The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.

The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 107-118). Also available in electronic version.

Best Practices: Antibiotic Stewardship and the Implementation of Evidence-Based Guidelines During Upper Respiratory Infections Among Rural, Primary Care Patients Sandra Kay Johnson, Doctor of Nursing Practice Candidate East Tennessee State University College of Nursing Abstract Antimicrobial resistance is a challenging clinical issue seen in the treatment of upper respiratory infections (URIs). Purpose/Aims: The purpose of this project is to implement an antibiotic stewardship (AS) program using antibiotic prescribing guidelines for URIs and clinic-wide patient education. The aim was to reduce antibiotic overuse. Processes: This quality improvement (QI) project was conducted at a rural clinic. Participants included the nurse practitioners and office staff. The target population included adult patients, ages 18 and up, who presented with upper respiratory symptoms. URI prescribing guidelines were used to compare antibiotic prescribing practices pre- and post-intervention. The initiative includes a provider education session and educational videos, posters, and scientific literature, which is available to those assessing the clinic. Educational materials were from the Centers for Disease Control and Prevention, the United States Department of Health and Human Services, and the National Center for Emerging Zoonotic Infectious Diseases. The Knowledge-to-Action framework was used to translate research into practice and data was collected through the administration of questionnaires and the review of EMRs. Results: Preliminary results suggest patient preferences, outside of guidelines, may influence prescribing behaviors. Limitations: The sample of providers was small and may not be characteristic of a larger group. Conclusions: This QI initiative was driven by the need to improve practice and educate patients regarding AS during upper respiratory-related illnesses. Preliminary evidence shows interventions such as patient education, implementation of guidelines, and identification of barriers and facilitators are all vital components of AS.

Antibiotics are commonly prescribed and requested for viral illnesses despite evidence-based research studies and societal guidelines that advise against this practice. Literature has indicated that antibiotic decision-making comes from a provider's experience or exposure to illness, uncertainty of illness, or from being pressured by the patient. Nurses and advanced practice nurses are important participants in the antibiotic stewardship initiative. The purpose of this project was to examine potential knowledge deficits responsible for inappropriate antibiotic prescribing at a rural urgent care clinic in the southeastern United States, which when addressed could promote an educational in-service to decrease the number of antibiotics prescribed during a high-volume cough, cold, and flu months. The health belief model was used as a foundational model and a knowledge, attitude, and practice survey to collect data. Antibiotic prescribing habits were evaluated in the preintervention group (n = 250) and a year later in the postintervention group (n = 265). Antibiotic prescribing decreased positively from 80% to 70% and watchful waiting also increased positively from 4% to 30%; X-² (1) = 12.302, p = .000. The increase in educational awareness from these results can support a decrease in inappropriate antibiotic prescriptions, which prevents the emergence of antibiotic-resistant bacteria, contributing to positive social change.

The reduced efficacy of antibiotics in treating common infections is one of the most pressing health concerns of the 21st Century. Increasing evidence links the widespread use of antibiotics in livestock production to the transfer of bacteria carrying antibiotic resistance genes to the broader environment. It is therefore critical to understand the persistence and dissemination of resistance in agricultural soils to understand potential threats to consumers. The goal of this large-scale agricultural field experiment was to identify the effects of crop (lettuce, radish) and fertilizer type (inorganic, compost, raw manure) on the incidence and persistence of antibiotic-resistant fecal coliforms, a common family of fecal indicator bacteria used to track the environmental spread of antibiotic resistance. Soil samples were collected eight times over a 120-day period and analyzed for fecal coliforms utlizing a suite of MacConkey agars supplemented with different antibiotics (ceftazidime, clindamycin, erythromycin, sulfamethoxazole, and tetracycline). Given the number of samples with resistant fecal coliform concentrations below the limit of detection, analyses to identify the effects of soil treatment and crop relied on Zero-inflated Poisson Regressions. Antibiotic-resistant culturable fecal coliforms were recoverable from soils across all treatments immediately following application, though persistence throughout the experiment varied by antibiotic. Sulfamethoxazole- and tetracycline-resistant fecal coliforms were nondetectable after Day 1; this was expected, as the cattle supplying the manure amendments were not treated with these antibiotics or similar analogs. Clindamycin- and erythromycin-resistant fecal coliforms were nondetectable after 42 days but rebounded on Day 90 in the soil; both of these drugs were of the same antibiotic class as the ones used to treat the dairy cattle during the manure collection period. Ceftazidime-resistant fecal coliform levels were consistently high throughout the duration of the growing season. No statistical differences were observed between root and aboveground crops. Results suggest that soils amended with raw or composted dairy manure are at risk of contamination with antibiotic resistant fecal coliforms; however, composting decreased the antibiotic resistant fecal coliform levels of the macrolide (erythromycin) and lincosamide (clindamycin) antibiotic classes administered to the dairy cattle (cephapirin and pirlimycin). / Master of Science / The reduced efficacy of antibiotics in treating common infections is one of the most pressing health concerns of the 21st Century. Increasing evidence links the widespread use of antibiotics in livestock production to the transfer of bacteria carrying antibiotic resistance genes to the broader environment. It is therefore critical to understand the persistence and dissemination of resistance in agricultural soils to understand potential threats to consumers. The goal of this large-scale agricultural field experiment was to identify the effects of crop (lettuce, radish) and fertilizer type (inorganic, compost, raw manure) on the incidence and persistence of antibiotic-resistant fecal coliforms, a common family of fecal indicator bacteria used to track the environmental spread of antibiotic resistance. Over the course of 120 days, samples were collected from field plots to identify if there were antibiotic-resistant bacteria (ARBs) in the soil. This study was partially motivated as a means to evaluate the Federal Drug Administration’s Food Modernization Safety Act updated manure treatment guidelines in decreasing potential pathogenic bacteria in soils used to grow vegetables for human consumption. Antibiotic-resistant bacteria were recoverable from soils across all fertilizer types immediately following application, though persistence throughout the experiment varied by antibiotic tested. From the above findings, compost amended soils had greater quantities of total and ceftazidime-resistant bacteria. However, composting did show a significant decrease in the antibiotic-resistant bacteria levels found in the same antibiotic classes, macrolides and lancosimides, that the dairy cattle were treated with at the beginning of this study.

Antibiotic resistance is a growing human health threat, making infections more difficult to treat and increasing fatalities from and cost of treatment of associated diseases. The rise of multidrug resistant pathogens threatens a return to the pre-antibiotic era where even the most common infections may be impossible to treat. It is estimated that the majority of global antibiotic use, and use in the U.S., is dedicated towards livestock, where they are used to promote growth, treat, or prevent disease. Given that exposure to antibiotics selects for antibiotic resistant bacteria (ARBs) and can stimulate the horizontal transfer of their associated antibiotic resistance genes (ARGs), it is important to examine livestock operations as a reservoir of resistance. Correspondingly, there is growing interest in identifying how agricultural practices can limit the potential for spread of antibiotic resistance through the "farm to fork continuum," starting with antibiotic use practices, manure management and land application and ending with the spread of ARBs and ARGs present onto edible crops and serving as a route of exposure to consumers. This study focused specifically on the effect of composting on the prevalence of ARBs and ARGs in cattle manure. Three composting trials were performed: small-scale, heat-controlled, and large-scale. The small-scale composting trial compared dairy and beef manures, with or without antibiotic treatment (treated beef cattle received chlortetracycline, sulfamethazine, and tylosin while treated dairy cattle received cephapirin and pirlimycin), subject to either static or turned composting. The heat-controlled composting trial examined only dairy manure, with or without antibiotic treatment, subject to static composting, but using external heat tape applied to the composting tumblers to extend the duration of the thermophilic (>55°C) temperature range. The large-scale composting trial examined dairy manure, with or without antibiotic treatment, subject to static composting at a much larger scale that is more realistic to typical farm practices. Samples were analyzed to assess phenotypic resistance using the Kirby Bauer disk diffusion method and by diluting and plating onto antibiotic-supplemented agar. Genetic markers of resistance were also assessed using quantitative polymerase chain reaction (qPCR) to quantify sul1 and tet(W) ARGs; metagenomic DNA sequencing and analysis were also performed to assess and compare total ARG abundance and types across all samples. Results indicate that composting can enrich indicators of phenotypic and genetic resistance traits to certain antibiotics, but that most ARGs are successfully attenuated during composting, as evidenced by the metagenomic sequencing. Maintaining thermophilic composting temperatures for adequate time is necessary for the effective elimination of enteric bacteria. This study suggests that indicator bacteria that survive composting tend to be more resistant than those in the original raw manure; however, extending the thermophilic stage of composting, as was done in the heat-controlled trial, can reduce target indicator bacteria below detection limits. Of the two ARGs specifically quantified via qPCR, prior administration of antibiotics to cattle only had a significant impact on tet(W). There was not an obvious difference in the final antibiotic resistance profiles in the finished beef versus dairy manure composts according to metagenomics analysis. Based on these results, composting is promising as a method of attenuating ARGs, but further research is necessary to examine in depth all of the complex interactions that occur during the composting process to maximize performance. If not applied appropriately, e.g., if time and temperature guidelines are not enforced, then there is potential that composting could exacerbate the spread of certain types of antibiotic resistance. / Master of Science / Antibiotics are drugs that are used to treat bacterial infections by killing the bacteria that cause the infection. Bacterial infections now exist that are resistant to several antibiotics; which are extremely difficult and costly to treat. Many antibiotics are used in the agriculture industry where they are used to promote growth, treat, or prevent disease in livestock animals. The antibiotics may then cause an increase in antibiotic resistance in bacteria by encouraging changes to the DNA of the bacteria which allow them to survive in the presence of antibiotics that would normally kill them. These DNA segments are called antibiotic resistance genes. Once developed, bacteria can share resistance genes among themselves, allowing for single bacteria that can resist several types of antibiotics. For this reason, it is important to see if it is possible to prevent the spread of antibiotic resistance from animal agriculture to people. One way that people could be affected would be if produce were exposed to resistant bacteria when grown in soil that had been fertilized with manure or compost. This study looks at the impact of composting on the presence and amount of antibiotic resistance genes in composted cattle manure. Three composting trials were performed: small-scale, heat-controlled, and large-scale. The small-scale composting trial compared dairy and beef manures, with or without antibiotic treatment, with or without regular turning during composting. The heat-controlled composting trial examined only dairy manure, with or without antibiotic treatment, without regular turning during composting, but using external heat to maintain high temperatures. The large-scale composting trial examined dairy manure, with or without antibiotic treatment, without regular turning during composting, but at a larger scale that is more realistic to how composting is actually performed on farms. Antibiotic resistance of compost bacteria was tested by growing bacteria on nutrient-dense plates containing antibiotic disks and measuring how much each antibiotic prevented the growth of the bacteria, in terms of the diameter about each disk where bacteria did not grow. Individual target resistance genes were measured throughout the study by using a method called qPCR. Metagenomic analysis was performed to identify all of the genes, especially resistance genes, in each of the samples. Results v show that composting may increase antibiotic resistance in bacteria that survive the composting process, but that most resistance genes are themselves reduced. The key to successful composting is maintaining high temperatures for as long as possible; this is necessary to kill off infectious bacteria. Extending the high temperature (>55°C) phase of composting is a potential method for improving the effectiveness of composting in eliminating pathogens and destroying resistance genes. Results were not significantly affected by whether antibiotics were given to the cattle and were not different between dairy or beef cattle. Based on these results, composting is a promising method of reducing resistance genes in composted manure, but further research is necessary to maximize performance. If not performed correctly, composting could have the opposite effect and be detrimental.

Mitomycin C (MMC), obtained from Streptomyces caespitosus, a clinically useful antitumour antibiotic, is the archetypical quinone bioreductive alkylating agent. The reductive activation mechanism of MMC, involves quinone reduction sequentially activating electrophilic sites in the drug molecule (C-l and C-lO for MMC). This research project was designed to investigate the role of the C-lO in alkylation processes by preparing compounds in which the electrophilicity at C-l is much reduced by substituting a cyclopropane for the aziridine ring. The resulting pyrrolo[I,2-a]indole, cyclopropamitosenes, could on reductive activation, by either 1- or 2-electron processes, followed by elimination of the carbamate, generate a powerful electrophile capable of alkylating DNA (or other nucleophiles) at C-lO .. A range of compounds was prepared utilising the azidocinnamate decomposition route to substituted indoles and an intramolecular [3 + 2] cycloaddition strategy was employed to synthesise the pyrrolo[I,2-a]indole nucleus. The rapid ring opening of cyclopropylcarbinyl radicals is briefly outlined. The reduction-initiated ring opening of the cyclopropane ring is investigated, thereby establishing its relevance to the potent bioreductive anticancer action of the cyclopropamitosenes, novel analogues of MMC. The design and synthesis of fused [I,2-a]indoles without the cyclopropane ring, is examined for comparative purposes. The key step in the synthesis is the formation of the [I,2-a ]indole nucleus via a radical cyclisation. Biological data were recorded for the cyclopropamitosenes and correlated with their structures.

No description available.

No description available.