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Humans can be exposed to waterborne bacterial pathogens and numerous outbreaks have been reported involving these microorganisms around the world. Many different enteric pathogens can be found in source waters used for drinking water. Assessing these pathogens and their possible threat to public health has always been important. Waterborne pathogens can be difficult to detect, and despite a large variety of recognized microbial detection techniques, the cause of many outbreaks has not been unidentified. Effective and rapid pathogen detection techniques are required to achieve reliable data for microbial source water quality, outbreak investigations, and for drinking water treatment efficacy monitoring. Bacteria have long been detected using classical culture-based methods, with the rationale that living cells are able to grow/replicate. However, many pathogenic bacteria in source waters may turn into viable but not culturable (VBNC) cells and are thus undetectable by growth-based methodologies. Alternatively, PCR-based techniques have been developed to detect both non-culturable and culturable bacteria. Yet with these techniques, post-death DNA persistency can inaccurately overestimate the number of viable cells. This problem may be circumvented by an alteration to the PCR procedure that is reported to be able to block PCR amplification of DNA that originates from dead cells. This alteration involves a chemical pre-treatment step prior to PCR using a photoreactive intercalating dye, propidium monoazide (PMA). In this research, a successful modification was made to the PMA-PCR method that can result in substantial suppression of the PCR signal from dead cells, and provide results that can more accurately measure bacterial pathogen viability. PMA-PCR was applied to high concentrations (1 × 107 cells mL-1) of heat-killed cells of Salmonella enterica and Campylobacter jejuni. Using PMA-PCR in combination with primers that amplified a relatively short fragment of the S. enterica invA gene (119 bp), only a 3-log reduction of the dead cell PCR signal was obtained. Similarly, for C. jejuni using PCR primers that amplified a relatively short fragment of DNA (174 bp of cpn60 gene), only a 1-log reduction of the PCR signal was observed for dead cells. Therefore, PMA treatment followed by PCR amplification of short DNA fragments resulted in incomplete signal inhibition of heat killed Salmonella and Campylobacter. To further investigate how PCR conditions can affect the ability of PMA to inhibit PCR amplification, primers were then used that could amplify a larger fragment of DNA. PCR amplification of a longer DNA fragment (1614 bp of invA gene for S. enterica and 1512 bp of cpn60 gene for C. jejuni) strongly suppressed the signal (7 log reduction) for both heat-killed Salmonella and Campylobacter. For UV-treated S. enterica and C. jejuni, short amplicon PMA-PCR showed no or very low PCR signal reduction, in part due to intact membranes directly after UV irradiation. Long amplicon qPCR, however, resulted in dead cell signal removal and PMA pretreatment had no effect on PCR signal suppression. This study used quantitative PCR and the PMA-PCR viability assays to evaluate the levels and occurrences of four groups of pathogenic bacteria in surface water samples from two locations on the Grand River, Ontario, Canada, to demonstrate the reliability of the PMA-PCR technique for the enumeration of viable cells. The bacterial groups investigated included S. enterica, thermophilic Campylobacter, Escherichia coli O157:H7, and Arcobacter butzleri. Small numbers of dead cells (not more than 0.5 log 100 mL-1) were present, detected as the difference between PMA-PCR and PCR without PMA treatment. In this particular river, pathogen enumeration by PCR was only slightly influenced by false positive signal detection due to the presence of dead cells or extracellular DNA and reliable bacterial pathogen detection could be attained by PCR without PMA pretreatment. Viable A. butzleri were detected at elevated concentrations (up to 4.8 log cells per 100 mL) in the Grand River. Arcobacter has not been previously studied in the Grand River and this is one of the few studies that have quantitatively assessed Arcobacter in the environment. This suggests that additional research is required on the pathogenicity of this organism and its occurrence in water. In the next stage of this research, both the improved viability assay (long amplicon PMA-PCR) and conventional quantitative PCR were applied to investigate the survival trends of selected enteric bacterial pathogens including Yersinia enterocolitica, S. enterica, C. jejuni, and A. butzleri. The target bacteria were inoculated into sterile or non-sterile river water to study the impact of background microbiota on cell survival. These experiments were perfomed at 3 different temperatures (5, 15, and 25°C) and at high/low dissolved oxygen (DO) concentrations (for C. jejuni, and A. butzleri only) to evaluate the effect of these potential environmental stresses on bacterial survival trends. The results indicated that the autochthonous microbiota in river water had a significant effect on the bacterial die-off. Although lower temperatures enhanced bacterial survival in non-sterile river water, it was found that PCR may overestimate the effect of temperature on survival and that the PCR viability assays (PMA-PCR) could more accurately measure the impact of temperature. The survival of viable C. jejuni was adversely affected by high DO levels only at a low temperature (5°C) and this effect was observed only when the PMA-PCR viability assay was applied. A. butzleri survival was not affected by water DO levels. This research provides an improved understanding of viable/active enteric waterborne bacteria and their survival in the aquatic microcosms as well as reliable data to better elucidate the effect of environmental factors on the occurrence of pathogenic bacteria. It can also offer valuable information for microbial risk assessments used by regulators and decision makers.

Depuis quelques années, des Toxi-Infections Alimentaires Collectives causées par la contamination de produits frais, comme les laitues, par des bactéries pathogènes entériques (Salmonella, Escherichia coli productrice de shigatoxines -ou STEC-) apparaissent de plus en plus nombreuses. La présence de ces bactéries dans cet environnement inhabituel est un risque sanitaire émergent majeur, d'autant plus que les bactéries entériques, pathogènes ou non, présentent fréquemment des résistances aux antibiotiques. Afin d’étudier la persistance des bactéries antibiorésistantes ou pathogènes sur des laitues, la caractérisation de plasmides de résistance portés par des souches de E. coli issues d’environnements aquatiques contaminés a été réalisé pour, par la suite, étudier leur implication potentielle dans l’adhésion des souches-hôtes sur différentes variétés de laitues. L’étude de la survie et de l’adhésion de souches de E. coli environnementales et de laboratoire, transformées avec les plasmides d’intérêt, sur de jeunes plants de laitues a permis de mettre en évidence trois points : 1) plus le temps de contact entre bactéries et feuilles augmente et moins la survie bactérienne est importante ; 2) il existe une différence de survie et d’adhésion selon les variétés de laitues étudiées ; 3) il existe une différence de survie et d’adhésion entre les souches de laboratoire et les souches environnementales, ces dernières étant en meilleur état métabolique et montrant une adhésion plus importante durant les 11-12 jours d’expérimentation. Après ces constatations de persistance des E. coli antibiorésistantes en conditions contrôlées, des études en champs sur 4 exploitations maraîchères normandes, possédant des itinéraires techniques différents, ont été réalisées. La recherche de pathogènes entériques, Salmonella et STEC, a été effectuée sur les laitues et une recherche de E. coli, témoin de contamination fécale, a été réalisée sur les laitues ainsi que dans l’eau d’irrigation d’un des sites. Les résultats révèlent une qualité microbiologique satisfaisante des parcelles étudiées (selon l’arrêté européen N°2073/2005) bien que des E. coli aient été régulièrement retrouvées au niveau des laitues, dont certaines antibiorésistantes. L’analyse de l’eau d’irrigation a montré la présence continue de E. coli, dont des souches présentant des profils d’antibiorésistance communs à ceux retrouvés sur les laitues, montrant que l’eau d’irrigation est l’une des sources critiques de contamination des végétaux en champs. / In recent years, foodborne diseases caused by fresh products contaminated, such as lettuce, with enteric pathogenic bacteria (Salmonella, Shigatoxin-producing Escherichia coli-or STEC-) increasingly. The presence of these bacteria in this unusual environment is a major emerging health risk, especially since enteric bacteria, whether pathogenic or not, are frequently resistant to antibiotics. To study the persistence of antibiotic-resistant or pathogenic bacteria on lettuce, the characterization of resistance plasmids carried by E. coli strains from contaminated aquatic environments was carried out in order to study their potential involvement in adhesion of host strains on different varieties of lettuce. The study of the survival and adhesion of environmental and laboratory E. coli strains, transformed with the plasmids of interest, on young lettuce plants allowed to highlight three points: 1) more time contact between bacteria and leaves increases and less bacterial survival is important; 2) there is a difference in survival and adhesion depending on the varieties of lettuce studied; 3) there is a difference in survival and adhesion between laboratory strains and environmental strains, the latter being in better metabolic state and showing greater adhesion during the 11-12 days of experimentation. After the persistence of antibiotic-resistant E. coli strains under controlled conditions, field studies on 4 Normandy vegetable farms, with different technical itineraries, were carried out. The search for enteric pathogens, Salmonella and STEC, was carried out on lettuce and a search for E. coli, a control of fecal contamination, was realized on the lettuce as well as in the irrigation water of one of the sites. The results reveal a satisfactory microbiological quality of the agricultural plots studied (according to the European decree N ° 2073/2005) although E. coli strains were regularly found at the lettuce level, including some antibiotic resistant. Analysis of the irrigation water showed the continued presence of E. coli strains, including strains with common antimicrobial resistance profiles to those found on lettuce, showing that irrigation water is one of the critical sources of plant contamination in the field.