Streptococcus pneumoniae (pneumococcus) is a commensal gram-positive colonizer of the human nasopharynx capable of causing diseases including otitis media, pneumonia, bacteremia, and meningitis. Although it is often a harmless colonizer, there is a high rate of mortality and morbidity among the immunocompromised, elderly, and young children. While these infections can often be treated with antibiotics, resistance to numerous antibiotics is increasing. Antibiotic resistance is a well-studied dilemma; however, little information is known of how bacteria take up certain antibiotics. Because most antibiotics cannot diffuse freely across the bacterial cell wall, we hypothesize that metabolite transport proteins participate in the uptake of certain classes of antibiotics.

Manure is a commonly used soil fertilizer, but there are concerns that this practice could affect the spread of antibiotic resistance genes (ARGs) from farm to fork. A microcosm-scale study evaluated the effect of prior antibiotic use (manure-based soil amendments generated from dairy and beef cattle with or without antibiotic administration), composting, and soil type on the quantity of ARGs and the microbial community composition of dairy and beef manure applied soil. ARGs were analyzed through novel metagenomic techniques and quantitative polymerase chain reaction of sul1, tet(W), and 16S rRNA gene, while the microbial community composition was determined via 16S rRNA amplicon sequencing. The results indicated that while prior antibiotic administration elevated the relative abundance of ARGs and changed the microbial community of raw manure applied soils, composting reduced this effect. However, compost applied soils still had a higher relative abundance of ARGs than the unamended soils and occasionally soil applied with raw manure of untreated cattle. Soil type may be a mediating factor as there were differences observed between the three soil types (sandy loam, silty clay loam, and silty loam) with sandy loam amended soils often having the least attenuation of ARGs. As the relative abundance of ARGs was still elevated and the microbial community composition still significantly different from the unamended soils after 120 days, these results suggest that 120 days is not a long enough waiting period between biological soil amendments and crop harvest for ARG dissipation. / Master of Science / Antibiotics are lifesaving drugs that kill infection-causing bacteria. However, bacteria are living organisms and can adapt to stresses, such as antibiotics. When antibiotics are used, not all of the targeted bacteria are necessarily killed, and populations of resistant bacteria can survive. Resistant bacteria can not only continue to grow, but can also share their resistance capabilities with other unrelated bacteria through the transfer of antibiotic resistance genes (ARGs). ARGs are segments of DNA encoding mechanisms for the bacteria to survive antibiotic attack, such as pumping antibiotics outside of the cell or strengthening the cell wall so antibiotics cannot enter. The transfer of ARGs to human pathogens is of utmost concern, as it can cause once treatable diseases to turn deadly. Antibiotics are thus a double-edged sword because they can save lives on one hand, while their overuse or misuse can undermine their effectiveness by increasing antibiotic resistance. In the U.S. and many other countries, the biggest user of antibiotics is the livestock industry. Thus, there is growing interest in possible routes by which antibiotic resistance can spread from agriculture to humans. While some previous work has been done on direct contact with animals and meat products, less attention has been paid to the potential role raw produce grown in soils fertilized with manure-based amendments. This study thus sought to determine which factors impact ARG levels in soil. Questions of interest included: What is the effect of composting raw manure prior to soil application? Does prior treatment of cattle with antibiotics matter? Does the soil type influence the levels of ARGs? Do the ARG composition and microbial community composition respond similarly to such factors? These and other questions were evaluated in a controlled environment by simulating amended field conditions in small glass jars (microcosms) containing mixtures of different soils and manure-based amendments. Three different soils were amended with one of the following manure-based amendments: raw manure from antibiotic administered cattle, composted manure of antibiotic administered cattle, raw manure from cattle not given antibiotics, composted manure of untreated cattle, and no amendment. This experimental setup was done in duplicate, one for treatments from dairy cows and one for the beef steer treatments. The experiment lasted 120 days, as that is a current standard for how long organic farmers must wait between manure application and crop harvest. Samples were taken throughout the 120-day experiment, and the quantity of targeted ARGs was determined by analyzing the DNA through qPCR, while the overall ARG profile was studied using a new tool, called metagenomics. To identify the kinds of bacteria present in the samples (microbial community composition), the 16S rRNA gene, which is a universal gene in organisms, was targeted and sequenced via amplicon sequencing. The results of these analyses indicated that administering antibiotics to cattle and then subsequently amending soil with their manure was associated with the highest levels of ARGs compared to the other treatments, but composting reduced the effect of prior antibiotic use. Depending on the ARG, composting decreased ARG levels relative to the other treatments, but in some instances, it increased ARGs compared to soils with raw manure of untreated cattle. Even after composting, there were still higher levels of ARGs in the soil than unamended soils. Different soil types did react differently to the amendments, but more research is needed. All of the treatments resulted in different changes to the microbial community composition and did not return to the unamended soil’s community structure even after 120 days. Overall, based on these results, ARGs and the microbial community do not return to the initial condition within 120 days, which is a recommended wait time between amendment and harvest, while composting and soil type appear to be mediating factors. Additional research is needed to further advance understanding of potential mitigation options and to benchmark them to defined and measureable risk endpoints.

Antibiotic resistance is a major threat to human health. Clinical situations are the main focus for antibiotic resistance research, but understanding the spread of resistance in the environment is also vital. A major contributor to this spread is wastewater from combined sewer overflow (CSO) events. The effect of CSO events on antibiotic resistance in the James River near Richmond, Virginia was studied using genomic and microbiological approaches. The abundance of genes associated with resistance to quinolones (qnrA) and tetracycline (tetW) was strongly correlated with the presence of fecal indicator bacteria (E. coli abundance) as well as total nitrogen and phosphorus loads, which suggests an anthropogenic source of these genes. Abundance of the blaTEM gene, which confers resistance to β-lactam antibiotics, was elevated during CSO events and increased with precipitation and river discharge. Bacteria isolated during a CSO event were resistant to more antibiotics and had higher multi-drug resistance when compared to isolates from a non-event. This study demonstrated that CSO events are contributing to the spread of antibiotic resistance.

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

The overuse and misuse of antibiotics to treat bacterial infections, the release of unmetabolized residuals into the sewer system, and the incomplete removal antibiotic residues by wastewater treatment plants (WWTPs) pose a severe threat to human health. The accumulation of antibiotic residue induces selective pressure on the bacterial population, resulting in the spread of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in water. This study investigated the degradation of different types of ARGs in water matrices using a wide variety of treatment technologies. Real wastewater samples were collected from a WWTP in urban Philadelphia and the presence of single and multidrug-resistant bacteria and resistance genes were investigated using molecular-based techniques. Subsequently, an analytical method was developed and validated for the detection and quantification of the ARGs against a range of antibiotics, such as tetracycline (TCN), ciprofloxacin (CIP), and levofloxacin (LVX). Finally, to remove the ARGs from water matrices, different conventional and advanced oxidation processes were applied. At the very onset, conventional treatment processes such as chlorine treatment was used to inactivate the E.coli resistant strains. It was observed that chlorination can potentially deactivate the ARBs by applying a lower dose and contact time. However, the effectiveness of chlorine treatment in removing all types of ARGs from water matrices was limited. For instance, no significant degradation of extracellular ARGs (e-ARGs) was observed in DI water during chlorine treatment. Subsequently, a peracetic acid (PAA) based treatment process was used to degrade the genomic and plasmid-encoded ARGs from the water matrices. Similar to chlorine treatment, no significant changes were observed in the degradation of extracellular ARGs (e-ARGs) in DI and WW. Then, the degradation kinetics of ARGs across different types (gyrAR, tetAR, qnrSR) and forms (chromosomal, plasmids) were evaluated using the Ultraviolet (UV) disinfection process. Compared to chlorination and PAA, UV treatment showed better removal efficiencies for the degradation of different types of e-ARGs in DI water. The degradation profile of e-ARGs showed 1-4 log reductions at a UV fluence of 900 mj/cm2. The i-ARGs showed similar degradation rates as compared to e-ARGs in phosphate buffer saline (PBS) at the same UV dosage. On the other hand, the regrowth potential of ARBs at low UV dosage (60–180 mJ/cm2) showed the evidence of damage repairment after several hours of exposure to light (photoreactivation) and dark conditions, making it susceptible again to the resistance spread. To resolve this issue, process parameters were optimized, and no regrowth of the ARBs were found from the higher fluence from 300 to 600 mJ/ cm2. Later, UV/ H2O2 based AOP was applied to evaluate the degradation and deactivation of the same resistant genes. The addition of H2O2 during the UV treatment produces strongly reactive •OH radicals during the treatment and showed considerable improvements in e-ARGs degradation (1.2-5 logs) compared to UV treatment alone. However, this AOP showed minimal contribution to i-ARG degradation (1-2.4 logs), possibly due to the scavenging of •OH radicals by the cellular components in PBS. In contrast to PBS, the wastewater matrix moderately enhanced the gene degradation during the treatment. In terms of plasmid degradation, the conformational differences of the supercoiled structures showed 1.2-2.8 times slower degradation rates than chromosomal ARGs. In addition, the degradation kinetics of the free residual ARGs (f-ARGs) were assessed during the treatment to reduce the AMR dissemination risk from the treated sample. This study also examined the potential of ozone (O3) based oxidation process to degrade and deactivate the extracellular and intracellular ARGs, and MGE (plasmid, intl-1) from E.coli ARBs. The degradation kinetics of the ARGs across different sizes (118-454 bps) and types were evaluated in different water matrices (DI water, PBS, and WW), and showed a significantly higher removal for chromosomal, and plasmid encoded ARGs than other treatment technologies. For the e-ARGs in DI water, 3.8-5.2 logs removal was observed at ozone dosage of 2.0 × 10-2 M.s. i-ARGs in PBS and wastewater showed nearly similar degradation (3.8-5 logs) during O3, indicating the elimination of i-ARGs was not dependent on the cellular components and effluent organic matter. Moreover, an analysis of environmental DNA (eDNA) from wastewater was conducted to examine the degradation of DNA and ARGs for different storage periods and temperatures (-20°C, 0°C, 4°C, 22±0.87°C). Result indicated that water samples kept at -20°C and 0°C showed the best performance in preventing the DNA concentration and gene degradation over time. Additionally, the effectiveness of different preservatives (Longmire buffers: LB1 and LB2, benzalkonium chloride at 0.1%, 0.01%) were investigated in preserving the DNA integrity and the gene degradation at an ambient temperature. It was found that the Longmire buffer (LB1) exhibited lowest gene degradation during the three-week storage period. In summary, this research provided a comprehensive assessment on the degradation of e-ARGs, i-ARGs, and free ARGs from water using different treatment technologies (i.e., UV, UV/H2O2, O3, PAA, chlorine). Additionally, this study suggested valuable information on optimizing the process parameters of the selected methods and developed a comparative assessment of removing the ARGs from the water matrix (DI/PBS, WW). The estimation of Electrical Energy per Order (EEO, kWh/m3) during UV and ozone treatments provided a comparison of the energy consumption for ARGs degradation in the water. Overall, the findings of this study can be useful for evaluating different types and forms (chromosomal, plasmid) of ARG degradation from water matrices and can help to reduce the risk of AMR dissemination in the environment. / Civil Engineering

No description available.

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.

Raw vegetables can sometimes be the source of outbreaks of human illness; however the potential for fresh vegetables to serve as a vehicle for antibiotic -resistant bacteria is poorly understood. Antibiotics and antibiotic-resistant bacteria have been shown to persist in manure of animals administered antibiotics, and in compost generated from this manure, where there is the potential for their transfer to produce. The purpose of this study was to determine the survival of antibiotic-resistant bacteria on raw, peeled, carrots after washing with commonly used chemical sanitizers. Multi-drug resistant E. coli O157:H7 and Pseudomonas aeruginosa were inoculated into a compost slurry of composted manure from dairy cattle, with and without prior administration of antibiotics, and used to inoculate carrot surfaces prior to the washing studies. This approach provided defined model antibiotic-resistant pathogens present within a background microbial community simulating potential carry over from manure-derived fertilizer. Carrots (n=3, 25g) were air-dried and stored at 4 °C until washing with tap water, XY-12 (sodium hypochlorite, 50 ppm free chlorine) or Tsunami 100 (peroxyacetic acid/hydrogen peroxide, 40 ppm free paracetic acid), according to manufacturer's directions. A second batch of carrots representing each inoculation x wash condition (n=3) were individually packaged for storage at 2 °C for 1,7, and 14 days, or 10 °C for 7 days and enumerated on those day intervals to recover bacteria from the surfaces of washed carrots. The resulting previously washed and stored carrots were subject to serial dilution and plated onto corresponding agar to enumerate total aerobic bacteria (R2A), aerobic bacteria tolerant or resistant to antibiotics (antibiotic-supplemented R2A), E. coli (Eosin Methylene Blue), and Pseudomonas spp. (Pseudomonas Isolation Agar). In addition, the tetA gene was quantified from the carrot samples as a measure of the effect of sanitizers and storage on an antibiotic resistance gene known to be carried by the inoculated bacteria.Inclusion of sanitizer in the wash water significantly reduced the absolute numbers of inoculated bacteria (E.coli and Pseudomonas) as well as populations of bacteria capable of growth on the R2A media containing cefotaxime (10μg/mL), sulfamethoxazole (100μg/mL), or tetracycline (3μg/mL). Comparable reductions in the inoculated P. aeruginosa resistant to tetracycline (PIA T, 4μg/mL), bacteria resistant to cefotaxime (10μg/mL) and tetracycline (3μg/mL) occurred after washing with XY-12 or Tsunami 100. The sanitizer effectiveness may be bacterial dependent, as evident by larger absolute reductions of the inoculated E. coli (EMB) and bacteria grown on sulfamethoxazole (100μg/mL)-amended plates after washing with Tsunami 100 compared to washing with tap water or XY-12. Re-growth of both the inoculated and native compost-associated bacteria was inhibited by storage at 2 °C, as there were no significant differences in the log CFU/g values on the various media (total aerobic bacteria, bacteria on antibiotic-amended plates, E. coli inoculum, P. aeruginosa inoculum) during the 14-day storage period. However, temperature abuse at 10 °C resulted in significant re-growth of native Pseudomonas, compared to storage at 2 °C. A sanitizer-associated interaction between re-growth and temperature was also observed for bacteria resistant to clindamycin (25μg/mL) and cefotaxime (10μg/mL), with substantial re-growth occurring only on carrots washed with Tsunami 100. There was no significant re-growth of the inoculated E. coli O157:H7 at either temperature. Results indicate that some bacterial populations are reduced by post-harvest washes and that temperature abuse of fresh produce may result in increases in antibiotic-resistant bacterial populations. / Master of Science in Life Sciences

The concentration of triclosan in antiseptics, disinfectants and preservatives in products exceeds the minimal lethal levels. Extensive use of triclosan and antibiotics results in bacterial resistance to their active ingredients. The precise relationship between use and resistance, however, has been challenging to define. The aim of the study was to identify and determine antibiotic resistance profiles of triclosan tolerant heterotrophic plate count bacteria isolates from sewage influent and effluent. R2 agar supplemented with triclosan was utilised to isolate the triclosan resistant bacteria. To determine the minimum inhibitory concentration (MIC), organisms were incubated for 24 hours at selected concentrations of triclosan. Polymerase chain reaction (PCR) amplification of the 16S rRNA region was done to identify isolates. An assay for cross resistance to various antibiotics was performed. Determination of enhanced resistance to antibiotics by adding antimicrobials to the medium will be performed by using three antibiotics. High performance liquid chromatography was conducted to quantified levels of triclosan persistent in sewage water. Forty-four isolates were resistant to levels of triclosan ranging from 0.25 mg/l to 0.5 mg/l. Minimum inhibitory concentration values of these isolates ranged from 0.125 mg/l to >1 mg/l of triclosan. 16S rDNA methods were used and five main genera namely, Bacillus, Pseudomonas, Enterococcus, Brevibacillus and Paenibacillus were identified. Cell wall targeting antibiotics showed more pronounced relation with the triclosan concentration. Relation to triclosan concentration is not as apparent with the antibiotic targeting protein synthesis. Combination of antimicrobials indicated that at certain triclosan concentrations synergism or antagonism is observed. The importance of applying the correct concentration and combination of antimicrobials is observed. Levels of triclosan were found throughout the sewage water. HPLC values indicated the presence of triclosan at post-grid removal and effluent of the WWTP. The triclosan concentrations decrease through the WWTP but small concentrations enter our water bodies. The presence of bacterial species that are resistant to high concentrations of triclosan and multiple antibiotics enter our natural water bodies and is cause for concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The concentration of triclosan in antiseptics, disinfectants and preservatives in products exceeds the minimal lethal levels. Extensive use of triclosan and antibiotics results in bacterial resistance to their active ingredients. The precise relationship between use and resistance, however, has been challenging to define. The aim of the study was to identify and determine antibiotic resistance profiles of triclosan tolerant heterotrophic plate count bacteria isolates from sewage influent and effluent. R2 agar supplemented with triclosan was utilised to isolate the triclosan resistant bacteria. To determine the minimum inhibitory concentration (MIC), organisms were incubated for 24 hours at selected concentrations of triclosan. Polymerase chain reaction (PCR) amplification of the 16S rRNA region was done to identify isolates. An assay for cross resistance to various antibiotics was performed. Determination of enhanced resistance to antibiotics by adding antimicrobials to the medium will be performed by using three antibiotics. High performance liquid chromatography was conducted to quantified levels of triclosan persistent in sewage water. Forty-four isolates were resistant to levels of triclosan ranging from 0.25 mg/l to 0.5 mg/l. Minimum inhibitory concentration values of these isolates ranged from 0.125 mg/l to >1 mg/l of triclosan. 16S rDNA methods were used and five main genera namely, Bacillus, Pseudomonas, Enterococcus, Brevibacillus and Paenibacillus were identified. Cell wall targeting antibiotics showed more pronounced relation with the triclosan concentration. Relation to triclosan concentration is not as apparent with the antibiotic targeting protein synthesis. Combination of antimicrobials indicated that at certain triclosan concentrations synergism or antagonism is observed. The importance of applying the correct concentration and combination of antimicrobials is observed. Levels of triclosan were found throughout the sewage water. HPLC values indicated the presence of triclosan at post-grid removal and effluent of the WWTP. The triclosan concentrations decrease through the WWTP but small concentrations enter our water bodies. The presence of bacterial species that are resistant to high concentrations of triclosan and multiple antibiotics enter our natural water bodies and is cause for concern. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015