Molecular Detection of Enterohemorrhagic Escherichia coli O157:H7 and Its Toxins in Beef
3.3. Molecular Identification of E. coli
3.3.1. Bacteria and Food Sample Preparation for PCR
1. Grow the bacterial strain in trypticase soy agar plates for 18 h at 37°C.
2. Pick a well-isolated colony and resuspend the bacterial cells in sterile PBS to a concentration of 105colony-forming units (CFU)/ml and use as a DNA template for PCR.
3. To identify E. coli from food samples, inoculate the homogenized food samples as detailed in Subheading 3.1. in TSB and incubate at 37°C for 6 h (see Notes 8–10).
4. Remove 1 mL/of the enriched food homogenate and wash once with sterile PBS and resuspend in 1 mL/of PBS.
5. Filter 400 µL of the PBS-washed sample through 5-µm ultrafree tubes (cat. no.
UFC3 0GV; Millipore, Bedford, Mass.) and centrifuge. This step will eliminate any particulate matter that might inhibit PCR reaction (seeNotes 11 and 12).
6. The 5-µm filtrate should then pass through 0.2 µm ultrafree tubes (cat. no.
SE3P009E4; Millipore) and centrifuge to remove bacteria only. This step will eliminate any dissolved matter that might inhibit PCR reaction.
7. Resuspend the 0.2-µm trapped materials in 400 µL of sterile PBS. Use 10-µL volumes of samples as the template for a PCR assay without extracting DNA.
8. All centifugation conditions are: 10,000g, 4°C, for 10 min.
3.3.2. Multiplex PCR
1. Prepare DNA template as described in Subheading 3.3.1. and use 10-mL as the template.
2. Add 1-mL (final concentration, 1 mM) of various synthesized oligonucleotide primers specific for uidA (UAL-754 and UAR-900), O157:H7-specific uidA (PT-2 and PT-3), SLT-I (LP-30 and LP-31) and II (LP-43 and LP-44) genes into the PCR reaction mixture.
3. 10X PCR buffer: Chemicals (Tris-HCl, 100 mM; MgCl2, 15 mM; KCl, 500 mM;
pH 8.3) dissolved in appropriate solution provided by the manufacturer will be used.
4. Deoxynucleotide triphosphates (dNTP): Make up a single solution containing 0.2 mMof dATP, dGTP, dCTP, and dTTP and use appropriate concentration as described in Subheading 3.3.2., item 7.
5. Primer sets that are used to amplify uidA(UAL-754 and UAR-900), O157:H7- specific (PT-2 and PT-3), SLT-I (LP-30 and LP-31), and SLT-II (LP-43 and LP-44) gene-specific fragments are as follows:
64 Venkateswaran UAL-754 5' AAA ACG GCA AGA AAA AGC AG 3'
UAR-900 5' ACG CGT GGT TAC AGT CTT GCC 3'
PT-2 5' GCG AAA ACT GTG GAA TTG GG 3'
PT-3 5' TGA TGC TCC ATC ACT TCC TG 3'
LP-30 5' CAG TTA ATG TGG TGG CGA AGG 3' LP-31 5' CAC CAG ACA ATG TAA CCG CTC 3' LP-43 5' ATC CTA TTC CCG GGA GTT TAC G 3' LP-44 5' GCG TCA TCG TAT ACA CAG GAG C 3'
6. TaqDNA polymerase: Supplied by the manufacturer at a concentration of 5 U/µL is used.
7. 1X Reaction mix: Forty-nine microliters of the reaction mix is required for each sample. To make 49 µL of this mix, combine 5.0 µL of 10X PCR buffer, 4.0 µL of 0.2 mM dNTP, 1 µL each of various primers (1.0 µM), 0.125 µL of Taq DNA polymerase (5 U/µL) with 38.875 mL of DNAse- and RNAse-free distilled water.
8. Mineral oil: Molecular biology grade mineral oil purchased from any manufac- turer can be used. Recent advancements are made in various thermal cyclers where mineral oil is not necessary.
3.3.3. PCR and Electrophoresis Conditions
1. Amplification: Using a DNA thermal cycler, program for 30 cycles consisting of 30 s at 94°C, 1.5 min at 58°C, and 2.5 min at 72°C, with a final extension step at 72°C for 7 min.
2. Electrophoresis: Remove 15-µL aliquots of each PCR mix and analyze for various amplification products by submarine gel electrophoresis on 2% agarose gels.
3. Run the electrophoresis for 50 min at 100 V.
4. Stain the gel with ethidium bromide for 15 min.
5. Visualize the stained bands by UV transillumination and photograph.
6. Include a suitable molecular size marker (100 bp ladder; Gibco-BRL) in each gel.
4. Notes
1. The majority of E. coliisolates shows typical metallic sheen colonies on EMB agar plates. However, S. dysenteriae mimics E. coli on EMB agar plates.
Similarly, gas may be produced from lactose at 44.5°C by E. coli O157:H7.
E. coliO157:H7 did not exhibit `-glucuronidase activity. However, some heat labile-toxin producing E. coli (ATCC 43886) strains may not produce fluores- cence on 4-methylumbelliferyl-`-D-glucuronide-supplemented commercial agar that contains lactose. The absence of sorbitol fermentation by O157:H7 is a characteristic phenotype used to isolate E. coliO157:H7 from clinical and food specimens. Although useful, confirmation with O157 and H7 antisera is required, as since other bacteria share this serotype and because there are strains of O157:H7 that can ferment sorbitol. As both S. dysenteriae andS. sonneii do not utilize sorbitol, they show green coloration and mimic E. coliO157:H7 in these agars. Therefore, biochemical characteristics alone will not differentiate E. coli O157:H7 from other toxigenic and nontoxigenic strains.
Enterohemorrhagic E. coli 65 2. Antibodies to the O157 antigen are used in many assays to detect O157:H7 in clinical and food samples. Cross-reaction of somatic antigen O157 and flagellar antigen H7 between O157 and O25, O26, O78, O111 as well as between H7 and H11, H–is established. These tests, however, provide no information on the toxin types produced by the isolates and are not specific, as the O157 antigen is present in other E. coli species. Also, anti-O157 sera often cross-reacts with Citrobacter freundii, E. hermanii and other bacteria. Analyses of food products with anti-O157 serum have recognized O157 isolates that neither produced SLT nor were of the H7 serotype. Furthermore, production of SLT toxins is not confined to E. coli O157:H7 strains and these toxins are produced in other serogroups of E. coli.
3. The standard bioassays used for identification of pathogenic E. coli, such as cytopathic effects on Y-1 adrenal cells and rabbit ileal loop, are not readily adapt- able for screening large numbers of E. coli isolates.
4. The uidAgene that is responsible for `-glucuronidase activity is a good marker for the differentiation of all types of E. coli strains from other group of coliforms, but species of the genus Shigella also possesses this gene.
5. Analysis of amplification products showed that all reference strains of O157:H7 serotype were correctly identified simultaneously with the SLT type known to be produced by these strains (seeFig.1, lanes 2–5). As anticipated, no products were amplified from wild-type E. coli (see Fig. 1, lane 1), whereas the expected toxin gene-specific products, but not O157:H7-specific products, were amplified from the SLT-producing non-O157: H7 serotypes examined (seeFig. 1, lanes 6–14).
6. The type of SLT identified by the multiplex PCR assay correlated well with the Vero cell toxicity data. Among non-E. coli strain, only S. dysenteriae exhibits an SLT I amplicon. The Shiga toxin of S. dysenteriae type 1 is almost identical to the SLT I of O157:H7; therefore, this is not unexpected.
Although the multiplex PCR assay will not discriminate between S. dysenteriae type 1 and non-O157:H7 EHEC serotypes that produce only SLT I, the O157:H7-specific primers readily distinguish S. dysenteriae type 1 species from O157: H7 isolates.
7. The major advantage of this method over existing assays is that it can identify the types of SLT encoded by the strain and at the same time discriminate other SLT- producingE. coli from O157:H7, predominant serotype implicated in disease.
8. When whole bacterial cells are used, 102CFUE. coli in 10-µL PCR mixture is necessary to amplify the PCR bands. However, a minimum of 106CFU/g is needed to amplify specific PCR products from food.
9. A 6-h incubation of contaminated food in a normal bacteriological medium would allow proliferation of 102 CFUE. coli/g initial inoculum to a detectable level.
Similarly, if the food homogenate is incubated overnight (16 h) at 37°C in shaking condition, a initial inoculum of 1 CFUE. coli/g slurry would attain a requisite density (>109 CFU/g), thus producing all PCR amplicons.
10. PCR amplification is possible, even when E. coliand other coliforms are in a ratio of 109:1.
66 Venkateswaran
11. Food particles and other unknown metabolic by-products may be inhibitory for PCR reaction. Hence, a two-step filtration procedure is necessary to remove any PCR inhibitory substances (seeSubheading 3.3.1., steps 5–7).
12. The two-step filtration procedure is successful for identifying appropriate PCR amplification products in various other food-borne pathogens directly from food enrichment culture without extracting DNA. Some examples are Salmonella, Vibrio cholerae, V. parahaemolyticus, Bacillus cereus groups, Camplyobacter spp. etc.
Fig. 1. Agarose gel electrophoresis of amplicons generated by multiplex PCR from E. coli strains isolated from various outbreaks. Lane 1, typical E. coli ATCC 25922;
lane 2, O157:H7 strain producing SLT-I, lane 3, O157:H7 strain producing SLT-I and SLT-II;lane 4 and5, O157:H7 strains producing SLT-II; lane 6, virulent strain not producing SLT-I or SLT-II; lanes 7–9, strains other than O157 serovar producing SLT-I; lanes 10–14, strains isolated from urinary tract and veterinary infections;
lane M, 100-bp marker. Number to the left of the gel are molecular sizes (base pairs).
Detecting L. monocytogenes with NASBA 67
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From: Methods in Biotechnology, Vol. 14: Food Microbiology Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ