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Staphylococci are opportunistic pathogens responsible for a range of infections. Many staphylococcal species are frequently found to be resistant to antibiotics. The environment is considered a potential reservoir of genes conferring antibiotic resistance, which known as the 'resistomes'. Monitoring the dissemination of antibiotic resistant staphylococci is instrumental to mitigating this global health risk. The overall aim of this study was to generate informative data regarding dissemination of antibiotic resistance in environmental and public settings. This included looking into the distribution, epidemiology characteristic and transfer of oxacillin resistant determinant mecA; gaining an insight into genomic features that contribute to multiple antibiotic resistance and pathogenicity of one S. epidermidis isolate; and understanding the stress responses in mediating oxacillin resistance in S. aureus. The use of MALDI-TOF MS allowed identification of staphylococci to species level. MALDI-TOF MS data were used for taxonomic analysis of staphylococci, and taxonomic data were then combined with isolation sites and antimicrobial susceptibility profiles to aid the understanding of dissemination of environmental resistant staphylococci. The widespread dissemination of antibiotic resistant staphylococci in the environment was demonstrated. 12% of staphylococci harboured mecA gene. Community associated SCCmec types IV and V were more prevalent than nosocomial associated SCCmec types I, II, and III in the environment. 52% of SCCmec were non-typable. In addition, 14 new environmental S. epidermidis MLST types were reported. 9 antibiotic resistant determinants that were responsible for the resistant to 7 antimicrobial classes have been identified in environmental S. epidermidis 118 (G6_2). Proteomic analysis revealed that stress responses, including SOS response, stringent response and heat shock response, mediate oxacillin resistance in S. aureus. These results demonstrate widespread multiple drug resistance in different staphylococcal species isolated from non-healthcare environments. This uncontrolled dissemination of multidrug resistant bacteria poses a potential public health threats.

Rising global antibiotic resistance has caused concerns over sources and pathways for the spread of contributing factors. Majority of the antimicrobials used in the U.S. are involved in veterinary medicine, primarily with livestock rearing. Animal manure land application integrates livestock farming and agroecosystems. This manure contains antibiotic resistant elements (AREs) (resistant bacteria, resistance genes, and veterinary antibiotics) that contribute towards antimicrobial resistance. Altering manure application techniques can reduce surface runoff of other contaminants such as excess N and P, pesticides, and hormones, that can impact water quality. Conventional tillage practices in the U.S. has reduced or stopped, making subsurface injection of manure a promising option when compared to surface application. Our research compared manure application methods, manure application seasons, cropping system, and manure-rainfall time gaps to gauge the impact on AREs in the environment. Two field-scale rainfall simulation studies were conducted along with one laboratory study. Using the injection method lowered concentrations of manure associated AREs entering surface runoff. When manure was surface applied and rainfall occurred 7 d after application, 9-30 times less resistant fecal coliform bacteria (FCB) entered surface runoff when compared to 1 d time gap for that broadcast method. Within a day of manure application, antibiotic resistance gene (ARG) profiles in soil began to differ from each other based on manure application and soil ARG richness in all manure-amended soil increased compared to the background. Runoff from injection plots contained 52 ARGs with higher abundance compared to runoff from surface applied plots. ARGs in the former were more correlated to soil and more correlated to manure in the latter. The highest antibiotic concentrations were in the injection slit soil of those plots. Antibiotic concentrations in samples corresponded positively to concentrations of resistant FCB and ARGs, and there was a positive correlation between resistant FCB and their associated ARGs (Spearman's ρ = 0.43-0.63). A CRIISPR-Cas12a assay for quantification of ARGs in environmental samples was just as precise as conventional methods. There is also potential for in-situ detection. These combined results can hopefully help farmers improve manure management practices that mitigate spread of AREs to surrounding water, crops, and soil. / Doctor of Philosophy / Rising global antibiotic resistance cause concerns over sources and pathways for the spread of contributing factors. Most of the antimicrobials used in the U.S. are involved in veterinary medicine, especially with livestock rearing. Overuse of antibiotics that are medically important to human medicine compromises the effectiveness of our medicines. Animal manure contains antibiotic resistant elements (AREs) such as resistant bacteria, resistance genes, and antibiotics) that contribute towards resistance issues. Once these AREs enter the environment, they can be taken up by crops, runoff into surface water or leached into ground water, or even reside within the animal products we consume. Altering manure application techniques is beneficial for nutrient conservation but also potentially for reducing ARE spread. With our research, we compared manure application methods, manure application seasons, cropping systems, and manure-rainfall time gaps to find ways to balance the need for manure application and the spread of resistance. We used two field-scale rainfall simulation studies along with one laboratory study. Overall, using the injection method resulted in significantly lower concentrations of manure associated AREs entering surface runoff. When manure was surface applied and rainfall occurred 7 d after application, less resistant fecal coliform bacteria (FCB) entered surface runoff when compared to the 1 d time gap for broadcast methods. Within a day of manure application, antibiotic resistance gene (ARG) profiles in soil began to differ from each other and soil ARG totals in all manure applied soil increased compared to the background. Runoff from injection plots contained more soil ARGs and runoff from surface applied plots containing more manure associated ARGs. The subsurface injection method also caused highest antibiotic concentrations in the injection slit soil of those plots. High antibiotic concentrations in samples generally meant high concentrations of resistant FCB and ARGs, and resistant FCB were also found with their associated ARGs as well. A CRISPR-Cas12a assay for quantification of ARGs in environmental samples was just as precise as conventional methods. There is also potential for onsite detection. These combined results can hopefully help farmers improve manure management practices that mitigate spread of AREs to surrounding water, crops, and soil.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ludek Zurek / Transfer of bacteria, including antibiotic resistant strains between companion animals and people is likely due to close physical contacts. However, surveillance programs on prevalence of antibiotic resistance are focused mainly on food animals and very little is known about the role of companion animals in the development and spread of antibiotic resistant bacteria. For this study, enterococci were chosen as model organism due to intrinsic and acquired antibiotic resistance and several virulence traits that make them the 3rd most important nosocomial pathogens. In addition, increased fecal shedding of antibiotic resistant bacteria from stressed animals has been reported from studies on food animals. To determine whether the gut microbiota of pet animals serves as a reservoir of clinically important enterococci, 360 enterococcal isolates from two groups: healthy group and pyoderma (stressed) group with 9 dogs in each were identified and screened for resistance to 10 antibiotics and 4 virulence traits. The transferability of resistance determinants and clonality of selected isolates were assessed by horizontal gene transfer assays and pulsed-field gel electrophoresis, respectively. In addition, overall diversity of bacteria as well as antibiotic and metal resistance genes in feces of healthy dogs was assessed by tag-encoded parallel pyrosequencing and microarray analysis, respectively. The most prevalent enterococcal species identified was E. faecalis: healthy group (70.5%); pyoderma group (44.0%). In the pyoderma group, antibiotic resistance and virulence traits (esp, gelE) were more frequent than in the healthy group; however, the overall prevalence of antibiotic resistant strains was low (< 37%) in both groups. The most prevalent resistance genes were tet(M)and tet(S). The antibiotic resistance traits were transferable in-vitro in E. faecalis (tetracycline, erythromycin, doxycycline) and E. faecium (tetracycline). Genotyping revealed less diverse E. faecalis community in pyoderma infected dogs. Pyrosequencing (~7,500 sequences per dog) revealed Firmicutes as the dominant phylum and most common genera included Turicibacter, Lactobacillus, Ruminococcus, Clostridium, and Fusobacterium. Two phyla Lentisphaerae (<1%) and Fibrobacteres (<1%) are reported for the first time from healthy dogs. Microarray data revealed the presence of several tetracycline, erythromycin, aminoglycoside, and copper resistance genes; however, most of these originated from one animal with history of chronic skin infection two year prior to our sampling. Higher prevalence of antimicrobial resistance in pyoderma infected dogs may be related to stress; however, this requires further investigation. In conclusion, based on our data, healthy and pyoderma infected dogs do not represent an important reservoir of clinically significant antibiotic resistant microbiota.

Thesis advisor: Roche P. John / The growing threat of antibiotic resistance makes it extremely important that citizens be informed about the risks posed by antibiotic-resistant bacteria, and measures with which they can reduce these risks. The print media are major sources of such information for members of the public. In the present study, articles from major newspapers in the United States and Canada appearing between 1998 and 2002 were surveyed to determine the extent to which mention was made of antibiotic resistance and the risks associated with antibiotic resistance, the contextual precision with which this information was communicated, and the extent to which information was presented about causes, and risk-reduction measures, associated with antibiotic resistance. The majority of articles surveyed mentioned antibiotic resistance, but most failed to mention associated risks (i.e., the risk of illness and/or the risk of mortality). Articles that did report risks, did so only at a low level of contextual precision. A relatively low percentage of articles mentioned causes of antibiotic resistance, and even fewer mentioned risk reduction measures. These findings suggest that the print media could improve the efficacy with which they inform the public about issues associated with antibiotic resistance. / Thesis (BS) — Boston College, 2003. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Faecal pollution regularly contaminates surface waters, introducing microorganisms, including bacteria and bacteria resistant to antibiotics, to coastal waters. People can come into contact with these potentially harmful microbes when they enjoy recreational activities in the sea. Understanding the risk to bathers of acquiring infections from the sea is important for developing effective intervention strategies to protect human health. This thesis consists of four original studies which aim to answer the question ‘are bacteria in the coastal zone a threat to human health’? First, we describe a systematic review on the risk of acquiring infections from recreational use of coastal waters. Synthesising risk estimates of reporting various symptoms of ill health, we quantify this risk as well as appraise the evidence that these infections are acquired from bathing in coastal waters. The results of the second study - a large online survey - corroborate these findings and provide updated estimates of risk for UK bathers. Third, we assess the risk of ingesting antibiotic resistant bacteria among UK coastal water users. In the final study, we measured the prevalence of faecal carriage of antibiotic resistant bacteria among a highly exposed group – surfers, and in an unexposed group (non-surfers). We conclude that despite improvements made to the collection, treatment and discharge of sewage, and initiatives to communicate water quality to members of the public in recent years, people who bathe in coastal waters are still at an increased risk of adverse health outcomes, whether this is experiencing symptoms of ill health, or exposure to and colonisation by antibiotic resistant bacteria.

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Introduction and Justification: Diarrheal disease is a leading cause of death in children in low- and moderate-income countries. Food, food waste, and water are all vehicles that can promote the spread of diarrheal disease-causing bacteria like Campylobacter, Salmonella enterica, and E. coli. Resistance to commonly used antibiotics is on the rise, making them difficult to manage. This study aimed to determine prevalence and antibiotic resistance profiles of Campylobacter, S. enterica, and E. coli isolated from food, food waste, and water samples obtained from the Chobe Region of Botswana. In addition, the survival of two common pathogens, E. coli and C. jejuni, on kale, a type of leafy green commonly consumed raw, was determined. Methods: Samples were collected from the Chobe region of Botswana in 2022 including water from the local river, food (produce, beef, pork, and poultry) from local vendors, and food scraps from the landfill. Food samples were enriched in the appropriate selective media: Brilliant Green Bile Broth for E. coli, Bolton Broth for Campylobacter, and Rappaport Vassiliadis Broth for S. enterica. Water samples were collected using modified USEPA methods1103.1 and 1604, E.coli isolation was performed by plating on RAPID E.coli2 agar and incubation at 37°C for 2h and 44°C for 16-22h. Campylobacter, S. enterica, and E. coli were isolated from meat, poultry, and water samples before being sent to Virginia Tech, while enriched bacterial pellets from the produce were shipped for screening and isolation at Virginia Tech. E.coli were confirmed by PCR detecting the phoA gene (all E. coli), and classified as pathogenic through screening for the eae (present in enterohemorrhagic and enteropathogenic E.coli), stx1 and stx2 (present in enterohemorrhagic E. coli) and est1b ( present in Enterotoxigenic E.coli) genes. Campylobacter isolates were confirmed using a genera-specific PCR while S. enterica isolates were confirmed using invA primers. These enrichment and primer sets were tested as part of a study to determine the survival of E. coli O157:H7 and C. jejuni on kale during a 21-day shelf life. E. coli and S. enterica isolates were subjected to antibiotic resistance testing using the Kirby-Bauer Disk Diffusion method. Results: Methods for detection of inoculated E. coli O157:H7 on kale indicated survival for the majority of the shelf-life (up to 19 d), in comparison, C. jejuni was undetectable by day 13 using enrichment and PCR or plating. From the Botswanan samples, E. coli was isolated from 20% of produce, 49% of meat, and 84.7% of water. Salmonella was only isolated from produce samples (2.4%, 7/294). Resistance was uncommon among the Salmonella isolates with only one isolate being resistant to chloramphenicol. No Campylobacter were isolated from the screened produce, meat, or food waste. E. coli resistant to 3 or more classes of antibiotics (MCR) were identified in 15.5% of produce, and 22.2% of meat isolates. Isolation of E. coli or Salmonella from meat was not associated with a particular food type. In contrast, isolation of E. coli was more common from certain types of vegetables and fruits. Antibiotic-resistant E. coli were isolated more commonly from beef, poultry, and pork than from produce. Multi-class resistant E. coli were isolated from fruits, greens, soil associated, and above ground associated vegetables, beef, and poultry. Water samples were collected from the same time period as the food samples. E. coli isolation, especially pathogens (based on eae presence) was more frequent from environmental water samples collected during the wet season compared to the dry season. Water samples collected during periods of increased rainfall were more likely to contain E. coli isolates, especially pathogens. S. enterica and Diarrheagenic E. coli isolates, especially MCR isolates, pose a significant risk of illness to consumers. Strategies to reduce the circulation of these pathogens in foods and water sources are needed. / Master of Science in Life Sciences / People can get sick with diarrheal diseases after consuming contaminated food and water. These illnesses are difficult to treat and control when the bacteria causing them are resistant to antibiotics. Campylobacter, Salmonella, and diarrheagenic E. coli are three types of bacteria that can cause illness from food and water. These illnesses disproportionately affect people, especially children, in low-and moderate-income countries like Botswana. Little is known about the prevalence of Campylobacter, Salmonella, and diarrheagenic Escherichia coli in Botswana. This study aimed to determine the prevalence of these microorganisms as well as how resistant they are to different types of antibiotics. Samples from produce, beef, poultry, pork, and recreational water sources were collected in the Chobe region of Botswana over the course of 2022. Food samples were collected from different vendors and food scraps were obtained from the landfill. E. coli and Salmonella were isolated out of meat and water samples in Botswana while produce samples were shipped as mixed cultures to Virginia Tech where E. coli, Salmonella, and Campylobacter isolation or confirmation was done. Once the target bacteria were isolated, their resistance to certain antibiotics was tested. Salmonella was only found in produce from samples collected during October-December. No Campylobacter was found from produce, meat, or food waste. More E. coli was isolated from fruit or vegetable food waste collected from the landfill than from produce bought at local vendors. E. coli was obtained from meat purchased from local vendors more often than samples from the landfill. E. coli was found more often in water when there was more rainfall. E. coli that causes illness was also more likely to be obtained during the wet seasons. Resistant E. coli that could not be killed by the screened antibiotics, were classified as multi-drug resistant when it was resistant to more than three antibiotics. Rainfall, season, and the food source influenced if E. coli isolates were likely to be multi-drug resistant. While more research is needed to determine how these bacteria are moving in the environment and gaining resistance to antibiotics, the findings of this study show they are present in the environment and require further research.