The primary objectives of this research were to demonstrate that: 1.) antibiotic resistant bacteria can promote the survival of antibiotic sensitive organisms when grown simultaneously as biofilms in antibiotics, 2.) community-level multiple antibiotic resistance of polymicrobial consortia can lead to biofilm formation despite the presence of multiple antibiotics, and 3.) biofilms may benefit plasmid retention and heterologous protein production in the absence of selective pressure. Quantitative analyses of confocal data showed that ampicillin resistant organisms supported populations of ampicillin sensitive organisms in steady state ampicillin concentrations 13 times greater than that which would inhibit sensitive cells inoculated alone. The rate of reaction of the resistance mechanism influenced the degree of protection. Spectinomycin resistant organisms did not support their sensitive counterparts, although flow cytometry indicated that GFP production by the sensitive strain was improved. When both organisms were grown in both antibiotics, larger numbers of substratum-attached pairs at 2 hours resulted in greater biofilm formation at 48 hours. For biofilms grown in both antibiotics, a benefit to spectinomycin resistant organism’s population size was detectable, but the only benefit to ampicillin resistant organisms was in terms of GFP production. Additionally, an initial attachment ratio of 5 spectinomycin resistant organisms to 1 ampicillin resistant organism resulted in optimal biofilm formation at 48 hours. Biofilms also enhanced the stability of high-copy number plasmids and heterologous protein production. In the absence of antibiotic selective pressure, plasmid DNA was not detected after 48 hours in chemostats, where the faster growth rate of plasmid-free cells contributed to the washout of plasmid retaining cells. The plasmid copy number per cell in biofilms grown without antibiotic selective pressure steadily increased over a six day period. Flow cytometric monitoring of bacteria grown in biofilms indicated that 95 percent of the population was producing GFP at 48 hours. This research supports the idea that ecological interactions between bacteria contribute to biofilm development in the presence of antibiotics, and demonstrates that community-level multiple antibiotic resistance is a factor in biofilm recalcitrance against antibiotics. Additionally, biofilms may provide an additional tool for stabilizing high copy number plasmids used for heterologous protein production.

Surface mucus of a freshwater fish, Alburnus alburnus (bleak), caught from Lake Mogan, situated in south of Ankara, was collected in different seasons. The total cultivable bacteria were enumerated by spread plate method on nine different media. Bacteria were isolated based on colony morphologies and pigmentation. A total of sixty bacterial isolates obtained. The mucus-dwelling bacteria were first tested for resistance against ampicillin and kanamycin / then streptomycin and chloramphenicol were added to the experimental set up. The resistance levels of isolates were determined in terms of four antibiotics by tube dilution method. About 90% of the isolates were resistant to chloramphenicol, about 84% to kanamycin, about 88% to streptomycin and about 98% to ampicillin. These high levels of antibiotic resistance are rather interesting from a standpoint that the lake has no record of antibiotics exposure of any sort. The plasmid isolations were carried out to determine if the multiple antibiotic resistance could be attributed to plasmids for starting assumption. But we found no direct relationship between the presence of plasmids and multiple antibiotic resistance. Our study indicated that multiple antibiotic resistance at high levels is among the current phenotypes of the fish mucus-dwelling bacterial populations in Lake Mogan.

To combat the ever-growing clinical burden imposed by antibiotic-resistant pathogens, multiple-antibiotic treatments are increasingly being considered as promising treatment options. The impact of multiple-antibiotic treatments on the evolution of resistance is not well understood however, and debate is ongoing about the effectiveness of various multiple-antibiotic treatments. In this thesis, I investigate how aspects of multiple-antibiotic treatments impact the rate of evolution of antibiotic resistance in the opportunistic human pathogen Pseudomonas aeruginosa. In particular, I look at the impact of interactions between antibiotics in combination on the evolution of resistance, and how creating heterogeneity in the antibiotic environment by rotating the antibiotics used may change the rate of evolution of resistance. I characterise the interactions present in 120 combinations of antibiotics and find that the type of interaction can be predicted by the mechanism of action of the antibiotics involved. I investigate the effect of a subset of these combinations on the evolution of antibiotic resistance. My results refute the influential but poorly-evidenced hypothesis that synergistic combinations accelerate the evolution of resistance, even when synergistic combinations have the same inhibitory effect on sensitive bacteria as additive or antagonistic antibiotic combinations. I focus on a combination of the antibiotics ceftriaxone and sulfamethoxazole and test whether it is more effective in preventing the evolution of resistance than predicted by the inhibitory effect of the combination on sensitive bacteria. I do not find the combination to be more effective than predicted. Finally, I create heterogeneous antibiotic environments by rotating the antibiotic present at different rates. For the first time in a laboratory setting, I test how varying the rate of fluctuation in the antibiotics present in a heterogeneous antibiotic environment impacts the rate of evolution of resistance. Unexpectedly, I find the rate of evolution of resistance increases with increasing levels of antibiotic heterogeneity.

Decreasing surface water quality in South Africa has become an issue of concern as the population grows, industrial and agricultural activities expand, and environmental pollution increases. Wastewater treatment plants and other anthropogenic activities are liable for releasing raw and inadequately treated effluents into the surface water. Extensive pollution accompanied by the use of disinfectants, pesticides, and other chemical pollutants has been attributed to increased antimicrobial resistance in bacteria such as Escherichia coli in surface water, increasing environmental antibiotic resistance spread. The research aimed to determine water quality and prevalence of antibiotic-resistant bacteria in Naauwpoortspruit River, eMalahleni, Mpumalanga Province. Five sampling sites were selected along the Naauwpoortspruit River and monitoring was done for seven consecutive months. Samples were collected and analysed for physicochemical, microbiological parameters, and susceptibility profile of antibiotic-resistant bacteria using standard methods. Pearson correlation analysis was used to assess the path and strength of the relationship between physicochemical and microbiological parameters in the study area. Results of physicochemical and microbial parameters showed variation throughout the selected study sites. The results revealed a pH range of 4.45 – 7.9 and electrical conductivity levels range of 58.63 - 113.3 mS/m for the different sampling sites during the study period with lower levels detected during the winter period and higher levels in the summer period. Also, water samples showed a high total dissolved solids levels range of 381.1 – 736.45 mg/L and biochemical oxygen demand range of 67.1 – 168 mg/L for the different sampling sites during the study period. The Naauwpoortspruit River had higher levels of ammonia of 33.4 mg/L at Point A during the winter period as compared to 15 mg/L in the summer period. Heavy metals results showed that mercury range of 0.01 – 0.065 mg/L and copper range of 0.001 – 0.0035 mg/L were not compliant with aquatic ecosystem guidelines at all selected sites throughout the study period. The foremost finding of this study was that E. coli were present in all the selected sites at concentrations (>100 cfu/100ml). Elevated concentrations of 5.4 x 103 and 4.2 x 103 cfu/100ml for the total and faecal indicator bacteria were detected from sites downstream to 2.2 x103 and 2.35 x103 cfu/100ml for sites upstream river, in the rainy months. During the dry season, total coliforms, and faecal coliforms concentration of 0.4 x103 to 0.65 x 103 cfu/100ml were detected downstream and 0.25 x 103 and 0.5 x 103 cfu/100ml from upstream, respectively. The physicochemical and microbiological parameters measured at selected sites exceeded acceptable limits and proved unsuitable for applications such as full and intermediate recreational activities, and aquatic ecosystems. The variation in physicochemical parameters results was influenced by both natural processes and human activities such as salinity and Acid Mine Drainage (AMD) within the Naauwpoortspruit River. Using the Kirby-Bauer disc diffusion method, E. coli and faecal coliforms were tested for resistance to antibiotics; ampicillin (10 μg/ml), kanamycin (30 μg), streptomycin (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), ox tetracycline (30 μg), erythromycin (15 μg/ml) and norfloxacin (10 μg). More than 60% of faecal coliform were resistant to at least four of the tested antibiotics and between 60 - 80% of the E. coli isolates were resistant to β lactam. The highest microbial antibiotic resistance (MAR) index value was observed at Site D (0.38 for E. coli) which showed multi-antibiotic resistance. Site D is characterized by wastewater treatment, power generation industries, and agriculture activities. The highest level of MAR observed at Site D indicates the need to control extensive pollution and constantly monitor the changing trends in antimicrobial resistance patterns of these waterborne pathogens. Statistical analysis showed that the development of microbiological parameters loads has a strong correlation with physicochemical parameters due to the association of sampling sites in the river environment. This study shows that the aquatic ecosystem needs constant monitoring to establish their conditions, impacts of pollution activities within the catchment, and input information into sustainable management of the water resources. / Environmental Sciences / M. Sc. (Environmental Science)