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.

Abstract The current rise in antibiotic resistance and lack of discovery of new antibiotics in recent years has caused an antibiotic crisis. A strategy for overcoming this crisis is to relook at previously discarded antibiotics and with medicinal chemistry, use their structure as a scaffold for the development of a new antibiotic. Edeines are a group of antimicrobials discovered by Kurylo-Borowska in 1959 that were discarded for toxicity and this work aimed to use edeine for the structural basis in the development of a novel antibiotic. Using both solution phase and solid phase peptide synthesis ꞵ-tyrosine-L-isoseryl-diaminopropionic acid was synthesized to develop a synthetic strategy for the peptide synthesis of Edeine A. Solid phase peptide synthesis techniques were used to synthesize two simplified edeine analogues which demonstrated that the amino acid 2,6-diamino-7-hydroxylazaleic acid was necessary for antimicrobial activity. A synthetic strategy for the synthesis of 2,6-diamino-7-hydroxylazaleic acid (DAHAA) using ring closing metathesis and aminohydroxylation was developed and starting materials were synthesized. Using solid phase synthesis and the strategy for synthesizing DAHAA, a library of edeine analogues could be made using parallel synthesis and assayed for specific antimicrobial activity. This could lead to the development of a lead antibiotic which used edeine as a structural scaffold, therefore a novel antibiotic could be clinically used without large scale resistance present in the environment like the currently used antibiotics. / Thesis / Master of Science (MSc)

The escalating threat of antimicrobial resistance has intensified the exploration of alternative treatments, with bacteriophage (phage) therapy emerging as a potential substitute for antibiotics. While strictly lytic phages rapidly kill bacteria, temperate phages can also go dormant in their hosts. Accordingly, despite their prevalence, they are considered unsuitable for therapy. My systematic review of phage therapy in urinary tract infections (UTIs) highlighted this. This review motivated me to explore how the potential of these phages could be leveraged. Chapter 3 introduces a novel strategy to do so, exploring whether the fluoroquinolone antibiotic ciprofloxacin could synergize with temperate phages. This innovative strategy exploits the ability of the antibiotic to awaken dormant temperate phages, driving a potent synergy (≥8 log reduction) able to result in bacterial eradication. This is a potential breakthrough in the use of phages. Chapter 4 expands on this finding, establishing that a synergy exists across various drug classes with diverse mechanisms of action. Surprisingly, the synergy extends beyond antibiotics triggering the bacterial SOS-response known to wake temperate phages and also includes protein synthesis inhibitors, offering a new approach to influence the phage lysis-lysogeny decision. Chapter 5 explores the identified synergy in antibiotic-resistant models, focusing on the impact of antibiotic resistance on the effect of combining temperate phages with antibiotics. While the majority of cases demonstrated synergy comparable to the absence of antibiotic resistance, an exception was noted in the acetylation-resistant models for both gentamicin and ciprofloxacin. These resistance genes abolished synergy with the temperate phage, emphasizing the importance of the resistance mechanism within temperate phage antibiotic synergy (tPAS). In conclusion, this thesis underscores the lack of interest in temperate phages for therapy and demonstrates a scalable strategy to overcome the major barriers to their use. I uncover the mechanisms underlying the synergy and show that these concepts are applicable even in the context of resistance to the synergizing antibiotic. These findings propose a remarkable shift in how antimicrobial therapy approaches are viewed. / Thesis / Doctor of Philosophy (PhD) / In the past decade, interest in viruses that only target bacteria (called “phages”) and their capacity to treat antibiotic-resistant infections has surged. Beginning with our systematic review on UTI treatments involving phages, we observed that none of the included studies explored the therapeutic use of the dormancy-capable “temperate” phages. This finding serves as motivation for the subsequent chapters of the thesis. In Chapter 3 I discovered temperate phage-antibiotic synergy (tPAS). An inventive strategy involving antibiotics activates temperate phages, demonstrating substantial synergy in eliminating bacterial infections and offering a potential breakthrough against antimicrobial resistance. In Chapter 4 I found that this synergy extends beyond antibiotics that result in a response to DNA damage within the bacteria (SOS response) to include protein synthesis inhibitors, providing an innovative approach to combat bacterial infections. Finally, in Chapter 5 I extended the study to antibiotic-resistant models. Across a wide array of mechanisms for antibiotic resistance, all but two supported the synergy we observed with temperate phage. This highlights the importance of the resistance mechanism in temperate phage antibiotic synergy (tPAS). The finding of this thesis paves the route for potentially integrating temperate phages into mainstream medical practices alongside antibiotic interventions.

Amikacin-, vancomycin- or amikacin/clindamycin-impregnated gel was placed subcutaneously on either side of horses' necks a total of 6 times each. Interstitial fluid was collected at 0, 4, 8, 12 and 24 hours, and days 2 through 10, via capillary ultrafiltration probes placed within the incision (0cm) and 1.5cm laterally. Plasma or serum was collected at days 0, 1 and 7. Biopsy samples were obtained at the completion of the study. A histomorphologic score was assigned to each sample, and the differences in mean scores between treatment (gel) and control incisions were assessed using Wilcoxon signed rank test. Amikacin and vancomycin samples were analyzed via fluorescence polarization immunoassay; clindamycin samples were analyzed via high performance liquid chromatography. Concentrations greater than 2000 times the MIC of vancomycin and clindamycin, greater than 1000 times the MIC of amikacin, and greater than 800 times the MIC of amikacin (amikacin/clindamycin gel) were obtained at 0cm. Mean concentrations remained above MIC for vancomycin and clindamycin for 10 days (0cm) and 8 days (1.5cm); for 9 days (0cm) and 7 days (1.5cm) for amikacin gel; and for 9 days (0cm) and 5 days (1.5cm) for amikacin (amikacin/clindamycin gel). Mean plasma amikacin and vancomycin concentrations were negligible; serum clindamycin concentrations were greater than MIC (0.52µg/ml and 0.63µg/ml) at 24 hours and 7 days respectively. There were no significant differences in histomorphologic scores between treatment and control incisions. Cross-linked dextran gel is a safe, effective alternative for local antibiotic delivery in horses, with substantially high local concentrations and minimal systemic absorption for amikacin- and vancomycin-impregnated gels. / Master of Science

No / A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.

Antibiotic resistance is a quickly emerging public health crisis, accounting for more than 700,000 annual global deaths. Global human antibiotic overuse and misuse has significantly expedited the rate at which bacteria become resistant to antibiotics. A renewed focus on discovering new antibiotics is one approach to addressing this crisis. However, it alone cannot solve the problem: historically, the introduction of a new antibiotic has consistently, and at times rapidly, been followed by the appearance and dissemination of resistant bacteria. It is thus crucial to develop strategies to improve how we select and deploy antibiotics so that we can control and prevent the emergence and transmission of antibiotic resistance. Current gold-standard antibiotic susceptibility tests measure bacterial growth, which can take up to 72 hours. However, bacteria exhibit more immediate measurable phenotypes of antibiotic susceptibility, including changes in transcription, after brief antibiotic exposure. In this dissertation I develop a framework for building a paper-based cell-free toehold sensor antibiotic susceptibility test that can detect differential mRNA expression. I also explore how long-term lab evolution experiments can be used to prospectively uncover transcriptional signatures of antibiotic susceptibility. Paper-based cell-free systems provide an opportunity for developing clinically tractable nucleic-acid based diagnostics that are low-cost, rapid, and sensitive. I develop a computational workflow to rapidly and easily design toehold switch sensors, amplification primers, and synthetic RNAs. I develop an experimental workflow, based on existing paper-based cell-free technology, for screening toehold sensors, amplifying bacterial mRNA, and deploying sensors for differential mRNA detection. I combine this work to introduce a paper-based cell-free toehold sensor antibiotic susceptibility test that can detect fluoroquinolone-susceptible E. coli. Next, I describe a methodology for long-term lab evolution and how it can be used to explore the relationship between a phenotype, such as gene expression, and antibiotic resistance acquisition. Using a set of E. coli strains evolved to acquire tetracycline resistance, I explore how each strain's transcriptome changes as resistance increases. Together, this work provides a set of computational and experimental methods that can be used to study the emergence of antibiotic resistance, and improve upon available methods for properly selecting and deploying antibiotics. / 2023-03-17T00:00:00Z

Outputs of human-derived chemicals and constituents to the environment, and shifts in these outputs, can result in unintended consequences to human and ecological health. One such shift is the advent of the modern antibiotic era, in which mass production and outputs of antibiotics, which are mostly naturally-derived microbial defense compounds and include a few synthetic antimicrobials, has profound implications for contributing to the spread of antibiotic resistance. Antibiotic resistance arises from mutations and/or sharing of antibiotic resistance genes (ARGs) among bacteria via horizontal gene transfer, with carriage of ARGs by pathogenic bacteria of particular concern to human health. While most attention to stopping the spread of antibiotic resistance has been devoted to the clinic, it is critical to consider the environmental origin, ecology and pathways by which antibiotic resistance spreads in order to develop comprehensive strategies to combat antibiotic resistance. In particular, wastewater treatment plants (WWTPs) represent a potentially key critical control point given that they receive antibiotic resistant bacteria (ARB) and ARGs from the population, which are then routed to activated sludge biological treatment, consisting of high density, highly active microbial populations. The research projects described in this dissertation aimed to explore the occurrence of ARGs in WWTPs, particularly WWTPs in developing countries representing the extremes of what is expected to be encountered in terms of potential to spread antibiotic resistance, and to improve and apply novel technologies for monitoring key markers of antibiotic resistance in WWTPs and affected environments. The pathogen Staphylococcus aureus and a corresponding ARG (methicillin resistance mecA gene) were chosen as model biocontaminants of concern due to their environmental and public health relevance. The results reported in Chapters 3-5 advance the knowledge of bio(nano)sensing techniques and highlight areas of promise and challenge. The results reported in Chapter 2 provided insight into the baseline levels of ARGs expected in a highly impacted WWTP in India, thereby highlighting the magnitude and global scale of the problem of antibiotic resistance as well as the need for innovative solutions. / Ph. D.