Investigating the Prevalence and Persistence of Different Escherichia Coli Genotypes in an Agriculturally Influenced Aquatic Ecosystem

Abstract

In several regions of Canada, including Saskatchewan, surface water such as streams and rivers are utilized as primary sources of irrigation water for fresh produce. Surface water can be exposed to faecal pollution from various sources such as storm water runoff, failing septic systems, and agricultural waste runoff. The use of faecally contaminated water in irrigating fresh produce may lead to food-borne outbreaks when enteric pathogens are present in the faeces. The ability to predict the presence of faecalderived pathogens is therefore important in assessing the human health risks associated with the use of open source irrigation water. Escherichia coli has long been used as an indicator of recent faecal pollution and pathogen presence. However, the long term survival and possible growth of E. coli in the environment suggests it may have limitations as a faecal indicator bacteria (FIB). To date, the mechanisms underlying the persistence of E. coli in the environment are not well understood. Therefore, this study was conducted to investigate its limitations as a faecal indicator bacteria including its prevalence, and a possible mechanism of persistence in the environment and compare its suitability as an indicator bacteria with the recently proposed indicator Bacteroidales in relation to enteric pathogens. Laboratory-based microcosms were designed to study the persistence of E. coli and identify the biofilm candidates in wastewater influent spiked with pig and cow manure. Microcosm experiments identified B1 as a dominant genotype along with subgenotype A0. Biofilm formation was recognized as a possible mechanism of E. coli persistence in the environment. DNA fingerprinting analysis of the two dominant genotypes and their subsequent biofilm formation tests demonstrated that the strains capable of producing substantial biofilms are genetically more diverse. A subsequent field study was carried out at sampling sites along the Qu’Appelle River and Wascana Creek, which are two key sources of irrigation water for several vegetable producers in Southern Saskatchewan. High levels of E. coli that exceed the recommended limit for irrigation water (≥ 100 MPN per 100 mL) in Canada have been frequently detected near three irrigation water pumping sites. A sudden increase in E. coli counts in all three sites was observed preceding storm and rainfall events. Phylogenetic analysis of E. coli genotypes over two irrigation seasons also identified B1 as the prevalent genotype regardless of sampling sites and season. A significant association of E. coli genotypes with their sites of isolation was also observed. The enteric pathogens Salmonella and Campylobacter were also detected in irrigation water but their presence does not correlate with the levels of E. coli. The decreasing concentrations of E. coli in a binary logistic regression was found to have significant predictive value for samples that were positive for the pathogens analysed. These findings highlight that exclusive use of E. coli concentrations is not an adequate method to predict pathogen presence. The use of host-associated Bacteroidales markers as alternative faecal indicators and their association with E. coli was also investigated. There was a significant positive correlation observed between the increasing concentrations of ruminant specific Bacteroidales marker (BacR) and the presence of Campylobacter spp. in the samples analyzed. A significant correlation between the BacR marker to E. coli subgenotypes B23 and D1 was observed, which indicates that these genotypes may be associated with ruminant faecal contamination.