Salmonella spp. is a human pathogen that has been reported in catfish, but with conflicting results. Salmonella spp. was isolated from live catfish, catfish products and the processing environment during catfish production, followed by evaluation of their antibiotic resistance and clonal similarities. Distinction of Salmonella spp. was increased by lowering background microflora with the addition of the antimicrobial novobiocin to the agar media. More than ten Salmonella serotypes were isolated from catfish and catfish products, such as live, chilled fillets, frozen fillets, and conveyor belts in catfish processing facilities. The isolates that were recovered include Salmonella ser. Typhimurium, Barranquilla, Mbadaka, Putten, Infantis and Thompson among others. The number of isolated Salmonella spp. and serotypes varied between sampling in catfish facilities. Clonal similarities of Salmonella spp. were found within sampling but did not show persistency among sampling periods, suggesting the opportunistic nature of the pathogen. Salmonella ser. Typhimurium was the most predominant isolate in live catfish and similarities were found within sampling but were not persistent among sampling periods. Antimicrobial resistant Salmonella was identified from the recuperated isolates. All Salmonella spp. isolates, showed resistance to erythromycin, vancomycin and rifampin regardless of the serotype, but resistant genes were absent suggesting that resistance was due to the pathogen’s biological nature. These results suggest that it is possible to recover Salmonella spp. in catfish products, but its opportunistic nature makes it difficult to predict the source or incidence of this pathogen.

Prophylactic and therapeutic antibiotic treatments have the potential to increase excretion of antibiotic resistance genes (ARGs) by dairy cattle through selection pressure on the gut microbiome. The objective of these studies was to evaluate the effect of cephapirin benzathine administered prophylactically at the end of lactation and pirlimycin hydrochloride administered therapeutically during a clinical mastitis infection on the abundance and relative abundance of ARGs in dairy cow feces. For prophylactic treatment using cephapirin benzathine, nineteen end-of-lactation cows were used. Treatment cows (n = 9) received cephapirin benzathine as an intramammary infusion prior to dry-off, and control cows (n =10) received no antibiotics. All cows received an internal non-antibiotic teat sealant. Fecal grab samples were collected for each cow on d -2 (baseline, used as covariate), d 1, 3, 5, 7, and once per week until d 49. Fecal samples were collected in sterile containers, then freeze-dried and subject to DNA extraction. The abundance of ampC, blaCMY-2, ermB, sul1, tetO, tetW, integrase-specific gene int1, and 16S rRNA were quantified using quantitative polymerase chain reaction (qPCR). The genes ampC and blaCMY-2 encode resistance to ß-lactam antibiotics, ermB to macrolides, sul1 to sulfonamides, tetO and tetW to tetracyclines, and int1 a class-1 integrase gene that facilitates horizontal transfer of ARGs across bacteria. The 16S rRNA gene was used as a representation of bacterial population. Absolute abundance was defined as number of ARG copies per gram of freeze-dried feces, while relative abundance was defined as ARG copy numbers per copy of 16S rRNA gene, which is indicative of the proportion of bacteria carrying ARGs. Non-normal data were logarithmically transformed and were statistically analyzed using PROC GLIMMIX in SAS 9.2. Abundance and relative abundance of sul1 and blaCMY-2 were below the limit of quantification in most samples and therefore not suitable for statistical comparisons. The int1 gene was not detectable in any sample. There were significant interactions between treatment and day for the abundance and relative abundance of ampC, tetO, and tetW. The abundance and relative abundance of ampC increased with time in control cows while remaining constant in antibiotic treated cows through the dry period. Antibiotics may act to stabilize the gut microbiome in response to diet and housing changes. There was a significant main effect of treatment for ermB with a significantly greater proportion of bacteria carrying ermB in control cows when compared to antibiotic treated cows. The tetracycline resistance genes tetO and tetW behaved similarly with a significant treatment by day interaction for the abundance and relative abundance of both genes. The relative abundance of both tetO and tetW were greater in control cows when compared to antibiotic treated cows on days 3, 5, 7, and 14. The abundance of both tetO and tetW resistance genes increased in antibiotic treated cows from day 1 to 49. There was also a significant increase in tetW relative abundance when comparing day 1 to 49. Administering long-acting antibiotics as intramammary dry treatment changed fecal bacteria composition during the dry period perhaps by stabilizing GI bacteria through dietary and housing changes. However, the use of prophylactic dry cow treatment does not uniformly or predictably lead to changes in fecal ARGs. In a second study, after clinical mastitis detection and identification, 6 lactating dairy cows received therapeutic mastitis treatment (pirlimycin hydrochloride as an intramammary infusion). Fecal grab samples were collected from each cow on d 0, 3, 9, and 12. Collection and analytical methods were as previously described. Abundance and relative abundance of sul1 and blaCMY-2 were again below the limit of quantification and therefore not suitable for statistical comparison. The int1 gene was not detected in any sample. The abundance of 16S rRNA genes decreased with day and relative abundance ermB, tetO, and tetW increased with day. There was no significant effect of day on the relative abundance of ampC or the abundance of ampC, ermB, tetO, and tetW in feces of cows with clinical mastitis. Administering fast-acting antibiotics as therapeutic intramammary mastitis treatment to dairy cows increased the relative abundance (gene copies per 16S rRNA) of selected ARGs but not the total abundance of ARGs in feces. The use of antibiotics for prevention and treatment of bacterial infections does not uniformly or predictably increase ARGs. / Master of Science

No description available.

The relentless evolution of antibiotic resistance in pathogens is one of the most pressing medical concerns of the 21st century. Antibiotic resistance and antibiotic drugs originated in environmental bacteria, where they have been integral to their evolution for millions of years. The application of antibiotics in medicine and agriculture has selected for mobilization and dissemination of resistance genes in pathogens. Understanding their evolution here will aid in combating their evolution in pathogens. This work expands the known mechanistic, functional, and genetic diversity of resistance (i.e. resistomes) in environmental bacteria. I systematically parse the extensively drug-resistant resistome of Paenibacillus sp. LC231, which was sampled from an underground ecosystem spatiotemporally isolated from the surface for over 4 Myr. Paenibacillus sp. LC231 was resistant to 26 of 40 drugs tested. Informatic annotation of resistance genes and functional genomes revealed 18 new resistance elements including five determinants without characterized homologs and three mechanisms not previously known to confer resistance. I investigated the resistome of Brevibacillus brevis VM4 to study the relationship between species diversity and resistance diversity in the Paenibacillaceae family, which includes Paenibacillus sp. LC231. I found that resistome diversity does not correlate with species diversity, consistent with horizontal transfer of resistance genes. In each of Paenibacillus sp. LC231 (MphI) and B. brevis VM4 (MphJ), I identified Mphs with unique substrate specifies. I identified the molecular determinants of substrate discrimination in MphI and in doing so, I developed a general strategy for understanding and predicting the functional evolution of resistance enzymes. Together, this work expands the known diversity of resistance that will enable better detection of resistance in pathogens. / Thesis / Doctor of Philosophy (PhD) / Infections caused by antibiotic resistant bacteria are a significant medical problem. Bacteria will always become resistant to antibiotic drugs. Understanding how resistance evolves is essential for increasing the effective lifetime of these drugs. Antibiotics have been naturally produced by bacteria for millions of years, which caused the spread of resistance in environmental bacteria. Medical and agricultural antibiotic use by humans caused resistance in environmental bacteria to transfer to pathogenic bacteria. My work expands the known causes of resistance in environmental bacteria so that we can better detect the causes of resistance in pathogens. In doing so, I demonstrate that multi-drug resistance is over 4 million years old and that environmental bacteria naturally transfer resistance genes. Furthermore, I develop a way to predict the evolution of new resistance functions by inferring their evolutionary histories.

In antibiotic therapy design, conventional wisdom holds that higher antibiotic dosages always leads to the observation of fewer bacterial cells, resulting in a monotonic decay in cell number as a function of increasing antibiotic dose; accordingly, throughout this thesis, we will call this phenomenon a monotone dose-response profile. When we analysed the evolution of antibiotic resistance mediated by the multi-drug efflux pump AcrAB-TolC in Escherichia coli to study if such a monotone dose-response is maintained at all times, our analysis showed that higher dosages can, in fact, lead to higher bacterial loads. This is because selection for drug resistance is mediated by the duplication of the genes, AcrAB-TolC, that encode the aforementioned efflux pump. As explained in detail below, our work highlights the idea that Darwinian selection on additional copies of AcrAB-TolC is a non-linear function of antibiotic dose and that the observed transition from monotone to non-monotone dose-response is a consequence of AcrAB-TolC being strongly selected at very specific dosages. We term this phenomenon an ‘evolutionary hotspot’. Next, we extended the above experimental system to solid media to study how selection on resistance mediated by AcrAB-TolC leads to a ‘spatio-genomic patterning’ effect that we call a ‘bullseye’. Using a bespoke culture device developed as part of this PhD, we show that spatial selection on resistance also depends non-linearly on the distance of the cell from an antibiotic source, and that the non-linearity can be multi-modal as a function of distance, and therefore also of antibiotic dose. This result also contradicts the aforementioned principle that higher antibiotic dosages necessarily lead to fewer bacterial cells. Following on from this, we then studied the ability of microbial competitors for resources to modulate the antibiotic sensitivity of a particular strain of E. coli, namely Tets , using a range of multi-species experiments. We measured the sensitivity to antibiotics of Tets both with, and without, one bacterial or fungal competitor. When that competitor was equally sensitive to the antibiotic, we observed that Tets was less sensitive to it, in part due to an ‘antibiotic sinking’ effect carried out by the competitor strain. However, when the competitor was not sensitive to the antibiotic, Tets was, accordingly, more sensitive than in the absence of competition. In this latter case, the competitor seemed to reduce the growth of Tets by carbon theft as part of a phenomenon known as ‘competitive suppression’. Moreover, this ecological effect is one that synergises with the action of the antibiotic. Finally, we turned to a study of an ecological trade-off motivated by ribosome-binding antibiotics. So, by manipulating the content of ribosomal RNA in the E. coli cell, a large and essential molecule that is bound by antibiotics such as tetracycline or erythromycin, we could subsequently manipulate what is known as a metabolic trade-off between growth rate and growth yield. The latter is the number of cells produced per molecule of carbon found in the extracellular environment of the bacterial population. Using glucose as carbon source we therefore constructed an empirical fitness landscape that shows how the optimum number of ribosomal rRNA operons depends on extracellular glucose concentration. Whilst this study does not relate directly to the presence of an antibiotic, it does show that by altering the number of operons in a manner that is known to affect antibiotic susceptibility, we can also mediate important growth parameters like cell yield, aka efficiency, and growth rate.

With regards to the potential severity of increased antibiotic resistance around the world it is puzzling that the global response to this issue has not been more comprehensive. In this thesis I will examine the political frames on ABR formulated by the global network ReAct in an attempt to understand why this is the case. The frames of an issue, that is how it is described politically in different ways, are crucial for agenda-setting. Moreover, framing is an important part of the work of transnational advocacy networks. Since the acknowledgement of an issue in terms of agenda-setting is an important part of a global response, the frames of transnational advocacy networks make up the focus of this thesis. My findings suggests that the existence of multiple frames on ABR to some extent helps us understand the lacking response to ABR. The construction of the frames in terms of causality, and inparticular a general vagueness in terms of responsibility, is however the main finding.

No description available.

No description available.

No description available.

Environmental pathogens, such as Escherichia coli and Streptococcus uberis, are currently the major cause of mastitis within dairy herds. This leads to severe financial losses, lower production rates and deterioration of the general health of the herd. E. coli mastitis is becoming a major threat to high milk-producing dairy herds. This is because of its increasing resistance to antibiotics, rendering antibiotic treatment regimes against E. coli infections mostly ineffective. The aim of this study was to develop a method to select mastitis causing E. coli isolates for the formulation of effective herd specific vaccines. Two methods, namely a genotyping method (Random Amplification of Polymorphic DNA; RAPD) and an antibiogram based method, were used. A dairy farm milking approximately 1000 Holstein cows in the Darling area, Western Cape Province, was selected for this study. The study was conducted over a period of 48 months and mastitis samples were analysed for mastitis pathogens. Antibiogram testing (disk diffusion method) and an in-house developed RAPD analysis method were used to analyse the E. coli isolates. A total of 921 milk samples were analysed from which 181 E. coli isolates were recovered. The number of all other common mastitis pathogens combined was 99 isolates (Streptococcus uberis 18, Streptococcus dysgalactiae 46, Streptococcus agalactiae 1, Staphylococcus epidermidis 21, Arcanobacterium pyogenes 13). All E. coli isolates, except for one, were resistant to at least three antibiotics. Antibiotic variance profiles were also highly erratic. The RAPD analysis revealed high levels of polymorphisms and clear epidemiological trends were observed over time. No similarities in the variance profiles between the antibiotic variance data and phylogenetic data were observed. Formalin inactivated autogenous vaccines were produced containing E. coli isolated from the herd. The vaccines were formulated using the RAPD or antibiogram data of the E. coli isolates. A total of 5 vaccines were formulated using RAPD data (Rvaccines) and one vaccine was formulated using antibiotic variance data (A-vaccine). The RAPD formulated vaccines were more effective than the antibiotic variance formulated vaccine. After each R-vaccination, the number of E. coli mastitis cases declined within the herd. The A-vaccinations seemed to have had no effect, which lead to a rise in E. coli mastitis cases. RAPD analysis on new emerging isolates was able to detect genetic variation from vaccine strains, which in turn facilitated the formulation of new updated vaccines with higher effectiveness than the previous vaccine. Mastitis data prior to and after the vaccination period revealed significant higher incidences of mastitis in the herd than during the vaccination period. This study demonstrated that sufficient sampling practices coupled with a reliable genotyping method, resulted in the formulation of updatable vaccines which were highly effective in controlling E. coli mastitis within the herd. / Thesis (M Environmental Sciences)--North-West University, Potchefstroom Campus, 2012