Manitoba Livestock Manure Management Initiative Project: MLMMI Tracing the Source of E. coli Fecal Contamination of Water Using rep-pcr.

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1 Manitoba Livestock Manure Management Initiative Project: MLMMI Tracing the Source of E. coli Fecal Contamination of Water Using rep-pcr. Final Report Submitted by: Dr. Paul Holloway Aug. 31, 2001 Dept. of Biology, The University of Winnipeg Introduction The objective of this project was to apply molecular biological methods to identify and differentiate between E. coli strains which originated from the feces of humans or various agricultural animals. The goal is to safeguard the drinking water supplies by enabling investigators to trace the source of fecal contamination to particular agricultural operations or to the activities of humans (ie. septic tanks, faulty sewage treatment). Bacterial Source Tracing (BST) has become a popular issue in the United States (Graves, 2001, Hartel et al., 2001) and due to the Walkerton tragedy and other municipal drinking water problems in Canada is also becoming more important here. Many methods have been used for BST including antibiotic resistance patterns, ribotyping, restriction fragment length polymorphism (RFLP) mapping, pulsed field electrophoresis, and PCR-based fingerprinting methods. We chose genetic fingerprinting using the polymerase chain reaction to amplify repetitive DNA sequences in the DNA of E. coli. This particular BST technique has been named rep-pcr fingerprinting. The polymerase chain reaction was applied to the molecular fingerprinting of individual E. coli strains. These E. coli strains were isolated from human, cattle, swine and poultry feces. The animal source of the E. coli strains were therefore known before the fingerprinting was done. In addition the technique was applied to the bacterial species Enterococcus faecalis. This species has been suggested as a replacement for E. coli as the indicator bacterium of fecal contamination since it survives longer in aquatic environments and does not grow outside of a mammalian host. The fingerprints generated were compared to each other to detect similarities. This comparison was graphically represented by a dendrogram, a tree-like structure where two strains more similar to each other than to other strains will lie at the end of adjacent branches. This was done blindly, the goal being to see if once the source of the strains was placed on the dendrogram that clusters of E. coli strains would appear. Each cluster would contain mostly strains of E. coli from one type of animal. With this data base new isolates of E. coli could be fingerprinted and entered into the dendrogram. The cluster into which the new isolate fell would reveal the type of animal source of that strain.

2 Procedures Used in the Study MLMMI project Tracing the source... 2 Sampling Fecal samples were obtained from The University of Manitoba Glenlea Experimental Farm. These include swine and cattle fecal samples. Additional cattle fecal samples were obtained from a commercial farm and from the herd at the Agriculture and Agri-Food Canada Brandon Research Centre. Additional swine samples were collected at several commercial swine husbandry operations. Poultry samples were collected from the Poultry Research Unit at the University of Manitoba and from a commercial farm. Human E. coli isolates were recovered from sewage collected at the West End Pollution Control plant and North End Pollution Control plant in the city of Winnipeg. Fecal samples were obtained with a sterile swab and transported in sterile tenfold diluted Nutrient Broth on ice. Isolation of E. coli E. coli was isolated from samples using standard methods (see Appendix 1 for details). Fecal samples were streaked onto selective agar. Sewage samples were filtered through 0.2 µm membrane filters which were incubated on selective media. Isolation of Enterococcus faecalis (Ent. faecalis) Ent. faecalis was isolated from samples using standard methods (see Appendix 2 for details). After streaking samples for E. coli isolation the Nutrient Broth was used to inoculate KF Streptococcal Broth for enrichment of Ent. faecalis. Characterization of E. coli strains. Presumptive E. coli isolates were identified using biochemical and morphological tests (see Appendix 1 for details). A total of 91 E. coli strains were collected: 17 from swine, 15 from chickens, 16 from humans and 43 from cattle. Characterization of Ent. faecalis Presumptive Ent. faecalis isolates were identified using biochemical and morphological tests (see Appendix 2 for details). A total of 68 Ent. faecalis strains were collected: 17 from swine, 15 from chickens, 18 from humans and 18 from cattle. Isolation of DNA from Bacterial Strains

3 MLMMI project Tracing the source... 3 DNA was isolated from E. coli by a conventional lysozyme-detergent method followed by phenol-chloroform extraction to purify the DNA (see Appendix 3). DNA was isolated from Ent. faecalis by a modified lysozyme-detergent method and purified by phenol-chloroform extraction (see Appendix 4). Extracted DNA was checked by gelelectrophoresis for quality and quantified by absorbance at 260 nm. E. coli DNA was relatively easy to obtain and all samples contained large quantities of high molecular weight DNA. DNA from Ent. faecalis was more difficult to obtain. The protocol had to be modified by increasing the amount of lysozyme used by five-fold to obtain sufficient cell lysis. Eventually all isolates yielded sufficient DNA. Rep-PCR Amplification of fingerprint fragments of DNA was obtained for all E. coli and Ent. faecalis strains. Rep-PCR for E. coli was accomplished by the protocol of Dombek et al., (2000) with minor modifications of the PCR amplification conditions (see Appendix 5 for details). Rep-PCR fingerprinting of Ent. faecalis was more difficult. Initial amplifications using either the BOX or REP primers yielded fingerprints with only a few bands, too few to permit differentiation between stains. By modifying the amplification protocols and combining amplification reactions using either BOX or REP primers individually sufficiently complex fingerprints were generated. Analysis of Fingerprints Rep-PCR fingerprints of individual strains were visualized by gel-electrophoresis of the amplification reactions followed by ethidium-bromide staining. Fingerprints were photographed for storage of the data. After all fingerprints were obtained a template of all observed different bands was generated (Fig. 1). Each fingerprint was scored with this template using a 1" for the presence of a particular band in the fingerprint and 0" for the absence of that band. A binary matrix was generated containing the information, for all strains, of the presence and absence of all bands (Table 1). This data was used by the SPSS statistics program to generate Jaccard similarity coefficients between every pair of isolates. The Jaccard coefficients were graphically represented by a dendrogram (Fig. 2) which was produced by the SPSS hierarchal comparison procedure. Details are given in Appendix 8.

4 Results MLMMI project Tracing the source... 4 During the course of this study a total of 91 E. coli and 68 Ent. faecalis strains were collected, identified and fingerprinted. The E. coli and Ent. faecalis strain fingerprints were analyzed separately. The presence or absence of particular DNA fragments in the genome of individual strains was detected by electrophoresis of rep-pcr reactions on agarose gels. This presence or absence was converted to a binary scoring system, one for presence and zero for absence, to allow statistical analysis. To make interpretation of similarities between E. coli strains more obvious a dendrogram was constructed using the SPSS statistics program (Fig. 2). In contrast to our hypothesis that E. coli strains independently isolated from the same type of animal would be more similar than E. coli strains isolated from other types of animals and also in contrast to the conclusions of Dombek et al (2000) we did not observe any significant clustering of E. coli strains by the type of animal from which they were isolated. That is to say that in Fig. 2 there is no significant clustering of the E. coli strain fingerprints into groups by animal source type. There does seem to be a clustering of E. coli strains isolated from cattle at the top of the figure (strains 71-63) and again near the bottom (strains 84-61). However the first cluster includes a human strain and all these E. coli isolates in both clusters were taken from the herd at the Agriculture and Agri-Food Canada Brandon Research Centre. This probably represents the spread and exchange of E. coli strains through out a group of animals in a restricted geographical location. Throughout the rest of the dendrogram and in every other subcluster E. coli strains originated from all four animal groups tested, there was no significant host animal clustering. E. coli populations in humans are generally regarded as clonal with between 100 and 1,000 clones circulating throughout the human population. In individuals clones are not stable, over time established E. coli clones disappear and new ones take their place. Less work has been done on the E. coli population structure of other mammals and other animals. If the situation in other animals was similar to humans then with four animals we would be dealing with between 400 and 4,000 E. coli clones circulating through these populations. It may be that with only 91 E. coli strains isolated and fingerprinted we do not have enough fingerprints to establish a typical pig, cow or human E. coli. This conclusion was also reached in a similar study of 81 E. coli isolates using a different but related genetic technique (Buchan et al., 2001). In that study the authors also concluded that a larger sample size may be necessary to determine whether or not the approach is valid and whether source tracing can be accomplished. Similar results were observed with the Enterococci strains isolated. Once the similarity matrix was calculated and displayed as a dendrogram (Fig. 3) it was observed that there was no significant clustering of Enterococcus faecalis strains by host animal group. As was the case with E. coli too few strains may have been tested to discern the outlines of any similarity between Enterococcus strains from the same animal source. In conclusion the results of this study failed to duplicate those of Dombek et al (2000) in that no significant similarity between E. coli or Enterococcus faecalis strains from the same animal host type were detected by rep-pcr fingerprinting. This technique is not yet ready or reliable for the identification of the source of fecal contamination of drinking water. Rep-PCR fingerprinting would be useful for tracing

5 MLMMI project Tracing the source... 5 particular strains believed to be involved in waterborne bacterial contamination as part of post-incident epidemiological study given the technique s high resolving power to discriminate between individual E. coli strains. It may be that with a much larger group of either E. coli strains or Enterococcus strains that clustering by animal source will become apparent. Along this line the University of Minnesota group now reports they have a database of over 1,000 strains (Dombek et al., 2001). It may also be possible to refine the predictive power of this technique by applying more sophisticated statistical analysis namely multi-variate analysis of variance. This is an approach we will be investigating with the data collected. It would also be worthwhile to try other methods of bacterial source tracing such as antibiotic resistance patterns and ribotyping to attempt to find similarities between strains from the same animal source. We placed the entire collection of isolates in -80 o C storage so they are available for any other research. References Dombek, P., Johnson, L., Zimmerley, S., and Sadowsky, M Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources. Appl. Environ. Microbiol. 66: Dombek, P., Johnson, L., Zimmerley, S., and Sadowsky, M Identification of sources of fecal E. coli in Minnesota watersheds using rep-pcr fingerprinting. American Society for Microbiology Annual Meeting. Abstract Q-316. Buchan, A., Alber, M., and Hodson, R Strain-specific differentiation of environmental Escherichia coli isolates via denaturing gel electrophoresis analysis of the 16S-23S intergenic spacer region. FEMS Microbiol. Letters. 35: Graves, A Determining sources of fecal pollution for a rural unsewered community. American Society for Microbiology Annual Meeting. Abstract Q-318. Hartel, p., Funk, A., Summer, J., Hill, J., and Frick, E Ribotype diversity of Escherichia coli isolates from the upper Chattahoochie River. American Society for Microbiology Annual Meeting. Abstract Q-322.

6 Appendices Appendix 1 Isolation and characterization of E. coli MLMMI project Tracing the source... 6 Fecal material was streaked onto mendo agar. Various quantities of sewage was filtered through 0.2 µm membrane filters and the filters were placed onto mendo agar. Inoculated plates were incubated at C for hours. Presumptive E. coli colonies appeared green with a metallic sheen on this medium. These colonies were streaked onto Trypticase Soya agar and incubated at 37 0 C for hours for to obtain pure cultures. Pure cultures were tested for the ability to ferment lactose with the production of gas in Phenol Red Fermentation Broth at 37 0 C for hours. Those strains which did ferment lactose with gas were further tested for the lack of ability to utilize citrate when grown on Simmons Citrate agar, production of indole, and lack of production of H 2 S. All lactose positive, indole positive, citrate negative and H 2 S negative isolates were Gram stained to detect Gram-negative short rods. These were presumed to be E. coli. Appendix 2 Isolation and Characterization of Enterococcus faecalis Fecal samples were enriched for Enterococci before isolation was attempted. After swabbing for E. coli 500 µl of the remaining transport medium was added to 5 ml of KF Streptococcal Broth and incubated at 37 o C for hours or until the media turned a yellow colour. A sample of this enrichment culture was streaked onto KF Streptococcal Agar supplemented with 0.01% TTC. This was incubated for 48 hours at 37 o C. Various quantities of sewage were filtered through 0.2 µm membrane filters and the filters were placed onto KF Streptococcal Agar (+ 0.01% TTC) and incubated for 48 hours at 37 o C. Presumptive Enterococcus colonies appeared dark red. These colonies were streaked onto Bile Esculin Agar and incubated at 37 o C for 24 hours. Colonies surrounded by a black colour of the medium were pure cultured by streaking onto Brain Heart Infusion Agar and incubated at 37 o C for 24 hours. Identification of the isolates as Enterococcus was made if the isolates grew in 6.5% NaCl nutrient broth at 37 o C, by growth on Brain Heart Infusion Agar at 45 o C (18 hours) and at 10 o C within 10 days. Strains were tested for the ability to ferment arabinose, mannitol and sorbitol (1% in Heart Infusion Broth with Phenol Red as the ph indicator). Presumed Enterococcus strains were Gram stained to ensure the culture were Gram-positive cocci. Appendix 3 Isolation of DNA from E. coli Isolation of genomic DNA from E. coli was performed by a conventional detergent-lysozyme technique. Cultures of each strain were grown overnight in Trypticase Soya broth at 37 o C with agitation. The overnight culture was diluted twenty fold into 6 ml of Trypticase Soya broth and grown for 4 hours at 37 o C with agitation. To harvest the E. coli cells 1.2 ml of the 4 hour culture was centrifuged and suspended Appendix 3 Isolation of DN

7 MLMMI project Tracing the source... 7 in 0.31 ml of HTE buffer (50 mm Tris-Cl, ph 8.0, 20 mm EDTA). To lyse the cells 0.35 ml of 2% sarcosyl in HTE buffer was added. After inverting the tubes containing the cells to mix the solutions the tubes were incubated at 37 o C for 15 minutes. Proteinase K was then added (35 µl of 10 mg/ml Proteinase K in 10 mm Tris-Cl, ph 8.0, 10 mm NaCl, 0.1 mm EDTA) and the tubes incubated for another 45 minutes at 37 o C. To purify the DNA the lysed cell solution was extracted with 0.7 ml of 1:1 bufferequilibrated phenol-chloroform. The resulting emulsion was separated by centrifugation and the aqueous layer transferred to a new tube. The aqueous layer containing the DNA was extracted twice with chloroform. DNA was then precipitated by adding 70 µl of 3 M sodium acetate and 0.7 ml of isopropanol. The tubes were centrifuged for 15 minutes, decanted and the DNA pellets washed in 70% ethanol and dried. DNA was dissolved in 50 µl of TE buffer (10 mm Tris-Cl, ph 7.5, 1 mm EDTA) and stored at - 20 o C until needed. The quality of the DNA was determined by gel electrophoresis through a 0.8% agarose gel at 60 V for one hour. Gels were stained with ethidium bromide and visualized by UV transillumination. Undigested lambda DNA was used as a size marker. DNA was quantified by measuring the absorbance at 260 nm of a diluted sample. Genomic DNA was diluted to a final concentration of 50 ng/µl and stored at -20 o C. Appendix 4 Isolation of DNA from Enterococcus faecalis Isolation of genomic DNA from Ent. faecalis began with a 5 ml overnight Brain Heart Infusion broth culture (37 o C with agitation). Overnight culture cells were then washed by centrifugation and suspension in TE buffer. The washed cells were then collected by centrifugation and suspended in 0.5 ml of a detergent lysis solution (6 mm Tris-Cl, ph 7.6, 1 M NaCl, 0.1 M EDTA, 0.5% Brij 58, 0.5% sarkosyl, 0.2% deoxycholate). Lysozyme and RNase were added to final concentrations of 2 mg/ml and 20 µg/ml respectively. This suspension was incubated at 37 o C for 2 hours. After cell lysis the solution was extracted twice with an equal volume of chloroform and to the aqueous layer containing DNA two volumes of ethanol were added to precipitate the DNA. Centrifugation was used to pellet the DNA which was washed with 70% ethanol, dried and suspended in 100 µl of TE buffer. DNA was stored at -20 o C until used. The quality of the DNA was determined by gel electrophoresis through a 0.8% agarose gel at 60 V for one hour. Gels were stained with ethidium bromide and visualized by UV transillumination. Undigested lambda DNA was used as a size marker. DNA was quantified by measuring the absorbance at 260 nm of a diluted sample. Genomic DNA was diluted to a final concentration of 50 ng/µl and stored at -20 o C. Appendix 5 Rep-PCR fingerprinting - PCR Amplification All PCR amplifications were performed under sterile air (0.45 µm filtered) flow in a cabinet. Reactions were performed in 200 µl thin walled tubes. PCR Supermix, a commercial premix of Taq polymerase, buffers, deoxynucleotides triphosphates and

8 MLMMI project Tracing the source... 8 stabilizers was used for all amplifications. To 45 µl of Supermix per tube were added 2 µl of 50 ng/µl genomic DNA as template, 2 µl of BOX A1R primer (0.3 µg/µl), 1 µl of 25 mm MgSO 4. The reaction mixture was overlaid with sterile mineral oil. For Enterococcus each strain was fingerprinted separately using either the BOX A1R primer or the ERIC 2I primer, both at 0.3 µg/µl in otherwise identical reaction mixtures. The PCR amplifications were performed in a RoboCycler The amplification cycle consisted of an initial 7 minute denaturation step at 96 o C followed by 35 cycles of denaturation; 96 o C for one minute, annealing; 58 o C for one minute and extension; 65 o C for 8 minutes and a final 8 minute 65 o C extension step. This was modified for Enterococcus fingerprinting to an initial 7 minute denaturation step at 96 o C followed by 35 cycles of denaturation; 96 o C for one minute, annealing; 40 o C for one minute and 20 seconds and extension; 65 o C for 8 minutes and a final 8 minute 65 o C extension step. All PCR reactions were frozen at -20 o C until used. Appendix 6 Rep-PCR fingerprinting - Electrophoresis Rep-PCR fingerprinting amplification products were separated by agarose gel electrophoresis. A gel concentration of 1.2% w/v agarose in TBE buffer (89 mm Tris-Cl, 89 mm borate, 1 mm EDTA) was used. For E. coli a volume of 15 µl of PCR reaction product was applied to the gel. For Enterococcus the BOX A1R and the ERIC 2I amplification reactions were combined (6.3 µl of each) and applied to the gel. The reaction products were electrophoresed at 60 volts for 90 minutes. Gels were stained with 0.5 µg/µl ethidium bromide. Bands were visualized by UV transillumination and photographed with Polaroid Polaplan 667 film. Appendix 7 Rep-PCR fingerprinting - Statistical Analysis After an initial inspection a template of all observed different bands was drawn up. Each individual fingerprint was scored for the presence or absence of each band in the template. A matrix of 1" for the presence of a band and 0" for the absence of a band was created for all strains. This matrix was entered into the SPSS statistics program and used to generate Jaccard similarity coefficients between every pair of isolates. To visually interpret the similarity between fingerprints the Jaccard coefficients were graphically represented by a dendrogram (Fig. 3) created using the SPSS hierarchical clustering procedure with the nearest neighbour clustering option.

9 Appendix 8 Media, Reagents and Equipment MLMMI project Tracing the source... 9 Media for the isolation, characterization and growth of both E. coli and Ent. faecalis were obtained from Difco (Detroit, MI). PCR Supermix and PCR primers were purchased from Life Technologies (Mississauga, ON). The PCR thermocycler was manufactured by Stratagene (La Jolla, CA). Reagent chemicals were obtained from VWR Scientific (Nepean, ON) except for ribose, Brij 58, sarkosyl and magnesium chloride which were purchased from Sigma (St. Louis, MO). Petri plates, PCR tubes, centrifuge tubes, culture storage tubes and pipet tips were obtained from VWR. Two Eppendorf pipettors were also purchased from VWR. Bacteriological membrane filters were from Pall-Gelman (Baltimore, MD). Proteinase K and Rnase were obtained from Roche (Baie d Urf, Que.).

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