showing plasmid of S. enterica serovar before treatment with dyes. KEY: Lane numbers 2, 9, 14, 20 and 24 from FMC Owerri, only lane 9 have plasmids with the Mwt. of 1.37 kb and lane 2 with two plasmids of Mwt. of 1.39 kb and 1.37 kb. Lane 28, 40,43,46,48 and 49 from FMC Umuahia with only lane number 28, 40 and 43 having plasmids of Mwt. of 1.37 kbs. Lane 64, 66, 67, 68 and 69 are from UNTH Enugu without plasmid. Lane 76, 77, 80, 81, 82, 83, 84, 85, and 90 are from FMC Abakaliki, with only lane numbers 76, 81 and 83 having plasmids of the same Mwt. of 1.37 kbs respectively, except lane number 85 with two plasmids of 1.39 and 1.37kbs. Lane T (positive control) is the S. enterica typed culture ATCC14028. Lane M is the lambda Hind III marker (0.12-23.1 kpb). 83 4.6. Distribution of resistant determinants of S. enterica in relation to patient’s age from the Southeast region of Nigeria. From Table 4.6 analysis shows the hospital record of the age range of individuals (patients) from whom S. enterica isolates were collected, the presence of plasmids harbored by the isolates, the presence of GyrA (Point mutation) and ParC (Double mutation) that codes for the quinolone resistant determining region (QRDR), bla-SHV, bla-TEM, and BlaCTX-M type which codes for the presence of Extended Spectrum ß- lactamase cefotaxime type (CTX-M type) enzyme on the genomic DNA of the isolates S.enterica. The age range were classified from; 0-10, 11-20, 21-30, 31- 40, 41-50, 51- 60, 61-70, 71years and above. Patients who refused to declare their age on the record sheet but specified to be Adult were grouped under 18 and above as shown on the Table 4.5. The analysis revealed the number of patients from whom the isolates were recovered their age range and whether male or female. A total of 9 (9%) of the patients as shown on the record sheet of the hospital had plasmid DNA harboured in the isolates recovered from them (both males and females). While 55 (55%) of the total isolates showed positive GryA (male and female), 14 (14%) of the isolates had ParC (male and female), and 13 (13%) positive BlaCTX-M type gene (male and female), and non harboured bla- SHV and bla-TEM, in the genomic DNA of the isolates S. enterica recovered from various age groups as shown on Table 4.6. Analysis revealed that the highest number of S. enterica isolates were recovered from patients between 18 and above, followed by the ages between 21- 30 and ages between 0- 10 years. Plates 4.6, 4.7, 4.8 and 4.9 show the agarose gel electrophoresis pattern, revealing the single PCR amplification of GyrA gene (gyrase enzyme) in S.enterica serovar. typhi from the hospitals in the Southeast region of Nigeria that is FMC Owerri (SO1-SO25), FMC Umuahia (SU26-SU50), UNTH Enugu (SE51-SE75) and FMC Abakaliki (SA76-SA100). Analysis revealed the PCR amplicons size 251 bp’s of GyrA gene mutation that codes for point mutation to fluoroquinolone, as recorded in isolates from Owerri, Umuahia, Enugu, and Abakaliki. The presence of this mutation in GyrA gene revealed the Quinolone Resistant Determining Region (QRDR) on the S. enterica serovar.typhi. This mutation in GyrA gene also codes for point mutation in the amino acid sequence of the S.enterica 84 chromosomal DNA sequence. Though this mutant genes (GyrA) alone does not rule complete resistance to fluoroquinolones (quinolones), without the presence of mutation in ParC (Topoisomerase IV enzyme) gene in the same isolate. Plates 4.10 and 4.11 show the agarose gel electrophoresis pattern, revealing the single PCR amplification of double mutation in ParC (Topoisomerase iv enzyme) on S.enterica from the hospitals in the Southeast region of Nigeria. From the result the isolates from FMC Owerri (Plate 4.10) on lane number 3, 18 and 19 having amplicons size (251 bp’s) in GyrA were also positive for mutation in ParC (Double mutation) genes in the DNA of S.enterica serovar.typhi (260 bp’s) in Plate 4.10. The isolates from UNTH Enugu had only Lane number 58 and 60 having positive amplicons for double mutation in ParC (Plate 4.10). Also from Plate 4.11 isolates from UNTH Enugu with lane numbers 72,73,74, 75 had amplicons size of 260 bp’s positive for mutation in ParC (Double mutation). Lane numbers 76, 77, 97, 98 and 99 are isolates from FMC Abakaliki also having amplicons positive for mutation in ParC (Plate 4.11). Fig. 4.1 shows the incidence rate of the resistance gene production and plasmid profile of S.enterica serovars in relation to gender. Isolates from the male patients (42) irrespective of their age produced more resistance genes of GyrA 24 (57.14%), ParC 6 (14.3%) and BlaCTX-M genes 7(16.7%), as against those from 58 female patients had GyrA 31 (53.4%), ParC 8(13.7%), and BlaCTX-M genes 6 (10.3%), while none of these genes was detected in bla-SHV, bla-TEM, and qnrB. However, the production of R-plasmids profilewas higher in the females (10.3%) than males (7.14%) from their respective S. enterica isolates. 85 Table 4.6. Distribution of resistant determinants of S. enterica in relation to patient’s age from the Southeast region of Nigeria. S/NO Age Limits No of patients Presence of Presence of Presence of Presence of Plasmids Gyr A Par C BlaCTX-M BlaTEM/BlaSHV/QnrB (yrs) (%) (M) (F) (M) (F) (M) (F) (M) (F) 1. 0-10 6 0 1 1 3 0 0 0 0 Not Detected 2. 11-20 5 0 1 2 2 1 0 0 0 Not Detected 3. 21-30 11 0 0 5 1 1 0 1 0 Not Detected 4. 31-40 6 0 0 1 2 1 0 0 0 Not Detected 5. 41-50 4 0 0 0 2 1 0 1 0 Not Detected 6. 51-60 6 0 1 0 1 1 0 0 0 Not Detected 7. 61-70 2 0 0 0 0 0 1 0 0 Not Detected 8. 71-Above 3 0 0 0 0 0 0 0 0 Not Detected 9. Adult (18-Above) 57 3 3 15 19 1 7 6 5 Not Detected Total 100 3 6 24 31 6 8 7 6 Nil in all Total Percentages 9 (9%) 55 (55%) 14 (14%) 13 (13%) KEY: M= Male Patients, F = Female Patients , Adult (18- Above) = Patients that are up to 18 years and above and refuse to disclose their actual age., GyrA = Gyrase A enzyme, BlaCTX-M = Beta lactamase cefotaxime-M class enzyme, bla-SHV = Beta- lactmase Sulfhydryl variable, bla-TEM = Beta-lactamase Temoniera, ParC= Topoisomerase iv enzyme,. Note; The QnrB and other ESBL checked were not detected on all the isolates (bla-SHV, bla-TEM) 86 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 bp’s 400 1000 600 400 200 Plate 4.6. Agarose gel electrophoresis pattern of PCR amplification products of GyrA genes from S. enterica. KEY: Lanes 1- 25 shows isolates from FMC Owerri. The amplicons size (251 bp’s) on lanes 3,9,11,13,14,15,16,18,19,21,22,23,24,25 shows positive GyrA(point mutagen) genes present in the DNA of S. enterica serovar.typhi. Lanes 26-31 show the isolates from FMC Umuahia having PCR amplicons on lane 26,28,30 and 31 positive for GyrA (point mutation) genes present in DNA of S. enterica serovar typhi. Lane M shows DNA molecular marker (100- bp ladder). 87 M 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 bp’s 1400 1000 600 400 200 Plate 4.7. Agarose gel electrophoresis pattern of PCR amplification products of GyrA genes from S. enterica serovar. typhi. KEY: Lanes 32- 50 shows isolates from FMC Umuahia. The amplicon size (251bp’s) on lanes 32,33,35,36,37,39,40,42,44,45,46,47,49 and 50 shows positive GyrA (point mutation) genes present in the DNA of S. enterica serovar.typhi. Lanes 51-64 show the isolates from UNTH Enugu and having PCR amplicons positive for GyrA (point mutation) genes on lanes 53, 54, 58, 59, 60, 61 and 62. Lane M shows DNA molecular marker (100- bp ladder). 88 M 65 66 6768 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 bp’s 1400 1000 800 600 300 Plate 4.8. Agarose gel electrophoresis pattern of PCR amplification products of GyrA genes from S. enterica. KEY: Lanes 65- 75 show isolates from UNTH Enugu with only lane 74 having amplicons size (251bp’s) positive for GyrA (point mutagen) genes in the DNA of S. enterica serovar.typhi. Lanes 76- 97 show the isolates from FMC Abakaliki, having PCR amplicons positive for GyrA (point mutation) genes in DNA of S. enterica serovar. Typhi on lane 76, 77, 78, 79, 82, 86, 87, 88, 91, 92, 93, 94, 95 and 96. Lane M shows DNA molecular marker (100- bp ladder). 89 M 100 99 98 bp’s 1400 1000 800 600 400 200 Plate 4.9. Agarose gel electrophoresis pattern of PCR amplification products of GyrA genes from S. enterica. KEY: Lanes 98-100 show isolates from FMC Abakaliki with only lane 98 having amplicon size (251 bp’s) positive for GyrA (point mutation) genes in the DNA of S. enterica serovar.typhi. Lane M shows DNA molecular marker (100- bp ladder). 90 M 1 2 3 4 5 6 15 16 17 18 19 20 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 bp’s 1000 800 600 300 Plate 4.10. Agarose gel electrophoresis pattern of PCR amplification products of ParC genes from S. enterica. KEY: Lanes 1-6 and lane numbers 15-20 show isolates from FMC Owerri with only lane number 3, 18 and 19 having amplicon size (260bp’s) positive for ParC ( Double mutation) genes in the DNA of S.enterica serovar.typhi. Lane number 50 shows only isolates from FMC Umuahia and was negative to the ParC genes. Lanes 51-70 show the isolates from UNTH Enugu with only Lane numbers 58 and 60 having positive amplicon for ParC genes in S.enterica. Lane M shows DNA molecular marker (100- bp ladder). 91 M 71 72 73 74 75 76 77 97 98 99 bp’s 900 800 700 500 400 300 Plate 4.11. Agarose gel electrophoresis pattern of PCR amplification products of ParC genes from S.enterica. KEY: Lanes 71-75 show isolates from UNTH Enugu with lane numbers 72, 73, 74, 75 having amplicon size (260 bp’s) positive for ParC (Double mutation) genes in the DNA of S.enterica serovar.typhi. Lane numbers 76, 77, 97, 98 and 99 are isolates from FMC Abakaliki also having amplicons positive for ParC. Lane M shows DNA molecular marker (100- bp ladder). 92 35 30 25 Male = 42 20 Female= 58 15 10 5 0 GyrA Plasmid BlaCTX-M ParC Bla- QnrB SHV/TEM Fig. 4.1. The incidence rate of qnrB, gyrA, parC, blaSHV, blaTEM, blaCTX-M and plasmids in S. enterica isolates from patients in Southeast region of Nigeria. KEY: GyrA = gyrase A, ParC= Topoisomerase iv, BlaCTX-M = B-lactamase cefotaxime –M type enzyme, N.D = Not detected 93 4.7. Antibiotics susceptibility of S. enterica in relation to their resistant determinants in the four (a-d) hospitals. Table 4.7 a shows the antibiotic susceptibility of S. enterica isolates in relation to their resistance genes, antigenic properties and ESBL’s production in the various hospitals. Isolates with code serial numbers SO1-SO25 were from the hospital in Owerri, the same applies to the hospitals in Umuahia (SU26- SU50) (Table 4.7 b), Enugu (SE51- 75) Table 4.7 c and Abakaliki (SA76- SA100) Table 4.7 d. From Table 4.6 a for Federal Medical Centre (FMC) Owerri 13 (52%) of the S.enterica were ESBL’s positive while 14 (56%) were positive for GyrA, 4 (16%) for BlaCTX-M, 2 (8%) for plasmid and 13 (12%) for ParC (topoisomerase IV enzyme) while none was accounted for in bla-SHV, bla-TEM gene and the quinolone resistant protein of the isolates. The same applies to FMC Umuahia, UNTH Enugu and FMC Abakaliki respectively as shown below on Table 4.7 b-d. 94 Table 4.7 a. Antibiotic susceptibility of S. enterica in relation to their resistant determinants in FMC, Owerri. Organism Presence of Presence of Presence of Presence of Presence Antibiotic Presence of Code GyrA genes ParC genes BlaCTX-M ESBL’S of Plasmids resistant S.typhi S/NO genes antigens O1. - - - - - 5 H &O O2. - - - - + 2 H &O O3. + + + + - 2 H &O O4. - - - - - 3 H &O O5. - - - + - 7 H &O O6. - - - - - 4 H &O O7. - - - - - 3 H &O O8. - - - - - 2 H &O O9. + - - + + 8 H &O O10. - - - + - 7 H &O O11. + - - + - 9 H &O O12. - - - + - 9 H &O O13. + - - - - 7 H &O O14. + - + + - 10 H &O O15. + - + + - 6 H &O O16. + - - + - 7 H &O O17. - - - + - 8 H &O O18. + + - -l - 10 H &O O19. + + - - - 10 H &O O20. - - - + - 8 H &O O21. + - - - - 5 H &O O22. + - - - - 3 H &O O23. + - - + - 4 H &O O24. + - + + - 6 H &O O25 + - - - - 4 H &O Percentage Prevalence 14 (56%) 3 (12%) 4 (16%) 13 (52%) 12 (8%) KEY: GyrA = gyrase A, ParC= Topoisomerase iv, BlaCTX-M = ß-lactamase cefotaxime –M type enzyme, ESBL’s = Extended Spectrum ß-lacatamase enzyme, H = Flagellated antigen, O = Somatic antigen, O1- 25= Isolates code from Owerri, Nil = Negative , +ve = Positive, bla-SHV and bla-TEM = Not detected (not shown on the table). 95 Table 4.7 b. Antibiotic susceptibility of S. enterica in relation to their resistant determinants in FMC, Umuahia (continue) Organism Presence of Presence of Presence of Presence Presence Antibiotic Presence Code GyrA genes ParC genes BlaCTX-M of of Plasmids resistant of S.typhi S/NO genes ESBL’S antigens U26. + - - + - 5 H &O U27. - - + - - 6 H &O U28. + - + - + 7 H &O U29. - - - - - 3 O U30. + - - - - 6 O U31. + - - - - 1 O U32. + - - - - 6 O U33. + - - + - 7 H &O U34. - - - - - 4 H &O U35. + - - - - 5 H &O U36. + - - - - 4 H &O U37. + - - - - 5 H &O U38. - - - + - 6 H &O U39. + - + - - 6 H &O U40. + - + + + 11 H &O U41. - - - - - 5 H &O U42. + - - - - 4 H &O U43. - - - - + 5 H &O U44. + - + - - 9 H &O U45. + - - - - 3 H &O U46. + - - - - 5 H &O U47. + - - - - 4 H &O U48. - - - - - 7 H &O U49. + - + - - 11 H &O U50 + - + - - 7 H &O Percentage Prevalence 16 (72%) 0 (0%) 7 (28%) 4 (16%) 3 (12%) KEY: GyrA = gyrase A, ParC= Topoisomerase iv, BlaCTX-M = ß-lactamase cefotaxime –M type enzyme, ESBL’s = Extended Spectrum ß-lacatamase enzyme, H = Flagellated antigen, O= Somatic antigen, U26- 50= Isolates code from Umuahia, Nil= Negative , +ve = Positive, bla-SHV and bla- TEM = Not detected (not shown on the table). 96 Table 4.7 c: Antibiotic susceptibility of S. enterica in relation to their resistant determinants in UNTH, Enugu (continue) S/NO Presence of Presence of Presence of Presence Presence No of Presence GyrA genes ParC genes BlaCTX-M of of Plasmids antibiotic of S.typhi genes ESBL’S resistant antigens E51. - - - -l - 7 H &O E52. - - - - - 9 H &O E53. + - + + - 8 H &O E54. + - + - - 5 H E55. - - - - - 7 H &O E56. - - - - - 6 H &O E57. - - - - - 6 H &O E58. + + - - - 5 H E59. + - - - - 4 H &O E60. + + - - - 7 H &O E61. + - - - - 9 H &O E62. + - - - - 3 H &O E63. - - - - - 6 H &O E64. - - - - - 8 H &O E65. - - - + - 8 H &O E66. - - - + - 13 H &O E67. - - - + - 11 H &O E68. - - - + - 10 H &O E69. - - - + - 12 H &O E70. - - - - - 7 H &O E71. - - - - - 9 H &O E72. - + - + - 11 H &O E73. - + - - - 8 H &O E74. + + - - - 6 H &O E75 - + - - - 13 H &O Percentage Prevalence 8 (32%) 6 (24%) 2 (8%) 7 (28%) 0 (0%) KEY: GyrA = gyrase A, ParC= Topoisomerase iv, BlaCTX-M = ß-lactamase cefotaxime –M type enzyme, ESBL’s = Extended Spectrum ß-lacatamase enzyme, H = Flagellated antigen, O = Somatic antigen, E51- 75= Isolates code from Enugu, Nil = Negative , +ve = Positive, bla-SHV and bla-TEM = Not detected (not shown on the table). 97 Table 4.7d. Antibiotic susceptibility of S. enterica in relation to their resistant determinants in FMC, Abakaliki. Organism Presence of Presence of Presence of Presence of Presence Antibiotic Presence of Code GyrA genes ParC genes BlaCTX-M ESBL’S of Plasmids resistant S.typhi S/NO genes antigens A76. + + - + + 12 H &O A77. + + - + - 14 H &O A78. + - - - - 13 H &O A79. + - - - - 10 H &O A80. - - - - - 13 H A81. - - - + + 10 H &O A82. + - - + - 10 H &O A83. - - - + + 12 H &O A84. - - - - - 13 H &O A85. - - - + + 10 H &O A86. + - - - - 12 H &O A87. + - - - - 9 H &O A88. + - - - - 11 H &O A89. - - - - - 6 H &O A90. - - - - - 9 O A91. + - - - - 8 H &O A92. + - - - - 1 H &O A 93. + - - + - 9 H &O A 94. + - - + - 11 H &O A 95. + - - + - 7 H &O A 96. + - - - - 6 H &O A 97. - + - - - 6 H &O A 98. + + - + - 8 H &O A 99. - + - + - 9 H &O A100. - - - + - 12 H &O Percentage Prevalence 15 (60%) 5 (20%) 0 (0%) 12 (48%) 4 (16%) KEY: GyrA = gyrase A, ParC= Topoisomerase iv, BlaCTX-M = ß-lactamase cefotaxime –M type enzyme, ESBL’s = Extended Spectrum ß-lacatamase enzyme, H = Flagellated antigen, O = Somatic antigen, A76- 100= Isolates code from Owerri, Nil = Negative , +ve = Positive., bla-SHV and bla- TEM = Not detected (not shown on the table). 98 4.8 Percentage antibiotic susceptibility pattern of S. enterica in relation to the resistant determinants Table 4.8 shows the percentage of antibiotic susceptibility pattern of S. enterica. From the results obtained 87% of the isolates were amoxicillin/clavulanic acid resistant and 80% were chloramphenicol resistant followed by amoxicillin which has 80% resistant isolates, and co-trimoxazole, sparfloxacin, streptomycin and gentamycin which had 78, 78, 77 and 51% resistant isolates respectively. Levofloxacin and ceftriaxone had 78% of the S. enterica being susceptible to them, followed by cefotaxime (73%), ofloxacin (72%), sparfloxacine (71%), ciprofloxacine (71%) and ceftazidime 56% susceptibility. Out of the 100, the isolates tested for genes encoding GyrA coding for point mutation in their chromosomal DNA, 48% of the isolates harbouring GyrA in their gene were found to be resistant to amoxiclavulanic acid while 11% showed BlaCTX-M type gene and ParC, and 7% produced plasmids. Others were chloramphenicol resistant isolates having 45% habouring GyrA gene, 12% of the isolates having BlaCTX-M type, 14% of isolates having ParC gene and 7% of the isolates harbouring plasmids, while none of the isolates harboured qnrB, bla-SHV and blaTEM gene respectively in all the 14 antibiotics used against them in this study. Others were as shown in the Table 4.8. Fig.4.2. shows the graphical representation of the genetic constituents of the isolates of S. enterica and their resistance factors as related to each of the 5 selected antibiotics screened. Out of the twenty-nine (29) isolates of S. enterica resistant to ciprofloxacin, only fifteen (15) produced mutation in GyrA, eight (8) produced double mutation in ParC, one (1) produced BlaCTX-M and three isolates produced plasmids. Also out of twenty-two (22) isolates resistant to levofloxacin nine (9) produced mutation in GyrA genes, while ParC, BlaCTX-M , and plasmids were produced by only two (2) isolates, each. Other resistance gene produced by each S. enterica isolates resistant to other antibiotics such as ceftriaxone (CRO), cefotaxime (CTX-M), and amoxiclavulanic acid (AMC) were as shown graphically. 99 Table 4.8. Percentage antibiotic susceptibility pattern of S. enterica in relation to the resistant determinants . PERCENTAGE RESISTANT AND SUSCEPTIBILITY OF RESISTANT DETERMINANTS ISOLATES S/NO ANTIBIOTICS SENSITIVE R E SISTANT GyrA ParC BlaCTX-M Plasmid (%) (%) (%) (%) (%) (%) 1. Co-trimoxazole 30 µg 22 78 40 12 9 9 2. Chloramphenicol 30 µg 20 80 45 14 12 7 3. Sparfloxacin 10 µg, 22 78 43 12 10 7 4. Ciprofloxacin 10 µg 71 29 15 8 1 3 5. Amoxicillin 30 µg 20 80 46 12 9 7 6. Gentamycin 10 µg 49 51 27 11 7 6 7. Pefloxacin 30 µg 71 29 17 7 4 4 8. Ofloxacin 10 µg 72 28 16 7 4 3 9. Streptomycin 30 µg 23 77 41 11 12 7 10. Levofloxacin 5 µg 78 22 9 2 2 2 11. Ceftriaxone 30 µg 78 22 9 4 4 5 12. Cefotaxime 30 µg 73 27 9 5 4 5 13. Ceftazidime 30 µg 56 44 21 6 3 4 14. Amoxicillin/Clavulanic 13 87 48 11 11 7 acid 30 µg KEY: GyrA = gyrase A, Par C= Topoisomerase iv, Bla CTX-M = ß-lactamase cefotaxime –M type enzyme, % = Percentage .Note; QnrB, bla-SHV/ TEM were not detected on the isolates during PCR. 100 KEY Fig.4.2. Shows graphical representation of the genetic constituents, the number of antibiotics and resistant pattern of isolates that produced Plasmids or a type of mutation in a gene detected using PCR amplification, out of the total isolates resistant to each antibiotics. 101 Level of resistant gene produced by S.enterica 4.9 Phenotypic antibiotic resistance patterns of S. enterica from the four hospitals (a-d) in relation to Beta- lactamase and ESBLs production. Table 4.9 shows the phenotypic pattern in isolates producing ß-lactamase enzyme and Extended Spectrum β-lactamases (ESBLs). As shown in Table 4.9 a- d, some isolates which showed Beta-lactamase negative were still resistant to at least 3-10 antibiotics, yet they were ESBL negative also. Also some ßeta-lactamase positive isolates that were also ESBL positive were resistanct to only two (2) antibiotics. These discripancies may be due to chromosomal constituents which might be constitutive or inductive (i.e some Beta-lactamase act only when there are presence of Beta-lactam drugs (inducive) while some Beta-lactamase enzymes are constituents of the chromosomal DNA and other are plasmids mediated). Table 4.9 a-d also show classes of ßeta-lactamase and ESBL, which might belong to several classes like TEM1, BlaCTX-M , SHV, Metallo or Zinc- mediated Beta-lactamse etc. The phenotypic resistance was exhibited more on the isolates from the Federal Medical Centre (FMC) Abakaliki (SA76-SA100) Table 4.9 d. Fig. 4.3 shows the graphical representation of the genetic constituents of S. enterica isolates received from different hospitals in the Southeast region of Nigeria. From the study, analysis revealed that from the hospital in Owerri (1-25), two (2) isolates produced plasmids, while 14, 4 and 3 S. enterica isolates produced GyrA, BlaCTX-M and ParC respectively. From the hospital in Umuahia (26-50), 3, 18, and 7 S.enterica isolates produced plasmids, mutation in GyrA, BlaCTX-M gene, while none of the isolates produced double mutation in ParC gene. Other isolates from the hospital in Enugu (51-75) and Abakaliki (76-100) were also reported graphically as shown in the Figure 4.3. 102 Table 4.9a. Phenotypic antibiotic resistance patterns of S. enterica from FMC Owerri in relation to Beta- lactamase and ESBL’s production. CODE NUMBER OF RESISTANCE PHENOTYPIC PATTERN BETA S/NO ANTIBIOTICS LACTAMASE PRESENCE PRODUCTION OF ESBL’S O1. 5 SP,AM,STR,CAZ,AMC + - O2. 2 CH,AMC + - O3. 2 CH,AMC + + O4. 3 SXT,CH,CAZ - - O5. 7 SXT,CH,SP,AM,STR,CAZ,AMC + + O6. 4 LEV,CTX,CAZ,AMC + - O7. 3 SXT,CH,AMC + - O8. 2 SXT,CAZ + - O9. 8 SXT,CH,SP,CPX,AM,STR,CAZ,AMC + + O10. 7 SXT,CH,SP,CPX,AM,CAZ,AMC + + O11. 9 SXT,CH,SP,CPX,AM,GEN,STR,CAZ,AMC + + O12. 9 SXT,CH,SP,CPX,AM,GEN,STR,CAZ,AMC + + O13. 7 SXT,CH,SP,AM,GEN,STR,AMC + - O14. 10 SXT,CH,SP,AM,STR,LEV,CRO,CTX,CAZ,AMC + + O15. 6 SXT,CH,SP,AM,STR,AMC + + O16. 7 SXT,CH,SP,AM,STR,CAZ,AMC + + O17. 8 SXT,CH,SP,AM,GEN,STR,CAZ,AMC - + O18. 10 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR,AMC - - O19. 10 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR,AMC + - O20. 8 SP,AM,STR,LEV,CRO,CTX,CAZ,AMC + + O21. 5 SXT,SP,AM,STR,AMC + - O22. 3 AM,STR,AMC - - O23. 4 AM,STR,CAZ,AMC - + O24. 6 AM,STR,CRO,CTX,CAZ,AMC + + O25 4 CH,AM,CAZ,AMC + - KEY: SXT= co-trimoxazole 30 µg, CH= chloramphenicol 30 µg, SP= sparfloxacin 10 µg, CPX= ciprofloxacin 10 µg, AM= amoxicillin 30 µg, GEN= gentamycin 10 µg, PEF= pefloxacin 30 µg, OFX= ofloxacin 10 µg, STR= streptomycin 30 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, CAZ= ceftazidime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum ßeta Lactamase, - = Negative. 103 Table 4.9b. Phenotypic antibiotic resistance patterns of S. enterica from FMC Umuahia in relation to Beta- lactamase and ESBL’s production (continue). BETA- CODE NUMBER OF RESISTANCE PHENOTYPIC PATTERN LACTAMASE PRESENCE S/NO ANTIBIOTICS PRODUCTION OF ESBL’S U26. 5 CH,AM,STR,CAZ,AMC - + U27. 6 SXT.CH,SP,AM,STR,AMC + - U28. 7 SXT,CH,AM,GEN,PEF,OFX,STR + - U29. 3 AM,CAZ,AMC - - U30. 6 SXT.CH.SP,AM,STR,AMC - - U31. 1 AM + - U32. 6 SXT,CH,SP,AM,STR,AMC + - U33. 7 SXT,SP,AM,PEF,OFX,CAZ,AMC + + U34. 4 CH,GEN,STR,AMC - - U35. 5 SXT,CH,SP,AM,AMC + - U36. 4 SXT,CH,SP,AMC + - U37. 5 CH,SP,AM,STR,AMC - - U38. 6 SXT,SP,AM,STR,CAZ,AMC - + U39. 6 CH,SP,AM,GEN,PEF,STR + - U40. 11 SXT,CH,SP,AM,GEN,PEF,OFX,STR,CRO,CTX,AMC + + U41. 5 SXT,CH,SP,GEN,AMC, + - U42. 4 CH,SP,GEN,AMC, - - U43. 5 SXT,CH,SP,GEN,AMC + - U44. 9 SXT,CH,SP,AM,GEN,PEF,OFX,STR,AMC + - U45. 3 CH,SP,AMC + - U46. 5 SXT,CH,SP,STR,AMC + - U47. 4 SP,AM,STR,AMC + - U48. 7 SXT,CH,SP,PEF,OFX,STR,AMC + - U49. 11 CH,SP,GEN,PEF,OFX,STR,LEV,CRO,CTX,CAZ,AMC + - U50 7 SXT,CH,SP,AM,GEN,STR,AMC + - KEY: SXT= co-trimoxazole 30 µg, CH= chloramphenicol 30 µg, SP= sparfloxacin 10 µg, CPX= ciprofloxacin 10 µg, AM= amoxicillin 30 µg, GEN= gentamycin 10 µg, PEF= pefloxacin 30 µg, OFX= ofloxacin 10 µg, STR= streptomycin 30 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, CAZ= ceftazidime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum ßeta Lactamase, - = Negative. 104 Table 4.9c. Phenotypic antibiotics resistance patterns of S. enterica from UNTH Enugu in relation to Beta- lactamase and ESBL’s production (Continue). BETA CODE NUMBER OF RESISTANCE PHENOTYPIC PATTERN LACTAMASE PRESENCE S/NO ANTIBIOTICS PRODUCTION OF ESBL’S E51. 7 SXT,CH,SP,AM,GEN,STR,AMC + - E52. 9 SXT,CH,SP,AM,GEN,PEF,OFX,STR,AMC + - E53. 8 SXT,CH,SP,CPX,AM,GEN,STR,AMC + + E54. 5 SXT,CH,SP,STR,AMC + - E55. 7 SXT,CH,SP,AM,GEN,STR,AMC + - E56. 6 SXT,CH,SP,GEN,STR,AMC + - E57. 6 SXT,CH,SP,GEN,STR,AMC + - E58. 5 SXT,CH,GEN,STR,AMC + - E59. 4 SXT,AM,STR,AMC + - E60. 7 CH,SP,AM,PEF,OFX,LEV,AMC - - E61. 9 SXT,CH,SP,AM,GEN,PEF,OFX,STR,AMC - - E62. 3 SXT,AM,STR - - E63. 6 SXT,AM,OFX,STR,LEV,AMC + - E64. 8 AM,PEF,OFX,STR,LEV,CRO,CTX,AMC + - E65. 8 SXT,CH,SP,CPX,AM,STR,CTX,CAZ - + E66. 13 SXT,CH,SP,CPX,AM,PEF,OFX,STR,LEV,CRO,CTX,CAZ,AMC - + E67. 11 SXT,CH,SP,AM,PEF,OFX,STR,LEV,CRO,CTX,AMC + + E68. 10 SXT,CH,SP,AM,PEF,STR,LEV,CRO,CTX,AMC + + E69. 12 SXT,CP,SP,AM,PEF,OFX,STR,LEV,CRO,CTX,CAZ,AMC + + E70. 7 SXT,CH,SP,AM,GEN,STR,AMC + - E71. 9 SXT,CH,SP,CPX,AM,GEN,PEF,STR,AMC + - E72. 11 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR,CTX,AMC + + E73. 8 SXT,CH,SP,AM,GEN,STR,CTX,CAZ + - E74. 6 SXT,CH,SP,AM,STR,AMC + - E75 13 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR,CRO,CTX,CAZ,AMC + - KEY: SXT= co-trimoxazole 30 µg, CH= chloramphenicol 30 µg, SP= sparfloxacin 10 µg, CPX= ciprofloxacin 10 µg, AM= amoxicillin 30 µg, GEN= gentamycin 10 µg, PEF= pefloxacin 30 µg, OFX= ofloxacin 10 µg, STR= streptomycin 30 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, CAZ= ceftazidime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum ßeta Lactamase, - = Negative. 105 Table 4.9 d. Phenotypic antibiotic resistance patterns of S. enterica from FMC Abakaliki in relation to Beta- lactamase and ESBLs production (Continue). BETA CODE NUMBER OF LACTAMASE PRESENCE S/NO ANTIBIOTICS RESISTANCE PHENOTYPIC PATTERN PRODUCTION OF ESBL’S A76. 12 SXP,CH,SP,AM,GEN,PEF,OFX,STR,CRO,CTX,CAZ,AMC + + A77. 14 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR,LEV,CRO,CTX,CAZ,AMC + + A78. 13 SXT,CH,SP,CPX,AM,GEN,PEF,STR,LEV,CRO,CTX,CAZ,AMC + - A79. 10 SXT,CH,SP,CPX,AM,GEN,PEF,STR,CAZ,AMC + - A80. 13 SXT,CH,SP,CPX,AM,GEN,PEF,STR,LEV,CRO,CTX,CAZ,AMC + - A81. 10 SXT,CH,SP,CPX,AM,GEN,STR,CRO,CTX,CAZ + + A82. 10 SXT,CH,SP,AM,GEN,OFX,STR,LEV,CAZ,AMC + + A83. 12 SXT,SP,CPX,AM,GEN,PEF,OFX,STR,LEV,CRO,CTX,AMC + + A84. 13 SXT,CH,SP,CPX,AM,GEN,OFX,STR,LEV,CRO,CTX,CAZ,AMC + - A85. 10 SXT,SP,CPX,AM,STR,LEV,CRO,CTX,CAZ,AMC + + A86. 12 SXT,CH,SP,CPX,AM,GEN,STR,LEV,CRO,CTX,CAZ,AMC + - A87. 9 SXT,CH,SP,CPX,AM,STR,LEV,CAZ,AMC + - A88. 11 SXT,CH,SP,CPX,AM,GEN,OFX,STR,LEV,CAZ,AMC + - A89. 6 SXT,CH,AM,GEN,STR,AMC + - A90. 9 SXT,CH,SP,AM,GEN,STR,CTX,CAZ,AMC + - A91. 8 SXT,CH,SP,CPX,AM,GEN,PEF,OFX - - A92. 1 AMC + - A 93. 9 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR - + A 94. 11 SXT,CH,SP,CPX,AM,GEN,PEF,OFX,STR,CAZ,AMC + + A 95. 7 SXT,CH,SP,AM,GEN,CAZ,AMC + + A 96. 6 SXT,CH,SP,AM,GEN,AMC + - A 97. 6 SXT,CH,SP,CPX,AM,GEN + - A 98. 8 SXT,CH,SP,CPX,AM,GEN,STR,CAZ + + A 99. 9 SXT,CH,SP,CPX,AM,GEN,STR,CAZ,AMC + + A100. 12 SXT,CH,SP,AM,GEN,OFX,STR,LEV,CRO,CTX,CAZ,AMC - + KEY: SXT= co-trimoxazole 30 µg, CH= chloramphenicol 30 µg, SP= sparfloxacin 10 µg, CPX= ciprofloxacin 10 µg, AM= amoxicillin 30 µg, GEN= gentamycin 10 µg, PEF= pefloxacin 30 µg, OFX= ofloxacin 10 µg, STR= streptomycin 30 µg, LEV= levofloxacin 5 µg, CRO= ceftriaxone 30 µg, CTX= cefotaxime 30 µg, CAZ= ceftazidime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum ßeta Lactamase, - = Negative. 106 KEY Fig. 4.3 shows graphical representation of the genetic constituents on the isolates from different hospitals in the Southeast Region and the relative distribution. 107 No of genes /Plasmid detected on Isolates using PCR amplifications 4.10 Phenotypic antibiotic resistance pattern of S. enterica against five selected antibiotics in relation to resistance genes. Tables 4.10 a- d shows the phenotypic pattern of resistance to five selected antibiotics in relation to the plasmid, GyrA, ParC, and BlaCTX-M gene produced. Of the isolates SO1-25, some were resistant to not less than four of the antibiotics tested, but notably, the isolates did not produce plasmid, no mutation in GyrA and ParC gene, or BlaCTX-M gene. As stated in the earlier Tables 4.9 c in this study, an isolate was resistant to 13 antibiotics as shown in the phenotypic pattern obtained but the isolates exhibited the presence of ESBL only. With the isolates, SU26 – 50, Out of the 5 selected antibiotics tested, 4 (16%) of the isolates were resistant to at least one antibiotics screened without harbouring mutant gene in GyrA and ParC, BlaCTX-M , and the plasmid DNA. Such isolates were SU29, SU38, SU41 and SU48 (Table 4.10 b). The isolates SE51- 75 had the same trend of results except in some cases where up to 4-5 of the antibiotics selected were completely resisted by the isolates (Table 4.10 c). This may be implying that different types of enzyme apart from the ones detected were playing a major role in the phenotypic resistance pattern. Similar observations were made in SA76 - SA100 as shown in Table 4.10 d. . 108 Table 4.10 a. Phenotypic antibiotics resistance pattern of S. enterica against five selected antibiotics in relation to resistant genes from Owerri CODE NUMBER OF RESISTANCE PRESENCE PRESENCE PRESENCE OF PRESENCE S/NO ANTIBIOTICS PHENOTYPIC OF GryA OF ParC BlaCTX-M OF PATTERN PLASMID O1. 1 AMC - - - - O2. 1 AMC - - - + O3. 1 AMC + + + - O4. 0 NIL - - - - O5. 1 AMC - - - - O6. 3 LEV,CTX,AMC - - - - O7. 1 AMC - - - - O8. 0 NIL - - - - O9. 2 CPX,AMC + - - + O10. 2 CPX,AMC - - - - O11. 2 CPX,AMC + - - - O12. 2 CPX,AMC - - - - O13. 1 AMC + - - - O14. 4 LEV,CRO,CTX,AMC + - + - O15. 1 AMC + - + - O16. 1 AMC + - - - O17. 1 AMC - - - - O18. 2 CPX,AMC + + - - O19. 2 CPX,AMC + + - - O20. 4 LEV,CRO,CTX,AMC - - - - O21. 1 AMC + - - - O22. 1 AMC + - - - O23. 1 AMC + - - - O24. 3 CRO,CTX,,AMC + - + - O25 1 AMC + - - - KEY: CPX= ciprofloxacin 10 µg, LEV= levofloxacin 5 µg, CRO = ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum Beta Lactamase, Nil = negative, GyrA = gyrase A, ParC = Topoisomerase IV, Bla CTX-M = Beta-lactamase cefotaxime -M type enzyme, 109 Table 4.10b. Phenotypic antibiotics resistance pattern of S. enterica against five selected antibiotics in rellation to resistant genes from Umuahia (Continue). CODE NUMBER OF RESISTANCE PRESENCE PRESENCE PRESENCE PRESENCE S/NO ANTIBIOTICS PHENOTYPIC OF GryA OF ParC OF BlaCTX-M OF PATTERN PLASMID U26. 1 AMC + - - - U27. 1 AMC - - + - U28. 0 NIL + - + + U29. 1 AMC - - - - U30. 1 AMC + - - - U31. 0 NIL + - - - U32. 1 AMC + - - - U33. 2 AMC + - - - U34. 1 AMC - - - - U35. 1 AMC + - - - U36. 1 AMC + - - - U37. 1 AMC + - - - U38. 1 AMC - - - - U39. 0 NIL + - + - U40. 3 CRO,CTX,AMC + - + + U41. 1 AMC, - - - - U42. 1 AMC, + - - - U43. 1 AMC - - - + U44. 1 AMC + - + - U45. 1 AMC + - - - U46. 1 AMC + - - - U47. 1 AMC + - - - U48. 1 AMC - - - - U49. 4 LEV,CRO,CTX,AMC + - + - U50 1 AMC + - + - KEY: CPX= ciprofloxacin 10 µg, LEV = levofloxacin 5 µg, CRO = ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum Beta Lactamase, Nil = negative, GyrA = gyrase A, ParC= Topoisomerase IV, Bla CTX-M = Beta-lactamase cefotaxime -M type enzyme, 110 Table 4.10 c. Phenotypic antibiotics resistance pattern of S. enterica against five selected antibiotics in relation to resistant genes from Enugu (Continue). CODE NUMBER OF ANTIBIOTIC PRESENCE PRESENCE PRESENCE PRESENCE S/NO ANTIBIOTICS RESISTANCE OF GRY A OF ParC OF BlaCTX-M OF PHENOTYPIC PATTERN PLASMID E51. 1 AMC - - - - E52. 1 AMC - - - - E53. 2 CPX,AMC + - + - E54. 1 AMC + - + - E55. 1 AMC - - - - E56. 1 AMC - - - - E57. 1 AMC - - - - E58. 1 AMC + + - - E59. 1 AMC + - - - E60. 2 LEV,AMC + + - - E61. 1 AMC + - - - E62. 1 NIL + - - - E63. 2 LEV,AMC - - - - E64. 4 LEV,CRO,CTX,AMC - - - - E65. 2 CPX,CTX - - - - E66. 5 CPX,LEV,CRO,CTX,AMC - - - - E67. 4 LEV,CRO,CTX,AMC - - - - E68. 4 LEV,CRO,CTX,AMC - - - - E69. 4 LEV,CRO,CTX,AMC - - - - E70. 1 AMC - - - - E71. 2 CPX,AMC - - - - E72. 3 CPX,CTX,AMC - + - - E73. 1 CTX - + - - E74. 1 AMC + + - - E75 4 CPX,CRO,CTX,AMC - + - - KEY: CPX= ciprofloxacin 10 µg, LEV = levofloxacin 5 µg, CRO = ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum Beta Lactamase, Nil = negative, GyrA = gyrase A, ParC= Topoisomerase IV, Bla CTX-M = Beta-lactamase cefotaxime -M type enzyme, 111 Table 4.10 d. Phenotypic antibiotics resistance pattern of S. enterica against five selected antibiotics in relation to resistant genes from Abakaliki. CODE NUMBER OF ANTIBIOTIC RESISTANCE PRESENCE PRESENCE PRESENCE PRESENCE S/NO ANTIBIOTICS PHENOTYPIC PATTERN OF GryA OF ParC OF BlaCTX-M OF PLASMID A76. 3 CRO,CTX,AMC + + - + A77. 5 CPX,LEV,CRO,CTX,,AMC + + - - A78. 5 CPX,LEV,CRO,CTX,,AMC + - - - A79. 2 CPX,AMC + - - - A80. 5 CPX,LEV,CRO,CTX,AMC - - - - A81. 3 CPX,CRO,CTX, - - - + A82. 2 LEV,AMC + - - - A83. 5 CPX,LEV,CRO,CTX,AMC - - - + A84. 5 CPX,LEV,CRO,CTX,AMC - - - - A85. 5 CPX,LEV,CRO,CTX,AMC - - - + A86. 5 CPX,LEV,CRO,CTX,AMC + - - - A87. 3 CPX,LEV,AMC + - - - A88. 3 CPX,LEV,AMC + - - - A89. 1 AMC - - - - A90. 2 CTX,AMC - - - - A91. 1 CPX + - - - A92. 1 AMC + - - - A 93. 1 CPX, + - - - A 94. 1 CPX,,AMC + - - - A 95. 1 AMC + - - - A 96. 1 AMC + - - - A 97. 1 CPX - + - - A 98. 1 CPX + + - - A 99. 2 CPX,AMC - + - - A100. 4 LEV,CRO,CTX,AMC - - - - KEY: CPX= ciprofloxacin 10 µg, LEV = levofloxacin 5 µg, CRO = ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, +ve = Positive, ESBL= Extended Spectrum Beta Lactamase, Nil = negative, GyrA = gyrase A, ParC= Topoisomerase IV, Bla CTX-M = Beta-lactamase cefotaxime -M type enzyme, 112 4.11 Prevalence of Beta-lactamase linked phenotypic resistance for the five selected antibiotics in relation to enzymatic production by S. enterica Table 4.11 shows the prevalence of ßeta-lactamase linked resistance against the 5 selected antibiotics and their enzymatic production of ESBL’s, mutation in GyrA and ParC, and BlaCTX-M gene from S. enterica. The result showed phenotypic resistance pattern of the isolates to only one type of the antibiotics tested and the number of isolates involved. The isolates reduced in number in their resistance to only two antibiotics as compared to the one resistance to only one antibiotic. From the Table 4.11, a total of 87, 27, 29, 22, and 22% of the isolates were resistant to only one of either of these antibiotics; AMC, CTX, CPX, LEV, and CRO respectively, as compared to 23, 23, 20, and 13 % of the isolates to two different types of antibiotics namely CPX and AMC, CTX and AMC, LEV and AMC, and CPX and CTX respectively. The Table 4.11 also shows that only Nine (9) of the isolates were resistant to the whole five selected antibiotics tested, out of which 8 (88.8%) produced ßeta-lactamase, 4 (44.4%) had mutation in GyrA genes, 2 (22.2%) were ESBLs positive, 1 (11.11%) had double mutation in ParC gene while no isolate had BlaCTX-M gene. Note: The qnrB gene, bla-SHV and bla-TEM were not detected in this study. 113 Table 4.11. Prevalence of Beta-lactamase linked phenotypic resistance for the five selected antibiotics in relation to enzymatic production by S. enterica RESISTANCE RESISTANCE NUMBER OF BETA GryA ESBL ParC BlaCTX-M GROUP PATTERN RESISTANT LACTAMASE (%) (%) (%) (%) ISOLATES (%) SINGLE AMC 87 70 (80.4) 48 (55.2) 31 (35.6) 3 (3.45) 6 (6.89) CTX 27 24 (88.8) 9 (33.3) 14 (51.8) 1 (3.70) 0 (0.0 ) CPX 29 24 (82.7) 15 (51.7) 13 (44.8) 2 (6.89) 0 (0.0 ) LEV 22 19 (86.4) 9 (40.9) 10 (45.5) 0 (0.0 ) 0 (0.0 ) CRO 22 20 (90.9) 9 (40.9) 12 (54.5) 0 (0.0 ) 0 (0.0 ) CPX, AMC 23 21 (91.3) 12 (52.2) 9 (39.1) 3 (13.0) 1 (4.35) DOUBLE CTX, AMC 23 22 (95.6) 9 (39.1) 12 (52.2) 0 (0.0 ) 0 (0.0 ) LEV, AMC 20 19 (95) 9 (45) 10 (50) 1 (5.0) 0 (0.0 ) CPX, CTX 13 11 (84.6) 4 (30.8) 5 (38.5) 0 (0.0 ) 0 (0.0 ) LEV, CTX, AMC 18 16 (88.8) 6 (33.3) 9 (50) 0 (0.0 ) 0 (0.0 ) TRIPLE LEV, CTX, CRO 17 15 (88.2) 6 (35.2) 9 (52.9) 0 (0.0 ) 0 (0.0 ) CRO, CTX, AMC 21 19 (90.4) 9 (42.8) 11 (52.4) 1 (4.76) 2 (9.52) CPX, CTX, AMC 11 10 (90.9) 9 (81.8) 3 (27.3) 1 (9.09) 0 (0.0 ) CPX, CRO, CTX 11 10 (90.9) 4 (36.4) 3 (27.3) 0 (0.0 ) 0 (0.0 ) CPX, LEV, AMC 10 9 (90) 5 (50) 2 (20) 0 (0.0 ) 0 (0.0 ) QUADRUPLE LEV, CRO, CTX, AMC 17 15 (88.2) 6 (35.3) 9 (52.9) 0 (0.0 ) 2 (11.76) CPX, CRO, CTX, AMC 10 9 (90) 4 (40) 2 (20) 1 (10.0) 0 (0.0 ) PENTRUPLE CPX, LEV, CRO, CTX, 9 8 (88.8) 4 (44.4) 2 (22.2) 1 (11.1) 0 (0.0 ) AMC KEY: CPX = ciprofloxacin 10 µg, LEV= levofloxacin 5 µg, CRO = ceftriaxone 30 µg, CTX= cefotaxime 30 µg, AMC= amoxicillin/clavulanic acid 30 µg, ESBL= Extended Spectrum Beta Lactamase, bla-SHV, qnrB and bla-TEM= not detected. 114 4.12 Curing of antibiotic resistance in S. enterica isolates with ethidium bromide and acridine orange. The Nine strains of S.enterica (SO2, SO9, SU28, SU40, SU43, SA76, SA81, SA83, and SA85) habouring plasmids and either of GyrA gene or BlaCTX-M gene were subjected to various concentrations of ethidium bromide and acridine orange and thereafter were tested against the MIC’s of the five selected antibiotics (amoxi/clavulanic acid ceftriaxone, cefotaxime, levofloxacin and ciprofloxacin), results showed that all the strains produce susceptibility as determined according to CLSI standard. As shown on the Appendix III. Plate 4.12 shows the plasmid profile of S. enterica seravar. typhi after treatment with ethidium bromide at concentration of 1.25 µg/ml. Strains SO2, SO9, SU28 SU40, SU43, SA76, SA81, SA83 and SA85 harbouring low molecular weight plasmid of 1.37 kbs were completely cured, similarly as the strains in lane number 2 and 85 carrying 1.39 kbs plasmid DNA were also cured. Plate 4.13 shows the plasmid profile of the Nine S. enterica serovar. typhi exposed to acridine orange at of 2.50 µg/ml. The plate indicates the absence of plasmid as evident in the susceptibility of the previously resistant strain, of S. enterica seravar. typhi to the MICs of the five antibiotic tested. 115 M 2 9 14 20 24 28 40 43 46 48 49 64 66 67 68 69 76 77 80 81 82 83 84 85 90 kpb’s 23.13 9.42 6.54 4.36 2.32 2.03 0.56 Plate 4.12. Agarose gel electrophoresis pattern of S. enterica serovar. typhi after exposure to Ethidium bromide (mutagen). KEY: Lane 2, 9, 14, 20 and 24 from FMC Owerri. Lane 28, 40, 43, 46, 48 and 49 from FMC Umuahia. Lanes 64, 66, 67, 68 and 69 are from UNTH Enugu. Lane 76, 77, 80, 81, 82, 83, 84, 85, and 90 are from FMC, Abakaliki. Lane M is the lambda Hind III marker (0.12-23.1 kpb). 116 M T 2 9 14 20 24 28 40 43 46 48 49 64 66 67 68 69 76 77 80 81 82 83 84 85 9 0 kpb’s 23.13 9.42 6.54 4.32 2.32 2.03 Plate 4. 13. Agarose gel electrophoresis pattern of S. enterica serovar. typhi after exposure to Acridine orange (mutagen). KEY: Lanes 2, 9, 14, 20 and 24 from FMC Owerri. Lanes 28, 40, 43, 46, 48 and 49 from FMC Umuahia. Lane 64,66,67,68 and 69 are from UNTH Enugu. Lane 76, 77, 80, 81, 82, 83, 84, 85, and 90 are from FMC Abakaliki. Lane T (positive control) is the S.typhi typed culture ATCC14028. Lane M is the lambda Hind III marker (0.12-23.1 kpb). 117 4.13. Analysis of variance (ANOVA) to determine the treatment effect of the antibiotics on the human isolates of S. enterica. Table 4.12 shows the ANOVA calculated using two- way grouping according to Martin et al. (2000), From the Table 4.12 it can be deduced that the block (drugs) degree of freedom of 3 and 12 is 3.49. While the treatment degree of freedom of 4 and 12 is 3.26 at p>0.05 level of significance, thus showing that the calculated F- value of block (7.21) and treatment (20.07) is higher than the tabulated F-value of block (3.49) and treatment (3.26). This is an indication that the null hypothesis of no treatment effect should be rejected. Thus there is enough evidence to suggest real significant differences in the individual performance of the five selected antibiotics drugs on the isolates of S.enterica (see appendix III, for the analytical calculation). 118 4.14: BLAST analysis of the gene sequencing and alignment of S. enterica from Southeast Nigeria Table 4.13 a-b shows the result of the sequence alignment of the gene resulting from the PCR amplifications of the DNA complete genome of the isolate from the various hospitals ( SO3, SO14, SA96 and SU33, SA98 as shown in Table 4.13a and Table 4.13b respectively. From the Table 4.13a, isolate SO3 had 95% and 94% maximum identity with the strains found in the gene bank ( Salmonella enterica subsp. enterica serovar. Typhimurium str. UK-1 chromosome, complete genome and Salmonella enterica subsp. enterica serovar. Typhi str. CT18, complete genome) with the accession number NC016863 and NC_003198 after 100% blast hits (query coverage) on the BLAST soft ware respectively. Also isolate SA98 (Table 4.13b) had 99% and 94% identity with the strain found in the genebank (Salmonella typhimurium strain 580 GyrA gene, partial cds and Salmonella enterica subsp. enterica serovar.Typhimurium strain ATCC 307) with the acession number EF059893.1 and CP009102.1, after 97% blast hits (query coverage) on the BLAST soft ware. Another gene (isolate SO14) sequenced was found to be 89% identical to a strain, Salmonella enterica subsp. enterica serovar Typhi strain B/SF/13/03/195, complete genome. Others were as shown on Table 4.13a-b. Fig.4.4 and 4.5 shows the Fast Minimum Evolution and Neighboring joining Taxonomic tree of some strains sequenced including their relatedness was as stated in Fig.4.4 and 4.5 and Appendix IV for isolates SO3, SO14, SU33, SA96, and SA98. 119 Table 4.12: Analysis of variance (ANOVA) to determine the treatment effect of antibiotics on the clinical isolates of S. enterica VARIATION D. F S. SQUARE MEAN SOURCE SQUARE F-VALUE BLOCK 3 209.75 69.9 7.21** 4 TREATMENT 778.3 194.57 20.07** ERROR 12 116.3 9.69 TOTAL 19 1104.55 KEY: ** = Highly Significant Treatment Effect, S.S = Sum of Square, D.F = Degree of freedom. 120 Table 4.13a: BLAST Analysis of the Gene sequencing and Alignment of S. enterica from Southeast Nigeria. Sequenced Description of significant alignment Acession No. of Max. Total Query E- Max. gene aligned gene score score coverage value Identity SO3 1.Salmonella enterica subsp. enterica serovar NC016863.1 174 174 100% 3e-43 95% Typhimurium str. UK-1 chromosome, complete genome 2.Salmonella enterica subsp. enterica serovar NC_003198.1 169 169 100% 2e-41 94% Typhi str. CT18, complete chromosome 3.Salmonella enterica subsp. enterica serovar Typhi str. Ty2 chromosome, complete NC_004631.1 169 169 100% 2e-41 94% genome 1.Salmonella enterica subsp. enterica serovar SO14 CP012151.1 147 147 100% 2e-35 89% Typhi strain B/SF/13/03/195, complete genome 2.Salmonella enterica subsp. enterica serovar CP012091.1 147 147 100% 2e-35 89% Typhi strain PM016/13, complete genome AY302588.1 274 274 100% 1e-73 92% 1.Salmonella enterica subsp. enterica serovar Typhi isolate 9618-2K DNA gyrase subunit SA96 A (gyrA) gene, partial cds 268 KTI62085.1 268 100% 6e-72 91% 2.Salmonella enterica subsp. enterica serovar Typhi strain CMCSTDY DNA gyrase II (gyrA) gene, partial cds Key: SO3,SO14= Isolates from owerri, SA96= Isolates from Abakiliki, BLAST = Basic Local Alignment Search Tool, Query length of SO3 = 109nts, Query I.D of SO3 = /c/2251., Query length of SO14 = 118nts, Query I.D of SO14 = /c/_47953. , Query length of SA96 = 196nts, Query I.D of S96 = /c/_82899. 121 Table 4.13b: BLAST Analysis of the Gene sequencing and Alignment of S. enterica from Southeast Nigeria. Sequenced Description of significant Acession No. of Max. Total Query E- Max. gene alignment aligned gene score score coverage value Identity SA98 1. 263 Salmonella typhimurium strain 580 GyrA EF059893.1 363 97% 1e-99 99% gene, partial cds 2.Salmonella enterica subsp. enterica serovar Typhimurium genome assembly LN829401.1 307 307 97 5e-83 94% NCTC13348, chromosome 3.Salmonella enterica subsp. enterica CP009102.1 307 97 5e-83 94% serovar Typhimurium strain ATCC 13311, 307 complete genome SU33 1.Salmonella enterica subsp. enterica 246 KC773840.1 246 96% 2e- 65 92% serovar Typhi strain 21g DNA gyrase subunit A (gyrA) gene, partial cds 2.Salmonella enterica subsp. enterica HQ176354.1 246 100% 2e-65 91% serovar Typhi strain ST33 GyrA (gyrA) 246 gene, partial cds Key: SA98= Isolates from Abakiliki, SU33= Isolates from Umuahia, BLAST = Basic Local Alignment Search Tool, Query length of SA98 = 208nts, Query I.D of SA98 = /c/_221473, Query length of SU33 = 184nts, Query I.D of SU33 = /c/_65033. 122 Figure 4.4: Isolate SO3, Taxonomic Neighboring joining tree 123 Figure 4.5: Isolate SO14, Taxonomic Fast Minimum Evolution tree 124 CHAPTER FIVE DISCUSSION 5.1 Discussion Typhoid fever is a disease that has been associated with human since close to the first appearance of hominids and may have first infected human ancestors anywhere from 200,000 to two million years ago (Mustaq, 2006). Typhoid fever is a systemic infection caused by Salmonella enterica Serotype typhi. It is a highly adapted human specific pathogen with remarkable mechanism for persistence in its host (Mustaq, 2006). The authentication of the S.enterica isolates used in this study, applying the conventional biochemical test and Microbact identification techniques, showed that they were Gram negative tiny rods, showing deposit of black residues on Salmonella Shigella agar and deoxycholate agar, ferment mannose and glucose, and also utilised carbon from citrate as the sole source of carbon as reported by Cheesbrough (2006). In this study, uniform numbers of isolates were collected from each of the four hospitals. The isolates recovered from the wards and departmental units in each hospital vary, but the number of isolates recovered from the female patients was higher than the once recovered from the male. These agree with the report of Cheesbrough (2006). In this study, generally the reports of In-patients units (IPU) recorded higher number of S. enterica isolates than the General out patient department (GOPD), suggesting that the patients on admission were more prone to getting infected with the organism nosocomially than GOPD patients. Among the IPU isolates of S. enterica therefore, there is the possibility of clonal dissemination of individual multidrug resistant S. typhi according to Mustaq (2006). Out of the 100 isolates of Salmonella enterica tested in this study against the antibiotics normally used for treatment in the hospitals studied, some were found to exhibit varying degrees of resistance cutting across different classes, namely; β-lactams, chloramphenicol, aminoglycosides, quinolones (fluoroquinolones) and the sulphonamides. It could be deduced that the S. enterica was resistant to more than three classes of antibiotics used in this study, thereby making the organism multi-drug resistant (MDR). This report agrees with those of Deak et al. (2015), Yah (2010), Brusch (2010) and Mustaq (2006). Thong et al. (2000), Connerton et al. (2000) and Mirza et al. (2000) have reported the multiple 125 outbreaks of infections with resistant strains of Salmonella spp. in India, Pakistan, Bangladesh, Middle East and Africa. In this study, the highest number of S. enterica isolates were recovered from patients between 18 years and above, followed by the ages between 21- 30 years, also supporting the reports of Brusch (2010) and Mustaq (2006). Brush et al. (2010) stated that genes for antibiotic resistance in S.typhi were acquired from Escherichia coli and other Gram negative bacteria via plasmids. They further stated that the plasmid contains cassettes of resistance genes that are incorporated into a region of the Salmonella genome called integron. Some of these plasmids also carry multiple cassettes and immediately confer resistance to multiple classes of antibiotics. This may be the reason why the isolates of S. enterica were resistant to more than three different classes of antibiotics and could also explain the appearance of multi-drug resistant (MDR) strains of S. typhi (Brush et al., 2010). According to Mandal et al. (2010) the original indication of chloramphenicol was in the treatment of typhoid. Currently, however there is a universal presence of multi-drug resistant Salmonella typhi as observed in this study such that chloramphenicol is seldom used for this indication except when the organism is known to be sensitive. Based on the CLSI standard used for interpreting the sensitivity screening results in this study, few of the isolates of S.enterica were sensitive to chloramphenicol. This has shown that the cause of resistance to these antibiotics was due to both the plasmid and chromosomal mediating enzymes. Enzymes such as β-lactamase and Extended spectrum β- lactamase were detected to account for the antibiotic resistance observed in this study, manifested in different forms of enzyme inhibition and resistant mutants, noticeable especially against cephalosporins and floroquinolones. These antibiotics have been recommended as replacements for chloramphenicol by most authorities for the treatment of typhoid fever. Some authors have reported the effectiveness of fluoroquinolones (Nathan et al., 2005; Noel et al., 2007; Brush et al., 2010) against susceptible organisms with better cure rate than cephalosporins. These workers further reported the emergence of resistance to first generation fluoroqninolones in many parts of Asia (Nelson et al., 2007; Brush et al., 2010). Brush et al. (2010) reported that third generation cephalosporins have been used in regions with high fluoroquinolone resistance rates, particularly in South Asia and Vietnam. However, in this study, both the third generation cephalosporin and the fluoroquinolone still maintained their choice as effective antibiotics for the treatment of chloramphenicol- 126 resistant and multi drug resistant strains of S. enterica serovar typhi in this part of Nigeria. The results obtained showed that among the antibiotics tested ceftriaxone (a third generation cephalosporin) and levofloxacine (a third generation fluoroquinolone) were each effective on 78 of the isolates out of the total 100 isolates, creened for sensitivity; cefotaxime was effective against 73 of the isolates, followed by ofloxacin, pefloxacin, ciprofloxacin and ceftaxidime with 72, 71, 71, and 56 isolates of S. enterica respectively. Analysis revealed that not all the isolates were β-lactamase positive, as only 80% of the isolates produced β-lactamase, while 36% of the isolates were positive to extended spectrum β-lactamases. Also, it was found that only 28 isolates had both the conventional β-lactamase and the ESBLs co-existing. According to Yujuan and Ling (2006), Valverde et al. (2008), and Yah (2010) resistance to broad-spectrum β- lactams was highly mediated by ESBL enzyme, which has been increasing the world health problems in clinical settings, coupled with the hydrolytic activity of the plasmid mediated β-lactamases on β-lactams (Yah, 2010). In this study, plasmids were not found in most of the isolates of S.enterica yet; they were resistant to a wide range of antibiotics including the third generation cephalosporins and fluoroquinolones. Thus, the resistance found in them may not be due to plasmid. This study reported low molecular weight plasmids which were recovered from 9 (9%) of the resistant isolates of which were recovered mainly from adult patients, but only one patient, between the age range of 0-10years and the age range of 11-20 years in which the isolates were recovered from, had plasmids. According to Soge et al. (2005) the first CTX-M-type β-lactamases were identified as plasmid-encoded enzymes in clinical isolates from the Enterobacteriaceae, and all the 30 isolates of Klebsiella pneumoniae in their study produced at least one β-lactamase and 17 (57%) produced CTX-M β-lactamase (Soge et al., 2005). Furthermore, reports have also shown that the resistance of gastroenteric Salmonella strains to antimicrobial agents was largely due to the production of extended-spectrum β- lactamases (ESBLs) encoded on plasmids, as well as on chromosomes (David and Frank, 2000; Yujuan et al., 2006 and Yah, 2010). In this study amoxicillin/ clavulanic acid was expected to be effective on the isolates because this combination of antibiotic is known to be a β- lactamase inhibitor, but the reverse was the case, as it was observed that out of the 100 S. enterica serovars isolate 87% of the organism were resistant to amoxicillin/clavulanic acid. It was also found that out of the 87 127 isolates of S. enterica resistant to amoxicillinl/clavulanic acid, 70 (80.4%) produced β-lactamase, while 31(35.6%) were positive for ESBL and were resistant to some of the cephalosporin tested in this study. This may be due to the fact that some isolates will not exhibit β-lactamase unless the enzyme has been induced, by exposing them to beta-lactam antimicrobial. This mechanism is achieved when the drug comes in contact with the organism; this initial contact will trigger the reaction or production of this enzyme by the organism as the organism tends to initiate resistance. It has been reported that beta-lactamases that hydrolyze extended spectrum cephalosporins have an oxyimino side chain which include cefotaxime, ceftriaxone and ceftaxidime (Philippon et al., 2002). Also reports stated that such ESBLs confer resistance to these antibiotics and related oxyiminino β-lactams (Philippon et al., 2002). According to Woodford et al. (2006), CTX-M beta-lactamase (class A) enzymes were named for their greater activity against cefotaxime than other oxyimino β-lactam substrates e.g ceftazidime, ceftriaxone or cefepime which are examples of plasmid acquisition of β-lactamase genes normally found on the chromosome of kluyvera species, a group of rare pathogenic commensal organism (Woodford et al., 2006). Woodford et al. (2006) stated that these enzymes are not very closely related to TEM or SHV beta-lactamases in that they show only approximately 40% identity with these two commonly isolated β-lactamases relative to CTX-M genes (Woodford et al., 2006). Thus, in this study in order to detect the type of ESBL that confers resistance on some of the third generation cephalosporins, a universal BlaCTX-M , SHV and TEM type primers were used for the Polymerase Chain Reaction (PCR) analysis on the DNA of the isolates of S.enterica. It was found that at least 13 (13%) of all the S. enterica isolates produced BlaCTX-M type genes while only 9 (9%) of the S. enterica had plasmids. According to Woodford et al. (2006) more than 80 CTX-M enzymes are currently known and are mainly found in strains of S. enterica serovar. typhimurium and E. coli. They further stated that despite their name CTX-M enzymes, a few of the enzymes are more active on ceftazidime than on cefotaxime. Interestingly, the enzyme produced by blaCTX-M gene detected in this study was more active on ceftazidime in view of the fact that out of the 44 isolates of S. enterica that were resistant to ceftazidime alone, 3 (6.8%) produced blaCTX-M type enzyme, while out of the 27 isolates with resistance to cefotaxime alone, 4 (14.8%) had the gene producing the enzyme. 128 According to the classification of Richmond and Sykes in 1987 as reported by Onyenwe et al. (2011) chromosomal cephalosporinase is inducible, and cannot be inhibited by clavulanic acid, thus having the ability to affect all cephalosporin drugs (including other generations) while the class 2 that is also chromosomally mediated is constitutive and is inhibited by clavulanic acid but affect only the penicillins. In this study, 87% of the isolates were resistant to amoxiclavulanic acid. Out of 13 isolates positive for blaCTX-M type gene, only 2 harbored plasmids, while all the 9 isolates carrying R- plasmids produced β-lactamase but only 2 of them were positive for blaCTX-M type gene. This implies that most of the blaCTX-M type genes were found in the chromosomes, all produced β-lactamase emzymes and all of them were also resistant to clavulanic acid inhibition, based on Richmond and Sykes classification. It could be deduced that the chromosomal broad- spectrum β- lactamases in the class 1 chromosomal-mediated cephalosporinase and most of the resistance encountered in this study were mostly chromosomal as few plasmids were found in the isolates. The results obtained in this study showed that some of the isolates that harbored plasmids and produced β-lactamase were resistant to high percentage of the antibiotics, such as isolate SO9 from a female adult patient in the General out – patient department (GOPD) unit that was resistant to 8 (57.1%) out of the 14 different antibiotics studied. The results also showed that isolate SA76 recovered from a male adult patient and isolate SA83 recovered from a female (58 years) adult patient both in the GOPD unit were resistant to 12 (85.7%) of the antibiotics. Analysis revealed that isolate SU40 recovered from a female adult patient in the In-patient units (IPU), was resistant to 11 (78.5%) antibiotics while isolate SA85 from female adult in IPU unit and isolate SA81 from 6 years old female patients in National Health Insurance Scheme unit (NHIS) were both resistant to 10 (71.4%) of the antibiotics each. Analysis showed that antibiotic resistant isolates of S. enterica harboring plasmid- mediated β-lactamase were still prevalent around the Southeast region of Nigeria as shown in this study, but chromosomal mediated β-lactamase was higher among the resistant isolates. This study showed that the S. enterica isolated from the male patients (42) irrespective of their age group produced more resistant genes of GyrA 24 (57.14%), ParC 6 (14.3%) and BlaCTX-M genes 7 (16.7%), as against the isolates from female patients (58) infected with S. enterica that had GyrA 31 (53.4%), ParC 8 (13.7%) and blaCTX-M genes 6 (10.3%). 129 The isolates of S. enterica harbouring plasmids were prevalent in the female patients, as the 10.3% of the isolates from them were found to be higher than the 7.14% of isolates harboring plasmid and producing β-lactamase in isolates from male patients. The S. enterica isolates in this study were resistant to the three first line drugs used for the treatment of typhoid; chloramphenicol (80%), co-trimoxazole (78%) and amoxicillin (80%). This report is in line with the reports of Brusch et al. (2010), where antibiotic resistant strains of S. typhi and S. paratyphi inactivated chloramphenicol via acetylation involving an enzyme that carries chloramphenicol acetyl transferase type I and multi drug resistant strains that may carry dihydrofolate reductase type VII, which confers resistance to trimethoprim. It has also been reported that amoxicillin and trimethoprim-suphamethaxazole were effective alternatives to chloramphenicol-resistant isolates till the end of 1990’s (Mustaq, 2006). According to Nathan et al. (2005) and Griggs (2007) chloramphenicol remains the drug of choice in the treatment of meningitis caused by Neiseria meningitis in patients with severe penicillin or cephalosporin allergy. Due to the increased resistance to the first line antibiotics, drugs such as cephalosporins and the fluoroquinlones have been recommended by most authorities for the treatment of typhoid fever. It has also been recommended (Nathan et al., 2005; Griggs, 2007) that in case where the origin of infection is unknown, a combination of a first- generation fluoroquinolone and a third-generation cephalosporin should be used. This study showed that 78% of the S. enterica serovar. typhi were susceptible to levofloxacin (a third-generation fluoroquinolone), 71% to ciprofloxacin (a second generation fluoroquinolone), 71% nd nd to pefloxacin (a 2 generation fluoroquinolone) and 72% to ofloxacin (a 2 generation fluoroquinolone). Interestingly, the third generation cephalosporins tested were active against S. enterica isolates in decreasing order of 78% > 73% > 56% for ceftriaxone, cefotaxime and ceftazidime respectively. The MIC obtained for the five drugs selected for testing; levofloxacin, ciprofloxacin, amoxicillin/clavulanic acid, ceftriaxone and cefotaxime retained their efficacy as the current drugs of choice in the treatment of typhoid fever, except amoxicillin/clavulanic acid (Parry and Beeching, 2009; Brusch et al., 2010). Screening for the gene constituents of the isolate, showed that out of 27 isolates of S. enterica that were resistant to only cefotaxime, 24 (88.8%) produced β-lactamase, 9 (33.3%) GyrA gene (topoisomerase II enzyme that codes for point or single mutation) and only one produced mutation in parC (topoisomerase IV enzymes) that codes for 130 double mutation. Also 87 isolates were resistant to amoxicillin/clavulanic acid among which 70 (80.4) isolates produced β-lactamase, 48 (55.2%) had single mutation in GyrA and 3 (3.45%) with double mutation in parC. Moreover, out of the 22 isolates resistant to ceftriaxone 20 (90.9%) produced β- lactamase, 9 (40.9%) had single mutation in GyrA gene, but none had double mutation in ParC gene. Of the 29 isolates of S. enterica resistant to ciprofloxacin 24 (82.7%) produced β-lactamase, 15 (51.7%) had GyrA and 2 encoded ParC. Finally, out of 22 isolates resistant to only levofloxacin 19 (86.4%) produced β-lactamase enzyme, 9 (40.9%) had single mutation in GyrA and none in ParC genes. These trend of results showed that there have been some forms of resistant mutants among the S. enterica serovar family, because the presence of mutations in GyrA gene in the isolates showed that resistance to fluoroquinolone is evolving in an ominous direction according to Brusch et al. (2010). This implies that the patients from whom the isolates were obtained could have been on fluoroquinolone drug for treatment. It has been recognized that S.typhi most commonly develops fluoroquinolone resistance through specific mutations in GyrA and ParC which codes for the binding region of DNA gyrase and topoisomerase IV respectively. In this study the failure rates of ceftriaxone and cefotaxime were (22%) and (27%) respectively just as levofloxacin and ciprofloxacin had 22% and 29% respectively against the isolates tested, suggesting mutational resistance and clonal spread. The single and double mutation in GyrA and ParC respectively in this study correlates with the works of Randal et al. (2005) and Brusch et al. (2010). According to Brusch et al. (2010), a single point mutation in GyrA confers partial resistance while a mutation in ParC coupled with a single GyrA mutation confers full in vitro resistance to fluoroquinolone. Remarkably, Ackers et al. (2000) found no resistance against ciprofloxacin, ceftriaxone and gentamycin when 350 isolates of Salmonella were tested, even though 16% of the isolates were multidrug resistant Salmonella typhi (MDRST). Nadeem and Colleagues in 2002 reported from Pakistan that 69% of isolates were found to be MDRST (Nadeem et al., 2002). In another report from Bahawalpur Pakistan, 53.8% of isolates were found to be MDRST and all the strains were sensitive to fluoroquinolone and the third generation cephalosporins (Munir et al., 2001). According to Munir et al. (2001) 28 isolates were exposed to all the three first-line anti-salmonella drugs, out of which 18 (64.3%) isolates turned out to be MDRST. In this study, 79% of the isolates were MDRST, showing a high resistance profile in the 131 Southeast region of Nigeria. Concerning ciprofloxacin and ceftriaxone, the results obtained were contrary to the works of Ackers et al. (2000) and Nadeem et al. (2002), but support the works of Mustaq (2006) who reported that out of 18 MDRST four of the isolates were resistant to ciprofloxacin and four to ceftriaxone while two were resistant to both drugs, and that the overall resistance to ciprofloxacin was 19.2%, ceftriaxone 17.9% and cefotaxime 21.1% out of 76 isolates. This agrees substantially with the findings in this study whereby resistance rates for ciprofloxacin, ceftriaxone and cefotaxime were 29%, 22% and 27% respectively. Threlfall and Ward (2001) reported decreased sensitivity to ciprofloxacin which supports the findings in this study and suggested possible alternatives as ceftriaxone and cefotaxime which correlates with the data obtained in this study, showing that ceftriaxone and cefotaxime had better activity against the isolates of S.enterica, than ciprofloxacin. Mustaq (2006) reported that resistance to trimethroprime/sulphamethoxazole was 94.2%, chloramphenicol 65.3%, and amoxiclavulanic acid 42.5% all of which were first line anti-Salmonella drugs. These reports clearly supported the results obtained in this study with 78%, 80% and 87% of the isolates were found to be resistant to co- trimoxazole, chloramphenicol and amoxicillin/ clavulanic acid respectively. As rightly stated by Mustaq (2006), the pattern of S.typhi resistance was changing rapidly and that Multi-Drug Resistant Salmonella Typhi resistant to ciprofloxacin and ceftriaxone was a major threat in the developing world, including the Southeast part of Nigeria as revealed in this study. It might be interesting to note that S.enterica had high susceptibility rate of 78% to levofloxacin, a racemic isomer of ofloxacin, even though some resistant mutant strains were detected in the present work. These resistant strains of S.enterica were found to harbor mutant genes in the gyrase A region (topoisomerase II enzyme) and ParC (topoisomerase IV enzyme) gene in their chromosomes and few produced plasmids which are of low molecular weight of about 1.37- 1.39kbs. This appears to be the first documented report of levofloxacin resistant S.enterica mutants in Southeast Nigeria. This high incidence of fluoroquinolone resistant S. enterica serovar. typhi could be attributed to some factors, namely: i. the isolates were from the In-patients Units, which means that these patients have been hospitalized before the organism was isolated from their stool sample, therefore might have been infected with resistant strain of nosocomial origin as reported by Martin et al. (2004) and Varma et al. (2005). 132 ii. the isolates were from a University affiliated medical centre (Federal Medical Centre Owerri) and University Teaching Hospital (UNTH Enugu) that treat patients with severe infection, indicated in the fact that the isolates from GOPD were the second highest S. enterica isolates recovered from the patients. This could be indicative that these patients would have been using these antibiotics (fluoroquinolone) inappropriately. Hakanem et al. (2001) stated that transferable resistance against quinolones was sometimes rare in bacteria invivo, but clonal resistance due to mutation in chromosomal gene remains the potential mechanisms, accounting for high level of reduced fluoroquinolone susceptibility in Southeast Asia. This report agrees with the findings in this study whereby chromosomal mediated resistance genes for mutation, clonal resistance and R-plasmids were detected. Statistical analysis of results obtained on the treatment effect of five selected antibiotics using Analysis of Variance (ANOVA) by two way grouping according to Martin et al. (2000) revealed significant differences at (p=0.05) level of significance (n=100) in the individual performance of the drugs on the isolates of S. enterica. Curing analysis carried out in this study using two dyes, ethidium bromide and acridine, orange showed that the effect of ethidium bromide at 0.625 µg/ml and 1.25 µg/ml were more pronounced than the acridine orange used at different concentrations (5.0, 2.5, 1.25, 0. 625, 0.3125 µg/ml) revealing ethidium bromide as a more effective curing agent than the acridine orange even at the reduced concentration of the ethidium bromide. Acridine was effective on Salmonella enterica only when its concentration was increased to 2.5 µg/ml, thus supporting the reports of Adeleke et al. (2002) on the use of acridine dyes as mild agents of curing for recognizing resistant plasmids in resistant S. aureus. The emergence of mutation- based resistance may be fostered by selection pressure caused by the use of antibiotics agents in either human medicine or agriculture, according to Parveen et al. (2007), corroborating the alarming increase in quinolone resistance observed during the past few years among food borne pathogens. In this study Salmonella enterica recognized as a food or water borne pathogen, showed increased quinolone chromosomally-mediated resistance and exhibited several mutations in their genes, an indicative of poor hygiene in the part of the patients, implicated in the study. This could therefore, have produced the increased level of mutation discovered among these S. enterica strains studied. It was reported by 133 Parveen et al. (2007) and Griggs (2007), that enrofloxacin (a fluoroquinolone used in agriculture) can select Salmonella mutants, resistant to nalixidic acid and fluoroquinolones. The results obtained in this study were in line with the above statement as up to 48% of the Salmonella enterica serovars were found to possess mutation in the gyrase A gene which codes for point mutation in the Quinolone Resistant Determining Region (QRDR), showing chromosomal- mediation and thus, created an increase in resistance to fluoroquinolone drugs. The Minimun Inhibitory Concentrations (MIC’s) of five selected antibiotics (ciprofloxacin, levofloxacin, amoxil/clavulanic acid, ceftriaxone and cefotaxime) obtained in this study increased from 1.56 µg/ml – 12.0 µg/ml in levofloxacin and ciprofloxacin, cefotaxime and ceftriaxone, except in few highly resistant mutants which had their MIC’s at 25.0 µg/ml. The MICs of amoxillin/ clavulanic acid were observed to be very high ranging from 12 µg/ml – 25 µg/ml except in few cases where some isolates were susceptible with the MIC of 6.0 µg/ml-12.0 µg/ml. Thus in this study, the MIC for levofloxacin was within 1.5 µg/ml and 3.0 µg/ml against 41% and 25% of the isolates, ciprofloxacin was within 1.5 µg/ml and 3.0 µg/ml against 43% and 23% of the isolates, cefotaxime was within 1.5 µg/ml and 3.0 µg/ml against 8% and 25% of the isolates, and ceftriaxone was within the same range against 56% and 15% of the isolates tested respectively. These showed that each of the five drugs had its MIC at 1.5 µg/ ml against the isolates indicating some level of susceptibility to the range of drugs selected. These results fall within an indication of 4 µg/ml as the MIC break point by CSLI (2007) and CLSI (2011) on quinolones, especially ciprofloxacin. The cephalosporins used in this study showed equal match in the treatment of S. enterica, especially ceftriaxone. About 78% of the isolates were susceptible to the drug while 56% of the isolates had MIC 1.5µg/ml, indicating ceftriaxone as one of the best against Salmonella infection in adult and children, as they are also more tolerated than ciprofloxacin in the case of the children. These agree with the most recent professional guidelines for the treatment of typhoid fever in South Asia, issued by the India Association of Pediatrics (IAP) in October, 2006 according to Brusch et al. (2010). According to the guidelines, for empiric treatment of uncomplicated typhoid fever, the IAP recommends cefixime and as a second- line agents, azithromycin, but for complicated typhoid fever they recommended ceftriaxone. 134 This indication agrees with the findings in this study because a very good number of the S. enterica isolates screened were positive to ESBL production, with most of the isolates found having their resistant genes located on chromosomes rather than on plasmid. Based on the trend of results obtained, recommendation could be made to physicians to prescribe the combination of fluoroquinolone and third generation cephalosporins, especially the ceftriaxone, as supported and stated by many authors such as Montaz et al. (2002) that according to the data from their study in Egypt, neither fluoroquinolone nor third generation cephalosporins resistance has emerge. Montaz et al. (2002) further reported that there could be a resurgence of quinolone resistance unless the use of this drug is restricted. Interestingly, the workers recorded a resurgence of chloramphenicol- susceptible S.typhi strains agreeing with the findings in this study that 20% of the isolates screened show susceptibility to chloramphenicol. This could be attributed to the previous long time restricted use of chloramphenicol to Salmonella treatment in Nigeria by health workers and possibly, the patients who indiscriminately use them. It was interesting to note that the level and degree of resistance vary globally and there are geographical variations in the epidemiology of Salmonella infections. In this study, Some isolates possessed no resistance gene or mutation in either gyrA or ParC in the chromosome, while some produced no β-lactamase and no plasmids but were only ESBL posistive strains, yet they were resistant to more than four antibiotics which included levofloxacin, ceftriaxone, cefotaxime, and amoxillin/clavulanic acid. Secondly, If these particular strains of Salmonella enterica serovars were resistant to cefotaxime as observed in this study, then it could be attributed to either the BlaCTX-M type gene detected (ESBL), but for the fact that it could not be inhibited by the action of amoxillin/ clavulanic acid which is a Beta-lactamase inhibitor, then this type of resistance may be chromosomal. Thirdly, no mutation in gyrA and ParC genes was detected on some isolates in the case of fluoroquinolone resistant strains after specific primers were used for the amplification of the determinant gene. Harbottle et al. (2006) suggested that there is a continuing need for increased surveillance of antimicrobial –resistance phenotypes in Salmonella isolates of animal and human origin on a global basis (Harbottle et al., 2006). According to Haugum et al. (2006) and Deak et al., (2015), important mechanisms for quinolone-resistance are mutations accumulating in the genes encoding the DNA gyrase and topoisomerase gyrA, gyrB, ParC, Par E. In this study only the mutant 135 genes encoding gyrA and parC were detected in the topoisomerase enzyme. Report from Eaves et al. (2004) stated that in Salmonella and E.coli, the majority of mutations in DNA gyrase are found between residues 67 and 106 in gyrA, in a region called the quinolone- resistant determining region (QRDR) while in ParC mutations in Salmonella have been found between residues 57 and 84 (Eaves et al., 2004). Hopkins et al. (2005) indicated that the mutations may only be required to achieve high level resistance. In this study, at least three (3) S. enterica serovars harboring plasmids were found to be resistant to some groups of antibiotics (SA81, SA83, and SA85 isolate) including 2 members of the fluoroquinolone (ciprofloxacin and levofloxacin) without harboring any resistance gene or mutation in their gyrA and parC regions in the chromosomes, hence an indication of plasmid- mediated quinolone resistance (PMQR). According to Hopkins et al. (2005) and Haugum et al. (2006) such resistance was reported only in Klebsiella and E.coli. Earlier studies have observed that in Salmonellae, the relative frequency of different mutations in gyrA was dependent on the quinolone antibiotics used for selection (Levy and Manshall, 2004). According to Lindtedt et al. (2004) it was discovered that a geographically dependent distribution of GyrA mutation was at codons 83 and 87 in S. hadar while Haugum et al. (2006) stated that the position and type of amino acid substitution in gyrA varied with the serovars. It has also been reported that among S.typhi isolates obtained in the United States between 1999 and 2006, 43% were resistant to at least one antibiotic. In this study, 98 % of the S.enterica serovars isolates were resistant to at least one of the 14 antibiotics tested. According to Hirose et al. (2002) fluoroquinolones have become the first-line drugs for the treatment of typhoid, active against isolates of Salmonella species. However, several reports have declared treatment failures when these antimicrobials were used to treat Salmonella infections caused by strains with reduced fluoroquinolone susceptibility (Hakanem et al., 2001), in agreement with the present study. Several clinical treatment failures after the administration of ciprofloxacin and other fluoroquinolone to patients with typhoid fever due to strains with decreased susceptibility to the fluoroquinolone have also been reported by Threlfall and Ward (2001). In this study, the mutations responsible for the fluoroquinolone resistance in the gyrA and ParC genes of the Salmonella enterica serovars were investigated. The sequences for the Quinolone Resistance Determining Region (QRDR) of the gyrA gene of the 136 isolates which showed reduced susceptibility to some fluoroquinolone were detected. There was single mutation at the Ser-83 – Tyr, Ser-87- Gly and Ser-83- Phe, while some were found in Asp-87- Gly or Asp-86- Gly in ParC gene. The sequence analysis also revealed that some of the positions of the amino acids in the gyrA mutation were identified as Asp-87- Asn or at Ser-83- Tyr. The gene (from isolate SA98) sequence alignment revealed that the S. enterica characterised in this study had 99% similaritiy or identity to the typed gene of Salmonella typhimurium strain 580 GyrA gene, partial cds (conservative domains) and 94% identical to the ATCC (American Typed Culture Centre) strain known as Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 307 found in the genebank respectively. Also in this study, some NCTC13348 (National Centre for Typed Culture) strains chromosome were found to be 94% identical to the quinolone resistant gene of the isolate sequenced in this study. Another gene (isolate SO3 having point and double mutation at position 83 and 87 codon) sequenced was also observed to be 94% identical to a strain, Salmonella enterica subsp.enterica serovar Typhimurium str. DT104, which is also similar to the gene of the clinical isolate reported by Hirose et al. (2002), which caused nosocomial infections in the United States with high level of fluoroquinolone resistance as seen in this study, according to the Fast Minimum Evolution and Neighboring joining tree, taxonomic analysis (Zheng, et al., 2000; Aleksandr et al. 2008) . These reports are in line with those reports of Hirose et al. (2002) where a single mutation at either the Ser-83 or the Asp-87 codon was found after sequencing the genes. According to Hirose et al. (2002), this indicated that gyrA mutations are of principal importance for the fluoroquinolone resistance of serovars typhi and para typhi A, among the salmonellae. Alterations at positions 83 and 87 of the gyrA amino acid sequence have also been described previously for Salmonella strains by Hirose et al. (2002). Double mutations at position 83 or 87 of the gyrA amino acid sequence were also reported in clinical isolates of serovars Schwarzengrund, which caused nosocomial infections in the United States and also exhibited ciprofloxcin resistance as earlier stated (Hirose et al., 2002). Nevertheless, these workers stated that strains with high level fluoroquinolone resistance due to double mutations at codons in positions 83 and 87 in the gyrA amino acid sequence have not been found in clinical isolates of serovar Typhi and Paratyphi A. In this study, however, double mutations were detected in ParC from S. enterica isolates SO3, 18, 19 strains and SE58 and 60 strains (Plate 4.10). Also double 137 mutation in ParC was detected from SE72, 73, 74, 75, and SA76, 77, 97, 98 and 99 strains (Plate 4.11), though in few cases of the clinical isolates. It could be deduced that the difference in fluoroquinolone resistance between two closely related species may be explained by differences in outer membrane permeability’s for fluoroquinolones and differences in active efflux activities (Hirose et al., 2002). Fluoroquinolone resistance in S.enterica is usually mediated by at least one mutation in a DNA topoisomerase gene as observed in this study. According to Gaind et al. (2006) and Deak et al., (2015) there was a single gyrA mutation in Ser-83-Phe or Ser- 83- Tyr which was associated with reduced susceptibility to ciprofloxacin with MIC’s of 0.125- 1.0 mg/L. In this study, there was reduced susceptibility to ciprofloxacin and levofloxacin with MIC ranging from 1.56- 12.5µg/ml in both drugs which also correlates with the mutation in gyrA, supporting Gaind et al. (2006) and Deak et al., (2015). Futhermore, in this study it was observed that there was a slight increase in MIC of ciprofloxacin and levofloxacin which is ≥ 1.56µg/l, when an additional mutation occurred in ParC gene in the amino acid position of Asp-87- Gly or Asp- 86- Gly with MIC range between 1.56- 12.5µg/ml leading to reduced susceptibility of the two drugs, thus correlating with the report of Gaind et al. (2006), that an additional mutation in ParC, as seen in this study was observed in Ser-80- ile, Ser-80- Arg, Asp- 69- Glu or Gly-78-Asp and was accompanied by an increase in ciprofloxacin MIC which ≥0.5mg/L. Gaind et al. (2006) further showed that three mutations conferred ciprofloxacin resistance, two in gyrA (Ser-83 –Phe and Asp-87- Asn or Asp -87 –Gly) and one in ParC. In this study, four was observed, three in gyrA (Ser-83 – Tyr, Ser-87- Gly and Ser-83- Phe) and one in ParC (Asp-87- Gly or Asp-86- Gly). Haugum et al. (2006) also indicated gyrA codon 83 and 87 as the main targets for mutation in Salmonella enteritidis. Based on the results obtained, the number of isolates with reduced susceptibility to fluoroquinolone such as ciprofloxacin and levofloxacin appeared to be increasing. Of a particular note was the emergence of quinolone resistance in some clones of the widespread Salmonella enterica serotype Typhimurium definite phage type 104 as earlier reported by Hakanem et al. (2001). According to Gaind et al. (2006) the presence of plasmid-borne integron in ciprofloxacin-resistance (though not determined in this study) may lead to a situation of untreatable enteric fever. Nevertheless, the fluoroquinolones such as ciprofloxacin, and the cephalosporins such as ceftriaxone are best indicated for the treatment of 138 severe S. enterica serovars in adult while ceftriaxone and cefotaxime are best recommended for children based on the reports in this study. 139 CHAPTER SIX CONCLUSION AND RECOMMENDATIONS 6.1 Conclusion The original indication of chloramphenicol was in the treatment of typhoid and chloramphenicol may have been effective against a wide variety of Gram negative bacterial (Mandal et al., 2010), including most anaerobic organisms (Falagas et al., 2008). However the almost universal occurence of multi-drug resistant Salmonella typhi meant that it is seldom used for this indication except when the organism is known to be sensitive (Mandal et al., 2010). There have been reports by Hakanem et al. (2001) that in this era of frequent international connections, microbes may be easily transmitted from one place to another. Correspondingly, factors furthering the emergence and spread of antimicrobial resistance in any country may soon have an impact on resistance of bacterial pathogens, or even of normal human flora, in faraway regions, even different continents. Thus, on this basis, the emergence of antimicrobial resistance in any part of the world may have a global bearing and thus deserves universal attention (Hakanem et al., 2001). Chloramphenicol is no longer a first line agent for any indication in both developed and developing nations like Nigeria. In low income countries like Nigeria unfortunately, chloramphenicol is still widely used mainly because it is cheap and readily available. The most serious adverse effect associated with its treatment is bone marrow toxicity. This may occur in two distinct forms- bone marrow suppression, a direct toxic effect of the drug, usually reversible. Secondly, aplastic anaemia which is idiosyncratic (rare, unpredictable and unrelated to dose) are generally fatal according to Mustaq (2006). Arising from this study, fluoroquinolones such as levofloxacin should only be used in patients who have failed at least one prior therapy, and also reserved for use in seriously ill patients who may require immediate hospitalization as specified by Shin et al. (2003), Jonhson and Johnson (2004) and Janssen Pharmaceuticals (2008). 140 6.2. Recommendations This study has highlighted the problem of bacterial resistance. Levofloxacin, ciprofloxacin (fluoroquinolone) and the cephalosporins (ceftriaxone and cefotaxime) were seen as possible drugs of choice in this study. Therefore the combination of ciprofloxacin and cefriaxone or levofloxacin and cefotaxime would be recommended for the treatment of Salmonella infections. It is also recommended that these drugs should be administered with utmost care and caution to avoid further resistance. According to Shin et al. (2003) and Fraundfelder and Fraundfelder (2009), the overuse of antibiotics has given rise to a breed of super- bacteria that are resistant to antibiotic entirely. This research correlates with and supports the report of Linder et al. (2005) that fluoroquinolone, including levofloxacin had become the most commonly prescribed class of antibiotics to adult. Based on this study carried out in the Southeast part of Nigeria, fluoroquinolone and the third generation cephalosporins would be regarded as the drugs of choice for the treatment of chloramphenicol- resistant Salmonella enterica and other multi- drug Salmonella species. Alternatively, doctors and Pharmacist who prescribe and dispense these drugs indiscriminately in the hospitals and government healthcare centers, should be discouraged. Also, hand washing in the hospital or at home is also recommended as a useful, safe and aseptic technique to patients to prevent diseases such as Typhoid fever caused by Salmonella enterica species. The control of indiscriminate intake of antibiotics such as fluoroquinolones and cephalosporins in animals and humans respectively can help in the control of MDRST; thus the low prevalence of Salmonella due to strict control programs result in a relatively low frequency of multi-drug resistance isolates. The degree of communication between veterinary organizations and health care providers in the various hospitals and tertiary health institutions is important for effective management of the menace of multidrug resistance. The government at all levels should be aware of the danger of the spread of antibiotic resistance and continue to enforce the existing laws guiding the sale and use of antibiotics. Also, the use of lactic acid bacillus (LAB) as probiotics in the treatment and management of the infections caused by this pathogen would assist in mitigating the high level of resistance from this organism. 141 Lastly, the establishment of bioscience and research institutes in Nigeria higher institution of learning, especially in Southeast Nigeria will aid the molecular studies of this type. 142 REFERENCES Ackers, M.L., Puhr, N.D., Tauxe, R.V., Mintz, E.D. 2000. Laboratory Based Surveillance of Salmonella Serotype Typhi Infections in the United States. Antimicrobial resistance on the Rise. Journal of American Medical Association 283: 2668-2673. Adeleke, O.E., Odelola, H.A. and Oluwole, F. A. 2002. Curing of antibiotic resistance in clinical strains of Staphylococcus aureus. African Journal of Medical and Pharmaceutical Sciences 6: 19- 25. Adeleke, O.E., Adepoju, T.J., and Ojo, D.A. 2006. Prevalence of Typhoid Fever and antibiotic susceptibility pattern of its causative agent of Salmonella typhi. Nigerian Journal of Microbiology 20.3:1191-1197. Ajibade, V.A., Aboloma, R.I. and Oyebode, J.A. 2010. Survival and antimicrobial Resistance of Salmonella enterica isolated from Bathrooms and Toilets following Salmonellosis in Some Homes in Ado-Ekiti. Research Journal of Agriculture and Biological Sciences 6.5 : 637-640. Albrecht,R.2007.USA:FDA.http://www.accessdata.fda.gov/drugsatfda_docs/appletter. Albrecht,R.2008.USA:FDA.http://www.accessdata.fda.gov/drugsatfda_docs/appletter. Aleksandr, M., George, C., Yan, R., Thomas, L.M., Richa, A., Alejandro, A.S. 2008. Database indexing for production MegaBLAST searches. Bioinformatics 24:1757-1764. Ambrose, P.G. and Owens, R.C. 2000. Clinical usefulness of Quinolones. Seminars in Respiratory and Critical Care Medicine (Medscape). i+23pp. Barclay, L. 2007. CDC issues new treatment recommendations for gonorrhea. Medscape i +5pp. 143 Batchelor, M., Hopkins, K., Threlfall, J.F., Clifton-Hadley, A., Stallwood, A.D., Davies, R.H., and Liebana, E. 2005. blaCTX-M Genes in Clinical Salmonella Isolates Recovered from Humans in England and Wales from 1992 to 2003. Antimicrobial Agents Chemotherapy 49 .4: 1319–1322 Bayer Corporation. 2001. HHS, Bayer agree to Cipro purchase. USA: U.S. Department of Health & Human Services. i+2pp. Bonnet, R. 2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrobial Agents and Chemotherapy 48:1-14 Bradford, P. A. 1999. Automated thermal Cycling Is Superior to Traditional Methods for Nucleotide sequencing of blaSHV Genes. Antimicrobial Agents and Chemotherapy 43.12: 2960-2963. Bradford, P. A. 2001. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clinical Microbiology Review 48:933-951. Bradley, J.S., Wassel, R.T., Lee, L., and Nambiar, S. 2009. Intravenous ceftriaxone and calcium in the neonate: assessing the risk for cardiopulmonary adverse events Pediatrics 123.4: 609–613. Brusch, J.L., Garvey, T., Schmitt, S.K. 2010. Typhoid Fever: Treatment & Medication. World Health Organization (WHO) guidelines updated. i+5 pp. Cahill, J.B., Bailey, E.M., Chien, S., and Johnson, G.M. 2005. Levofloxacin secretion in breast milk: A case report. Pharmacotherapy 25.1: 116-118. Carrie, A.G. and Kozyrskyj, A.L. 2006. Outpatient treatment of community-acquired pneumonia: evolving trends and a focus on Floroquinolones. The Canadian Journal of Clinicals 13.1: e102-111. nd Cheesbrough, M. 2006. District Laboratory Pratice in tropical countries. 2 ed. (2). Cambridge University, press. U.k. clxxxii+187 pp 144 Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. Wayne (PA): The Institute; (document M100-S17). CLSI - Clinical and Laboratory Standards Institute, Guidelines. 2011. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard M31-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa. Clinical and Laboratory Standards Institute. 2014. Performance standards for antimicrobial susceptibility testing, 24th informational supplement. M100-S24 Clinical and Laboratory Standards Institute, Wayne, PA. Cloeckaert, A., and Chaslus-Dancla, E. 2001. Mechanisms of quinolone resistance in Salmonella Veterinary Reservation 32:291-300. Connerton, P., Wain, J., Hien, T.T. 2000. Epidemic typhoid in Vietnam: molecular typing of multiple-antibiotic- resistant Salmonella enterica serotype typhi from four outbreaks. Journal of Clinical Microbiology 38: 895- 897. Cooper,J.G., Harbore, K., Frost, S.K., Skadberg, Ø. 2005. Ciprofloxacine interacts with thyroid replacement therapy. British Medical Journal 330.7498: 1002. Corbett, K.D. and Berger, J.M. 2004. Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annual Review of Biophysics and Biomolecular Structure 33:95-118. Cui, S., Li, J., Sun, Z., Hu, C., Shaohong J., Guo, Y., Ran, L., Ma, Y. 2008. Ciprofloxacin-Resistant Salmonella enterica Serotype Typhimurium, China, EID Journal Home 14: 3. David, L.W., Frank, G.R. 2000. Surveillance of antimicrobial resistance in Salmonella, Shigella and Vibrio cholerae in Latin America and the Caribbean:A collaborative project. Canadian Journal of Infectious Diseases 11.4:181-186. 145 Deak, E., Hindler, J.A., Skov, R., Sjölund-Karlsson, M., Sokovic, A., Humphries, R.M. 2015. Performance of Etest and Disk Diffusion for Detection of Ciprofloxacin and Levofloxacin Resistance in Salmonella enterica. Journal of Clinical Microbiol. 53.1: 298–301. Diaz, F., Bayona-Bafaluy, M.P., Rana, M., Mora, M., Hao, H., and Moraes, C.T. 2002. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Reservation 30.21: 4626-4633. Dolui, S.K., Das, M. and Hazra, A. 2007. Ofloxacin-induced reversible arthropathy in a child. Journal of Postgraduate Medicine 53.2: 144-145. DrugBank. 2009. Showing drug card for Levofloxacin.Canada. http://www.drugbank.ca/drugs. Eaves, D.J., Randall, L., and Gray, D.T. 2004. Prevalence of mutations within the quinolone resistance-determining region of gyrA,gyrB,parC,and parE and association with antibiotic resistance in quinolone-resistant.Salmonella enterica. Antimicrobial Agents and Chemotherapy 48:4012-4015. Falagas, M.E., Grammatikos, A.P., Michalopoulos, A. 2008. Potential of old- generation antibiotics to address current need for new antibiotics. Expert Review of Antimicrobial and Infectious Therapy 6.5: 593-600. Fey, P.D., Safranek, T.J., Rupp, M.E., Dunne, E.F., Ribot, E., Iwen, P.C., Bradford, P.A., Angulo, F.J., and Hinrichs, S.H. 2000. Ceftriaxone-resistant Salmonella infection acquired by a child from cattle. New England Journal of Medicine 27:342.17:1242-1249. Fraunfelder, F.W., and Fraunfelder, F.T. 2009. Diplopia and fluoroquinolones. Ophthalmology 116.9: 1814-1817. Friedrich, L.V., and Dougherty, R. 2004. Fatal hypoglycemia associated with levofloxacin. Pharmacotherapy 24.12: 1807-1812. 146 Gaind, R., Paglietti, B., Murgia, M., Dawar, R., Uzzau, S., Cappuccinelli, P., Deb, M., Aggarwal, P., Rubino, S. 2006. Molecular characterization of ciprofloxacin-resistant Salmonella enterica serovar Typhi and Paratyphi A causing enteric fever in India. Journal of Antimicrobial Chemotherapy 58.6:1139-1144. Galanis, E., Lo Fo Wong, D.M., Patrick, M.E., Binsztein, N., Cieslik, A., Chalermchikit, T., Aidara-Kane, A., Ellis, A., Angulo, F.J., Wegener, H.C. 2006. World Health Organization Global Salm-Surv; Web-based surveillance and global Salmonella distribution, 2000-2002. Emerging Infectious Disease 12.3:381-388. Gautam, V., Gupta, N.K., Chaudhary, U., Arora, D.R. 2002. Sensitivity pattern of Salmonella serotypes in Northern India. Brazilian Journal of Infectious Disease 6:281-287. George, A., Jacoby, M.D., and Luisa Silvia Munoz-Price, M.D. 2005. Mechanisms of disease: The New beta-Lactamases. New England Journal of Medicine 352:380-391. Grépinet, C., Guillocheau, E., Berteloot, A., Vachée, A., Herbin, O., Gautier, S. 2008. l'association des centres régionaux de pharmacovigilance. Drug-induced fever during treatment with levofloxacin: a case-report. Therapietics 63.4: 341-343. Griggs, K. 2007. Frog killer fungus 'breakthrough. BBC News. http://news.bbc.co.uk/2/hi/science/nature/. Guerrant, R.L., Van Gilder, T., Steiner, T.S., Thielman, N.M., Slutsker, L., Tauxe, R.V. 2001. Practice guidelines for the management of infectious diarrhea. Clinical and Infectious Disease 32:331-351. Gupta, A. 2009. Clinical Ophthalmology: Contemporary Perspectives. Elsevier. India.+ 112pp. 147 Gupta, A., Fontana, J., Crowe, C., Bolstorff, B., Stout, A.,VanDuyne, S., Hoekstra, M.P., Whichard,J.M., Barrett,T.J., and An-gulo,F.J. 2003. Emergence of multidrug-resistant Salmonella enterica serotype Newport infections resistant to expanded-spectrum cepha-losporins in the United States. Journal of infectious Disease 188:1707-1716. Hakanen, A., Kotilainen, P., Huovinen, P., Helenius, H., and Siitonen, A.2001. Reduced Fluoroquinolone Susceptibility in Salmonella enterica Serotypes in Travelers Returning from Southeast Asia. Journal of Antimicrobial Chemotherapy 7: 6. Harbottle, H., S., Thakur, S. Z. and White, D.G. 2006. Genetics of antimicrobial resistance. Animal Biotechnology 17:111-124. Haugum, K., Aas, L., and Lindstedt, B.A. 2006. Effect of quinolone antibiotics and chemicals on mutation types in Salmonella enterica serovars Enter itidis, Hadar and Virchow.Division for Infectious Diseases Control, Norwegian Institute of Public Health, N-0403 Oslo, Norway. i+5pp. Hawkey, P.M. 2003. Mechanisms of quinolone action and microbial response. Journal of Antimicrobial Chemotherapy 51:29-35. Helms, M., P. Vastrup, P., Gerner-Smidt, and Molbak. K. 2002. Ex-cess mortality associated with antimicrobial drug-resistant Salmonella Typhimurium. Emerging infectious Disease 8: 490-495. Hirano, T., Yasuda, S., Osaka, Y. 2008. The inhibitory effects of fluoroquinolones on L-carnitine transport in placental cell line BeWo. International Journal of Pharmaceutics 351.1-2: 113-118. Hirose, K., Tamura, K., Sagara, H., and Watanabe, H. 2001. Antibiotic susceptibilities of Salmonella enterica serovar Typhi and S. enterica serovar paratyphi A isolated from patients in Japan. Antimicrobial Agents and Chemotherapy 45:956-970. 148 Hirose, K., Hashimoto, A., Tamura, K., Kawamura,Y., Ezaki, T., Sagara, H. and Watanabe, H. 2002. DNA Sequence Analysis of DNA Gyrase and DNA Topoisomerase IV Quinolone Resistance-Determining Regions of Salmonella enterica Serovar Typhi and Serovar Paratyphi A. Antimicrobial Agents and Chemotherapy 46.10: 3249-3252. Hooper, D.C. 2001. Emerging mechanisms of fluoroquinolone resistance. Emerging Infectious Disease 7.2: 337- 341. Hopkins, K.L., Davies, R.H., Threlfall, E.J. 2005. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: Recent developments. International Journal of Antimicrobial Agents 25:358-373. Huang, Q., and Fu, W.L. 2005. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clinical Chemistry and Laboratory Medicine 43.8: 841-842. Hunt, M. 2007. Levofloxacin: dysglycemia and liver disorders. Canadian adverse reaction newsletter (Canada: Health Canada Newsletter). 17:1. Ivanoff, B. 1995. Typhoid fever: global situation and WHO recommendations. South Asian Journal of Tropical Medicine and Public Health 26.2:1-6. Ivanov, D.V., and Budanov, S.V. 2006. Ciprofloxacin and antibacterial therapy of respiratory tract infections (in Russian). Antibiotics Khimioterapy 51.5: 29-37. Jacobs, M. 2005. Worldwide Overview of Antimicrobial Resistance. International Symposium on Antimicrobial Agents and Resistance. i+ 20 pp. Jam, S., and Chen, J. 2006. Antibiotic resistance profiles and cell surface components of Salmonellae. Journal of Food Protection 69:1017-1023. Janssen Pharmaceuticals. 2008. Highlights of prescribing information. USA:FDA.http://www.accessdata.fda.gov/drugsatfda_docs/label. 149 Johnson and Johnson. 2009. Analysis of Sales by Business Segments. (PDF). Shareholder. +27 pp. Johnson and Johnson, 2004. United States Securities and Exchange Commission. USA Shareholder.com. xxviii +29pp. Kaplowitz, N. 2005. Hepatology highlights. Hepatology 41: 227. Karageorgopoulos, D.E., Giannopoulou, K.P., Grammatikos, A.P., Dimopoulos, G., Falagas, M.E. 2008. Floroquinolones compared with beta-lactam antibiotics for the treatment of acute bacterial sinusitis: a meta-analysis of randomized controlled trials. Canadian Medical Association Journals 178.7: 845-54. Kim, J.Y., Jung, H.I., and An, Y.J. 2006. Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C beta-lactamase. Molecular Microbiology 60.4: 907-916. Kim, H.B., Park, C.H., Kim, C.J., Kim, E.C., Jacoby, G.A. and Hooper, D.C. 2009. Prevalence of plasmid-mediated quinolone resistance determinants over a 9-year period. Antimicrobial Agents and Chemotherapy 53:639-645. Kollef, M.H. 2009. New antimicrobial agents for methicillin-resistant Staphylococcus aureus. Critical Care Resuscitation 11.4: 282-286. Lamb, E. 2008. Top 200 Prescription Drugs of 2007. USA: Pharmacy Times. http://www.pharmacytimes.com/issues/articles. Lardizabal, D.V. 2009. Intracranial hypertension and levofloxacin: a case report. Headache 49.2: 300-301. Larkin, C, Poppe, C., McNab, B., McEwen, B., Mandi, A., and Odumeru, J. 2004. Antibiotic resistance of Salmonella isolated from hog, beef, and chicken carcass samples from provincially inspected abattoirs in Ontario. Journal of Food Protection 67:448-455. 150 Le Saux, N. 2008. The treatment of acute bacterial sinusitis: no change is good medicine.Canadian Medical Association Journals 178.7: 865-866. Lee, J.H., Jung, H.I., and Jung, J.H. 2004. Dissemination of transferable AmpC-type β-lactamase (CMY-10) in a Korean hospital. Microbiology Drug Resistant 10: 224-230. Levy, S.B., and Marshall, B. 2004. Antibacterial resistance worldwide: Causes, challenges and responses. National Medicine 10:S122-S129. Linder, J.A., Huang, E.S., Steinman, M.A., Gonzales, R., and Stafford, R.S. 2005. Fluoroquinolone prescribing in the United States: 1995 to 2002. The American Journal of Medicine 118.3: 259-268. Lindstedt, B.A., Aas, L., and Kapperud, G. 2004. Geographically dependent distribution of gyrA gene mutations at codons 83 and 87 in Salmonella Hadar, and a novel codon 81 Gly to His mutation in Salmonella Enteritidis. Acta Pathologica, Microbiologica et Immunologica Scandinavica. 112:165-171. Liu, H., and Mulholland, S.G. 2005. Appropriate antibiotic treatment of genitourinary infections in hospitalized patients. American Journal of Medicine 118.7A: 14S-20S. Logue, C.M., Sherwood, J.S., Olah, P.A., Elijah, L.M., and Dockter.M.R. 2003. The incidence of antimicrobial-resistant Salmonella,pp. on freshly processed poultry from US Midwestern processing plants. Journal of Applied Microbioliology 94:16-24. MacDougall, C., Guglielmo, B.J., Maselli, J. and Gonzales, R. 2005. Antimicrobial drug prescribing for pneumonia in ambulatory care. Emerging Infectious Disease 11.3 : 380- 384. Mandal, S., Mandal, M.D., and Pal, N.K. 2010. Reduced minimum inhibitory concentration of chloramphenicol for Salmonella enterica serovar typhi. Indian Journal of Medical Sciences 58:16-23. 151 Martin, L.J., Fyfe, M., Dore, K., Buxton, J.A., Pollari, F., and Henry, B. 2004. Increased burden of illness associated with antimicrobial-resistant Salmonella enterica serotype Typhimurium infections. Journal of Infectious Disease 189:377-384. Martins, O.O., and Igwemma, A.A. 2000. Applied Statistical Techniques for nd Business and Basic Sciences. 2 . Skillmark media Ltd., Owerri, Nigeria. ccxvi + 216-304. McIntyre, J., and Choonara, I. 2004. Drug toxicity in the neonate. Biology of Neonate. 86.4 : 218-221. Mirza, S., Kariuki, S., Mamun, K.Z., Beeching, N.J., and Hart, C.A. 2000. Analysis of plasmid and chromosomal DNA of multidrug resistant Salmonella enterica serovar typhi from Asia. Journal of Clinical Microbiology 38:1449- 1452. Moloney, G.P., Kelly, D.P. and Mack, P. 2001. Synthesis of Acridine -based DNA Bis-intercalating Agents. Molecules 6: 230-243. Momtaz, O. W., Robert, F., Tharwat, F.I. ,Hoda, M.,Joseph, L.M., Frank, J.M. 2002. Trends of Multiple-Drug Resistance among Salmonella Serotype Typhi Isolates during a 14-Year Period in Egypt. Infectious Diseases Society of America. 35.10:1265-1268. Moubareck, C., Daoud, Z., Hakime, N.I., Hamze, M., Mangeney, N., Matta, H., Mokbat, J.E., Rohban, R., Sarkis, D.K., Populaire, F.D. 2005. Journal of clinical Microbiology. 43.7 :3309-3313. Munir, T., Lodhi, M., Butt, T., Karamat, K.A. 2001. Incidence and Multidrug Resistance in Typhoid Salmonellae in Bahawalpur Area. Pakistan Armed Forces Medical Journal 51.1: 10-13. Murray, R. K., Granner, D. K., Mayes, P.A., Rodwell, V. W. 2006. Protein Synthesis and the Genetic Code. Harper's Illustrated Biochemistry (27 ed.). McGraw-Hill Medical. i+378pp. 152 Mushtaq, M.A. 2006. What after ciprofloxacin and ceftriaxone in treatment of Salmonella typhi. Pakistan Journal Medical Sciences 22 : 151-154. Muto, C.A., Jernigan, J.A., Ostrowsky, B.E., Richet, H..M., Jarvis, W.R., Boyce, J.M., Farr, B.M. 2003. SHEA guideline for preventing nosocomial transmission of multidrug-resistant strains of Staphylococcus aureus and enterococcus. Infectious Control and Hospital Epidemiology 24.5: 362- 386. Nadeem M, Ali N, Achkzai H, Ahmed I. 2002. A profile of Enteric fever in adults at Quetta. Pakistan. Journal of Pathology 13.1: 12-7. Nardiello, S., Pizzella, T., and Ariviello, R. 2002. Risks of antibacterial agents in pregnancy (in Italian). Le Infezioni in Medicina. 10.1: 8-15. Nathan, N., Borel, T., and Djibo, A. 2005. Ceftriaxone as effective as long-acting chlroramphenicol in short-course treatment of meningococcal meningitis during epidemics: a randomised non-inferiority study. Lancet. 366.9482: 308-313. National Toxicology Program. 2005. Executive summary Ethidium Bromide: Evidence for possible Carinogenic Activity” http://ntp.niehs.nih.gov. Nelson, J.M., Chiller, T.M., Powers, J.H., and Angulo, F.J. 2007. Floroquinolone- resistant Campylobacter species and the withdrawal of fluoroquinolones from use in poultry: a public health success story. Clinical Infectious Disease 44.7: 977-980. Noel, G.J., Bradley, J.S., and Kauffman, R.E. 2007. Comparative safety profile of levofloxacin in 2523 children with a focus on four specific musculoskeletal disorders. Pediatrics Infectious Disease Journal 26.10: 879-891. Olesen, I., Hasman, H. and Aarestrup, F. M. 2004. Prevalence of b-lactamases among ampicillin resistant Escherichia coli and Salmonella isolated from food animals in Denmark. Microbial Drug Resistance. 10: 334-340. 153 Oliphant, C.M., and Green, G.M. 2002. Quinolone : a comprehensive review. American. Family Physician 65.3: 455-464. Onyenwe, N.E., Mbata, T. I. and Adeleke, O. E. 2011. Comparative analysis on the curing effect of acridine orange (mutagen) on beta - lactamase producing Staphylococus aureus, from bovine and human origin. African Journal of Microbiology Research 5.25: 4278-4290. Onyenwe, N.E., Adeleke, O.E., Mbata, T.I., Udeji,G.N. 2012. The level of beta- lactamase linked antibiotic resistance in bovine and human isolates of Staphylococcus aureus. International Research Journal of Microbiology 3.11: 345-351. Owens, R.C., and Ambrose, P.G. 2005. Antimicrobial safety: focus on fluoroquinolones.. Clinical infectious diseases : An official publication of the Infectious Diseases Society of America. 41.2: S144-S157. Parry, C.M., Hien, T.T., Dougan G., White, N.J., and Farrar, J.J. 2002. Typhoid Fever. New England Journal of Medicine 347: 1770-1782. Parry, C.M., and Beeching, N.J., 2009. Treatment of Enteric fever. New England Journal of Medicine 228: b1159. Parveen S., Taabodi M., Schwarz,J.G., Oscar,T.P., harter-dennis,J.,' and whites,D.G. 2007. Prevalence and Antimicrobial Resistance of Salmonella Recovered from Processed Poultry. Journal of Food Protection 70.11: 2466-2472. Paterson, D. L., Hujer, K. M., Hujer, A. M., Yeiser B., Bonomo, M. D., Rice, L. B. and Bonomo, R. A. 2003. Extended-spectrum beta-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type beta-lactamases. Antimicrobial Agents and Chemotherapy 47: 3554–3560. Pegler, S., and Healy, B. 2007. In patients allergic to penicillin, consider second and third generation cephalosporins for life threatening infections. British Medical Journal 335.7627: 991-991. 154 Philippon, A., Arlet, B., and Jacoby, G.A. 2002. Plasmid-determined AmpC-type β- lactamases. Antimicrobial Agents and Chemotherapy. 46: 1-11. Pichichero, M.E. 2006. Cephalosporins can be prescribed safely for penicillin-allergic patients . The Journal of family practice 55.2: 106-112. Piñeiro-Carrero and Piñeiro, A. 2004. Liver. Pediatrics 113.4: 1097-1106. Randall, L.P., Coldham, N.G., Woodward, M.J. 2005. Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation. Journal of Antimicrobial Chemotherapy. 56(4): 619-623. Richard, L. S., and Ronald, S. G. 2009. Infectious Diseases of the Female Genital Tract. Lippincott Williams & Wilkins. pp. 403. Robicsek, A., Jacoby, G.A., and Hooper, D.C. 2006. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infectious Disease 6.10: 629-640. Roy, P., A., Dhillon, S., Lauerman, L.H., Schaberg, D.M., Bandli, D. and Johnson, S. 2002. Results of Salmonella isolation from poultry products, poultry environment, and other characteristics. Avian Disease 46:17-24. Sanofi- aventis, 2008. “NegGram” USP USA: FDA. http:// www. access data. fda. gov/ drug.satfda_docs/label. Schaumann, R., Rodloff, A. C. 2007. Activities of Quinolones Against Obligately Anaerobic Bacteria (PDF). Anti-Infective Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Infective Agents) (Bentham Science Publishers) 6.1: 49-56. Schneider, K. 2008. Stockholders’ Newsletter Bayer AG. http://www.stockholders- newsletter-. 155 Shin, H.C., Kim, J.C., Chung, M.K. 2003. Fetal and maternal tissue distribution of the new fluoroquinolone DW-116 in pregnant rats. Comparative Biochemistry and Physiology part C, Toxicology and Pharmacology 136 (1): 95-102. Sissi, C., and Palumbo, M. 2003. The quinolone family: from antibacterial to anticancer agents. Current Medicinal Chemistry. Anti-cancer Agents 3.6: 439-450. Soge, O.O., Queenan, A.M., Ojo, K.K., Adeniyi, B.A., and Roberts, M.C. 2005. CTX- M-15 extended-spectrum β-lactamase from Nigerian Klebsiella pneumonia. Journal of Antimicrobial Chemotherapy 57: 24–30. South Australia Department of Health . 2008. Health in all Policies. Public Health Bulletine 5.1: 1-55. Stacy, J., and Childs, M.D. 2000. Safety of the Fluoroquinolone Antibiotics: Focus on Molecular Structure. (USA: FQresearch). Infect Urology 13.1: 3-10. Stork, C.M. 2006. Antibiotics, antifungals, and antivirals. In Nelson LH, Flomenbaum N, Goldfrank LR, Hoffman RL, Howland MD, Lewin NA. Goldfrank's Toxicologic Emergencies. New York: McGraw-Hill. + 847pp. Surolia, N. and Surolia, A. 2001. Triclosan offers protection against blood stages of malaria by inhibiting Groyl-ACP reductace of Plasmodium falciparium. National Medicine 7:167-173. Tacconelli, E., De Angelis, G., Cataldo, M.A., Pozzi, E., and Cauda, R. 2008. Does antibiotic exposure increase the risk of methicillin-resistant Staphylococcus aureus (MRSA) isolation. A systematic review and meta-analysis. Journal of Antimicrobial Chemotherapy 61.1: 26-38. Thong, K.L., Bhutta, Z.A., and Pang, T. 2000. Multidrug-resistant strains of Salmonella enterica serotype typhi are genetically homogenous and coexist with antibiotic-sensitive strains as distinct, independent clones. International Journal of Infectious Disease 4:194-7. 156 Threlfall, E.J., and Ward, L.R. 2001. Decreased Susceptibility to Ciprofloxacin in Salmonella Enterica serotype Typhi, United Kingdom. Emerging Infectious Disease 7: 448-450. Threlfall, E.J. 2002. Antimicrobial drug resistance in Salmonella : problems and perspectives in food- and water-borne infections. FEMS Microbiology Review 26:141-148. Valverde, A., Grill, F., Veresa, M.C., Pintado, V., Fernando, B., Rafael, C., and Cobo, J. 2008. High rate of intestinal colonization with extended-spectrum- lactamase-producing organisms in household contacts of infected community patients. Journal of Clinical Microbiology 46.8: 2796-2799. Varma, ,J.K., Molbak, K., Barrett, T.J., Beebe, J.L., Jones, T.F., and Rabatsky-Ehr T. 2005. Antimicrobial-resistant nontyphoidal Salmonella is associated with excess bloodstream infections and hospitalizations. Journal of Infectious Disease 191:554-61. Von Wurmb-Schwark, N., Cavelier, L., Cortopassi, G.A. 2006. A low dose of ethidium bromide leads to an increase of total mitochondrial DNA while higher concentrations induce the mtDNA 4997 deletion in a human neuronal cell line. Mutation and Resistance 596.1-2: 57-63. Vonberg, R. 2009. Clostridium difficile: a challenge for hospitals. European Center for Disease Prevention and Control. Institute for Medical Microbiology and Hospital Epidemiology i+20pp. Wang, J.C. 2002. Cellu lar roles of DNA topoisomerases: A molecular perspective. Nature Reviews and Molecular Cell Biology 3:430-440. White, D.G., Datta, A., McDermott, P., Friedman, S., Qaiyumi, S., Ayers, S., English, L., McDermott, S., Wagner, D.D., and Zhao, S. 2003. Antimicrobial susceptibility and genetic relatedness of Salmonella serovars isolated from animal-derived dog treats in the USA. Journal of Antimicrobial Chemotherapy 52.5:860-863. 157 White, D.G., Datta, A., McDermott, P., Friedman,S., Qaiyumi, S., Ayers, S.,English, L., McDermott, S., Wagner, D.D., Zhao, S. 2007. Antimicrobial susceptibility and genetic relatedness of Salmonella serovars isolated from animal-derived dog treats in the USA. England Journal of Medicine and Chemotherapy 345.16:1147-1154. Woodford, N., Ward, E., and Kaufmann, M.E. 2006. Molecular characterization of Escherichia coli isolates producing CTX-M-15 extended spectrum B- lactamase (ESBL) in the United Kingdom. Health Protection Agency xi +19 pp. Yah, S.C., Chineye, H.C., Eghafona, N.O. 2007. Multi antibiotics-resistance plasmid profle of enteric pathogens in pediatric patients from Nigeria. Biokemistri 19.1: 35-42. Yah, S.C. 2010. Plasmid-Encoded Multidrug Resistance: A Case study of Salmonella and Shigella from enteric diarrhea sources among humans. Biological Resistance 43: 141-148. Yujuan, J., Ling, J.M. 2006. CTX-M-producing Salmonella spp. in Hong Kong: an emerging problem. Journal of Medical Microbiology 55: 1245-1250. Zhao, S., McDermott, P.F., Friedman, S., Qaiyumi, S., Abbott, Kiessling, J.C., Ayers, S., Singh, R., Hubert, S., Sofos, J., and White, D.G. 2006. Characterization of antimicrobial-resistant Salmonella isolated from imported foods. Journal of Food Protection 69 : 500 - 507. Zheng, Z., Scott, S., Lukas, W., and Webb, M. 2000. A greedy algorithm for aligning DNA sequences. Journal of Computer Biol. 7.1-2: 203-214. 158