Microbiology
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Item ENVIRONMENTAL HYGIENE AND MICROBIOLOGICAL ASSESSMENT OF FOOD SERVICE ESTABLISHMENTS IN SELECTED BOARDING HIGH SCHOOLS IN IBADAN, NIGERIA(2023-07) ADEBAYO, K.ASchools’ Food Service Establishments (FSEs) have been incriminated in numerous foodborne diseases outbreaks globally and have been linked to the environment and food handling procedures in the establishments. Despite this, FSEs in Nigerian boarding schools have been poorly investigated. In order to provide baseline data for infection control, this study was designed to assess environmental hygiene and food handlers’ Knowledge, Attitude and Practices (KAP) and investigate food-related microbial contamination from selected boarding schools’ FSEs in Ibadan, Nigeria.Observational checklist and interviewer-administered questionnaire were used to evaluate environmental hygiene parameters, food handlers’ KAP in four schools’ FSEs out of forty-three schools by inclusion criteria and balloting. Swabs from Food Contact Surfaces (FCS): utensils and surfaces; 20 food handlers’ hands and samples of Ready to-Eat (RTE) foods were examined for Aerobic Plate Count (APC), Total Coliform (TC), Faecal Coliform (FC) and selected important foodborne pathogens counts using standard methods. Isolated bacteria were characterised phenotypically and subjected to 16S rRNA sequencing. Antibiotic susceptibility testing was determined using disc diffusion and E-strip techniques based on CLSI and EUCAST standards, respectively. Data were analysed using descriptive statistics and ANOVA at α0.05. Schools FSEs’ compliance mean scores for environmental hygiene parameters were 82.2, 56.8, 52.7 and 65.6% for toilets, dining areas, kitchens and observed food handlers at work, respectively. The food handlers had good knowledge (61.9%), positive attitude (81.4%) to ensure food safety, but poor hygiene practices (52.6%) which differed significantly among schools (p=0.012, χ2=10.15). Major unsanitary practices observed were: use of basins and buckets for dish washing, uncovered solid waste receptacles, non-availability of sanitising agents and inadequate handwashing. Mean logCFU/cm2 of APC for counter tops, chopping boards, grinders, trays and knives were 5.59±1.56, 4.38±2.62, 4.01±0.77, 2.47±2.23 and 2.38±1.75, respectively. Food handlers’ hands’ mean logCFU/cm2 of APC, TC, FC, Staphylococcus and Bacillus species were 3.10±1.78, 2.62±1.23, 2.80±1.74, 1.94±1.04 and 1.97±1.39, respectively. Seventy-eight percent of RTE foods conformed to acceptable limit of < 4logCFU/g for APC. The distribution of bacteria from schools FSEs were 62.0% (FCS), 19.0% (food handlers’ hands) and 19.0% (RTE foods). The identified food-related bacteria were Alcaligenes faecalis, Achromobacter xylosoxidans, Bacillus cereus, Ochrobactrum anthropi,viiiProteus mirabilis, Serratia marcescens, Staphylococcus saprophyticus and Bordetella species. Alcaligenes faecalis resistance (%) to cefixime, cefuroxime, ceftazidime, gentamicin, augmentin, nitrofurantoin, ofloxacin and ciprofloxacin were 76.2, 71.4, 66.7, 61.9, 57.1, 42.9, 4.8 and 4.8, while for Bacillus cereus, they were 85.7,100.0, 57.1, 85.7, 28.6, 57.1, 0.0 and 0.0, respectively. The minimum inhibitory concentration of colistin for Alcaligenes faecalis ranged from 1.5 µg/mL to >256 µg/mL which was highly significant (F=9.194, p<0.05) compared to other antibiotics. Two Bacillus cereuswere resistant to imipenem, 81.0% were multi-antibiotic resistant, while none of the identified bacteria showed resistance to piperacillin/tazobactam.Food contact surfaces and food handlers’ hands were grossly contaminated. The presence of colistin-resistant Alcaligenes faecalis and resistance of Bacillus cereus to imipenem in boarding schools’ food service establishments is a serious public health concern. These findings will be useful in policy formulation and the development of food safety guidelines in boarding schools.Item INDIGENOUS BACTERIAL DIVERSITIES IN TWO AGRICULTURAL SOILS AS INFLUENCED BY CHEMICAL AND ORGANIC HERBICIDES(2024-06) NWOKOCHA, A.GAgricultural soils as habitats for beneficial soil bacteria are usually invaded by weeds, an occurrence which has detrimental effects on crops. Chemical herbicides mostly employed for weed control adversely affect soil bacteria. Previous studies have reported chemical herbicides’ effects on soil bacterial diversity suppressing microbial growth, however, information on soil types, and organic herbicides that could be an alternative is limited. Therefore, this study was designed to investigate the effects of chemical and organic herbicides on bacterial diversity in Alfisol and Inceptisol. Soils (Alfisol and Inceptisol) were collected from research farms at University of Ibadan, Institute of Agricultural Research and Training, National Horticultural Research Institute, and International Institute of Tropical Agriculture. In a screen house experiment using a factorial arrangement in a completely randomised design, Atrazine and S-Metolachlor (AS-M), Isopropyl Amine (IA), and Acetic Acid (AA) herbicides were applied as pre-, post- and organic herbicides, respectively on the soils. In the field experiments, AS-M, IA and AA herbicides were also applied to the Alfisol and Inceptisol. All herbicide-treated soils were sampled at 4 and 8 weeks and bulked to determine physico-chemical parameters and herbicides persistency of the treated and untreated soils using standard methods. The effect on bacterial diversity in herbicide treated Alfisol and Inceptisol were determined by DNA extraction from the soil samples using 16S rRNA amplicons sequenced on illumina miseq. Data were analysed using descriptive statistics, QIIME 2, and ANOVA at α0.05. Untreated Alfisol pH (6.20±0.12), organic carbon (3.90±0.04g/kg) and clay content (112.20±0.55g/kg) were significantly higher than Inceptisol pH (5.50±0.15), organic carbon (1.90±0.01g/kg) and clay content (103.90±0.81g/kg). At week 4, pH and organic carbon were significantly higher in Alfisol (6.30±0.21: 0.40±0.06g/kg) than Inceptisol (5.40±0.21; 0.10±0.06g/kg). Herbicides’ persistency was higher in Alfisol than Inceptisol. Intermediate products (acetamide) in AA-treated soil (Alfisol: 30.2%; Inceptisol: 25.0%) < 2-amino-3-4- dihydro-4-4-dimethyl-6-pyrimidinone (Alfisol: 48.7%; Inceptisol: 35.3%) in IA < atrazine (Alfisol: 50.9%; Inceptisol: 37.1%) in AS-M at week 4, with a similar trend Alfisol: 22.1%, Inceptisol: 19.2%; Alfisol: 42.6%, Inceptisol: 33.2%; Alfisol: 43.9%, Inceptisol: 35.1% at week 8. Alfisol had significantly higher bacteria taxa of 79% than 21% in Inceptisol. Methylomicrobium, Saccharopolyspora, Domibacillus, Blatococcus, Fuctibacillus, Limnobacter, Sneathiella, Nocardiopsis, Aquisphaera, and Stenotrophomonas were found in Alfisol and Inceptisol. However, Limnobacter and Methylomicrobium; Sneathiella and Nocardiopsis; Aquisphaera and Saccharopolyspora; were the bacterial genera that survived in AS-M, IA and AAtreated-soils, respectively. Significant effect in relative abundance of bacterial genera Chthoniobacter (Alfisol: 0.5%, 0.4%, 0.3%, 0.6%: Inceptisol: 0.3%, 0.2%, 0.3%, 0.5%) and Pedosphaera (Alfisol: 0.6%, 0.6%, 0.5%, 0.9%, Inceptisol: 0.7%, 0.5%, 0.5%, 0.9%) were observed at week 4, Chthoniobacter (Alfisol: 0.5%, 0.4%, 0.4%, 0.6%: Inceptisol: 0.3%, 0.3%, 0.4%, 0.5%) and Pedosphaera (Alfisol: 0.6%, 0.8%, 0.5%, 0.9%, Inceptisol: 0.3%, 1.2%, 0.9%, 1.3%) at week 8 in untreated and soil treated with AS-M, IA and AA, respectively. Alpha and beta diversity indicated significant differences in the relative abundance of bacteria between Alfisol and Inceptisol. The relative abundance of bacteria diversity in Alfisol and Inceptisol were reduced by chemicals but increased by organic herbicidesItem Candidaemia or candidasis: controversy of staphylococcus sexually transmitted infection?(2016-01) Ogunshe, A. O.Herbal medications are becoming increasingly popular but a most-extraordinary claim by traditional/herbal medical practitioners relates to a Gram-positive bacterium, Staphylococcus, which has been depicted as a deadly sexually transmitted disease that manifest in the form of worms and other symptoms; with contributory roles including infertility, sexual dysfunction and impotency. They further boasted that they are the only ones that possessed the remedy (herbal) for the Staphylococcus sexually transmitted scourge. In the absence of distinguishing phenotypic taxonomic tools, Staphylococcus and Candida spp. may be confused for each other. However, Staphylococcus is a bacterium and not an infection; therefore, there must be more to the traditional medical practitioners’ boasts in ability to cure an infection that was not an infection in the first place. In conclusion, the common sense is that candiaemia or candidiasis is most likely the misdiagnosed sexually transmitted Staphylococcus disease, which is of significant human clinical health issue.Item Phytochemical screening and microbial inhibitory activities of ficus capensis(2012-01) Adebayo-Tayo, B. C.; Odeniyi, A. O.Ficus plant components have application in traditional medicine because of the myriad uses they have been subjected to. The ease of application is based on the secondary metabolites this plant contains. The challenges faced by modern medicine especially in the complete cure of microbially-associated diseases through abrupt and unpredictable genetic mutations in the presence of conventional drugs informed the investigation of the microbial inhibitory activities of the stem, root and leaf parts of F. capensis against test disease causing microorganisms. The phenolic, alkaloid and tannin phytochemical fractions were highest in F. capensis bark extract (180, 165 and 155 μg/ml respectively) followed by that contained in the stem extract (100, 90 and 85 μg/ml respectively). While Streptococcus faecalis and Pseudomonas mirabilis were resistant to many different antibiotics (87.5%), they were effectively inhibited by all concentrations of ethanolic F. capensis extracts. The minimum inhibitory concentration of ethanolic extracts ranged from 25% leaf and stem extract concentration respectively (4mm) against S. faecalis and (2mm) against P.mirabilis. All test isolates were 100% susceptible to ethanol extract growth inhibition..Item Candidaemia or candidasis: controversy of staphylococcus sexually transmitted infection?(2016-01) Ogunshe, A. O.Herbal medications are becoming increasingly popular but a most-extraordinary claim by traditional/herbal medical practitioners relates to a Gram-positive bacterium, Staphylococcus, which has been depicted as a deadly sexually transmitted disease that manifest in the form of worms and other symptoms; with contributory roles including infertility, sexual dysfunction and impotency. They further boasted that they are the only ones that possessed the remedy (herbal) for the Staphylococcus sexually transmitted scourge. In the absence of distinguishing phenotypic taxonomic tools, Staphylococcus and Candida spp. may be confused for each other. However, Staphylococcus is a bacterium and not an infection; therefore, there must be more to the traditional medical practitioners’ boasts in ability to cure an infection that was not an infection in the first place. In conclusion, the common sense is that candiaemia or candidiasis is most likely the misdiagnosed sexually transmitted Staphylococcus disease, which is of significant human clinical health issue.Item DEGRADATION OF POLYAMIDE-6 BY MICROBIAL ISOLATES FROM SOLID WASTE DUMPSITES IN LAGOS STATE, NIGERIA(2012-10) SANUTH, H. A.Polyamide-6 (also known as nylon-6) is one of the biodegradation-resistant synthetic polymers used in the manufacturing of commodity plastic materials. The environmental effects of the persistence of this material in landfill and on surface water bodies pose global problems that endanger public health. Knowledge of the microbial distribution and pattern of their interaction with these plastics will provide the biological resources and scientific basis for the development of sustainable disposal and treatment method. Therefore, microbial degradation of polyamide-6 and its monomers were investigated. Soil samples were randomly collected at five spots to a depth of 15cm and mixed to form composites from each of the three selected dumpsites at Olusosun, Abule-Egba and Isheri-Igando in Lagos state. Microbiological analysis was carried out on the samples on bimonthly intervals over a period of eight months. The fungal and bacterial isolates were screened for their ability to utilize ε-caprolactam (the monomer of the nylon-6) as a sole source of carbon and nitrogen for growth on a synthetic solid medium and were phenotypically characterized. The 16SrRNA gene sequences were used for the molecular typing of the isolates. The isolates with the best growth within 120hrs in ε-caprolactam medium were tested for nylon-6 degradation. Intermediate products in the culture medium were monitored using High Performance Liquid Chromatography (HPLC) while biodegradation of the nylon-6 was monitored using Fourier Transform Infrared Spectroscopy (FTIR), average number molecular mass (Mn) and weight loss. An un-inoculated experiment served as the control. Data obtained were analysed using ANOVA. A total of 64 bacteria and 22 fungi were isolated from the soil samples. Sixteen species of the bacterial isolates made up of the genera Pseudomonas (5), Alcaligenes (3), Corynebacterium (2), Leucobacter (2), Bordetella (1), Proteus (1), Providencia (1) and Lysinibacillus (1) were capable of utilising ε-caprolactam as the sole source of carbon and nitrogen up to a minimum inhibitory concentration of 20 gL-1. The fungi were identified as five species of Aspergillus and a species of Penicillium. Proteus vulgaris utilized 97.2%, Bordetella petrii 92.5%, Pseudomonas aeruginosa (NTS1) 90.5% and Alcaligenes faecalis (2ABA2) 82.3% of 10 gL-1 ε-caprolactam within 120hrs compared to the control experiment. The HPLC analysis of the culture supernatant revealedthe presence of 6-aminohexanoic acid, ε-caprolactam and some un-identified oligomers as the degradation products of the nylon-6 fibre. The changes observed in the FTIR spectra of different functional groups confirmed the effect of microbial degradation of the nylon-6 fibre. Aspergillus niger was the most efficient of the fungi isolates in degrading the polyamide-6. It achieved 29.77 % reduction in polyamide-6 Mn and 23.95 % weight loss. Moreover, P. aeruginosa (NTS1) degraded Polyamide-6 with 12.82 % reduction in Mn and 5.23% weight loss. These changes were found to be significant at p = 0.05. Aspergillus niger and Pseudomonas aeruginosa degraded nylon-6 fibre and this serves as baseline information for the bio-treatment of the nylon polymer.Item CHARACTERIZATION OF β-GALACTOSIDASE BY LACTIC ACID BACTERIA FROM MILK AND TRADITIONALLY FERMENTED MILK PRODUCTS FROM IBADAN(2014-01) PARKHA, O. A.Lactose intolerance (a condition in which man elucidates an immune reaction towards the presence of lactose due to inability to produce enzyme lactase) is a major nutritional deficiency among some adult consumers of milk and other dairy products worldwide. β–galactosidase hydrolysis of milk is one of the promising enzymatic applications in dairy industries for reducing lactose intolerance of milk products. However, plant and animal sources cannot meet the high demand of the enzyme in food industries. Hence, the aim of this study was to characterize β–galactosidase production by Lactic Acid Bacteria (LAB) isolated from locally fermented milk products. Raw milk from Sokoto Gudali was collected from Fulani settlement in Ojoo, Ibadan along with some fermented milk products (‘’Nono’’ and ‘’Wara’’). LABs were isolated from them and identified using conventional methods. The ability of the isolates to hydrolyze 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was used to screen for β–galactosidase production. Isolates with the best β-galactosidase production were selected. The enzyme was extracted and optimization of growth conditions (temperature, pH, nitrogen, carbon sources, inoculums size and inoculums age) for β-galactosidase production was carried out using o-nitrophenyl-β-D-galactopyranoside (ONPG). The enzyme produced was characterized using pH, temperature, metal and non-metal ions, and inhibitors. Purification of the enzyme was carried out using dialysis and chromatographic methods. Hydrolytic effects of the purified β–galactosidase were determined in different concentrations of lactose using standard method. Data were analyzed using descriptive statistics. The isolated bacteria were identified as Lactobacillus plantarum (G11, E13 and E36), L. brevis, L. casei, L. lactis, Leuconostoc lactis, Streptococcus sp, and Bacillus subtilis. Lactobacillus plantarum (G11) had optimum growth value of 4.2 at 20°C and pH 7.0 with maximum enzyme value of 6.2U/mL at 30 hrs. The optimal β-galactosidase production occurred at neutral pH and 6% inoculum size. The best inoculums age varied between 18 hrs and 36 hrs. The best carbon source for enzyme production was raffinose with maximum value of 0.3U/mL while minimum activity was found in fructose with 0.2U/mL. The best nitrogen source was NH4NO3 with maximum value of 0.5U/mL and yeast extract had minimum value of 0.1U/mL. β–galactosidase activity increased with increase in molar concentration of the mono-valent chloride ions in which the highest was recorded in KCl at maximum value of 0.08U/mL while the minimum value of 0.001U/mL was obtained by NaCl at a concentration of 0.2 mmol respectively. The best sulfate ion was CuSO4 with maximum activity value of 0.2 U/mL at 0.2 mmol concentration and minimum value of 0.007 U/mL at 0.1 mmol by ZnSO4. The best enzyme inhibitor was KCN with maximum activity of 0.2U/mL at 0.2 mmol. The specific activity of β-galactosidase was 292.5 U/mg, 104.2 U/mg and 585.46 U/mg for G11, E13 and E36 respectively. The hydrolytic effects of the purified β-galactosidase showed a maximum yield of 35.8% glucose, 19.3% galactose and 35.3% glucose and 18.5% galactose at 80% and 60% lactose concentration respectively. β-galactosidase produced by Lactobacillus plantarum strain achieved lactose hydrolysis and could be of potential application for production of low lactose dairy products for consumption by lactose intolerant people.Item BIOREMEDIATION OF HEAVY METALS CONTAMINATED SOIL FROM A STEEL ROLLING COMPANY IN IBADAN(2016-06) OYINLOYE, I. A.The accumulation and persistence of Heavy Metals (HMs) in soil poses serious environmental challenges. These HMs may leach and impair surface and ground water quality as well as bioaccumulate in plants. The burden of HMs in the environment can be reduced by Organic Amendment (OA) stimulated bacterial remediation. However, there is dearth of information on the treatment of HMs contaminated soil with OA stimulated microorganisms. This study was designed to bioremediate HMs contaminated soil using cattle dung slurry stimulated bacterial inoculum. Heavy metal contaminated soil samples were purposively collected from the surrounding of a steel rolling mill in Alomaja, Oluyole Local Government Area, Ibadan, Nigeria. Cattle dung slurry used as OA was collected from a commercial animal pen in Bodija Market, Ibadan. Bacteria were isolated from the soil using pour plate agar technique and identified using biochemical and molecular techniques. The isolates were screened to select high HMs tolerant strains used in remediation following standard techniques. The soil and OA samples were analysed for HM and sterilised using appropriate techniques. The sterilised soil was mixed with OA in ratio 5:1 and remediated with selected bacterial strains and thereafter Corchorus olitorius was planted for eight weeks using potted experiment. The experimental set up was completely randomised design of 16 groups consisting of sterilised mixture with three isolates (A, B, and C) singly and in combinations while Sterilised Soil (SS) only and Unsterilised Soil (US) with OA served as controls and each had five replicates. Agronomic parameters (Plant Height (PH), Stem Diameter (SD) and Number of Leaves (NL)) were monitored weekly under screen house conditions for eight weeks. Plants and composite soil for each study group were analysed for HMs thereafter. Data were analysed using descriptive statistics and ANOVA at α0.05. The soil was heavily contaminated with heavy metals, especially lead and chromium with concentrations of 1505.1-2333.6 and 1526.0-1678.7 mg/kg, respectively. Thirty-six bacteria isolates were identified as Pseudomonas (19), Proteus (5), Alcaligenes (5), Enterobacter (3), Providencia (2) and Bacillus (2). Alcaligenes aquatilis (A), Pseudomonas mucidolens (B) and Bacillus cereus (C) had high tolerance for HM (400-450 µg/mL) and were used for remediation. The results obtained for the PH, SD and NL in un-amended soil revealed a significant difference between ABC that had 7.84±0.69 cm, 1.35±0.00 mm, 5.8±0.45 and SS that had 4.10±0.55 cm, 1.33±0.04, 5.0±0.0, respectively. While in OA treated soil, BC had 9.24±1.78 cm, 1.35±0.00 mm, 5.60±0.55 and US had 22.94±4.30 cm, 2.20±0.20 mm and 9.40±1.82 for PH, SD and NL, respectively. Post bioremediation analysis of the soil samples revealed a reduction in the concentration of lead from 2333.55 to 20.8 and 22.6 mg/kg in B and ABC while chromium reduced from 1678.7 to 1.8 mg/kg in B. Postharvest analysis of C. olitorius revealed that percentage crude fibre, dry weight (g), lead and chromium concentration (mg/kg) in ABC and SS were 21.72±0.99, 0.99±0.17; 20.61±0.78, 0.63±0.24; 10.90±0.85, 4.15±0.64 and 9.15±0.64, 4.00±0.14, respectively. Pseudomonas mucidolens effectively remediated heavy metals contaminated soil and can be employed in the treatment of such contaminated environment.Item SELECTION, PRODUCT CHARACTERISTICS AND METABOLITE SPECTRUM OF A COMMON STARTER CULTURE FOR FUFU AND USI PRODUCTION(2015-07) OYINLOLA, K. A.Fermented cassava products like fufu and usi (edible starch) are important staple foods in many African homes. Natural fermentation time is usually long resulting in slower acidification process and inconsistent nutritional composition of products which could be overcome with the use of starter culture. However, most available starter cultures are used for single food fermentation and are uneconomical. This necessitates the development of a starter culture for multiple related food products to reduce cost. Hence this study was designed to produce a common starter culture for the production of fufu and usi. Cassava varieties TME 30572, TME 4(2)1425 and TME 50395 were obtained from the International Institute of Tropical Agriculture, Ibadan and landraces from Bodija market. Fresh, peeled, chipped and grated cassava tubers were spontaneously fermented in the laboratory. Lactic Acid Bacteria (LAB) were isolated from the fermenting mash and identified phenotypically. Genotypically identified starters were selected based on screening for starch hydrolysis, linamarase and pectinase enzyme production, antimicrobial compound production and rate of acidification using standard methods. The starters were utilised singly and randomly combined to initiate fermentation for production of fufu and usi. Un-inoculated fermentation mash served as control. Rate of production of organic acids, various sugars, metabolic enzyme assays, nutritional and anti-nutritional content of the resulting mashes were monitored using standard procedures. Best starter was applied in the final production of fufu and usi. Shelf-life of the products were evaluated and compared with the control. Data were subjected to descriptive statistics and ANOVA technique at p=0.05. Ninety-eight LABs were identified as Lactobacillus plantarum (50.0%), L. acidilactici (12.2%), L. brevis (11.3%), L. fermentum (10.3%), L. delbrueckii (8.2%), L. mesenteroides (6.0%), and L. lactis (2.0%). Screened isolates did not hydrolyse starch but produced pectinase, linamarase alongside hydrogen peroxide, diacetyl and lactate with a rapid decrease in medium pH (6.5 - 3.6). Selected potential starters were genotypically identified as L. pentosus F2A (A), L. plantarum subsp. argentolarensis F2B (B), L. plantarum F2C (C), L. plantarum U2A (G) and L. paraplantarum U2C (I). The best starter combination CGI gave significant reduction in fermentation pH (7.1 - 3.7) and lactic acid ranged between 0.04mg/ mL and 6.9mg/mL. Sugars produced include xylose (3.2µg/mL), arabinose (1.4µg/mL), fructose (26.2µg/mL), glucose (30.3µg/mL) and sucrose (99.7g/mL). Enzyme assay revealed peak amylase (10.1U/mL) and pectinase (4.4U/mL) activities at 24 hours as well as linamarase (0.8U/mL) at 48 hours in fufu, whereas, in usi, highest linamarase (0.7U/mL) and pectinase (1.0U/mL) activities were recorded at 72hours with no amylase activity. The CGI-produced fufu and usi had significant reduction in phytate (0.3-0.1mg/g and 0.3-0.27mg/g), tannin (35.4-34.0mg/g and 35.4-32.3mg/g), cyanide (0.1-0.05mg/g and 0.1-0.0mg/g), and moisture (7.3%-5.1% and 7.3%-5.4%) content while total protein content increased (1.0-1.3% and 1.0-1.8%) respectively. Starter fermented fufu and usi had shelf-life of five days while control had three days. The selected starter was able to ferment both fufu and usi to yield products with improved nutritional content, better shelf-life and reduced anti-nutritional composition. This could be employed in the production of indigenous fermented foods.Item MOLECULAR CHARACTERISATION OF SOME MULTI-DRUG RESISTANT SALMONELLA ENTERICA OF HUMAN ORIGIN IN SOUTHEAST NIGERIA(2016-01) ONYENWE, N. E.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 (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.
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