Theses and Dissertations

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Open Access
Bacteriological Studies of Open Fractures at the Obafemi Awolowo University Teaching Hospitals Complex (O.A.U.T.H.C), Ile-Ife
(2015-06-26) Daniels, Folashade Victoria
This study was aimed at isolating the bacteria associated with open fractures, enumerating the antibiotic sensitivity profiles and production of enzyme with virulent potential which could increase the virulence/pathogenicity of such microorganism. Fractures were examined according to Gustilo and Anderson's classification as grade I (2.1%), grade II (29.8%), grade IIIa (36.2%) and grade IIIb (31.5%). Samples were taken from superficial swab, superficial biopsies, deep swabs as well as deep biopsies making a total of four samples per patient at presentation, A total of 180 samples obtained on day 1, 42 samples were obtained on day 3 and 26 were obtained on day 7.Each sample was introduced onto thioglycolate broth to enhance the growth of anaerobic organism. Serial dilutions were made from these samples and plated out on nutrient agar. Incubation was allowed for 24 hrs at 37°C for the viable cell counts at each presentation. The samples in thioglycolate broth were then incubated for 24h at 37°C. Positive samples were plated out onto differential medium so as to identify some of the isolates and selective medium. Negative cultures that demonstrated fowl smells were plated out onto lactose egg yolk milk agar and neomycin blood agar and incubated anaerobically using Gas Pak for 24h at 37°C and extended to seven days and beyond to aid the growth of strict anaerobes. Discrete colonies were further characterized by standard methods. The results showed the prevalence of open fractures was higher in male 37(78.7%) than in females (21.3%).The results showed that tibial fractures constituted 66.1% and were mainly from road accident (RTA). A total of 203 bacterial isolate were cultured from the forty-seven patients with 53 from superficial swabs, (52) from superficial biopsies, (52) from deep swabs and (46) from deep biopsies were cultured. Gram negative bacteria isolates constituted 53.2% of total bacterial, Escherichia coli was the commonest with 26 (12.8%) followed by Salmonella spp and Pseudomonas aeruginosa occurring at the rate of 8.9%. Gram positive cocci constituted 58(28.6%) with Staphylococcus aureus constituting 31(15.3%) and S. epidermidis constituting 27(13,3%). A high incidence of antibiotic resistance was observed against penicillins such as amoxicillin and cloxacillin with values 72(68.6%) and 28(58.3%) respectively for superficial specimens and 57(58.2%) and 15(31.9%) for deep samples. Bacteria isolates cultured were relatively resistant to tetracycline with values 57(54.%) and 41(41.8%) in superficial and deep samples respectively but considerably sensitive to gentamycin and nutrofurantoin while quinolones especially ofloxacin were noted for high effectiveness and against screened bacterial isolates. 75% and 87.5% of Staphylococcus aureus cultured produced lipase and DNase respectively from deep samples while all the Pseudomonas aeruginosa stains produced DNase with 77.5% and 75% lipase production in superficial and deep samples respectively. In conclusion, both Gram-positive and Gram-negative organisms were relatively sensitive to gentamycin especially Pseudomonas aeruginosa which is known for high resistance against most antimicrobials. Ofloxacin was also highly effective against Gram negative organisms.
Open Access
The Effects of Bacterial Enzymes and Biosurfactants on Hydrocarbon Degradation in a Crude Oil Polluted Fresh Water
(2015-05-05) Olajide, Patience Orobosa
The present work was designed to identify extracellular enzymes and biosurfactants from five bacterial isolates and investigate their actions on crude oil degradation as a means of bioremediation of hydrocarbon pollution. The Five bacterial isolates including Proteus vulgaris, Bacillus subtilis, Pseudomonas aeruginosa, Sarcinae litoralis, and Alcaligenes viscolatis were selected for the study based on their relatively high efficiency for crude oil utilisation and their wide occurrence in the Niger Delta basin of Nigeria. The types of enzymes produced by the selected isolates were determined using standard API ZYM kit they were produced in spent media. The activity of the enzymes on crude oil biodegradation was investigated using the enzyme inhibition method. Biosurfactants were extracted from cultures grown in a neutral medium (pH 7.0) containing 2% glucose, shaken at 150 rpm at 37°C for 7 days. Qualitative drop-collapse, blue agar plate, haemolytic, emulsification activity (E24), oil spreading techniques as well as the swirling beaker test were used to confirm the production of biosurfactant. The effect of pH, temperature and salinity concentration on biosurfactant production was evaluated using standard instrumental methods. Biosurfactants were evaluated using acid-precipitation followed by extraction using chloroform-methanol (2:1). The anthrone positive fraction of the biosurfactants was identified by measuring extract absorption in an iodine-polysaccharide complex using a spectrophotometer NOVASPEC II, (Pharmacia Biotech) at wavelengths range of 380 to 700 μm. Enzyme systems detected with the API ZYM kit varied depending on the growth substrate used. Enzyme inhibition slowed down degradation of crude oil and the enzyme activity behaved similarly over time whereas the effect of catalase, cytochrome c oxidase and lipase increased the biodegradation of crude oil in the enzyme cultures medium. All the microorganisms investigated produced highest biosurfactant in glycerol medium (1.26-1.64) mg/1 during a 48 h of growth. Biosurfactants production was optimum at pH 6.2 and 7.2 and temperature of 37°C and 2% NaCl. All biosurfactants emulsified oil to varying degrees with varying emulsification index (E24) with over 60% emulsification activity. They were all stable with temperature between 15 and 90°C and pH range of 4.2-10.2. Only three isolates haemolysed blood agar and formed dark blue halos on agar plates indicating the production of glycolipids. The complex formed between the reaction of iodine with the polysaccharide of the biosurfactants had a maximum absorption wavelength between 380 to 420 μm. In conclusion, the enzymes and biosurfactants produced by the investigated bacterial isolates were effective in degrading crude oil.
Open Access
A Study of Antibiotics Susceptibility Profile of Clinical Bacterial Isolates in Ile-Ife and the Minimum Bactericidal Concentrations of Selected Antibiotics
(2015-05-04) Oladapo, Oluwabunmi Oluwatoyin
The study evaluated the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of selected antibiotics. This was with a view to determine the antibiotic resistance profile of selected bacterial isolates from clinical sources in Ile-lfe. Clinical specimens were taken from 196 patients from Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Seventh Day Adventist Hospital (SDAH), Obafemi Awolowo University Health Centre (OAUHC) and Osun State Comprehensive Health Centre (OSCHC) in Ile-lfe from July 2005 to June 2006. A total of 222 bacterial isolates were cultured from the specimens. Using the broth macrodilution method, the MIC and MBC of some commonly used antibiotics in the environment were assayed. The antibiotic susceptibility patterns were determined by the Kirby-Bauer disc-agar diffusion method. Descriptive statistics was used to analyse the data. The results showed that out of 222 isolates tested, 86.04% were resistant to amoxicillin, 74.77% to cotrimoxazole, 65.76% to tetracycline and 59.46% to augmentin. Of the 145 isolates tested with nitrofurantoin, nalidixic acid and ofloxacin. 48.96°/x, were resistant to nalidixic acid, 43.50% to nitrofurantoin, and 13.10% to ofloxacin. The results also showed that the incidence of multiple resistant bacterial isolates in the four hospitals ranged from 91.9% for OAUHC to 94.8% for OSCHC. These values were higher than those recorded in previous studies in the same environment. The ratio of MIC to MBC revealed that some of the antibiotics had lost their effectiveness against the organisms on which they were tested. Ofloxacin (a quinolone) was found to be the most effective against pseudomonas aeruginosa with MBC/MIC ratio of 1 while gentamicin (an aminoglycoside) was the most effective against Staphylococcus aureus with MBC/MIC ratio of 1. The study concluded that the phenomenon of bacterial resistance to antibiotics is increasing and has become widespread in Ile-Ife.
Open Access
Mechanism of Immunity against Campylobacter Jejuni in Mice
(2015-04-20) Ewuola, Kola Sambakeyu
The roles of B-lymphocytes, T-lymphocytes and macrophages in the mechanism of immunity against Campylobacter jejuni in mice were investigated. This was done in an attempt to proffer solution to the problem of diarrhea through the enhancement of the immune mechanism involved in the protection against this agent. The roles of B-lymphocytes, T-lymphocytes and macrophages were investigated by injecting different sets of mice with carrageenan, anti-C-jejuni serum, and anti-thy mocytic serum obtained from rabbit respectively after being injected with I ml of C-jejuni cell suspension containing approximately 4.6x104 cells were sacrificed at one-day, three- day, five- day, and seven-day intervals. The liver and the spleen were removed, homogenized, serially diluted, plated and incubated for bacterial cell count. The data collected were subjected to statistical analysis using student’s t-test. The result showed that higher bacterial populations were found in the organs (liver and spleen) of the carrageenan-treated mice than in the control mice within the first twenty four hours post infection. Since this substance was toxic, the high bacterial population organs indicated that the macrophages were rendered inactive. However, a fall in the bacterial population later in the course of infection was observed. In the liver of the mice that were given anti-thy mocytic serum, the activity of the T-lymphocytes was hindered till seventy two hour post infection as shown by high bacterial count in this organ. Thereafter, there was a decrease in bacterial population which implied that the T-Iymphocytes had become more active in fighting the bacteria as a result of the loss of inhibitory potency by the anti-thymocytic serum. In the spleen of experimental set of mice, the bacterial count remained lower than in the control (t=. 0 0 0 , p=1.000, P> 0.05) mice throughout the period of the experiment. In the passively immunized mice, growth patterns in both the liver and the spleen were similar to those of control within the first twenty four hours post infection ( t = 632, P= 561 P> 0.005) In the liver, an increase in bacterial population was observed after twenty four hours (t = 2.828, P =.047, P<0.05) while in the spleen, there was an increase in bacterial count Followed by a decrease in population (t= 5.330, p = 0.06 p<0.05). The result showed that at some stages in the course of infection, there existed significant difference (P<0.05) in the means of the bacterial populations in the liver and the spleen. The study concluded that macrophages. T-lymphocyte and antibody complemented one another in effecting immunity to this diarrhea-causing pathogen in the mice. This implied that the immune mechanisms involved were both cellular and humoral.
Open Access
Production and Characterization of Biosurfactants from Petroleum Degrading Aquatic Bacteria
(2015-03-20) Adedeji, Yewande Olufunto
This study was undertaken to produce, isolate and characterize biosurfactants from oil degrading bacteria using various carbon sources and determine the conditions under which specific oil degrading bacteria will produce optimally, biosurfactants required for optimal oil degradation. Six bacterial isolates, namely Bacillus subtilis, Proteus vulgaris, Pseudomonas putida, Pseudomonas oleovorans, Sarcinae litoralis and S58 (an unidentified bacteria) were maintained on nutrient agar slants and subcultured every forthnight. Standardized cultures (1.0 ml) of the bacteria isolates were inoculated into 100 ml capacity conical flasks each containing 50 ml mineral salts medium (MSM) and 0.2 % (v/v) Escravos light crude oil was aseptically added as the sole carbon source. The cultures were agitated in an incubator shaker at 37 °C for a week. The presence and activities of the biosurfactants produced by each bacterial isolate was detected using the swirling beaker method. The growth rates of the bacteria were measured using a spectrophotometer and the best condition of pH, temperature, salinity, concentrations of nitrogen, iron, and magnesium ions which each bacterial species produced optimally were studied. The biosurfactants were isolated and characterized spectrophotometrically between 380 nm and 700 nm and chemically by spraying the TLC chromatograms with specific reagent such as iodine. The result showed that the maximum absorption wavelength of the iodine-polysaccharide complex was almost the same for each biosurfactant from the various bacteria and ranged from 380 nm to 420 nm, which was characteristic of a polyglucose unit. The reducing sugars from all the samples proved to be glucose units. A long chain fatty acid was obtained from all the samples which exhibited Rf values close to that of Myristic and Stearic acids. The results further revealed that all the isolates emulsified oil in the swirling beaker test, indicating the presence of biosurfactants. The biosurfactant produced was composed of polyglucose units and fatty acid which is characteristic of glycolipids. During the growth of these bacteria on crude oil-MSM, the oil was degraded best at 28 °C and pH 6.8 and 7.2. Biosurfactant production increased at concentrations of 0.5 % w/v for NaCl and magnesium but decreased as the concentration was increased to 1.0 % w/v and later to 1.5 % w/v. Production of biosurfactant increased at a concentration of 1.0 % w/v for nitrogen. The bacteria produced biosurfactant when diesel was used as the sole source of carbon. It was concluded that the bacteria employed in this study would be useful in the biodegradation of oil in petroleum contaminated sites because of their ability to produce biosurfactant and emulsify crude oil.

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