Master of Science, The Ohio State University, 2023, Environment and Natural Resources

Antibiotic-resistant bacteria (ARB) pose a growing threat to public health by rendering antibiotic treatments ineffective and causing prolonged hospital stays, increased healthcare expenditures, and greater mortality. Of particular concern are those resistant to carbapenems, powerful antibiotics used for life-threatening infections. Carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem resistance genes are established in surface waters through hospital and wastewater effluent. However, little is known about their presence in aquatic wildlife. Transmission of CRE from aquatic to terrestrial systems could introduce CRE into animal operations and subsequently the human food supply. Therefore, it is crucial to understand pathways of dissemination within and between aquatic and adjacent riparian organisms. This study investigated the effects of (1) fish-based ecological network structure and (2) trophic position and diet composition on carbapenem resistance in linked river-riparian reaches surrounding six wastewater treatments plants discharging into medium-to-large rivers in Cincinnati, Columbus, and Cleveland, Ohio. Carbapenem resistance was present at all six sites, with CRE genes recovered from multiple environmental samples over two sampling years. Carbapenem resistance was not observed in any samples of aerial insectivorous birds feeding in sample reaches (Tree Swallow Tachycineta bicolor). 10.5% of fish isolates exhibited carbapenem resistance, and 4.67% of fish isolates yielded CRE genes. Model selection of negative binomial models revealed that nutrient concentrations and turbidity were strongly supported predictors of carbapenem occurrence in fish communities. Ecological network properties including linkage density (i.e., number of interactions per species), compartmentalization (i.e., degree of connectedness of subgroups within a network), and variability in the magnitude of interactions were also important predictors. Carbapenem resistance generally (open full item for complete abstract)

Committee: Stephen Matthews (Advisor); Thomas Wittum (Committee Member); S. Mažeika Patricio Sulliván (Advisor) Subjects: Ecology; Freshwater Ecology

Master of Public Health, The Ohio State University, 2017, Public Health

Antimicrobial resistance (AMR) is a growing concern in human and animal medicine and threatens public health on a global scale. Beef cattle are a reservoir of Salmonella and have been implicated in foodborne outbreaks of AMR Salmonella in the U.S. Understanding trends in AMR and factors which affect resistance to medically important antibiotics in beef cattle is vital to animal and human health. The USDA's National Animal Health Monitoring System administered farm management questionnaires to beef feedlots in major cattle-feeding states around the U.S. and collected fecal samples from 100 feedlots in 1994, 73 feedlots in 1999, and 68 feedlots in 2011 for pathogen and AMR testing. The objectives of this study were to 1) evaluate change in the prevalence of AMR in Salmonella over time from USDA beef feedlot studies conducted in 1994, 1999, and 2011 and 2) to explore farm management factors, including antibiotic use, associated with the odds of AMR in a Salmonella isolate. Logistic regression models were created for each of four antibiotics: amoxicillin-clavulanic acid, ampicillin, ceftiofur, and trimethoprim-sulfamethoxazole. Final models were selected via backward elimination to measure statistical associations between management factors and odds of a Salmonella isolate being resistant to the antibiotic. There was a significant increase in odds of resistance from 1994 to 1999 for all four antibiotics, amoxicillin-clavulanic acid (OR=32.6), ampicillin (OR=6.1), ceftiofur (OR=115.4), and trimethoprim-sulfamethoxazole (OR=17.4). Isolates from 1999 had significantly higher odds of resistance compared with isolates from 2011 for amoxicillin-clavulanic acid (OR=3.8) and trimethoprim-sulfamethoxazole (OR=35.0). Use of antibiotics tylosin, tetracyclines, and sulfas as health or production management tools did not approach significance (p=0.20) in any of the four models. Management practices associated with increased odds of resistance to at least one antimicrobial included: (open full item for complete abstract)

Committee: Gregory Habing DVM, MS, PhD (Advisor); Thomas Wittum MS, PhD (Committee Member); Michael Pennell PhD (Committee Member) Subjects: Agriculture; Animal Diseases; Public Health; Statistics; Veterinary Services

Master of Science (MS), Bowling Green State University, 2019, Biological Sciences

The research described here focuses on the phylogenetic characterization of water-derived pseudomonads and their antagonistic activity against multi-drug resistance (MDR) P. aeruginosa and Burkholderia species. Phylogenetic work was based on the gyrB housekeeping gene. Genetic techniques have been optimized and employed to identify genes associated with antimicrobial production via transposon (Tn) mutagenesis using a triparental mating system approach with Pseudomonas as the model organism. This study expands on theses previous studies in the lab to identify biosynthetic gene clusters (BGC) involved in production of novel antibiotics capable of inhibiting the growth of MDR pathogens. We utilize a previously optimized workflow to identify genes from environmental isolates involved in the inhibition of MDR P. aeruginosa and species within the Burkholderia cepacia complex (Bcc). We show that both MDR Bcc and P. aeruginosa pathogens were inhibit by environmental Pseudomonas strains. Out of 7,784 interactions, 210 of these were antagonistic. Superkillers (SK), defined as strains that inhibit ≥3 of MDR pathogens, were selected for optimization of Tn mutagenesis to identify gene cluster whose products inhibit these MDR pathogens. Only six out of the 24 SK's were capable of this process. Out of these six, three were selected for large scale mutagenesis to identify loss of inhibition (LOI) mutants. Four LOI mutations were found for strain S5F11, one of which had an insertion within a BGC predicted to produce an NRPS complex. Seven LOI mutants were found for S3E7. Although none of these insertions were identified within a BGC, genes have been identified that are observed to be heavily involved in antibiotic production. This study suggests that environmental Pseudomonas strains have the capacity to inhibit the growth of CF-derived MDR pathogens. Using Tn mutagenesis, we have identified novel loci that are associated with antibiotic production.

Committee: Hans Wildschutte PhD (Advisor); Timothy Davis PhD (Committee Member); Robert Huber PhD (Committee Member) Subjects: Biology

Doctor of Philosophy (Ph.D.), Bowling Green State University, 2017, Biological Sciences

The emergence of antimicrobial resistance bacteria has become a major threat to human society. The rapid spread of resistant pathogens and the associated loss of efficacy of available drugs needs to be met with the development of antibiotics and alternative treatments. Pseudomonas aeruginosa is an opportunistic human pathogen evolving resistance to many currently used antibiotics. Chronic lung infections with the bacterium P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Escalating this problem is that pharmaceutical companies have dropped drug development due to low profitability, thus making the efforts of drug discovery of prime importance. To address this global health threat research institutes have now stepped forward to aid in discovery of novel compounds. P. aeruginosa dominates the lungs during chronic infections in CF patients, yet it's abundance in non-human habitats such as water and soil is less compared to other diverse groups of pseudomonads. A trait that could contribute to such decreased abundance is bacterial competition from other Pseudomonas populations that dominate water and soil habitats. We hypothesized that environmental Pseudomonas from diverse soil and water habitats produce secondary metabolites capable of inhibiting the growth of CF derived P. aeruginosa. Here, we sought to determine if clinical isolates of P. aeruginosa are susceptible to competition by environmental pseudomonads which may provide a source of inhibitory factors. We have used a population based study in association with transposon mutagenesis, PCR techniques, whole genome sequencing and bioinformatic analysis to identify environmental Pseudomonas biosynthetic gene clusters (BGCs) and characterize antagonistic compounds that are effective against CF-derived P. aeruginosa. A total of five BGCs have been identified in this study from environmental Pseudomonas strains S4B6, S3F9 (soil-derived) and LE6C6 (water-derived) encoding (open full item for complete abstract)

Committee: Hans Wildschutte Dr. (Advisor); Robert McKay Dr. (Committee Member); Paul Morris Dr. (Committee Member); Scott Rogers Dr. (Committee Member); Kurt Panter Dr. (Other) Subjects: Biology; Microbiology

Master of Science, The Ohio State University, 2017, Food Science and Technology

There is growing concern as to the continued ability of antibiotics in clinical settings to be effective due to increases in antibiotic resistance in pathogens. This increase is a major threat to human health, with approximately 23,000 annually killed by untreatable bacteria in the United States alone. The environment is a potential reservoir for antibiotic resistance genes and soil is high in bacterial diversity. This study aims to analyze augmented soils, especially a pilot study on their resistome, to assess the potential risk of commercial soil in disseminating antibiotic resistant bacteria to humans and the environment. A study of fifteen commercial soils was conducted using culture-dependent and metagenomic methods, with six noncommercial garden and general environmental soils used for comparison. Serially diluted soil samples were plated on plate count agar (PCA) and individual colonies were picked and transferred to antibiotic-supplemented agar using the following types and concentrations: ampicillin (16g/L), tetracycline (100g/L), erythromycin (100g/L), and lincomycin (16mg/L). DNA was extracted from soil samples, pooled and sequenced by high throughput sequencing. Primers were designed for three resistance genes in relative high abundance, aac(3)-Id, catB6, and MacB. PCR amplification to detect these genes in individual samples was conducted. All samples studied lacked response to at least one antibiotic using culturing methods, on average 66% to ampicillin, 77% to lincomycin, 15% to erythromycin, and 9% to tetracycline. A total of 59 total phyla were represented in the pooled soil sample, representing 2118 different genera, with the vast majority of bacteria present belonging to the phylum Proteobacteria. aminoglycoside resistance genes presented at elevated levels. Aac(3)-Id was detected in five commercial and one noncommercial soil. catB6 was detected in one soil. The ratio of antibiotic resistance gene hits to total 16S rRNA was 0.023, consistent with (open full item for complete abstract)

Committee: Hua Wang Dr. (Advisor) Subjects: Food Science; Microbiology

Master of Science (MS), Bowling Green State University, 2024, Biological Sciences

Muco-obstructive illness is caused by any number of defects in the respiratory tract that result in viscous or static mucus. This creates an environment conducive for pathogen colonization with subsequent synergistic interactions that drive selection of a smaller set of apex pathogens, resistant to immune and antibiotic intervention. Successful control and potential resolution of such infections will require the development of new therapeutic strategies. One potential approach involves identification of bacterial viruses that target the dominant bacterial strains that manifest in patients with compromised respiratory functions. This study considers a set of bacteriophage specific for, and toxic to Burkholderia species that often dominate in such patient populations. I isolated a set of bacteriophage from environmental samples (including soil, human sewage, and agricultural waste for consideration. Some environmental isolates were capable of infecting pathogenic Burkholderia and a suite of lab adapted and pathogenic Burkholderia strains. As a result of this bacteriophage hunt, 15 potential bacteriophage isolates have been maintained, four of which have been sequenced and analyzed further. To do so, DNA was extracted from the purified and amplified isolates and sequenced. Thus, it was postulated that the novel bacteriophages “Clear “and “Cloudy” may be related to the P2-family of bacteriophage: whereas novel bacteriophage “P1P” contains elements similar to mu-like phage. Further research is necessary to detail the genome structure, genetic relationships, and gene function; however, these novel isolates reiterate the notion that pathogen-infecting bacteriophage are potentially isolated from the environment using non-pathogenic or pathogenic strains.

Committee: Raymond Larsen Ph.D. (Committee Chair); Hans Wildschutte Ph.D. (Committee Member); Simon Morgan-Russell Ph.D. (Committee Member) Subjects: Microbiology

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Bacterial pathogens are evolving resistance to all available antibiotics and are a leading cause of death worldwide. Thus, the discovery of new antibiotics that inhibit multi-drug resistant (MDR) pathogens is urgent. We hypothesize that environmental Pseudomonas strains that can inhibit the growth of MDR pathogens may be source of novel antibiotics that may have new or multiple mechanisms of action. We isolated 288 environmental Pseudomonas strains from the Portage River in northwest Ohio which represents a polluted water source. Pseudomonas strains were isolated and phylogenetically characterized using the gyrB housekeeping gene. To test our hypothesis, the environmental strains were competed against a collection of human and animal MDR pathogens including nine Burkholderia species, seven Staphylococcus aureus, and nine Pseudomonas aeruginosa strains, all isolated from lungs of cystic fibrosis patients, and five Aeromonas fish pathogens. Results from the 8,640 individual antagonistic assays showed that 115 of the environmental Pseudomonas strains demonstrated the ability to inhibit at least one pathogen. This suggests that these environmental strains may produce novel compounds which effectively inhibit these MDR pathogens. We performed transposon mutagenesis and screened for loss of inhibition mutants with strains J4D8, JA1H2, J1A5 and J3E11 to determine biosynthetic gene clusters involved in antibiotics production. Whole genome sequencing and arbitrary PCR coupled with antiSMASH were used to identify specific gene clusters involved in antagonistic activity. The compounds were predicted to include pyochelin, pyoverdine, MA026, lankacin C and cepacin A which are all derived from non-ribosomal peptides. Further analysis of these compounds is required to determine their novelty.

Committee: Hans Wildschutte Ph.D. (Committee Chair); Raymond Larsen Ph.D. (Committee Member); Christopher Ward Ph.D. (Committee Member) Subjects: Biochemistry; Bioinformatics; Biology; Biomedical Research; Microbiology; Molecular Biology

Bachelor of Science, Wittenberg University, 2022, Biochemistry/Molecular Biology

Antibiotic resistance in bacteria is becoming a significant concern, originating from the increased amount of antibiotics prescribed by doctors and patients failing to complete their course of treatment. Resistant bacteria are quickly adapting to new forms of antibiotics and are becoming more deadly. Based on the Centers for Disease Control and Prevention's 2019 estimate, in the United States approximately 2.8 million antibiotic resistant infections occur each year and about 90,000 deaths can be attributed to bacterial infections1. Therefore, it is essential that high school and undergraduate students interested in going into the medical field are introduced to the idea of antibiotic resistance. Several different methods of growing resistance to kanamycin and ampicillin in E. coli were created and tested to provide a laboratory experience to students at an introductory chemistry level. Multiple procedures were created using either solid or liquid medium, however, the most practical and successful procedure involved growing resistance in liquid medium in a single flask, which was left to grow over the course of a week. Additionally, the ability for already resistant strains to lose their inherent resistance was also tested through continually propagating cultures in liquid medium without antibiotic. It was found that, through this method, kanamycin resistance in E. coli could be lost in about three weeks or less.

Committee: Daniel Marous (Advisor); Michelle McWhorter (Committee Member); Margaret Goodman (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Microbiology; Molecular Biology

Bachelor of Arts, Wittenberg University, 2022, Chemistry

According to the CDC, in the United States, about 3 million antibiotic resistant infections occur each year with 35,000 deaths from bacterial infections. The large quantity of antibiotics that are prescribed to patients increases the ability of bacteria to develop this resistance, especially when patients do not complete their full course of medication. With the development of bacterial resistance becoming a global phenomenon, it is crucial that the public become more educated on this issue. Toward this end, a method has been developed that demonstrates the growth of antibiotic resistance and could be incorporated into a high school or undergraduate laboratory curriculum. This lab experience would be ideal for students who are considering a medical or scientific research career, allowing them to witness resistance firsthand. It would also serve to educate other individuals, making them aware of the general issues associated with bacterial resistance. Over the course of a week, resistance is gradually evolved in E. coli. The students would then determine the minimum inhibitory concentration of the resistant strain and compare their value to the wildtype level.

Committee: Daniel Marous (Advisor); Justin Houseknecht (Committee Member); Matthew Collier (Committee Member) Subjects: Biochemistry; Biology; Cellular Biology; Microbiology; Molecular Biology

Bachelor of Arts, Wittenberg University, 2019, Chemistry

The use of antibiotics to cure bacterial infections is one of the single most significant medical advancements in human history. This research focuses on the ability of bacteria to develop resistance to these antibiotics. The aminoglycoside antibiotic kanamycin was used to develop resistance in E. coli (ATCC 25922) as high above the minimum inhibitory concentration (MIC) as possible. Resistance was developed up to 2500 μg/mL of kanamycin, 100 times the MIC of 25 μg/mL. However, more frequently, resistance could be developed to 100 μg/mL. The antibacterial-resistant E. coli was then used in a cross-resistance study. This study aimed to find whether kanamycin resistance translated to resistance to other antibiotics. The kanamycin-resistant E. coli were tested against neomycin and gentamicin (both aminoglycosides) and ampicillin (a β-lactam). The kanamycin-resistance did not confer resistance to ampicillin, but resistance above the wild-type MIC was seen with gentamicin and to a larger extent with neomycin.

Committee: Daniel Marous (Advisor); Ray Dudek (Committee Member); Brian Yontz (Committee Member) Subjects: Biochemistry; Chemistry; Microbiology; Molecular Biology

Master of Science (MS), Bowling Green State University, 2020, Biological Sciences

Multidrug resistant (MDR) pathogens are predicted to cause more than ten million annual deaths worldwide by 2050, making bacterial infections the leading cause of death. Although bacteria are evolving resistance to all known antibiotics, no large pharmaceutical companies are involved in drug discovery due to high cost and low profitability; thus, no new antibiotics are available, and the current ones are increasingly becoming ineffective at treating MDR infections. Moreover, recent studies suggest that there are few remaining new antibiotics in the environment left for discovery. Since MDR bacterial infections are predicted to be a major crisis, a new or augmented therapy to treat infections is imperative. Bacteriophage therapy is an alternative solution and has been internationally used for over 100 years. In bacteriophage therapy, phage bind to bacteria through specific protein-protein interactions that result in narrow host range infectivity. Although this specificity is beneficial because the interaction precisely targets a single pathogen, it is also problematic because a single phage usually cannot infect multiple strains of the same bacterial species. The phage discovery process to treat a specific pathogen is both time consuming and phage can fall short of the ability to antagonize multiple infections. Thus, phage with broad host range killing phenotypes are more beneficial when using phage therapy to treat infections caused by a particular pathogen. Therefore, this study set out to isolate phage with broad host range killing phenotypes such that phage that could inhibit more than one MDR pathogen could be found. In this study, 29 phage that antagonize cystic fibrosis (CF) derived MDR Pseudomonas aeruginosa were isolated from equine fecal water, purified, characterized through host range assays, and shown to kill two to eight CF derived MDR P. aeruginosa strains. Since phage have been shown to drive bacterial evolution toward increased antibiotic susce (open full item for complete abstract)

Committee: Hans Wildschutte (Advisor); George Bullerjahn (Committee Member); Raymond Larsen (Committee Member) Subjects: Biology; Biomedical Research; Health; Microbiology; Therapy

PHD, Kent State University, 2018, College of Arts and Sciences / Department of Biological Sciences

In urban streams, contaminants such as antibiotics, ARGs, nutrients, and metals, co-occur with anthropogenic activities. These toxicants can have a profound effect on microbial community composition, which may, in turn, affect microbial community function. Such results suggest that the composition and function of microbial communities are consequences of their response to environmental stimuli. However, the differing physiological responses of microorganisms to environmental stressors suggests that community structure may be an important factor driving the community's functional responses to avoid or reduce the effects associated with physiochemical changes in the environment. With the projected increase in concurrent environmental stressors associated with the anthropogenic activity, there is a need to understand how microbial communities respond to compounded stressors. The overarching question for my dissertation is: how does chemical stress (in the form of heavy metals) interact with other environmental factors (including hydrologic conditions and nutrients) to impact microbial community structure and function in streams? To address this question, I utilized a combination of trait-based molecular approaches in tandem with biological and chemical field measurements and laboratory experiments. I looked at how microbial, specifically bacterial and diatom, community composition, structure, and function were affected by different stresses that are found within urban streams.

Committee: Laura Leff (Advisor); Christopher Blackwood (Committee Member); Xiaozhen Mou (Committee Member); Elizabeth Herndon (Committee Member) Subjects: Aquatic Sciences; Biology; Ecology; Environmental Science; Microbiology

MS, University of Cincinnati, 2018, Arts and Sciences: Biological Sciences

A survey for the presence of antibiotic-resistant E. coli was undertaken by collecting samples from seven geographically dispersed US wastewater treatment plants (WWTPs). Samples were collected at each WWTP in cool and summer months and cultured using selective media. The resulting E. coli isolates were characterized for resistance to imipenem, ciprofloxacin, cefotaxime and ceftazidime, presence of carbapenemase and extended spectrum beta lactamase (EBSL) genes, and phylogroups and sequence types (STs). The survey identified 322 antibiotic-resistant (AR) E. coli isolates and characterized the associated resistance profiles, with 65 of these being imipenem-resistant. Of the 65 carbapenem-resistant E. coli (CREC) isolates, 62% contained = 1, and 31% contained = 2, of nine studied carbapenemase and ESBL genes. The most common carbapenemase gene detected was blaVIM and two instances of blaKPC were found. However, the gene panel was negative for many of the CREC isolates. Carbapenem resistance was seen in pathogenic E. coli phylogroups (B2 & D) and STs (1193, 405, 648) as well as commensal phylogroups (A & C). The occurrence of CREC in wastewater is a potential concern because this matrix may serve as a reservoir for gene exchange and thereby increase the risk of AR bacteria (including CR) being disseminated into the environment and thence back to humans.

Committee: Brian Kinkle Ph.D. (Committee Chair); Dennis Grogan Ph.D. (Committee Member); Samuel Hayes Ph.D. (Committee Member) Subjects: Microbiology

Master of Science in Biological Sciences, Youngstown State University, 2018, Department of Biological Sciences and Chemistry

Stenotrophomonas maltophilia Oak Ridge strain 02 (S. maltophilia 02) was isolated from a heavy metal contaminated stream, East Fork Poplar Creek in Oak Ridge, Tennessee. This strain has high levels of resistance to both metals and antibiotics. All strains of S. maltophilia are gram-negative bacilli. Some strains are opportunistic pathogens that are resistant to high levels of metals and to antibiotics such as cephalosporins, quinolones, carbapenems, penicillins, and ß-lactam/ß-lactamase inhibitor combinations. S. maltophilia 02 contains two ß-lactamase genes, L1 and L2. In this study, the L2 ß-lactamase and its regulator, LysR, were cloned and sequenced. MICs were performed to test for antibiotic resistance against ampicillin, showing resistance even at 1500 µg/ml. In addition, L2 was also found to confer resistance to the antibiotic carbenicillin, but not to cefoxitin. By studying penicillin resistance mechanisms, it may be possible to develop strategies to combat drug resistance in pathogenic strains of S. maltophilia.

Committee: Jonathan Caguiat PhD (Advisor); David Asch PhD (Committee Member); Xiangjia Min PhD (Committee Member) Subjects: Biology; Molecular Biology

Master of Public Health, The Ohio State University, 2016, Public Health

Antimicrobial resistance (AMR) has become a central topic as it is a growing threat in human and animal health. Major surveillance systems, such as the National Antimicrobial Resistance Monitoring System (NARMS), are now established to monitor AMR and provide physicians, veterinarians, and scientists with valuable information to make informed decisions on policy and therapeutic treatment. However, there is a lack of comprehensive literature on AMR among nonhuman primates (NHP). This study aims to provide data on current antimicrobial use strategies and on the prevalence of AMR in zoonotic bacteria recovered from NHPs within biomedical research institutions. We focused on four zoonotic enteric bacteria: Shigella flexneri, Yersinia enterocolitica, Y. pseudotuberculosis, and Campylobacter jejuni. Fifteen veterinarians, seven biomedical research institutions, and four diagnostic laboratories participated, providing susceptibility test results across three years (1/2012 – 4/2015). Veterinarians primarily treated cases caused by S. flexneri, Y. enterocolitica, and Y. pseudotuberculosis with enrofloxacin, but treated C. jejuni cases with azithromycin and tylosin. All isolates were susceptible to their associated primary antimicrobials. However, high proportions of AMR was observed to other antimicrobials. S flexneri isolates were resistant to erythromycin (87.5%, 21/24), doxycycline (73.7%, 14/19), and tetracycline (38.2%, 157/411). Y. enterocolitica isolates were resistant to ampicillin (100%, 49/49) and cefazolin (93.6%, 44/47). No Y. pseudotuberculosis isolate (0/58) was resistant to any tested antimicrobial. C. jejuni isolates were resistant to methicillin (99.5%, 569/572) and cephalothin (97.5%, 557/571). Notably, resistance patterns were not shared between this study's NHP isolates and human isolates presented by NARMS. This study demonstrates that zoonotic bacteria recovered from NHP diagnostic samples are broadly susceptible to the antimicrobials used to treat t (open full item for complete abstract)

Committee: Gregory Habing (Advisor); Dondrae Coble (Committee Member); Julie Bower (Committee Member) Subjects: Animal Diseases; Animal Sciences; Animals; Biomedical Research; Occupational Health; Occupational Safety; Public Health; Veterinary Services

Master of Science, The Ohio State University, 2015, Veterinary Preventive Medicine

Cultivable bacteria are a fraction of the fecal microbiota, yet serve as the foundation of current antibiotic resistance (AR) research. The small percentage of bacteria that can be cultured may be a biased representation of the AR gene reservoir. As such, our first objective was to perform Real-Time Quantitative PCR (RT-QPCR) of whole fecal DNA extracts for quantification of tet (C), a gene that confers resistance to tetracyclines. A challenge of utilizing community DNA for estimation of gene concentration is selection of the appropriate denominator. In objective 1, we compared tet (C) concentrations based on 3 different denominators: fecal weight, 16S rRNA (the nucleic acid responsible for the bulk of the prokaryotic 30S ribosomal subunit) concentration, and community DNA concentration. Approximately 200 mg of feces were collected from 212 pigs. Total fecal DNA was extracted and quantification of tet (C), 16S rRNA, and total DNA was performed by Real-Time PCR. To evaluate the correlation of tet (C) concentrations using fecal weight, 16S rRNA concentration, and DNA concentration as denominators, Kendall's tau was calculated for each pair of estimates (tet(C)/mg feces: tet(C)/DNA concentration; tet (C)/mg feces: tet (C)/16S rRNA; tet (C)/DNA concentration: tet (C)/16S rRNA). There was significant correlation for tet (C) concentrations for all comparisons (tau > 0.69, p<0.05). This may suggest that, at least in comparing tet (C) concentrations in community DNA samples of swine of similar age, the use of the fecal weight as a denominator for concentration estimation is valid as proxy measures for bacterial concentration. In addition, the use of fecal weight represents significant cost savings as compared to the other measures evaluated. As such, these data establish the appropriate denominator for future studies of tet (C) concentrations in swine of similar age. In objective 2, the concentration of tet (C) in fecal community DNA from antibiotic free (ABF) pigs was (open full item for complete abstract)

Committee: Thomas Wittum MS, PhD (Advisor); Paivi Rajala-Schultz DVM, PhD, DACVPM (Committee Member); Armando Hoet DVM, PhD, DACVPM (Committee Member) Subjects: Veterinary Services

Doctor of Philosophy, The Ohio State University, 2014, Food Science and Technology

The rapid emergence of antibiotic resistance (AR) has become a major public health concern. Recent findings provided solid evidences suggesting that multiple risk factors contributed to AR development, enrichment, dissemination and persistence, and a comprehensive understanding of AR ecology is essential for targeted mitigation. Following the investigation of AR in aquaculture products from China, a study on AR in fish and aquaculture production-related samples from a U.S. fish farm with controlled practices and no history of antibiotic applications was further conducted to better understand the potential impact of aquaculture production practice on the prevalence of antibiotic resistant (ART) bacteria in the aquaculture ecosystem. Phenotypic resistant populations against sulfamethoxazole with trimethoprim (Sul/Tri), tetracycline (Tet), erythromycin (Erm) or cefotaxime (Ctx) were screened by conventional plating with the corresponding antibiotics, followed by population assessments with denaturing gradient gel electrophoresis (DGGE) and 16S rDNA next generation sequencing (NGS). Despite the absence of antibiotic application in the farm, our results showed that antibiotic resistant (ART) bacteria were abundant in all samples examined, including fish intestine, surface rinsing water, feed, pond water and mud samples. By NGS, a total of 569 genera were identified in Tetr and Ctxr bacteria from five types of samples. Certain correlations of ART bacteria between different types of samples were observed. Various AR genes measured by quantitative PCR (qPCR) were significantly more abundant in fish intestine and feed than farm environmental samples. Certain dominant ART bacteria subpopulations in feed and pond water were also identified in fish intestine. About 79% of 4747 ART isolates from aquaculture samples showed resistance to more than one antibiotic. Some ART isolates showed MIC of Sul/Tri, Tet, Erm or Ctx no less than 512 µg/ml. Various AR genes were detected in A (open full item for complete abstract)

Committee: Hua Wang (Advisor); John Litchfield (Committee Member); Gireesh Rajashekara (Committee Member); Zhongtang Yu (Committee Member) Subjects: Aquaculture; Food Science; Microbiology

Doctor of Philosophy, The Ohio State University, 2011, Food Science and Nutrition

The prevalence of antibiotic resistant (ART) bacteria in ready-to-eat foods including cheese products has been a major public health concern. The objectives of this study were to determine critical control points (CCP) in cheese fermentation for effective mitigation, and to characterize an ART isolate from the dairy environment to reveal potential mechanism(s) involved in the development, maintenance and dissemination of antibiotic resistance (AR). Pilot plant studies indicated that pasteurization effectively reduced ART bacteria in milk and AR can be effectively minimized and controlled with proper sanitation and processing controls. ART bacteria were not significantly amplified during ripening. However, antibiotic facilitated the proliferation of ART bacteria by inhibiting the growth of starter cultures, suggesting rapid and sufficient growth of starter cultures for acid production was important to control AR during chess making process. Results of assessing samples from commercial cheese manufacturing facilities indicated that approximately 104 CFU/ml of Tetr bacteria were found in a manufacturer-maintained starter/adjunct culture vat and representative isolates were identified to be Streptococcus thermophilus (tet(S)). Additional identified AR gene (tet(S), tet(M) and tet(L)) carriers included Streptococcus spp., Leuconostoc sp., Staphylococcus sp., Lactococcus sp., and Lactobacillus sp., from cheese samples collected during the manufacturing process. Results from a small scale survey indicated that the overall quantities of AR genes by real-time PCR in retail cheese products purchased in 2010 reduced compared to those from 2006, suggesting the effectiveness of targeted AR mitigation in related products. A dairy Enterococcus faecium isolate M7M2 carrying both tet(M) and tet(L) genes was further characterized and both tet(M) and tet(L) genes were found to be located on a 19.6 kb plasmid, which was stable (99% retaining rate) in the absence of antibiotic sele (open full item for complete abstract)

Committee: Hua Wang PhD (Advisor); Valente Alvarez PhD (Advisor); John Gunn PhD (Committee Member); Zhongtang Yu PhD (Committee Member) Subjects: Food Science

Master of Science, The Ohio State University, 2008, Food Science and Nutrition

Antibiotic resistance (AR) in pathogens is a worldwide human health concern. The potential of food and enteric commensals as carriers of AR determinants to humans need to be clarified. Gastrointestinal microflora of 14 healthy individuals were examined for the presence of tetracycline-resistant bacteria and tetracycline-resistance genes. Results revealed a resistant count ranging from 106-109 CFU-mL-1, with non-resistant counts ranging from 106-1010 CFU-mL-1. The tetM gene pool determined by TaqMan real-time polymerase chain reaction ranged from 107-109 tetM gene copy number per gram. From the isolates examined, 8 distinct plasmid profiles were observed. DNA-DNA hybridization indicated the plasmid carriage of the tetM gene in some of the isolates. The regular inoculation of the colon with ART bacteria through the food chain is hypothesized as a significant contributor towards the enrichment of antibiotic resistant ART isolates and AR genes in the gut.

Committee: Hua Wang PhD (Advisor); Macdonald Wick PhD (Committee Member); Jeff Culbertson PhD (Committee Member) Subjects: Food Science

Doctor of Philosophy, The Ohio State University, 2024, Food Science and Technology

Antibiotics are powerful tools to treat bacterial infections. However, the surge of antibiotic resistance (AR) is considered one of the top threats to food safety and global public health. Every year, close to 5 million deaths are associated with AR bacterial infections with around 1.27 million deaths directly attributed to AR bacterial infections worldwide. Besides the public health threat, around $60-100 trillion financial loss is expected by 2050 if no action is taken. AR commensals, while usually not directly causing death, can disseminate AR genes to pathogens, and thus indirectly negate effective therapy, making people vulnerable again. Thus, even the most common AR bacterial infections, like mid-ear infections and tooth extraction, may become life-threatening again. For decades, the overuse of antibiotics has been considered as the main cause of AR. Thus, limiting the use of antibiotics has been the primary control strategy worldwide. However, in reality, AR bacteria are still prevalent and persistent in human and animal hosts, as well as the environment even without antibiotic exposure. Humans and animals will get sick and inevitably need antibiotic treatment. AR is a complicated issue and there are multiple contributors to it. Besides the applications of antibiotics, it is now recognized that the rich gut microbiota residing in human and animal gastrointestinal tract system as a breeding ground have key roles in the AR ecology from evolution, dissemination, enrichment and persistence. Oral/nasal introduced AR bacteria proliferate and persist in the host gut. Gut-impacting (orally administered and biliary excreted) antibiotics further selectively enrich AR bacteria. The AR-rich human and animal feces have served as the most significant avenue impacting the AR gene pool in the ecosystem. Therefore, an improved understanding of AR persistent mechanisms and effective mitigation of the pre-existing, persistent AR bacteria in host GI tract system are essential fo (open full item for complete abstract)

Committee: Hua Wang (Advisor); Matthias Klein (Committee Member); Zhongtang Yu (Committee Member); Ye Xia (Committee Member); Michael Cressman (Committee Member) Subjects: Food Science; Health