Antimicrobial resistance (AMR) is concerning because it limits antimicrobial drug treatment options. AMR occurs by the overuse and misuse of antimicrobial drugs. In environmental settings, AMR can disseminate from places of high use, which leads to increased exposure to humans and animals. A previous study from our laboratory group showed extended-spectrum cephalosporinase-producing Escherichia coli/Klebsiella pneumoniae were isolated from fecal samples obtained in rural Cambodian community settings. Based on these isolates, this study has two aims. The first aim was characterization of selected Cambodian isolates with random amplification polymorphic DNA (RAPD) and antibiotic susceptibility test. From RAPD, the selected six isolates are diverse, except for C61 and C66 bacteria isolates with potential clonality. Additionally, the selected isolates are multidrug resistant (MDR) with reduced susceptibility to beta-lactams and fluoroquinolones. The second aim was to assess two developed methodologies, multiplex automated genome engineering (MAGE) and One-Step Curing Plasmid, by validation in bacteria laboratory strain and development for six Cambodian isolates. To modify AMR genetic elements, MAGE uses pMA7-SacB for homologous recombination with oligos for chromosomal gene disruption. Meanwhile, One-Step Curing Plasmid uses pFREE with the CRISPR/Cas9 system for plasmid and self-curing. Validation showed that MAGE can modify 8% of E. coli MG1655 with lacZ control screening oligos and almost 90% are cured from pFREE. Selected Cambodian isolates have antibiotic-resistance plasmids of IncR or IncFII replicon. For usage in Cambodian isolates, pFREE was modified to be pCAM-FREE by cloning IncR and IncFII plasmid as gRNA1 and gRNA5, respectively. Sequencing results showed pCAM-FREE have gRNA5. In conclusion, our study managed to characterize selected Cambodian isolates as MDR and diverse. In a laboratory strain, MAGE and One-Step Curing Plasmid are functional methods. Furthermore, pCAM-FREE was constructed to target IncFII and in the future, MAGE and pCAM-FREE could be tested in Cambodian isolates.

<p>Regulatory interactions evolve to incorporate new genomic material and contribute to bacterial diversity. These regulatory interactions are flexible and likely provide bacteria with a means of rapid environmental adaptation. In this thesis, the RpoS regulon is used as a model system to investigate the hypothesis that regulon composition and expression are modified according to environmental pressures. Several novel findings are presented, namely the distribution of RpoS homologs in bacteria, the flexibility of the RpoS regulon, and the effect of diverse environmental pressures on RpoS regulon expression. Based on phylogenetic and reciprocal best hits analyses, RpoS was determined to be conserved in gamma-, beta-, and delta-proteobacteria, likely because it confers a selective advantage in many bacterial niches. Regulon composition, however, was highly flexible. Even between species of the same class, <em>Escherichia coli</em> and <em>Pseudomonas aeruginosa</em>, only 12 of 50 orthologs were regulated in common by RpoS. RpoS regulon flexibility may thus be the result of adaptation to different bacterial habitats. Indeed, mutations in <em>rpoS</em> and differential regulon expression could be identified among environmental <em>E. coli</em> isolates collected from diverse sources. Among environmental <em>E. coli</em> isolates, RpoS mutant frequency was found to be 0.3%, and activity of KatE, a prototypical RpoS regulon member, was undetectable in some isolates despite the presence of functional RpoS. Modulated RpoS regulon expression among environmental <em>E. coli</em> isolates is consistent with environment as a key factor shaping regulatory interactions. Regulon flexibility was similarly apparent in oxidative stress regulons, OxyR and SoxRS, of <em>E. coli</em>. SoxRS regulon function is weakly conserved, possibly due to low selective pressure for a superoxide stress response regulon in some bacterial species. Environment, therefore, is a crucial element that defines the dynamics of regulatory networks.</p> / Doctor of Philosophy (PhD)

Campylobacter est l’agent pathogène zoonotique responsable de la majorité des gastro-entérites d’origine bactérienne chez l’homme. Les produits de volaille représentent la principale source d’infection; toutefois, l’exposition peut également découler de contacts directs avec les animaux ou avec l’eau. Une forte variation saisonnière est présente dans les cas rapportés, qui n’est toujours pas élucidée : les eaux environnementales, sources d’infection connues, sont soupçonnées. Cette étude transversale a été réalisée dans la région Sud-Est du Québec (Canada) où Campylobacter fut quantifié et génotypé à partir de différentes sources d’eau (eaux de captage, récréatives et usées) et de cas cliniques afin d’évaluer les risques potentiels posé par l’eau environnementale. Différents essais PCR en temps réel furent appliqués à l’eau environnementale et comparés: 2 ont été sélectionnés pour leur spécificité et sensibilité de quantification. Les courbes standards ont été calibrées en utilisant la PCR digitale pour déterminer précisément les concentrations. Les isolats environnementaux et cliniques furent comparés génétiquement en utilisant le CGF (« comparative genomic fingerprinting »). Les eaux usées étaient plus contaminées que les eaux de captage et récréatives (3.9Log, 1.7Log et 1.0Log cellules/L en moyenne, respectivement). Six pour cent des isolats d’eaux environnementales étaient génétiquement similaires (100 % homologie) aux isolats cliniques. Les cas cliniques de campylobactériose d’été montraient des isolats avec davantage de similarités génétiques avec les isolats retrouvés dans l’eau environnementale comparativement aux autres saisons (p<0.01). Les faibles concentrations et similarités génétiques entre les isolats d’eau et cliniques suggèrent un risque de transmission possible, mais faible. / Campylobacter is a zoonotic pathogen that is responsible for the majority of cases of bacterial gastroenteritis. Among the numerous Campylobacter transmission routes including direct contact, food and water, poultry consumption has been recognized as the major route. A strong seasonal variation in campylobacteriosis cases exists for reasons that are not well understood; environmental water is suspected to be involved. This cross-sectional study was conducted in the Southeastern region of Quebec (Canada), wherein Campylobacter from different waters (drinking water source, recreational and sewage) and clinical sources was quantified and genotyped in order to evaluate the potential risks posed by environmental water. Several real-time PCR assays were compared for specific application to environmental water: two were selected for their specificity and sensitivity of quantification. Standard curves were calibrated using digital PCR to accurately determine concentrations. Campylobacter isolates from clinical and water sources were genetically compared using CGF (comparative genomic fingerprinting). Sewage waters showed the highest Campylobacter concentrations, while drinking water source and recreational waters showed the lowest (average of 3.9Log, 1.7Log and 1.0Log cells/L, respectively). CGF revealed that 6% of water isolates were genetically similar (100% homology) to clinical isolates. Summer cases of campylobacteriosis revealed isolates showing more genetic similarities with environmental water isolates compared to other seasons (p<0.01). The low Campylobacter concentrations and genetic similarities between water and clinical isolates from the same region, suggests that these environmental waters pose a real, but low risk of transmission.