Yes / Aeromonas hydrophila is a well-known waterborne pathogen that recently was found to infect humans. Here, we report the draft genome of a freshwater isolate from a Malaysian waterfall, A. hydrophila strain M023, which portrays N-acylhomoserine lactone-dependent quorum sensing. / University of Malaya via High Impact Research Grants (UM C/625/1/HIR/MOHE/CHAN/01, A-000001-50001, and UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

Yersinia enterocolitica are potentially pathogenic bacteria transmitted through the fecal oral route. Typical disease symptoms include those associated with gastrointestinal disease, although infection can also lead to more serious and invasive illnesses, particularly in sensitive populations. Previous surveys have detected Y. enterocolitica in surface water in various parts of the world, and studies have found drinking untreated water to be a possible risk factor for Y. enterocolitica infection. Methods available for the detection of Y. enterocolitica have been developed primarily for food and clinical samples and have not been tested extensively with water. More commonly used methods include culture based isolation of Yersinia spp. and polymerase chain reaction (PCR) based detection of Y. enterocolitica. Reports suggest that culture based methods available for the isolation of Y. enterocolitica may not be effective for environmental samples. Strain isolation using culture based methods is important, so that further subtyping information can be obtained for epidemiological investigations. In contrast, PCR based detection is more rapid, of higher throughput, can be highly specific and can target pathogenic strains within a species. The overall objective of this work was to evaluate culture based and PCR based methods for the detection of Y. enterocolitica in water, and to examine its prevalence in the Grand River watershed in Southwestern Ontario, Canada. Surface water in this watershed is used to provide all or part of the drinking water for approximately 500,000 people, as well as for recreational purposes. It is also one of the most heavily impacted watersheds in Canada by both agricultural and urban activities. Culture based studies compared two selective agars and four enrichment broths. Results showed that Cefsulodin Irgasan Novobiocin (CIN) agar and modified tryptic soy broth (mTSB) had greater potential for recovering Y. enterocolitica from surface water. Consequently, enrichment in mTSB followed by growth on CIN agar was used to isolate Yersinia from the Grand River. Yersinia strains were isolated from 52 out of 200 (26 %) surface water samples collected over a 17 month period. No seasonal trends were observed in isolation rates. Species isolated were typically considered to be non pathogenic species, although recent evidence suggests they may have potential virulence to humans. The majority of these strains have been found by other groups in surveys of aquatic environments. PCR methods developed targeted two Y. enterocolitica virulence genes: the ail gene, located in chromosomal DNA; and the yad A gene, located on a virulence plasmid. In surface water collected from the Grand River, the ail gene target was detected in 121 samples out of 319 (38 %) over a 29 month period and the yadA gene target was detected in 44 samples out of 206 (21 %) over a 20 month period. Both genes were detected more frequently when the water temperatures were colder. PCR-based studies conducted were quantitative, which has not previously been done with water samples. The median and maximum concentrations in samples positive for the ail gene were 40 and 2,000 cells/100 mL, and in samples positive for the yadA gene were 32 and 3,276 gene copies/100 mL, respectively. Overall results demonstrated that culture based methods are less sensitive than PCR based detection methods for specific detection of pathogenic Y. enterocolitica, suggesting that previous culture based surveys may have underestimated their potential prevalence. Furthermore, potentially pathogenic Y. enterocolitica may be present in the Grand River watershed. While Y. enterocolitica is relatively easily inactivated by traditional disinfection methods used in drinking water treatment processes, it is possible their presence poses a concern for recreational users and individuals drinking untreated water. This study suggests that further investigation is necessary to evaluate possible health risks associated with the occurrence of potentially pathogenic Y. enterocolitica in the Grand River. This work assists with the development of methods and information gathering for an emerging waterborne pathogen that has not been surveyed in the Grand River watershed, nor quantitatively surveyed in any water previously. Findings provide important information for drinking water providers and public health investigations.

Yersinia enterocolitica are potentially pathogenic bacteria transmitted through the fecal oral route. Typical disease symptoms include those associated with gastrointestinal disease, although infection can also lead to more serious and invasive illnesses, particularly in sensitive populations. Previous surveys have detected Y. enterocolitica in surface water in various parts of the world, and studies have found drinking untreated water to be a possible risk factor for Y. enterocolitica infection. Methods available for the detection of Y. enterocolitica have been developed primarily for food and clinical samples and have not been tested extensively with water. More commonly used methods include culture based isolation of Yersinia spp. and polymerase chain reaction (PCR) based detection of Y. enterocolitica. Reports suggest that culture based methods available for the isolation of Y. enterocolitica may not be effective for environmental samples. Strain isolation using culture based methods is important, so that further subtyping information can be obtained for epidemiological investigations. In contrast, PCR based detection is more rapid, of higher throughput, can be highly specific and can target pathogenic strains within a species. The overall objective of this work was to evaluate culture based and PCR based methods for the detection of Y. enterocolitica in water, and to examine its prevalence in the Grand River watershed in Southwestern Ontario, Canada. Surface water in this watershed is used to provide all or part of the drinking water for approximately 500,000 people, as well as for recreational purposes. It is also one of the most heavily impacted watersheds in Canada by both agricultural and urban activities. Culture based studies compared two selective agars and four enrichment broths. Results showed that Cefsulodin Irgasan Novobiocin (CIN) agar and modified tryptic soy broth (mTSB) had greater potential for recovering Y. enterocolitica from surface water. Consequently, enrichment in mTSB followed by growth on CIN agar was used to isolate Yersinia from the Grand River. Yersinia strains were isolated from 52 out of 200 (26 %) surface water samples collected over a 17 month period. No seasonal trends were observed in isolation rates. Species isolated were typically considered to be non pathogenic species, although recent evidence suggests they may have potential virulence to humans. The majority of these strains have been found by other groups in surveys of aquatic environments. PCR methods developed targeted two Y. enterocolitica virulence genes: the ail gene, located in chromosomal DNA; and the yad A gene, located on a virulence plasmid. In surface water collected from the Grand River, the ail gene target was detected in 121 samples out of 319 (38 %) over a 29 month period and the yadA gene target was detected in 44 samples out of 206 (21 %) over a 20 month period. Both genes were detected more frequently when the water temperatures were colder. PCR-based studies conducted were quantitative, which has not previously been done with water samples. The median and maximum concentrations in samples positive for the ail gene were 40 and 2,000 cells/100 mL, and in samples positive for the yadA gene were 32 and 3,276 gene copies/100 mL, respectively. Overall results demonstrated that culture based methods are less sensitive than PCR based detection methods for specific detection of pathogenic Y. enterocolitica, suggesting that previous culture based surveys may have underestimated their potential prevalence. Furthermore, potentially pathogenic Y. enterocolitica may be present in the Grand River watershed. While Y. enterocolitica is relatively easily inactivated by traditional disinfection methods used in drinking water treatment processes, it is possible their presence poses a concern for recreational users and individuals drinking untreated water. This study suggests that further investigation is necessary to evaluate possible health risks associated with the occurrence of potentially pathogenic Y. enterocolitica in the Grand River. This work assists with the development of methods and information gathering for an emerging waterborne pathogen that has not been surveyed in the Grand River watershed, nor quantitatively surveyed in any water previously. Findings provide important information for drinking water providers and public health investigations.

In recent years, surface water used for domestic, industrial, and irrigation purposes in South Africa has deteriorated due to inadequate wastewater treatment, urban and agricultural runoff, and rural settlements with deficient sanitation. Access to safe drinking water and sanitation is a basic human right, and if waterborne pathogens are present in the water environment, they compose a human health risk. With some hydrological models, e.g., Hydrological Predictions of the Environment (HYPE), it is possible to model microbial water quality and predict how land use and climate changes affect recipient water sources. In this thesis, waterborne pathogen transport in South Africa is investigated using World-Wide HYPE (WWH), to increase the understanding of the largest sources affecting pathogen concentration in surface water and processes affecting pathogen transport. Initially, a literature study was performed with emphasis on finding the most suitable pathogen to simulate. Because of the amount of available data, the indicator microorganism, E. coli, was chosen. Observed E. coli concentrations in surface water were used to evaluate the conformity of the simulated concentration, and contributions from separate sources were analysed. A sensitivity analysis was performed to increase the understanding of process parameters affecting the transport of E. coli in WWH. The findings of this project show that the largest contributions of E. coli originate from humans with unsatisfactory waste management, where wastewater is partially released directly to surface water. The largest deviation in average E. coli load per year was obtained when altering t1expdec, which denotes the half-life time of the simulated microorganism. The half-life time was also the process parameter with the most significant effect on the simulated concentration. In addition, when the parameter that specifies the fraction of E. coli that is released directly to surface water was altered, which affects one of the largest E. coli sources, a large deviation in average E. coli load per year was observed. This finding shows the importance of estimating the load from contamination sources accurately. The conformity of simulated and observed E.coli load was acceptable, but the simulated discharge needs to be improved to achieve better conformity of the E. coli concentration in surface water. WWH has great potential to simulate waterborne pathogens, but further developments to improve the simulated discharge are encouraged to obtain more reliable results. / Under de senaste åren har kvaliteten av ytvattnet i Sydafrika försämrats på grund av bristfälliga vattenreningsverk, avrinning från urbana miljöer och åkermark och områden med undermålig sanitet. Att ha tillgång till rent vatten och fungerande sanitet är en grundläggande mänsklig rättighet och om patogener är närvarande utgör detta en hälsorisk för människor som kommer i kontakt med dessa smittoämnen. Det är möjligt att modellera vattens kvalitet med avseende på mikroorganismer och att förutse hur markanvändning påverkar kvaliteten i recipienten. I detta arbete har transporten av vattenburna patogener i Sydafrika undersökts genom World-Wide HYPE (WWH) med syftet att öka förståelsen av de största källorna som bidrar till ökande koncentrationer av patogener i ytvatten, samt att öka förståelsen av processerna som påverkar transporten. En litteraturstudie utfördes för att hitta en passande patogen att simulera, och på grund av mängd tillgängliga data valdes indikatororganismen E. coli. Uppmätt koncentration av E. coli i ytvatten i Sydafrika användes för att utvärdera överrensstämmelsen med simulerad koncentration, och bidrag från olika källor av E. coli analyserades. En kompletterande känslighetsanalys utfördes för att öka förståelsen om transportprocesserna i WWH. Resultatet visade att de största bidragskällorna av E. coli till ytvatten i modellen är människor med otillräcklig hantering av mänskligt avfall där genererat avloppsvatten delvis släpps ut direkt till ytvattnet. Från känslighetsanalysen visade det sig att den mest känsliga modellparametern var t1expdec som beskriver mikroorganismenshalveringstid. Det var också den processparameter som också hade störst påverkan på den simulerade E. coli koncentrationen. När parametern som bestämmer andelen av E.coli som släpps ut direkt till ytvatten varierades, som påverkar en av de största källorna, resulterade det också i stor förändring i genomsnittlig belastning av E. coli per år. Detta indikerar att det är viktigt att estimera bidragskällorna korrekt. Överrensstämmelsen mellan simulerad och uppmätt belastning av E. coli per dag var acceptabel men det simulerade vattenflödet bör förbättras för att uppnå en bättre överrensstämmelse mellan simulerade och uppmätta koncentrationer av E. coli. WWH har stor potential att modellera vattenburna patogener, men vidareutveckling av simulerade vattenflöden behöver utföras att få mer tillförlitliga resultat.