Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water.

TITLE PAGE

Development of a PCR protocol for the detection of Escherichia coli O157:H7 and Salmonella spp. in surface water

Silvia Bonetta1_{, Elena Borelli}2_{, Sara Bonetta}1_{, Osvaldo Conio}2_{, Franca Palumbo}2 _{and Elisabetta Carraro}1*

1_{Dipartimento di Scienze dell’Ambiente e della Vita, Università degli Studi del Piemonte Orientale “A.}

Avogadro”, via T. Michel 11, 15121 Alessandria, Italy; 2 _{Laboratori Iride Acqua Gas Srl, via Piacenza 54, 16138}

Genova

*_{Corresponding author Tel: +390131360261; fax:+390131360243} E-mail address: elisabetta.carraro@mfn.unipmn.it

Keywords: surface water, Salmonella, Escherichia coli O157:H7, PCR

ABSTRACT 1 2 3

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Escherichia coli O157:H7 and Salmonella are pathogenic microorganisms that can cause severe gastrointestinal

illness in humans. These pathogens may be transmitted in a variety of ways, including food and water. The presence of Salmonella and E.coli O157:H7 in surface waters constitutes a potential threat to human health when used for either drinking or recreation. As with most waterborne pathogens, Salmonella and E.coli O157:H7 are difficult to detect and enumerate with accuracy in surface waters due to methodological limitations. The aim of this study was to develop a protocol for the detection of Salmonella spp., E. coli O157:H7 and E. coli virulence genes (stx1, stx2 and eae) in water using a single enrichment step and PCR. In spiked water samples PCR results showed high sensitivity (<3 CFU/L) for both microorganisms. The protocol developed in this study has been applied in different surface waters in association with microbiological and physical analysis. The frequency of PCR positive samples was 33% for Salmonella and 2% for E.coli O157:H7 producing intimin (eae) and Shiga-like toxin I (stx1). Moreover, the finding of amplicons corresponding to eae and stx1 genes in the absence of

E.coli O157: H7 suggested the possible presence of other pathogenic bacteria that carry these genes (e.g. EHEC, Shigella strains). The results obtained showed that the developed protocol could be applied as a routine analysis

of surface water for the evaluation of microbiological risks.

INTRODUCTION 1

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Faecal contamination is one of the sources of environmental bacterial contamination and it is responsible for the presence of pathogens in natural environments. Human pathogens of enteric origin are, therefore, a potential contamination risk transmitted through soil, agriculture, water or sediments (Marucci et al. 2010). In this context,

Salmonella spp. and E. coli O157:H7 represent two important pathogens. E. coli O157:H7 has emerged as a

serious gastrointestinal pathogen in many countries. Although the predominant mode of its transmission to humans is via consumption of contaminated meat and other food, waterborne outbreaks have also been documented (Avery et al. 2008). Waterborne transmission of E. coli O157:H7 has been reported from both recreational water and contaminated drinking water (Hrudey et al. 2004; Nwachuku et al. 2008). One of the most serious outbreaks of E.coli O157:H7 occurred in the water supply system of the small farming community of Walkerton, Ontario Canada, in May 2000, when six people died and more than 2000 people fell ill (Hrudey et al. 2003). The epidemiologic research investigation identified drinking water as the culprit in this outbreak; possible explanations include that the water network could have been contaminated by surface waters, particularly during flood periods (Bertrand et al. 2007). Salmonellosis, one of the most important causes of acute enterocolitis due to contaminated foods, is one of the major public health problems in Europe, with a total of 131.468 confirmed cases reported in 2008 (EFSA 2010). Many serovars of Salmonella have been isolated in natural environments, contaminated by human and animal faeces, particularly in river waters, estuarine waters and seawaters (Ahmed et al. 2009; Touron et al. 2005).

It is important to note that the presence of pathogenic enteric microorganisms in surface waters, including

Salmonella and E.coli O157:H7, constitutes a potential threat to human health both for their drinking or

recreational use. Currently standard guidelines based upon microbial indicator concentrations (e.g. thermotolerant coliforms and enterococci) are used to protect the environment and prevent exposure of the public to pathogenic microorganisms. However, there are no established correlations between the prevalence and concentration of these faecal contamination “indicators” and specific pathogens, including E.coli O157:H7 and

Salmonella (Duris et al. 2009; Sugumar et al. 2008; Lemarchand et al. 2003). In this context, the research of

these specific pathogens is a matter of increasing importance regarding the safety and protection of drinking water sources. On the other hand, as with most waterborne pathogens, Salmonella and E.coli O157:H7 are difficult to detect and enumerate with accuracy in surface waters due to methodological limitations. The low concentration of these organisms in surface water and the high cost and effort of the current detection technologies are particularly problematic.

The use of polymerase chain reaction (PCR) has become increasingly popular an elective method to detect different pathogens in a great variety of samples. The PCR assay can detect minute amounts of specific DNA;

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however, this sensitivity can be influenced by physical dilution in aquatic environments and may result in pathogens escaping detection. Frequently, a pre-enrichment of samples is necessary to lower the detection limit and dilute any inhibitory substances present in the samples (Moganedi et al. 2007). Moreover, a preliminary enrichment step before gene amplification affords the advantage of exclusively detecting recoverable forms, and ruling out, from the analysis a priori, the detection of dead bacteria or free DNA from lysed bacteria (Touron et al. 2005). Detection limits using molecular methods such as PCR associated with an enrichment step may be lower when compared with conventional growth-based assays; these molecular methods also have the advantage of increased specificity (Feder et al. 2001). Achieving low detection limits in any environmental pathogen assay is of paramount importance, especially in water samples, where the presence of a single organism may result in human illness (Thompson et al. 2006).

The aim of this study was to develop a protocol for the detection of Salmonella spp., E. coli O157:H7 and E.

coli virulence genes (stx1, stx2 and eae) in water using a single enrichment step and PCR. This protocol was applied to the detection of these pathogens in different surface waters.

MATERIALS AND METHODS

Bacterial strains and culture media

E. coli O157:H7 strain NCTC 129 (non-toxigenic strain) and Salmonella typhimurium ATCC 14028 (Oxoid)

were used throughout this study. The strains were cultivated on Tryptic Soy Agar (Applichem, TSA) or in Tryptic Soy Broth (Applichem, TSB) at 37°C.

Sample processing and PCR analysis

The main steps of the protocol for E.coli O157:H7 and Salmonella spp. detection are summarised in Fig. 1. Briefly, 1 L of water sample was concentrated by filtration through 0.45 m pore size nitrocellulose filters (Millipore). The filters were then vortexed in peptone water (Oxoid) and the peptone water containing the filter was cultivated at 37°C for 18 h (enrichment step). Following incubation, 2 mL of peptone water was centrifuged at 4500 g for 20 min to recover bacteria. The bacterial pellet was resuspended in 700 l of Chelex-100 (20%, BioRad) and 4.5 l of proteinase K (20 mg/mL, Sigma). This mixture was incubated at 56°C for 30 min, and then at 95°C for 10 min. Samples were then centrifuged at 6000 g for 5 min, and the supernatant was used for PCR amplification. Multiplex PCR for E.coli O157:H7 and E. coli virulence gene detection was performed on

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extracted DNA using primers targeting genes associated with O157 antigen (primers RfbF and RfbR), H7 antigen (primers FLICh7-F and FLICh7-R), intimin (primers IntF and IntR) that mediates the intimate adherence

of the organism to host cells and Shiga-like toxins I (primers SLT-IF and SLT-IR) and II (primers SLT-IIF and SLT-IIR). The Shiga-like toxins identify the major known virulence traits of Escherichia coli O157:H7. Oligonucleotide sequences of primers used for the PCR amplification of E.coli O157:H7 are listed in Table 1. The amplification primer choice, PCR cycling conditions and agarose gel electrophoresis visualisation of PCR products were performed as described by Hu et al. (1999). In particular, an aliquot of 10 l of template DNA was added to 90 l of PCR mixture consisting of: 1X PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl), 0.2 mM of each dNTPs, 2.5 mM of MgCl2, 0.1 M of each Rfb primers, 0.1 M of each FLIC primers, 0.075 M of

each Int primers, 0.2 M of each SLT primers and 2.5 U of Taq Gold Polymerase (Applied Biosystems). The PCR program was conducted in a thermalcycler (Applied Biosystems) with the following parameters: initial denaturation step of 94°C for 30 s; touchdown procedure in which the annealing temperature was decreased from 59 to 48°C at a rate of 1°C every cycle; 23 additional annealing cycles at 59°C; extension at 72°C for 1 min and a final extension of 72°C for 7 min. For the detection of Salmonella spp., primers 139 and 141 were used to amplify a 284-bp sequence of the invA gene (Table 1). This gene is located on pathogenicity island 1 of

Salmonella spp., which encodes proteins of a type III secretion system as described by Collazo et al. (1997).

PCR mixture (25 l) containing 0.4 M of each primers, 200 M of each dNTP, 1X PCR Buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl), 1.5 mM MgCl2, 0.75 U of Taq Gold Polymerase (Applied Biosystems), and 5 l of

sample DNA was prepared according to Malorny et al. (2003). The incubation conditions were 95 °C for 10 min, followed by 38 cycles of 95 °C for 30 s, 64 °C for 30 s and 72 °C for 30 s. A final extension of 72 °C for 4 min was used. All PCR products were loaded onto a 2% agarose gel and run in 1% TAE buffer at 70 V for 1 h. Gels were stained with ethidium bromide solution and visualised with an UV transilluminator (Biorad).

PCR detection sensitivity in artificially contaminated water samples

In order to verify the sensitivity of the protocol, environmental surface water samples were collected (1 L) and artificially inoculated with different concentrations of E.coli O157:H7 and Salmonella typhimurium bacterial cells (experiment with filtration). The same pathogen concentrations were directly added to peptone water to evaluate the influence of the filtration procedure on bacterial recovery of both pathogens (experiment without filtration). In the experiments without filtration, a filter used with a non-contaminated surface water sample was added in the inoculated enrichment to simulate the presence of environmental PCR inhibitors.

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Sampling and microbiological analyses

Surface water samples (13 river samples, 45 samples total) were collected from the Brugneto Lake watershed located in the Liguria Region of Italy, during 1 year (Fig 2). The land in the watershed is sparsely occupied and used for livestock operation mainly in the spring and summer seasons. During the sampling period, approximately 700 cattle and calves, 70 sheep and 80 goats were present in the land. A wastewater treatment plant is not present in the watershed. Water samples were collected in sterile plastic bottles 20 cm below the water surface, transported on ice to the laboratory and tested within 24 h. One litre of each sample was used for the E.coli O157:H7 and Salmonella spp. detection using the PCR method described above. Moreover E.coli, enterococci, Clostridium perfringens, coliforms and Salmonella spp. were determined according to the Italian Official Methods for water samples (ISS 2007a). Briefly, the membrane filtration method was used to process the water samples for enterococci and C.perfringens enumeration. Serial dilutions of each sample were made and filtered through 0.45 µm pore size (47 mm diameter) nitrocellulose membranes (Millipore) and placed on either Slanetz-Bartley Agar or Clostridum perfringens Agar base (m-CP agar) for the isolation of enterococci or

C.perfringens, respectively. The Slanetz-Bartley agar plates were incubated at 37°C for 48 h and m-CP agar

plates were incubated at 44°C for 24 h. Suspicious enterococci were re-isolated in Bile-Esculin-Azide Agar and incubated at 44°C for 2 h. For C.perfringens detection, the colonies were confirmed after exposure to ammonium idroxyde vapours. Water samples were assayed for E.coli and coliforms with commercial Quanti-TrayTM ₂₀₀₀

(IDEXX Laboratories Inc.). In particular, for each sample, 100 mL of an appropriate dilution was added to Colilert Quanti-TrayTM _{dehydrated medium. The inoculated substrates were then poured into Quanti-Tray}TM

plates, sealed and incubated for 18-22 h at 37°C. For Salmonella detection, 1 L of each sample was filtered through 0.45 µm pore size (47 mm diamater) nitrocellulose membranes (Millipore). The membrane was placed into 100 mL of peptone water and pre-enriched for 16-20 h at 37°C. One hundred microlitres of each sample was then transferred into 10 mL of Rappaport-Vassiliadis broth (RV) and incubated for 18-24 h at 42°C. After enrichment in RV, samples were subcultured onto Rambach Agar plates by drop inoculation and incubated for 24-48 h at 37°C. Bacterial colonies with typical Salmonella morphology were tested by slide agglutination with

Salmonella antiserum. All media and reagents were from Oxoid. The turbidity was evaluated according to the

Italian Official Methods for water (ISS 2007b).

RESULTS 1 2 3

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PCR detection sensitivity in artificially contaminated water samples

Multiplex PCR analysis of pure cultures of non-toxigenic E.coli O157:H7 strain revealed three distinct bands of expected size, corresponding to H7, intimin and O157 (Fig 3a). As expected, no PCR amplicons were observed for Shiga-like toxin I and II genes. Also, the PCR primers used for the detection of Salmonella spp. have generated a specific PCR product (Fig 3b).

The results obtained with artificially contaminated water samples are shown in Table 2. The method tested (experiment with filtration) demonstrated a high sensitivity for both microorganisms, with levels as low as 3 CFU/L in the spiked surface water samples. The presence of PCR products was observed in all duplicate water samples for both pathogens, also at the lowest concentration of inoculum. As expected, no PCR amplicons were observed in unspiked samples. Results obtained also showed that sample filtration was not a limitating step for the sensitivity of the method, as the same results were found with or without filtration (Table 2).

Detection of E.coli O157:H7 and Salmonella spp. in surface water samples

The results of microbiological and physical analyses performed on surface water samples are reported in Table 3. The frequency of positive water samples for Salmonella using the molecular method developed in this study was 33% (14/42). In nearly all the rivers investigated (70%) at least one sample showed Salmonella contamination. In particular, all the samples collected in river A (Fig. 2) were positive for this pathogen. Using the culture method, only 7% (3/42) of water samples showed contamination by Salmonella.

The results obtained with PCR highlighted that only one sample (river A sample 1) was positive for E.coli O157:H7, showing the four bands corresponding to H7, O157, intimin and Shiga-like toxin I. The other water samples that contained the eae gene encoding for intimin (7%, 3/42) do not show the E.coli O157 and H7 genes, whereas the eae gene was associated in one sample to the stx1 gene (river H sample 3). 55% (23/42) of water samples were positive for the H7 gene. A total of 15 water samples (15/42 or 36%) revealed the presence of amplicons corresponding to Shiga like toxin I on the other hand, the presence of stx2 gene (Shiga like toxin II) was never found.

Regarding the microbiological parameters, the maximum concentrations of Clostridium perfringens, coliforms,

E.coli and enterococci were observed in river A (mean 5.767 CFU/100 mL, 185.158 CFU/100 mL, 114.633

CFU/100 mL, 3753 CFU/100 mL, respectively), while the other rivers showed a lower extent of contamination (ranging from 1 to 75 CFU/100 mL, 3 to 4.200 CFU/100 mL, 1 to 3.320 CFU/100 mL and 1 to 400 CFU/100 mL).

In general, no apparent relationship between concentration of microbiological parameters (E.coli, enterococci,

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C.perfringens, coliforms), turbidity and pathogens was observed.

DISCUSSION

The detection of Salmonella and E.coli O157:H7 is an important environmental issue because of the health problems that could be involved. In order to achieve successful detection in water samples, we have proposed a protocol based on a single enrichment step and the PCR. In recent years, the development of molecular methods for the amplification of specific gene sequences has produced rapid and sensitive techniques (PCR, PCR-ELISA, RT-PCR or Real-Time PCR) for detecting pathogens in waters (Ahmed et al. 2009; Liu et al. 2010; Mull et al. 2009; Touron et al. 2005). However, considering the low concentration of pathogens in water, an enrichment step is frequently required to improve the sensitivity. In fact, the use of the enrichment step can dilute inhibitory compounds produced by competing bacteria in the sample as well as aid the recovery of injured, stressed or lag-phase bacterial cells (Moganedi et al., 2007; Touron et al. 2005). Moreover, also the enrichment-free genetic procedure allows to reveal low concentrations of viable pathogens, but these methods are frequently not applicable in routine analysis due to the high cost and complexity of these techniques (e.g. Real-Time PCR). In this context, the protocol developed in this study, which includes an enrichment step and a subsequent PCR, showed high sensitivity, highlighting the pathogens’ presence even at very low concentrations. This result is particularly important for E.coli O157:H7 as the minimum infectious dose of this microorganism is as few as 10 cells (Dharmasiri et al., 2010). To our knowledge, the PCR conditions used in this study have never been applied in water monitoring. Sample filtration did not present a limitation to the sensitivity of the method because the same results were found with and without filtration. Similar findings were also reported by Bertrand et al. (2007) that evaluated the effect of a filtration procedure in water samples by processing different concentrations of

E.coli O157:H7 with and without a filtration step.

In addition to high sensitivity, the developed protocol shows other relevant advantages: i) it allows us to use the same enrichment broth for the simultaneous detection of both pathogens; ii) the enrichment broth (peptone water) can also be used for Salmonella detection in water with the cultural method as required in the Italian Official method (ISS, 2007a); and iii) the multiplex PCR protocol uses primer pairs for E.coli O157:H7 detection that can simultaneously reveal serotype O157:H7 and its virulence traits. This is particularly important considering the time consuming procedures for serotype identification and toxin profiling in the standard culture method, which can be replaced with a single PCR reaction.

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This method was applied to the detection of E.coli O157:H7 and Salmonella spp. in surface water samples, and the results obtained confirmed that PCR was more sensitive than conventional culture for Salmonella spp. detection, as observed in other works (Feder et al. 2001). In fact, 79% of PCR positive samples (11/14) were negative by culture method, whereas the opposite situation was never found. It is important to note that the different sensitivity between culture and PCR method could also be related to the presence of viable but nonculturable cells (Touron et al., 2005).

Although E.coli O157:H7 was revealed only in one sample, the presence of amplicons corresponding to intimin and Shiga-like toxin I genes highlighted the possible presence of other pathogenic bacteria. In fact the stx1/stx2 genes are widely distributed among E.coli (Shiga-toxin producing E.coli or verotoxin producing E.coli) and

Shigella strains, as well as other waterborne bacteria, due to their dissemination via bacteriophages (James et al.,

2001; Strauch et al., 2008). Consequently, the presence of stx1/stx2 genes in water samples may be presumptive but not definitive for enterohemorrhagic E.coli (EHEC). Moreover, the eae gene encoding for intimin is not unique to EHEC; in fact, it is a critical virulence factor for the enteropathogenic E.coli (EPEC), which are similar to EHEC but lack stx genes (Shelton et al. 2006; Kuhnert et al. 2000).

The results obtained highlighted that the monitored rivers do not represent a possible source of risk for

Escherichia coli O157:H7 except for river A, whereas Salmonella seems more widespread in the monitored

rivers. Except for river A, the presence of the microbial indicators seem to not be associated with the two pathogens monitored, underlining that the sole reliance of indicator bacteria, which do not persist in the environment, may be misleading in terms of water quality from a microbial perspective (Dorner et al. 2007). The low water quality observed in river A could be related to the presence of septic tanks located near the river. In conclusion, our molecular method proved to be sufficiently sensitive to detect low levels of E.coli O157:H7 and Salmonella contamination. This is promising evidence that this method can complement the current culture-based standard methods for detection of these pathogens in surface water samples. The developed techniques could be applied as routine analysis in surface water for the evaluation of microbiological risk.

ACKNOWLEDGMENTS

The authors thank the collaboration whit the Mediterranea staff that provided excellent logistical support. This study was supported by Mediterranea delle Acque S.p.A., Iride Group S.p.A.

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