The rise of β-lactam-resistant bacteria from agricultural settings, including food-producing animals and their related food products has become a significant public health concern. Consumption of food contaminated by such bacteria may cause infection as well as the transmission of resistance genes. Here we used a mouse model to assess the impact of different antibiotic treatments on the composition of the gut microbiota and any impact on the transfer of β-lactam resistance genes between donor and recipient bacteria. Mice were inoculated with β-lactam resistant Escherichia coli and an antibiotic-susceptible Salmonella Heidelberg strain. The mice were treated with either streptomycin, ampicillin or both antibiotics. Mouse feces were collected at regular intervals and processed using selective culture techniques to capture potential transfer of resistance genes. Gene transfer was confirmed by whole genome sequencing. DNA extracted from the feces was used for monitoring changes in microbial profiles by 16S rDNA sequencing. In the absence of antibiotic treatment, the inoculated bacteria were only transiently detected and no transconjugants were recovered from the mouse feces. In comparison, antibiotic treatment changed microbial profiles in the mouse gut, enhanced colonization of the bacterial isolates, and facilitated the transfer of the resistance genes into both S. Heidelberg and commensal E. coli recipient strains. The results of this study indicated that the use of multiple antibiotics may enhance infection of opportunistic β-lactam resistant bacterial pathogens relative to single antibiotics and pose a greater risk in terms of antibiotic resistance gene transfer. Such process might occur in clinical settings where patients are under prolonged antibiotic treatments. Information gained through this study together with future work will inform the development of new policies guiding the prudent use of antibiotics.

Background: Antimicrobial resistance (AMR) is a widely recognized threat to global health that has been accelerated by the overuse of antimicrobials. Policy and regulatory interventions offer a useful approach to bring about widespread change in antimicrobial use. Objectives: This dissertation aims to investigate and characterize the role of policy interventions on antimicrobial use, to understand which policy levers have been used, which interventions have proven to be effective, and to identify analytic strategies that assist researchers and policymakers in developing evidence-informed approaches to address human AMR. Methods: To investigate effectiveness of policy interventions, I developed a systematic review and evidence map and appraised the quality of research on government policies to reduce antimicrobial use. To evaluate the extent of policy action on AMR in the Canadian context, I conducted an environmental scan of AMR action between 2008-2018. Finally, to facilitate advances in the scientific evidence base on antimicrobial resistance policy, I developed a framework for planning, conducting, and disseminating AMR intervention research. Findings: Governments have a variety of policy options at their disposal to respond to the growing threat of AMR, however, most of these policy options have not been rigorously evaluated and their effectiveness is unknown. Some regulatory, communication, guidelines, and fiscal interventions have been effective in particular settings, however, the interventions were too disparate to generalize about the effectiveness of any particular policy approach and reporting gaps in pose challenges for interpreting and replicating study results. In Canada, policy interventions have been too few, too small and too uncoordinated to achieve our AMR goals. Building on these findings, I developed a framework for planning, conducting, and disseminating AMR intervention research that identifies challenges, areas for increased coordination with policymakers, and best-practices for overcoming common limitations in AMR research. Discussion: Governments around the world have committed to respond to AMR, however, rigorous evidence to inform their policy and regulatory strategies is lacking. As the threat posed by AMR grows, it is increasingly important to strengthen the scientific evidence base on AMR policy interventions, to learn from existing policies and programs, and integrate scientific evidence into the global AMR response.

The prevalence of antimicrobial resistance (AMR) phenotypes and genotypic characteristics (Class 1 integron and AMR gene cassettes) in commensal Escherichia coli (EC) and vancomycin resistant Enterococcus faecium (EF) isolated from humans and swine in a semi-closed, integrated farrow-to-fork population were evaluated in a crosssectional study. Our objective was to establish baseline antimicrobial resistance patterns and to evaluate the stability of isolate recovery phenotype within multiple grab samples per collection day and over multiple biweekly samples collected during a period of several months. This data will serve as a baseline for continuing longitudinal studies within the population. These continuing studies should produce the first comprehensive epidemiological data to document the transmission dynamics of antimicrobial resistance in the farrow-to-fork continuum. Outcome variables assessed included: phenotypic resistance in EC, pan-susceptibility, multi-resistance and genotypic resistance. Potential predictor variables included: 1) host species, 2) unit, 3) unit type, 4) housing cohort by species, and 5) time of day. There were significant differences (p<0.05) between host species with swine at higher odds for both single and multiple resistance. There were also differences in resistance based on unit location, unit-type, and housing cohort within both humans and swine. Our study found no significant differences (p>0.05) in resistance between swine workers and non-swine workers with the sole exception of resistance to cephalothin, with non-swine workers at 1.89 higher odds for resistance (p=0.02). A total of 17 VRE were isolated from human wastewater samples, and to the author’s knowledge these represent the first environmentally isolated VRE in the U.S. Several unique multi-resistance phenotypes were observed and future evaluation of AMR phenotype in continuing longitudinal studies provides a unique opportunity to study phenotypic patterns and dissemination through the study population.

Bacterial resistance to antibiotics has increased rapidly within recent years, and it has become a serious threat to public health. Infections caused by multi-drug resistant bacteria entail higher morbidity, mortality, and a burden to health care systems. The use of biocides, including silver compounds, may affect the resistance to both biocides and antibiotics and, thereby, can be a driving factor in this development. The aim of the following thesis was to investigate the frequency of silver resistance and the effects of silver exposure on bacterial populations being of clinical significance and from geographically different parts of the world. Furthermore, it explored the genetic background of silver resistance, and if silver could select directly or indirectly for antibiotic resistance. By a range of methods, from culture in broth to whole genome sequencing, bacterial populations from humans, birds and from the environment were characterized. The studies showed that sil genes, encoding silver resistance, occurred at a high frequency. Sil genes were found in 48 % of Enterobacter spp., in 41 % of Klebsiella spp. and in 21 % of all human Escherichia coli isolates with production of certain types of extended-spectrum beta-lactamases (CTX-M-14 and CTX-M-15). In contrast, silver resistance was not found in bird isolates or in bacterial species, such as Pseudomonas aeruginosa and Legionella spp., with wet environments as their natural habitat. One silver-resistant Enterobacter cloacae strain was isolated from a chronic leg ulcer after only three weeks of treatment with silver-based dressings. The in-vivo effects of these dressings were limited, and they failed to eradicate both Gram-positive and Gram-negative bacteria. The activity of silver nitrate in vitro was bacteriostatic on Gram-positive species such as S. aureus and bactericidal on Gram-negative species. In Enterobacteriaceae, sil genes were associated with silver resistance phenotypes in all but one case. Using whole genome sequencing, single nucleotide polymorphisms in the silS gene were discovered after silver exposure in isolates with expressed silver resistance. This resistance could co-select for resistance to beta-lactams, co-trimoxazole and gentamicin. The findings of this thesis indicate that silver exposure may cause phenotypic silver resistance, and it may reduce the susceptibility to mainly beta-lactams and select for bacteria with resistance to clinically important antibiotics.

Antimicrobial resistance and antimicrobial misuse are of global concern but affect more the resource-constrained settings. Limited research has been done in Peru in order to address these topics. We interviewed 256 physicians of two hospitals of Lima in order to assess the knowledge, attitudes, and practices about antimicrobial resistance and antimicrobials use. Most of them agreed that antimicrobial resistance is a problem worldwide and within Peru, but only 20% correctly estimated the level of resistance of Klebsiella pneumoniae to third generation cephalosporins. We also developed a network of nine hospitals in Lima to perform a resistance surveillance of key isolates causing bacteremia. We found during a one-year period that Staphylococcus aureus was the most frequently Gram positive isolated (22%). K. pneumoniae and Escherichia coli were the most frequent Gram-negative bacilli, >75% of both produced extended-spectrum β-lactamases (ESBLs). ESBL-producers microorganisms had also higher level of co-resistance to ciprofloxacin and gentamicin. Finally, to explore the antimicrobial resistance and molecular characteristics of methicillin-resistant S. aureus, we analyzed isolates from patients and healthcare workers (HCWs). Fifty percent of 338 blood isolates were methicillin-resistant (MRSA); one predominant multidrug-resistant MRSA strain was found in all hospitals (ST 5 spa t149-SCCmec I-, the Cordobes-Chilean clone. MRSA nasal carriage rate of 8.7%, was found among HCWs. The two most common clones circulating among HCWs were also the two predominant among patients with bacteremia. There is a need to implement cost-effective infection control policies to reduce the transmission of multidrug resistant microorganisms in these settings. / La résistance aux antibiotiques et leur utilisation inappropriée constituent un problème majeur de santé publique qui affecte particulièrement les pays à revenus faibles et intermédiaires. Les données concernant cette problématique sont très limitées au Pérou. Nous avons interrogé 256 médecins de deux hôpitaux à Lima afin d’évaluer leurs connaissances et leurs pratiques vis-à-vis de l’utilisation des antibiotiques et de la résistance aux antibiotiques. La plupart des médecins étaient conscientisés à la problématique de la multirésistance chez les entérobactéries, mais seulement 20% d’entre eux estimaient correctement le taux résistance de Klebsiella pneumoniae aux céphalosporines à large spectre au sein de leurs institutions. Au cours de ce travail, un réseau entre neuf hôpitaux de Lima a été implémenté afin de suivre la résistance aux antimicrobiens des souches isolées d’hémocultures. Au cours des années 2008-2009, le Staphylococcus aureus était la bactérie Gram-positive la plus fréquente (22%) ;K. pneumoniae et Escherichia coli représentaient les bactéries Gram-négatives les plus souvent isolées d’hémocultures. Parmi ces dernières entérobactéries, 75% produisaient des β-lactamases à spectre étendu (BLSE) avec un taux élevé de co- résistance à la ciprofloxacine et à la gentamicine. Les souches de S. aureus isolées d’hémocultures chez les patients hospitalisés et de dépistage chez les professionnels de santé (PS) ont été caractérisées pour leur profil de résistance aux antibiotiques et génotypées par biologie moléculaire. La moitié des 338 souches isolées d’hémocultures étaient résistantes à la méticilline (« MRSA ») et appartenaient à un clone prédominant disséminés dans ces différents hôpitaux, appelé le ST 5- spa t149-SCCmec I, ou le clone Cordobes-Chilien. Le taux de portage nasal de MRSA parmi les PS était à 8,7% ;les deux génotypes les plus fréquemment observés chez les PS appartenaient aux mêmes clones que ceux retrouvés majoritairement chez les patients hospitalisés. Cette observation suggère une transmission horizontale. L’implémentation de politiques de contrôle de l'infection est primordiale dans les établissements de santé au Pérou afin de réduire la transmission de micro-organismes multi-résistants. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

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Yes / The purpose of this workshop was to promote interdisciplinary learning and collaborations between UK and India in the area of bacteria and wounds, particularly when bacteria are increasingly antibiotic resistant. To this end we invited colleagues from India with clinical experience ofmanaging bacterially infected wounds in patients, particularly when antibiotic resistant (Dr Prashant Garg) and colleagues from India with expertise in developing new drug delivery systems (Prof Nikhil Singh and Dr Vamsi Venuganti) and together with UK delegates, spanning those with international experience in investigating bacterial infections (Prof Simon Foster), investigating how biofilms communicate (Prof Paul Williams), international expertise in discovering materials for antimicrobial resistance and for instruction of macrophages (Prof Morgan Alexander), expertise in developing responsive films for detecting infection in wounds using a visible signal (Prof Toby Jenkins) and other scientists working on the axis of detecting and treating infection in wounds. / MRC/DBT

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev Narayanan / The use of antimicrobials in animal feed has come under increasing scrutiny from the public and regulatory agencies. Direct-fed microbials (DFM) are considered valuable alternatives to antimicrobials in food animal nutrition. DFM are products containing live (viable microorganisms). Studies in Europe have reported antimicrobial resistance (AMR) in organisms used in DFM. This is of serious concern because of the potential for transferring resistance to pathogenic bacteria in the gut. The aim of the present study is to characterize phenotypic and genotypic AMR profiles for 20 different antimicrobials in bacterial strains isolated from 10 commercially available DFM used in. Two antimicrobial susceptibility testing methods, disc diffusion and broth micro-dilution based assay were performed. Enterococcus faecium isolates showed resistance towards metronidazole (n=9/9) with a MIC of > 32 μg/mL, erythromycin (n=5/9) with a MIC of ≥ 8 μg/mL, ciprofloxacin (n=2/9) with a MIC ≥ 4 μg/mL, ceftriaxone (n=6/9) with a MIC ≥ 0.25 μg/mL, rifampin (n=8/9) with a MIC of > 4 μg/mL, trimethoprim/sulfamethoxazole (n=4/9) with a MIC ≥ 1 μg/mL and clindamycin (n=5/9) with a MIC of > 0.5 μg/mL. A Propionibacterium freudenreichii isolate showed resistance towards kanamycin with a MIC of > 64 μg/mL. The same strain also had a MIC of 16 μg/mL for levofloxacin. Two Lactobacillus acidophilus were resistant to vancomycin (n=2/6) with a MIC ≥ 32 μg/mL. All the Lactobacillus species including L. acidophilus (n=6), L. casei (n=4) and L. plantarum (n=2) were resistant to metronidazole, MIC > 32 μg/mL. Two strains of Bacillus subtilis showed resistance to clindamycin, with an MIC of 4 μg/mL and erythromycin with an MIC of > 8 μg/mL, and one strain had no zone of inhibition for metronidazole (MIC > 32 μg/mL). Microarray analysis revealed resistance genes in E. faecium strains of 3 different DFM, including aminoglycoside resistance genes, ant(4’)-Ia, erythromycin resistance genes, ere(A2) and ermB, tetracycline resistance genes, tet39, tet31, tetK and tetC, and beta-lactam resistance gene, pbp5. Conjugation with filter mating showed erythromycin resistance gene transfer, msrC gene, from donor strains to a recipient strain (E. faecium 45-24). These studies show that AMR is prevalent among bacterial strains used as DFM in the cattle industry in the U.S., justifying further characterization, detection and observation of transferable antibiotic resistance between the same genus. .

Many studies have attempted to link antimicrobial use in food animal agriculture with an increased risk of antimicrobial-resistant (AR) bacterial levels in humans. Our data arise from longitudinal aggregated fecal samples in a 3-year cohort study of vertically integrated populations of human workers and consumers, and swine. Human and swine E. coli isolates (N = 2130 and 3485, respectively) were tested for antimicrobial susceptibility using the SensititreTM broth microdilution system. The associations between AR prevalence for each antimicrobial agent, multi-drug resistant E. coli, or multivariate AR E. coli, and the risk factors (host species, production type (swine), vocation (human swine worker versus non-worker), and season) in the study were assessed using generalized estimating equations (GEE), GLM with multinomial distribution, or GEE in a multivariate model using a SAS® macro to adjust for the correlated AR phenotypes. There were significant (p < 0.05) differences in AR isolates: 1) between host-species with swine at higher risk for ceftiofur, chloramphenicol, gentamicin, kanamycin, streptomycin, sulfisoxazole, and tetracycline. The prevalence of ciprofloxacin, nalidixic acid, and trimethoprim/sulfamethoxazole resistance were higher among human isolates, 2) swine production group was significantly associated with AR with purchased boars, nursery piglets, and breeding boars at a higher risk of resistance to streptomycin and tetracycline, and 3) human swine worker cohorts exhibited an elevated tetracycline prevalence, but lowered sulfisoxazole prevalence when compared to nonworkers. High variability among seasonal samples over the 3-year period was observed. There were significant differences in multiple resistance isolates between host species, with swine at higher risk than humans of carrying multi-resistant strains; however, no significant differences in multiple resistance isolates within humans by vocation or within swine by production group. The odds-ratios, adjusted for multivariate dependence of individual AR phenotypes, were increased relative to unadjusted oddsratios among 1) swine as compared to human for tetracycline (OR = 21.8 vs. 19.6), and 2) increased significantly among swine-workers as compared to non-workers only for tetracycline (OR = 1.4 vs. 1.3). Occupational exposure to swine-rearing facilities appears to be associated with an increased relative odds for the prevalence of tetracycline resistance compared to non-workers.

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