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THE PREVALENCE OF FURUNCULOSIS AND PLASMIDMEDIATED RESISTANCE OF ISOLATES OF STAPHYLOCOCCUS AUREUS FROM INFECTED INDIVIDUALS IN SOUTHWEST NIGERIA
(2012) Okunye, O.L.
Furunculosis, a cosmopolitan infection of human skin caused by Staphylococcus aureus, is recurrent among most infected individuals. It is characterized by a honey crusted ‘cropped’ latent boil with potential to recur in a susceptible host. It is a common colonizer of the skin with a remarkable ability to hydrolyse β-lactam antibiotics, degrade skin lipid barrier and spread within the skin loci. In this study, the gender and age distributions of furunculosis, the antibiotic susceptibility pattern of its causative agent and the genetic basis of the recurrency were determined. Exudates of ‘cropped-boils’ were obtained from 140 human volunteers (40 hospital reported and 100 non-reported cases of recurrent furunculosis) from different age range(1-100 years) for both genders within the six southwest states of Nigeria. The exudates were processed for isolation and identification of S. aureus by selective plating and biochemical tests. Antibiogram of the isolates was determined by disc-diffusion using multi-discs of eight standard antibiotics. Minimum Inhibitory Concentrations (MIC) of five selected antibiotics: amoxicillin-clavulanic acid, penicillin, ceftriaxone, cefuroxime and cloxacillin were determined by broth-dilution method. Detection of β-lactamase was carried out by cell-suspension iodometric method. Positive strains were processed for plasmid DNA isolation and molecular weight determination by lystostaphin cell lysis and agarose gel electrophoresis. Curing of R-plasmid DNA in selected bacterial strains was done by exposure to ethidium bromide prepared in 2-fold dilutions in nutrient broth from 6.25-100.00 μg/mL. Transfer of resistance was done by conjugation between some resistant strains as donors and Carolina-typed sensitive E. coli; E. coli 01 and E. coli 02 strains as recipients. The data were analysed using ANOVA at p = 0.05. A total of 102 isolates comprising seventeen from each of the six southwest states were identified and selected. The gender distributions were 46.0% females and 54.0% males, comprising of 20.0% hospital reported and 80.0% non-reported for the highest prevalence females and 5.0% hospital reported and 95.0% non-reported for males. Recurrent furunculosis had the highest prevalence in males within the age groups 11-50 years and in females, age groups, 11-70 years. The isolates exhibited the lowest resistance of 8.0% to amoxicillinclavulanic acid and 95.0% as the highest resistance to amoxicillin. Thirty of the isolates possessed β-lactamase in varying degrees out of which 29.0 were plasmid-borne. Of this number, 7.0 had multiple plasmid DNA of 2- 4 copies, ranging between 0.25 and 63.09 kb. The MIC of the antibiotics showed that the isolates were most susceptible to amoxicillin-clavulanic acid (3.90 – 250 μg/mL) and the highest resistance was recorded for penicillin G (7.8 – 500 μg/mL). Curing of R-plasmid DNA occurred at concentrations of 50.0μg/mL and 100.0μg/mL of ethidium bromide while conjugation was achieved in two out of seven competent cells, indicating a plasmid-borne resistance. The prevalence of furunculosis varied across the different age groups. The high level multidrug resistance elicited by the strains of Staphylococcus aureus isolated was established to be due to the associated transferable R-plasmid encoded β-lactamase.
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..
MICROBIAL DEGRADATION OF MAIZE COB AND ITS ASSESSMENT FOR USE AS BROILER FEED SUPPLEMENT
(2012-02) BAMIGBOYE, O. O.
Maize, Zea mays is extensively cultivated in Nigeria with estimated total grain yield of 1.37 million tonnes per year. The maize by-product which includes the cob is readily available. This project was designed to convert maize cob to value-added products in broiler feed through microbial degradation using batch fermentation. One hundred and fifty kilogrammes of maize cob samples were collected from four dump sites in a farmers’ market in Oyo town. Bacteria and fungi were isolated using nutrient agar and potato dextrose agar media respectively. Identification of isolates was done by conventional methods and cellulase enzyme assay carried out by dinitrosalicylic acid method. The isolates with cellulase activities were then used for solid state fermentation. The process was then sealed up in fabricated tanks for bulk fermentation using Bacillus subtilis and Aspergillus niger solely. The derivatives labelled Bacteria Enriched Cob Product (BECP) and Fungi Enriched Cob Product (FECP) were subjected to chemical and microbiological analyses. The BECP and FECP were each substituted at 18%, 50% and 82% inclusions in compounded feeds while commercial feed served as control. These were fed to four-week old broiler chicken for six weeks in a completely randomized experimental set up of seven groups with ten chicks per group. Mean Weight Gain (MWG), Feed Conversion Ratio (FCR), economic performance, haematological indices and organ histopathology were determined. Data was analysed using inferential statistics at p = 0.05. Thirty-three (33) isolates made up of 19 bacteria and 14 fungi had cellulase activity. These were the genera Bacillus (10), Pseudomonas (8), Proteus (1) Aspergillus (6), Penicillium (4), Rhizopus (1), Mucor (1), Botryotrichum (1) and Geotrichum (1). The best cellulase producers were Aspergillus niger, 1.7 mg/ml and Bacillus subtilis 0.7 mg/ml with optimal activity at 40oC, pH 4.0 and 28oC, pH 7.0 respectively. Cellulose degradation ability of fungi 17.3% was higher than bacteria 6.6% while the crude protein levels were 7.5% and 9.5% respectively. The MWG (18.0g) of chicks fed BECP (82%), was much lower than the control (34.3g), FECP (18%), 38.9g; BECP (18%), 37.6g; FECP (50%) 31.0g and BECP (50%), 30.7g. The feed cost per kilogram gain for FECP (28%), N185.7; BECP (18%), N208.4 and FECP (50%) N227.4 were lower than the control N235.9. The FCR for BECP (82%), 8.4 was higher than the control value of 3.6. The red blood cell count for all treatments (1.7 – 2.6 x 106/mm3) did not differ significantly. However, white blood cell count was significantly higher in chicken fed with 82%, BECP (27.2 x 103/mm3) and 82% FECP, 20.4 x 103/mm3 compared to the other diets (11.2 – 16.2 x 103/mm3). Only the kidneys and lungs of chickens fed with 82% BECP were severely congested. Maize cob contained cellulolytic bacteria and fungi capable of degrading it at different extents. Substitution of either bacteria enriched product or fungi enriched product up to 50% were economically suiTable and safe for use as broiler chicken feed supplement.
ISOLATION, CHARACTERISATION AND BIODEGRADATION ABILITY OF BACTERIA ISOLATED FROM SOIL CONTAMINATED WITH HYDROCARBONS
(2012-02) AYANDELE, A. A.
Contamination of land and water bodies by crude oil and refined petroleum products is a major challenge worldwide. Indiscriminate disposal of crankcase oil into the environment has increased hydrocarbon pollution in Nigeria. Microorganisms have been identified as major contributors in fighting pollution. The remediation ability of bacteria isolated from hydrocarbon contaminated organic rich soil has not been fully investigated. This research was designed to study in-situ genera and hydrocarbon degrading ability of bacteria isolated from an organic rich tropical soil deliberately contaminated with a Nigerian crude oil and crankcase used oil Bacterial enrichment for hydrocarbon degradation was carried out by deliberately contaminating garden soil samples collected from the Nursery of the Department of Microbiology, University of Ibadan. Top soil were collected and mixed with Forcados Blend crude and used crankcase oil at a mixed ratio of 5:1. Hydrocarbon degrading bacteria counts were obtained at two week intervals for ten weeks by sub-culturing on mineral salts oil agar supplemented with the hydrocarbons. Isolation was done by randomly selecting colonies of bacteria based on morphological and growth characteristics. Isolated bacteria were screened on sterile Hydrocarbon agar plates and were identified by classical methods. The DNA extraction and amplification of ten selected strains were carried out using molecular technique. Amplified DNA was digested by HaeIII and Rsal restriction enzyme and subjected to Restriction Fragment Length Polymorphism analysis and sequencing of the 16SrRNA.The BLAST search for the obtained sequences were made and phylogenetic tree of amplicons constructed using MEGA4.1. Plasmid presence, sizes and numbers in the isolates were determined. Hydrocarbon degradation rate by the bacteria isolates was determined by gravimetry and Gas Chromatography analysis using Flame Ionisation Detector. Hydrocarbon-utilising bacteria increased from 35 × 104 to 265 × 104 cfu/mL, while total bacteria count decreased from 245 × 104 to 123× 104 cfu/mL between the second to tenth week. Ten out of forty-two hydrocarbon-utilizing bacteria detected showed high crude and crankcase oil degrading ability. Phylogenetic analyses of the isolates showed high sequence identities (75-100%) in amplified genes when compared to those in the GenBank. The isolates belonged to four genera; Bacillus (5), Providencia (3), Proteus (1) and Alcaligenes (1). Utilization of complex hydrocarbons present in crude and crankcase oil by these isolates ranged between 51.9-77.0% and 42.4-75.8% respectively. Four out of the ten bacterial isolates contained plasmids of varying sizes. Bacillus OUE3 and Providencia OCR1 contained two plasmids each of sizes 2.57 kb and 2.0 kb, and 1.3 kb and 1.9 kb respectively, while Bacillus OUE6 and Providencia OCR2 contained a plasmid each. The percentage total degradation for polycyclic aromatic hydrocarbon ranged from 29.64 to 98.45% for crude and crankcase oil. About 25.2 to 91.7% and 98.2 to 99.6% of aliphatic groups were utilised by the isolates in crude and crankcase oil respectively within 20 days. Ten of the isolated bacteria could remediate hydrocarbon pollution from soil environment. Providencia sp. had the highest degradative ability.
PHYSIOLOGICAL, GENOMIC AND PRESERVATIVE PROPERTIES OF PEDIOCOCCUS ISOLATES FROM MEATS UNDER LOW TEMPERATURE STORAGE
(2012-06) DUYILEMI, O. P.
The need for meat preservation cannot be overemphasised. However, existing methods of meat preservation including the use of artificial preservatives have toxic side effects. There is a dearth of information on the use of Lactic Acid Bacteria (LAB), which is known as good preservatives for food, in the preservation of meat. Hence, the aim of this study was to examine the use of Pediococcus acidilactici and low temperature in improving the quality of beef, chicken and turkey meat samples. Samples of beef, chicken and turkey were obtained from open market retailers and stored for 28 days at 4oC, 2oC, -4oC and -15oC. From these, LAB were isolated and identified using conventional methods. Quantities of lactic acid and acetic acid were determined using high performance liquid chromatography, while diacetyl and hydrogen peroxide were determined by enzymatic methods. High production of lactic acid was used as a criterion for selecting five isolates of Pediococcus acidilactici and the 16S rDNA genes were amplified and sequenced. The isolates were checked for plasmid presence and tested for bacteriocin production using gel electrophoresis and agar well assay. Antimicrobials produced by the isolates were tested in vitro against known meat spoilage organisms: Staphylococcus aureus, Escherichia coli, Pseudomonas faecalis, Listeria monocytogenes, Salmonella typhimurium and Bacillus cereus. The isolates and their filtrates were applied to fresh meat at -4oC and -15oC. Microbial load, proximate and biochemical parameters of the meat samples were monitored at seven days interval for 28 days. Data were analysed using ANOVA at p = 0.05. One hundred and ten LAB isolates from beef [Lactobacillus (24), Pediococcus (6), Leuconostoc (9)], chicken [Lactobacillus (27), Pediococcus (4), Leuconostoc (2)] and turkey [Lactobacillus (31), Pediococcus (5), Leuconostoc 2] were identified. Fast freezing (-15oC) and freezing (-4oC) gave significantly lower LAB count (4.1 ± 0.03 - 5.1 ± 0.02 logcfu/mL) than those of chilling (2oC) and refrigeration (4oC) (4.2 ± 0.04 - 5.4 ± 0.02 logcfu/mL) in all the meat samples. Lactic acid and acetic acid production peaked at 30.7 g/L and 32.0 mg/mL respectively while diacetyl and hydrogen peroxide production peaked at 40.8 ng/L and 16.0 µg/L respectively. Sizes of the 16S rDNA from the five strains of P. acidilactici ranged from 145 bp to 161 bp. Band size of plasmid DNA ranged from 861 - 20643 bp. Bacteriocin inhibition zones ranged from 1.0 to 6.5 mm. The highest zone of inhibition of antimicrobial action was 12 mm against P. faecalis. Proximate and biochemical analyses gave lower values compared with control samples: pH (4.8 / 5.7), thiobarbituric acid (0.2 / 0.5 mg malonaldehyde/kg), free fatty acid (0. 2 / 0.5 KOH/g lipid), total volatile nitrogen (0.6 / 1.4 mgN/100) and crude fat (3.4 / 4.5 %); but increased crude protein (22.1 / 17.7 %). Lowest microbial load (total bacteria count 2.2 logcfu/ml, coliform count 1.4 logcfu/ml, fungal count 2.0 logcfu/ml) and highest LAB count (4.9 logcfu/ml) were observed on the 28th day. Pediococcus acidilactici with optimum physiological characteristics prolonged the keeping quality of meat under low temperature storage.
NUTRITIONAL AND BIOCHEMICAL CHARACTERISTICS OF LACTIC ACID BACTERIA-CHALLENGED SORGHUM AND ITS FERMENTATION PRODUCT
(2012-06) OJO, F. T
Sorghum is one of the major cereals malted for the brewing of beer. The conditions of transport and storage of this cereal predispose it to contamination by microbes thus affecting the quality of the end products. The contamination is usually controlled by treating the sorghum with chemicals, which however alter the chemical constituents of the sorghum. Therefore, the need to seek alternative functional methods of control of microbial contaminants becomes imperative. This study was aimed at investigating lactic acid bacteria as biocontrol agents against microbial pathogens of stored cereals for beer fermentation. Sorghum were obtained from Bodija market and from the Institute of Agricultural Research and Training, Ibadan. Lactic Acid Bacteria (LAB) were isolated from spontaneously-fermenting sorghum and identified using classical techniques. The abilities of the LAB strains to produce antimicrobials and their antagonistic activity against known cereal pathogens were used to select the best three strains for further work. The selected strains were applied singly and in combination at inoculum concentration of 2.3 x 104 cells/mL for five days to challenge sorghum seeds prior to malting and wort production. Sorghum wort was fermented for five days with Saccharomyces carlsbergensis. Physiological and nutritional characteristics of the unchallenged and challenged sorghum, and fermentative characteristics of the wort were determined using the European Brewery Convention methods. Data were analysed using ANOVA. One hundred and twenty seven strains of LAB were isolated and identified as Lactobacillus plantarum (32), Lactobacillus brevis (31), Lactobacillus fermentum (25), Lactobacillus delbrueckii (8), Lactobacillus casei (12) and Lactobacillus acidophilus (19). Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus casei produced high antimicrobial lactic acid (2.5±0.5g/L, 2.4±0.3g/L and 2.5±0.5g/L respectively) and had high inhibitory activities (17mm, 14mm and 17mm respectively). Lactobacillus brevis produced antimicrobial lactic acid with the highest mean concentration of 2.7±0.5g/L from local sorghum but was not used for further work because the inhibitory activity was low when tested against pathogenic organisms. All the LAB produced bacteriocin with antagonistic effects on all the pathogens tested, and Lactobacillus plantarum had the highest zone of inhibition (17mm) against Bacillus subtilis. All the LAB grew at temperature of 30oC, pH 5.0-5.5, high glucose and peptone concentration (1.5-2.0mg/ml). The malted untreated -sorghum had 13.2 % protein, 3.0 % crude fat, 1.9 % ash, 1.8 % crude fibre, 42.5 mg/g phytate, 36.0 mg/g tannin, 2.0 mg/g protein inhibitor and 16.0 IoBunits diastatic power. Fermentation of the unchallenged wort (pH 6.2) yielded ethanol content of 2.2 %. With LAB treatment, there was a reduction in protein (12.2 %), crude fat (2.1 %) and crude fibre (1.1%); and significant (p<0.05) reduction in antinutrients (phytate 32.7 mg/g, tannin 22.4 mg/g, protease inhibitor 0.0 mg/g and wort pH 4.2). There was also an increase in diastatic power (24.0 IoBunits). The subsequent fermentation produced 4.8 % ethanol. The microbial profiles of the challenged malted sorghum showed a steady decrease in Bacillus, Staphylococcus and Pseudomonas count compared with the unchallenged where they showed steady increase. Lactobacillus starter cultures reduced spoilage pathogens, antinutritional factors of sorghum during malting and improved the end products.
FERMENTATION OF COCOA (Theobroma cacao L.) POD HUSK AND ITS HYDROLYSATE FOR ETHANOL PRODUCTION USING IMPROVED STARTER CULTURES
(2012-07) IGBINADOLOR, R. O.
Fossil fuel, a main but dwindling energy source for automobiles, causes emission of environment unfriendly oxides of carbon. These contribute substantially to greenhouse gases which bring about climate change. There is therefore the need for sustainable source of energy like ethanol an environmental friendly bioenergy. Hence this study was aimed at the fermentation of cocoa pod husk for ethanol production. Isolates of yeast were obtained from sun-dried Cocoa Pod Husk (CPH), subjected to spontaneous submerged fermentation for 7 days. Five strains of Saccharomyces sp. (MX1, MX2, MX3, MX4 and MX5) with high frequency of occurrence were selected for further studies. The MX1 and MX2 were used for genetic modifications. Dried CPH was subjected to chemical analysis and pretreatment using particle size reduction and high pressure liquid hot water at 130oC for 30 minutes. Acid and enzymatic hydrolysis of the pretreated CPH was carried out using standard method. Products of the hydrolysis were analysed with high performance liquid chromatography. Two genes XL1 (xylose reductase) and XL2 (xylitol dehydrogenase) encoding pentose utilization were obtained from genomic DNA of Pichia stipitis (CBS 6054) using basic local alignment search tool. Primers of these genes were designed with Saccharomyces genome database, amplified with Polymerase Chain Reaction (PCR) and purified. The amplicon (genes) were ligated into plasmid vectors (pGAPZA and pVT100-U). Strains MX1 and MX2 were transformed with these construct using lithium acetate method. Physiological characterization of the selected unmodified yeast strains and the two genetically-modified strains was done under different environmental conditions including temperatures, pH and varied concentrations of acetic acid. The CPH hydrolysates were fermented for 120 hours using the unmodified and genetically-modified yeast strains respectively and the ethanol yield determined. Data were analysed using ANOVA. Twenty yeast isolates identified as Saccharomyces cerevisiae (80%) and Saccharomyces uvarum (20%) were obtained. Chemical composition of CPH included hemicellulose (13.9%) cellulose (18.6%) and lignin content (14.2%). Acid hydrolysis yielded 50.1% glucose, 11.97% xylose, 11.2% mannose while enzymatic hydrolysis gave 31.7% glucose, 4.8% mannose and 16.8% galactose. The inserted gene XL1 had 318 amino acids polypeptides while XL2 had 363 amino acid polypeptides. Restriction enzyme analysis and colony PCR confirmed the transformational integration of these constructs into Saccharomyces cerevisiae MX1 and MX2. The five isolates had optimal growth at 30 – 40oC and pH of 4.0 – 5.5. However the genetically-modified yeast strains were able to utilize xylose and arabinose carbon sources better than the unmodified types and also tolerated low concentration of acetic acid than the unmodified types. Ethanol production was highly significant (p0.05) in the modified starters (29.7g/L) than the unmodified strains (14.0g/L). Genetically-modified organisms performed better in ethanol production than the non-modified organisms. The application of genetic modification of microorganisms will aid the potential use of waste biomass like cocoa pod husk for bioenergy production and this will contribute significantly to reducing greenhouse gases associated with climate change.
RAFFINOSE METABOLISM AND UTILISATION BY L. PLANTARUM ISOLATED FROM INDIGENOUSLY FERMENTED CEREAL GRUELS FOR NUTRITIONAL IMPROVEMENT
(2012-08) ADEYEMO, S. M.
Most African foods used in weaning are usually cereal-based gruels fermented by Lactic Acid Bacteria (LAB). The food mainly supplies carbohydrate; excessive intake of which might cause malnutrition in growing children. Minimum dietary requirements of a child could be met through fortification with protein-rich supplements. Soybean is rich in dietary protein but contains some antinutritional factors and raffinose, an oligosaccharide responsible for gas formation, bloating and flatulence in weaning children. The use of microorganisms for hydrolysing raffinose has not been fully exploited in Nigeria. In this study, the use of LAB to hydrolyse raffinose, reduce antinutritional factors and improve nutritional composition of such food blends were investigated. Commercially- hawked “Ogi” (CO) samples, Local Varieties (LV) of sorghum and maize were obtained from Bodija market, Ibadan and Typed Varieties (TV)-Samsorg 40, Samsorg 41 and Ex-Kano from the Institute of Agricultural Research and Training, Ibadan. The LAB were isolated from spontaneously-fermenting cereal gruels and identified using standard methods. Nine strains of Lactobacillus plantarum were selected based on the abundant production of -galactosidase, and characterized by PCR amplification of 16SrDNA genes. Plasmid presence was determined using agarose gel electrophoresis and the effect of plasmid curing was monitored. The growth of the organisms and metabolites production in different carbon sources were monitored at 200C to 800C and pH of 3.0 to 9.6. Soyabean was pre-treated by milling, cooking and roasting while the relationship of the isolates to raffinose metabolism during fermentation was monitored daily for 5 days. Reducing sugar, residual oligosaccharides, nutritional, antinutritional factors and alpha-galactosidase were determined using UV-spectrophotometer following Association of Official Analytical Chemist procedures. Data were analysed using ANOVA at p=0.05. One hundred and twenty LAB isolates were obtained and identified as L. plantarum (35.8 %), L. fermentum (12.5 %), L. pentosus (7.5 %), L. acidophilus (15.8 %), L. casei (5.8 %), L. brevis (6.7 %), L. cellobiosus (6.7 %), L. jensenii (5.0 %) and L. reuterii (4.2 %). Analyses of the nine L. plantarum isolates revealed high sequence identities (97.0 %). These isolates exhibited significant differences in utilization of raffinose at varying concentrations of 0.2 -1.0 mg/mL, while isolates obtained from LV performed better than those from TV and CO. Fermentation reduced the oligosaccharide content of the soyabean by 74.6 % while the reducing sugars increased by 65.0 %. Fortification of the gruel with soyabeans using uncured L. plantarum strains improved the nutritional quality (protein: 8.4 to 17.8 %, fat: 3.6 to 12.9 %, ash: 2.0 to 3.8 %, Fe: 6.4 to 10.7 mg/100g and Ca: 156.7 to 211.0 mg/100g), and a significant reduction in antinutritional factors (Tannin: 1.9 to 0.1 mg/g, Phytate: 1.2 to 0.1 mg/g and Trypsin Inhibitor : 1.2 to 0.0 mg/g) was observed after fermentation . Oligosaccharide content, reducing sugar, nutritional and antinutritional composition and organoleptic attributes of the end product were significantly affected by plasmid curing. Utilisation of raffinose by Lactobacillus plantarum from local food sources reduced antinutritional factors and oligosaccharides in soybeans. Nutritional quality of cereal gruels were improved by inclusion of Lactobacillus plantarum.
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.
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.
MICROBIAL DEGRADATION OF POLYURETHANE
(2014-02) OKOLIE, B. I.
Polyurethane is a non-easily degradable synthetic polymer used as packaging material. Its presence and durability in the environment pose global disposal and degradation problem. Polyurethane releases toxic substances with carcinogenic or mutagenic potential during burning in dumpsites. An alternative to burning of waste materials is the use of microorganisms to degrade them. However, little is known about microbial degradation of polyurethane. Therefore, microbial degradation of polyurethane was investigated as an alternative treatment and management method. Polyurethane samples were collected from a dumpsite in the University of Ibadan and rubbish-bins of two fast foods outlets within Ibadan metropolis. The packs were buried at depths ranging from 15-70 cm in the garden soil of Microbiology Department, University of Ibadan. They were exhumed at predetermined intervals between the dry and rainy seasons of June 2010-June 2012 for isolation of microorganisms. The microbial isolates were screened for ability to utilise polyurethane as nitrogen and/or carbon source using mineral salts medium. Selected polyurethane-utilising microorganisms as carbon and nitrogen sources were characterised and identified using standard microbiological procedures and the advanced bacterial identification software database. Biodegradation study was carried out on sterilised soil in the laboratory and garden soil in the field with the best six polyurethane-utilising bacteria. This was done using complete randomised block design with 4x3x2 factorial experiment for isolates combinations (A: Pseudomonas alcaligenes E14+ Providencia pseudomallei D25, B: Enterobacter amnigenus D12+ Vibrio sp. C32, C: Pseudomonas aeruginosa E32 + Providencia pseudomallei D21 and D: Consortium of the six bacteria) and biostimulation treatments (cassava peels, potato peels, no peels) at 1 and 3 month periods. Changes in the functional groups of degraded polyurethane samples were determined using Fourier transform infrared spectroscopy. Weight loss of polyurethane samples was monitored by measurement. Data were analysed using ANOVA at p = 0.05. Of the 106 bacterial isolates obtained, 94 utilised polyurethane as carbon, nitrogen or both with highest occurrence (26.0 %) at 70 cm depth. Eighty-seven per cent of the isolates were obtained during the rainy season. Fifteen bacteria isolates that utilised polyurethane as carbon and nitrogen sources were: Pseudomonas (4), Corynebacterium (1), Providencia (2), Enterobacter (2), Comamonas (2), Micrococcus (1), Arthrobacter (1), Vibrio (1) and Bacillus (1). Fungi isolates could not utilise polyurethane. Percentage degradation of polyurethane with potato peels, cassava peels and no peels was respectively 91.0, 33.0 and 57.2 % in laboratory and 35.9, 0.0 and 76.3 % in field. Ether peak was removed by B, C and D in the field biostimulated with cassava peels. Carbonyl peak area was reduced by 87.6 % with D biostimulated with potato peels in the laboratory and changes in the functional groups were significant. The highest weight losses were 22.5 and 15.0 % for the field and laboratory studies after one month. The isolated bacteria degraded the polyurethane by removal of resistant functional groups. Thus they could be used for degradation and management of polyurethane in the Nigeria environment.
PERFORMANCE CHARACTERISTICS OF FUNGAL DEGRADED RICE BRAN AND PALM KERNEL CAKE SUBSTITUTED IN Clarias gariepinus FEEDS
(2014-02) ALUKO, J. F.
Fish farming enterprise has increased in recent times due to public demand for fish protein. However, rising costs of fish feed ingredients have greatly reduced the profit margin of local fish farmers. Cheap, readily available and locally sourced ingredients may be better substitutes to the costly fish feeds. The aim of this study was to evaluate the performance of fermented Palm Kernel Cake (PKC) and Rice Bran (RB) as substitutes in the diet of Clarias gariepinus. Fungi were isolated from Unfermented PKC (UPKC), RB (URB) and from Naturally-Fermented PKC (NFPKC) and RB (NFRB) ligno-cellulosic substrates using Potato Dextrose Agar (PDA). Isolates were identified and screened for enzyme production (amylase and cellulase) using standard methods. They were then subjected to physico-chemical (pH, temperature, incubation period, carbon and nitrogen sources) analysis to obtain optimal conditions for enzyme production. Proximate analysis of fungi-fermented samples was carried out to select the starters used in degrading the ligno-cellulosic substrates. Starters that gave the highest protein contents were selected. Starter-Degraded PKC (SDPKC) and RB (SDRB) were analysed for proximate and mineral compositions, amino acid profile, anti-nutrition factors, vitamins and heavy metals. The degraded and undegraded ligno-cellulosic bulks were substituted in the diets of fingerling and compared with a standard fish feed as control for three months. Fish performance characteristics [(Mean Weight Gain (MWG), Specific Growth Rate (SGR)] and Feed Conversion Ratio (FCR)) were evaluated. Data were analysed using ANOVA at p=0.05. Ten fungi isolates obtained were Aspergillus fumigatus, Aspergillus niger, Aspergillus clavatus, Aspergillus tamarii, Aspergillus terreus, Aspergillus versicolor, Rhizopus stolonifer, Rhizopus oryzae, Rhizopus sp. and Trichoderma sp. Highest amylase (63.2units/mg) at pH 6.0, 50.0oC, Vmax 3.9 (UI/mL) and Km 1.3 (g/mL) was produced by Aspergillus niger which grew best on wheat bran (8.0 mg/g) as carbon source and ammonium nitrate (4.6 mg/g) as nitrogen source. Highest cellulase (3.1Units/mg) at pH 8.0, 20.0oC, Vmax 0.9 (UI/mL) and Km 0.8(g/mL) was produced by Rhizopus oryzae which grew best on RB (7.3 mg/g) as carbon source and potassium nitrate (1.6 mg/g) as nitrogen source. Selected starters, Aspergillus clavatus and Aspergillus tamarii, gave protein contents of 6.5% and 17.9%. Protein contents of NFPKC, NFRB, UPKC and URB were 5.7, 22.0, 5.2 and 16.0% respectively. Highest zinc composition (520.0µg/g) was recorded in SDPKC and SDRB with reduction in amino acid profile (16.7 – 0.03µg/g). Fermentation decreased oxalate (24.1-18.2 µg/g) among the anti-nutritional factors and had no effect on the vitamin contents, while no heavy metal was detected in the feeds. The MWG of fish fed with URB and UPKC ranged from 18.9g - 35.8g and that of SDRB and SDPKC ranged from 12.8 - 43.3g while that of the control feed was 31.5g. Fish fed SDPKC gave the highest SGR of 1.3g/day and the lowest FCR of 1.4. Significant difference in SGR was observed between fish fed with SDPKC and UPKC. Fungi degraded palm kernel cake and rice bran supported the growth of Clarias gariepinus better than the control feed and could be good sources of fish feed components.
SCREENING FOR BIOACTIVE COMPOUNDS IN FIVE SELECTED NIGERIAN MEDICINAL PLANTS AGAINST SOME PATHOGENIC MICROORGANISMS
(2014-03) ONAJOBI, I. B.
The search for new drugs to combat infectious diseases has stimulated the interest of scientists globally due to emergence of Multi Drug-Resistant (MDR) microorganisms. Indigenous medicinal plants are potential reservoir of bioactive compounds from which new active drugs could be obtained, but are yet to be adequately exploited. This study was therefore designed to screen for new bioactive compounds from some indigenous medicinal plants against selected MDR microorganisms. Harungana madagascariensis Lam. Ex Poir and Enantia chlorantha Oliv. barks, Senna alata Linn., Gossypium hirsutum Linn. and Alstonia bonnie De Wild leaves were collected from a farmland in Idi-Ayunre, Ibadan and authenticated at Forest Research Institute of Nigeria. Ethanol extracts from the leaves and barks were tested against reference strains of Escherichia coli, Bacillus subtilis, Salmonella typhi, Shigella flexneri, Pseudomonas aeruginosa and Staphylococcus aureus, Microsporum canis, Candida albicans, Candida glabrata, and Aspergillus flavus using Agar well diffusion method. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extracts were determined using tube dilution method. Extract purification was carried out using column chromatographic techniques. Chemical structures of compounds obtained were determined using Mass spectroscopy, Ultraviolet spectroscopy, Infra-Red spectroscopy and (1D and 2D) Nuclear Magnetic Resonance. Purified compounds which showed the best antimicrobial activities were tested against MDR clinical strains of P. aeruginosa and S. aureus compared to standard antibiotics. The compounds’ antioxidant Radical Scavenging Activity (RSA), and in vitro alpha-glucoxidase enzyme inhibition activity was measured at 400 nm using Ultraviolet-Visible absorption spectrophotometer with arcabose as positive control. Data were analysed using ANOVA at p = 0.05. Harungana madagascariensis, E. chlorantha and S. alata crude extracts showed broad spectrum antibacterial activity, inhibiting all the tested bacterial species with 24.3±0.3, 25.7±0.3 and 27.7±0.6 (mm) diameter zones of inhibition respectively. All the extracts except A. bonnie leave extract exhibited more than 90% inhibition against M. canis. Enantia chlorantha and H. madagascariensis bark extracts showed highest inhibition against C. glabrata (80%) and C. albicans (70%) respectively. The MIC of the extracts ranged from 5.0 to 20.0 mg/mL while the MBC ranged from 20.0 to 30.0 mg/mL. Minimum fungicidal concentration of 50.0 mg/mL of the extracts inhibited all the tested fungi. Five new prenylated anthranoids (Harundigin anthrone, kenganthranol D, E, F and G) from H. madagascariensis were identified. These purified compounds at 100 μg/mL inhibited all the tested microorganisms. The MDR S. aureus was susceptible to isolated kenganthranol G with MIC of 25 µg/mL, but was resistant to standard antibiotics used (oxacillin, amikacin, chloramphenicol, erythromycin, sulfamethozale and ciprofloxacin). Harungana madagascariensis exhibited most significant antioxidant activities against 1,1-Diphenyl-2-picrylhydrazyl radical with 92% RSA, IC50 (33.3±1.8) at 0.5 μg/mL. Harundigin anthrone and kenganthranol E were good inhibitors (97%) of alpha-glucooxidase at IC50 of 69.9±4.2 and 122.4±1.1 respectively as against arcabose with 59.1%. The prenylated anthranoids obtained from the plants exhibited antimicrobial and anti-enzymatic activities against multidrug-resistant S. aureus. They could serve as alternative source of bioactive compounds against resistant strains of S. aureus.
PLASMID PROFILE, METHICILLIN RESISTANCE DETERMINANTS AND CHARACTERISATION OF STAPHYLOCOCCUS SPECIES ISOLATED FROM CLINICAL AND COMMUNITY ENVIRONMENTS IN IBADAN
(2014-03) EZEAMAGU, C. O.
Methicillin-resistant staphylococcus (MRS) infections are of global concern in healthcare institutions and community settings with significant morbidity and mortality due to multi-drug resistance challenges. In Nigeria, most methicillin resistance detection was based on phenotypic method with scanty reports on molecular characterisation of MRS. In this study, molecular techniques were used to determine the presence of methicillin resistant gene (mecA) with its associated resistance determinants (vanA and blaZ) and plasmid profile of staphylococci isolated from clinical and community samples. Staphylococcus species from clinical (55) and community (53) samples were isolated from air, selected waste water drainages and human swabs (eye, semen, ear, high vagina swab, throat, urethra, wound, nostril, skin) in the University of Ibadan and University College Hospital. They were identified using standard microbiological procedures. The isolates identity was confirmed to genus level using 16S-rRNA specific primers and identified to species level by PCR-Restriction Fragment length Polymorphism supplemented with PCR species-specific-primers. The isolates were phenotypically screened for resistance to methicillin and other antibiotics by agar diffusion method. Multiplex PCR was used to assess presence of mecA and the resistant determinants while Simplex PCR was used to determine the origin of mecA isolates by detecting the presence of Panton-Valentine Leukocidin (PVL). Plasmid profiles of clinical (35) and community (19) isolates with multiple drug resistance were determined using standard procedures. Data was analysed by descriptive statistics. The organisms were identified as S. epidermidis (92.6 %), S. aureus (6.5 %) and S. xylosus (0.9 %). Phenotypic resistance to methicillin was 72.7 and 62.3 % in clinical and community isolates respectively. In the clinical isolates of S. epidermidis, 30.9, 32.7, 34.5, 40.0, 41.8, 60.0, 76.4, and 89.1 % were resistant to Chloramphenicol, Vamcomycin, Streptomycin, Erythromycin, Gentamycin, Tetracycline, Cotrimoxazole and Cloxacillin respectively. Correspondingly, in community isolates of S. epidermidis, 28.3, 3.8, 32.1, 50.9, 26.4, 58.5, 90.6 and 92.5 % were resistant to these antibiotics. In the clinical isolates of S. aureus, 3.6, 5.5, 5.5, 7.3, 7.3, 7.3, 9.1 and 9.1 % were resistant to Vamcomycin, Erythromycin, Chloramphenicol, Streptomycin, Gentamycin, Tetracycline, Cotrimoxazole and Cloxacillin respectively. In community isolates, 1.9 % S. aureus were resistant to Cotrimoxazole, Chloramphenicol, Erythromycin, Gentamycin and Streptomycin while 3.8 % were resistant to Cloxacillin. Among clinical isolates of S. xylosus, 1.8 % was resistant to all the antibiotics except Chloramphenicol and Streptomycin. All the strains lacked vanA gene, while only clinical isolates (3.6 %) had mecA when its specific primers were used and 5.5 % using its regulatory element specific primers in PCR. The blaZ gene was found in 16.4 % of clinical and 1.8 % of community isolates. There was no PVL in the isolates with mecA. Plasmid size of 23.13kb was found in 94.3 % of clinical and 84.2 % of community isolates. The detection of blaZ gene in community isolates showed that such resistance determinants predominantly found in clinical isolates are also emerging in the community isolates. Hence, setting up antibiotic surveillance system is necessary to minimize this trend.
MOLECULAR CHARACTERISATION OF ANTIBIOTIC-RESISTANT BACTERIA FROM SELECTED WATER DISTRIBUTION SYSTEMS IN SOUTHWESTERN NIGERIA
(2014-05) ADESOJI, A. T.
The presence of Antibiotic-Resistant Bacteria (ARB) in water sources and treated drinking water is an emerging public health issue. Antibiotic resistance genes and mobile genetic elements (integron and gene cassettes) have been reported as bases of resistance in ARB. Available data on ARB in southwestern Nigeria are based on phenotypic studies. Information on molecular basis of resistance in ARB is necessary to determine the mode of resistance transfer among bacteria. This research was therefore aimed at molecular characterisation of ARB in water distribution systems of dams in Southwestern Nigeria. Ninety-six water samples were purposively collected aseptically into sterile screw cap bottles from six selected water distribution systems of dams in Ife, Ede, Asejire, Eleyele, Owena-Ondo and Owena-Idanre in Southwestern Nigeria. Samples were collected four times between December 2010 and July 2011 from raw, treated and two randomly selected municipal distribution taps. Bacteria were isolated from water samples and characterised using 16S rDNA sequencing. Antibiotic susceptibility of isolates was determined using point inoculation method. Multi-Drug Resistant (MDR) bacteria were selected based on resistance to over three classes of antibiotics and resistance genes were characterised by PCR and microarray analysis. Class 1 integron was detected by PCR while variable regions of integrase positive isolates were sequenced to identify inserted gene cassettes. Data were analysed using descriptive statistics and ANOVA at p=0.05. A total of 292 bacteria isolates were obtained. The highest (11.0%) bacteria occurrence was from Owena-Idanre raw water while the lowest (0.3%) was obtained from Eleyele treated water. They were identified as α-proteobacteria (4), β-proteobacteria (39), γ-proteobacteria (131), bacteroidetes (4), actinobacteria (2), firmicutes (105) and uncultured bacteria (7). Resistance to tetracycline, sulfamethoxazole, ampicillin and streptomycin among the 191 found to be MDR bacteria were 62.8, 93.7, 90.0 and 52.8 % respectively. Significant variation was observed between percentage of MDR bacteria in Owena-Idanre dam (12.6%) and those from Ife dam (4.2%). The most frequent resistance genes detected among MDR bacteria were ant(3’)b (56/101), blaTEM (59/172), sul 2 (51/179) and tetA (23/120). The highest incidence of ant(3’)b (12.5%) was found among bacteria obtained from Eleyele raw water while tetA (13.0%) was from Owena-Ondo treated water. Other antibiotic resistance genes such as floR, tetJ, tetH, StrB, qnrA1, dfrA21 and aadE from bacteria isolated in Asejire treated water and Owena-Idanre municipal taps were detected with Microarray. Class 1 integron was found in 42 bacteria with the highest frequency (14.3%) in Eleyele raw water while 2.4% each were obtained from Ede, Asejire and Eleyele municipal taps. Variable region was detected from 69.0% of bacteria carrying class 1 integron. Gene cassettes identified in the variable region of class 1 integron include aadA1, bla CTX-M, dfrA, Sul 1 and qnr A1 encoding resistance to aminoglycoside, extended β-lactams, trimethoprim, sulfamethoxazole and quinolones respectively. The presence of multi-drug resistant bacteria carrying different antibiotic resistance genes, integrons and gene cassettes as revealed by molecular characterisation, in the water distribution systems could facilitate the transfer of antibiotic resistance among bacteria of public health significance, hence the need for surveillance.
ANTIMICROBIAL AND ANTI – INFLAMMATORY ACTIVITIES OF EXTRACTS OF FICUS THONNINGII BLUME (MORACEAE)
(2014-05) COKER, M. E.
Infectious diseases and the associated inflammation pose a serious health problem worldwide, accounting for about 50% of all deaths in tropical countries. This is further complicated by the frequent development of bacterial resistance to many chemotherapeutic agents. These problems have necessitated the continuous search for new and effective drugs from plant materials. Thus, Ficus thonningii Blume (Moraceae), a plant used ethnomedicinally in West Africa for the treatment of some microbial infections, was studied for its antimicrobial and anti-inflammatory activities. Dried leaves and stem bark of Ficus thonningii were screened for secondary metabolites. Successive gradient extraction was carried out on the pulverised plant parts using hexane, chloroform and methanol with Soxhlet apparatus. Antimicrobial activity of the extracts on Gram-positive (10) and Gram-negative (11) bacteria, and fungal (12) isolates was evaluated using agar-diffusion method. Antibiogram of the microorganisms was determined using established antibiotics. Bioassay-guided fractionation of crude extracts using column chromatography was done. Minimum Inhibitory Concentrations (MIC) and minimum bactericidal concentrations of the crude extracts, fractions and isolated compound were determined by agar-dilution. Bactericidal kinetics of the methanol leaf extract against Staphylococcus aureus and Escherichia coli at 2.5-10.0 mg/mL were determined. Structure elucidation of the bioactive compound was carried out using 1H-NMR, 13C-NMR, DEPT 135, COSY, UV and GC-MS spectroscopy. In vivo anti-inflammatory activity of leaf extract was evaluated using carrageenan-induced rat paw oedema with acetylsalicylic acid as the reference drug. Acute oral toxicity, haematological and histopathological evaluations were carried out to determine the safety profile of methanol leaf extract in rats. Statistical analysis was carried out using Student’s t-test at p = 0.05. Alkaloids, flavonoids, terpenoids and cardiac glycosides were detected in the plant extracts. Antimicrobial assay of crude extracts and fractions showed a broad spectrum activity on sensitive and multidrug-resistant strains with the leaf and stem bark extracts having similar antimicrobial activity. Hexane leaf extract and bioactive fractions gave MIC range of 78-625 µg/mL and 20-625 µg/mL respectively while methanol leaf extract and bioactive fractions gave 156-625 µg/mL and 39-625 µg/mL. Structure elucidation of the bioactive compound isolated from hexane leaf fraction revealed a triterpenoid with MIC range of 20-156 µg/mL (Gram-positive bacteria), 39-156 µg/mL (Gram-negative bacteria) and 10-78 µg/mL (fungi), while that of gentamicin and tioconazole were 5-30 µg/mL and 10-20 µg/mL respectively. Methanol leaf extract showed bactericidal activity in a concentration-dependent manner on the microorganisms, with a 100% bactericidal action at 10 mg/mL on Staphylococcus aureus and 84% on Escherichia coli within 4 hours. The anti-inflammatory activity of methanol leaf extract was 57.5% while that of acetylsalicylic acid was 93.2%. Acute oral toxicity of methanol leaf extract showed an LD50> 5g/Kg. Significant increases were observed in the red blood cell count and mean corpuscular haemoglobin value, while histopathological evaluation revealed no significant tissue pathological changes in the major organs. Extracts of Ficus thonningii leaves contain antimicrobial and anti-inflammatory agents. These could be useful in the development of safe chemotherapeutic agents for the treatment of relevant microbial infections and inflammation-prone diseases.
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.
OCCURRENCE, DIVERSITY AND ANTIMICROBIAL SUSCEPTIBILITY OF Listeria SPECIES FROM RETAIL RAW BEEF AND GOAT MEAT IN PORT HARCOURT, NIGERIA
(2015-09) ERUTEYA, O. C.
Listeriosis has emerged as a disease of public health importance because of its clinical severity, high fatality and challenges to its control including resistance to antibiotics. Outbreaks reported in developing countries including Nigeria were associated with consumption of meat and meat products. However, previous work on Listeria in Nigeria focused on environmental samples. Hence, the need to ascertain the presence of Listeria in meat and their response to antimicrobial agents. The study was designed to investigate the occurrence, diversity, virulence and antimicrobial resistance of Listeria species in raw beef and goat meat in Port Harcourt, Nigeria as well as control of Listeria monocytogenes using plant extracts. A total of 240 meat samples (122 beef and 118 goat), comprising flesh (72), kidney (60), intestine (38) and liver (70) were purchased from Choba, Rumuokoro and Rumuokwuta markets. Bacteria were isolated from the meat and tested for the presence of Listeria spp using Listeria selective media. Isolates were identified using conventional and molecular methods. The isolates were screened for susceptibility to selected antibiotics using disk diffusion method. Virulence genes and plasmid were screened using molecular methods. Thymus vulgaris L., Allium sativum L., Piper guineense Schum and Thonn, Monodora myristica (Gaertn) Dunal, Ocimum gratissimum L. and Xylopia aethiopica (Dunal) A. Rich were separately extracted with ethanol and water. Effects of the extracts (0.1-5.0%) were determined against L. monocytogenes (1× 108 cell/mL) in agar well diffusion, treated and untreated meat which served as control using standard methods. Data were analysed using descriptive statistics and ANOVA at p=0.05. Eighty-one (33.8%) samples were positive for Listeria spp. Listeria isolates identified were L. monocytogenes (4), L. innocua (20), L. ivanovii (4), L. seeligeri (72), L. welshimeri (139), and L. grayi (71). All isolated Listeria were susceptible to gentamicin and vancomycin but resistant to amoxicillin (100%), augumentin (100%), cloxacillin (100%), tetracycline (88.5%), oxacillin (73.6%), erythromycin (43.7%), chloramphenicol (43.7%) and cotrimoxazole (33.3%). Virulence genes such as inlJ and inlC were detected in L. monocytogenes. Aqueous extract of P. guineense and T. vulgaris as well as ethanol extract of T. vulgaris inhibited L. monocytogenes growth at concentrations ranging from 1.0-5.0%, 0.5-5.0% and 0.1-5.0%, respectively. The reduction of L. monocytogenes attached to meat pieces varied during a 15 minutes immersion, ranging from 0.8 to 1.4 log10cfu/g. After 4-day storage at 30oC, L. monocytogenes exhibited significant higher growth in control samples compared to extract treated samples with initial population in log10 cfu/g increasing from 5.4-6.6 to 9.1-10.2 and 5.3-6.5 to 8.5-9.9 respectively. There was no significant difference between control and treated samples stored at 10oC for 15 days, although initial population increased from 5.4-6.6 to 8.5-8.8 log10cfu/g and 5.2-6.5 to 8.2-8.5 log10cfu/g respectively.
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.
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.