Detection of Shiga toxin-producing Escherichia coli O157 in shellfish (Crassostrea gigas)
Ronan GUYON1 *, Francis DOREY1, Jean-François COLLOBERT2, Joel FORET1, Christelle GOUBERT1, Viviane MARIAU2, Jean-Paul MALAS1
RÉSUMÉ Détection d’Escherichia coli O157 producteurs de Shiga-toxines dans les huîtres (Crassostrea gigas).
Une campagne d’analyse, pour contrôler la qualité microbiologique des huîtres récoltées sur la côte du Calvados, a été entreprise en 1996 sur 150 échan- tillons. Les analyses portaient sur la recherche de salmonelles et d’Escherichia coli O157 ainsi que sur le dénombrement des coliformes fécaux et d’E. coli.
Les résultats d’analyses concernant les paramètres réglementaires (salmo- nelles et coliformes fécaux) étaient satisfaisants pour les 150 échantillons. Par ailleurs, une souche d’E. coli O157 fut détectée dans un échantillon présentant un taux de coliformes fécaux plus important que le reste de l’échantillonnage (276 UFC/100 g de chair). Des expériences de biologie moléculaire montrèrent que cette souche possédait trois gènes de pathogénicité : eae, EHEC-hlyA, et stx. Elle présentait alors les caractéristiques des souches entérohémorra- giques. D’après les résultats de l’identification moléculaire des variants des gènes stx, deux types de toxines pouvaient être exprimés : Stx1 et Stx2c. Au moment de cette étude, l’ensemble des huîtres pouvait légalement être mis sur le marché pour la consommation humaine.
Mots clés : STEC, coquillages, IMS, E. coli O157, Salmonella.
SUMMARY
One hundred and fifty oysters were tested for faecal coliform bacteria, Salmo- nella, E. coli and E. coli O157. All samples satisfied French regulations for the quality of live shellfish. However, one strain of E. coli O157 was recovered from one sample that contained 276 cfu of faecal coliform bacteria per 100 g of tis- sue, no Salmonella and no other E. coli. This strain tested positive for three pathogenicity genes: eae, EHEC-hlyA, and stx. According to the results of stx genes subtyping, we can conclude to the presence of stx1and stx2c. At the
1. Laboratoire départemental Frank Duncombe, 14053 Caen cedex 4, France.
2. Direction des Services vétérinaires du Calvados, 14040 Caen cedex, France.
* Correspondence:
r.guyon@cg14.fr
time of this study (1996), this sample could legally have been distributed for human consumption.
Key-words: STEC, Shellfish, IMS, E. coli O157, Salmonella.
1 - INTRODUCTION
Shellfish, particularly oysters which are eaten raw, often carry foodborne pathogens. Shellfish concentrate microbial pathogens in their tissues from the contaminated water because of its filter-feeding activities. Outbreaks associa- ted with the consumption of contaminated oysters are well-documented (RIPER, 1994; WILSON and MOORE, 1996). Shellfish quality is an important issue for public health and for consumer confidence in the fishing industry. The suitability of oysters for human consumption was assessed by testing for faecal coliform bacteria and Salmonella. Now, tests for E. coli are carried out.
Shiga toxin-producing E. coli O157:H7 (STEC) is a causal agent of haemorrha- gic colitis (HC), haemolytic uraemic syndrome (HUS) (CHAPMAN, 1995; GRIMONT, 1998; KARMALI et al., 1985; SIMMONS, 1997; TAYLOR et al., 1986). The STEC pathovar also includes other serotypes (CHAPMAN, 1995; GRIMONT, 1998; KARCH et al., 1997; SIMMONS, 1997; TARR and NEILL, 1996; TARR et al., 1996) but it is unclear whether non-O157 STEC are always pathogenic (GRIMONT, 1998; KAPER, 1998). Escherichia coli O157:H7 has been detected in water (MACGOWAN et al., 1989) and in many kinds of food: raw milk (CHAPMAN et al., 1993; MARTINet al., 1986), yoghurt (MORGANet al., 1993), mayonnaise (RAGHUBEERet al., 1994), cider (BRESSERet al., 1993; MILLERandKASPER, 1994) and beef (CARTERet al., 1987;
CDC, 1991; GRIFFINand TAUXE, 1993; RILEYet al., 1983; RYANet al., 1986). Identi- fication of a E. coli as having the O157:H7 serotype is not sufficient for classifica- tion of the bacterium as a pathogen. Escherichia coli O157:H7 virulence is mediated by at least three factors encoded by eae, EHEC-hlyA and stx. The eae gene encodes a 97 kDa membrane protein called intimin, which is involved in the attachment of bacteria to enterocytes. Its presence seems necessary to EHEC pathogenicity and plays a role in effacement lesions (destruction of microvilli) (GERMANIet al., 1997;GRIMONT, 1998; JERSEand KAPER, 1990; JERSEandKAPER, 1991; KAPER, 1998; LOUIEet al., 1993; YUandKAPER, 1992). The EHEC-hlyA pro- duct is an α-hemolysin-like factor belonging to the RTX (repeat in toxin) family (cytolysin) (BAUERandWELCH, 1996; SCHMIDTet al., 1995). EHEC-hlyA is located on a 90 Kb plasmid, pO157 (BARRETTet al., 1992; GRIMONT, 1998; KAPER, 1998;
SCHMIDTet al., 1995). The stx genes, which encode Shiga toxins may differ bet- ween variant strains: stx1and stx2are the most frequent genes, and stx2variants are known: stx2cand stx2e. Most STEC strains produce Stx2 with or without Stx1, few produce only Stx1. The stx genes are located in the genome of bacterio- phages which accounts for their frequent loss during laboratory tests (BASTIANet al., 1998; CALDERWOOD, 1994; GRIFFIN, 1995; GRIMONT, 1998; KAPER, 1998;
KARCHet al., 1992; O’BRIENet al., 1989; STROCKBINEet al., 1986).
In this paper we assess the contamination of oysters by E. coli O157:H7 and compare it with that for bacterial indicators of faecal contamination (faecal coli- form bacteria, E. coli and Salmonella).
2 - MATERIALS AND METHODS
2.1 Oyster sampling
Oysters (Crassostrea gigas) were obtained from a distribution centre. They came from a grade A zone of cleanliness or a purification basin. Shellfish were transported to the Frank Duncombe laboratory, within 2 h of being sampled, at low temperature (+ 4 to + 6°C).
2.2 Detection of faecal coliform bacteria, E. coli and Salmonella We tested for faecal coliform bacteria, E. coli and Salmonella. For each test, we homogenised 25 g of oyster tissue in 225 ml peptone-buffered water. We counted faecal coliform bacteria in broth as recommended by the French Minis- try of Agriculture (brilliant green lactose labelling) with the determination of the most probable number of bacteria by the three tubes method: three series of tubes with three appropriate dilutions of the sample. Each tube contained a Durham bell to appreciate the liberation of gas (positive reaction). The number of positive tubes in each serie was marked in a table and the most probable number was determined.
E. coli was counted according to the French norm Afnor NF V08-053. This test involved the inoculation of 1 ml of sample deep into semisolid PTX medium (AES laboratoires, Combourg, France). Further plates were inoculated with dilu- tions of the sample and were incubated at 44°C for 18 to 24 h. E. coli was detected as characteristic blue colonies.
Salmonella were detected using the Salmonella rapid test® (Oxoïd, Dardilly, France). In this test, the homogenised sample, after being enriched in peptone- buffered water, was incubated in the specific culture vessel consisting of two different tubes containing two culture media separated by a porous filter. Lower parts of tubes contain a selective medium and the upper parts, an indicator medium. If Salmonella were present, they migrated from the selective medium to the indicator medium, resulting in a change of colour. Positive results obtai- ned, should be confirmed by other tests, such as Salmonella Latex Test®
(Oxoïd) and by culture on SMID medium (BioMérieux, Marcy l’Étoile, France) at 37°C for 16 to 24 h. Salmonella were detected as characteristic pink colonies.
These assays were followed by identification of the Salmonella strains. An API 20E (BioMérieux, Marcy l’Étoile, France) test was carried out, along with serotyping of the suspected colonies.
2.3 Detection of Escherichia coli O157
Samples (25 g) of tissue were placed in peptone-buffered water for 6 h at 37°C. An immunomagnetic separation method (Dynabeads® anti-E. coli O157TM, Dynal Compiègne, France) (WRIGHTet al., 1994) was used to concen- trate the targeted E. coli presenting O157 surface antigens. Dynabeads are paramagnetic beads coated with a specific antibody raised against O157 anti- gen. Samples (1 ml) were incubated for 30 min, at room temperature, with 20 µl of Dynabeads suspension. Complexes of beads and bacteria were then concentrated using a magnetic particle concentrator, washed three times and
then were inoculated on selective media. For this study, two media were used, Sorbitol MacConkey Agar (SMAC) and Sorbitol MacConkey Agar supplemented with cefixin tellurite (CT-SMAC). Both types of plates were incubated at 37°C for 18 to 24 h.
Biochemical (API 20E, BioMérieux Marcy l’Étoile, France), oxydase (Stick oxydase unipath®, Oxoid Dardilly, France) and serological (Latex test E. coli O157®, Oxoid) tests were carried out on suspected colonies. All of them were sent to the National Reference Centre (Institut Pasteur, Paris, France) for confir- mation of E. coli O157:H7 serotype.
2.4 Detection of pathogenicity genes
Two PCR assays were performed to detect i) eae and EHEC-hlyA simulta- neously and ii) the stx genes. Sequences of the primers used in this work are given in table 1.
Table 1
Sequence of primers used in this study
Primer Sequence (5’ →3’) Reference Target gene Amplicon
size (bp) eae F GAC CCG GCA CAA GCA TAA GC PATONand PATON, 1998 eae 384 eae R CCA CCT GCA GCA ACA AGA GG
hlyA F GCA TCA TCA AGC GTA CGT TCC EHEC-hlyA 534
hlyA R AAT GAG CCA AGC TGG TTA AGC T
stx-Lin F GAA CGA AAT AAT TTA TAT GT LINet al., 1993 stx ≈900*
stx-Lin R TTT GAT TGT TAC AGT CAT
(*) The size of the stx-Lin PCR product depended on the stx variant
All PCR were performed in a final volume of 50 µl with 2 mM MgCl2. Cycling steps for the multiplex (eae and EHEC-hlyA) were: denaturation at 95°C for 5 min followed by 35 cycles (95°C for 1min, 65°C for 2.5 min and 72°C for 1.5 min). A final 10 min elongation was carried out at 72°C. The stx-Lin system was amplified as described by Lin et al., 1993: 94°C for 5 min; 94°C for 1 min, 43°C for 1.5 min and 72°C for 1.5 min for 35 cycles; 72°C for 7 min. PCR pro- ducts (15 µl) were analysed by electrophoresis in 2% agarose gel stained with ethidium bromide.
2.5 Identification of stx variants
Endonuclease restriction was carried out as described by BASTIAN et al.
(1998). The stx-Lin PCR products (12 µl) were incubated with 40 units of HincII (Sigma-Aldrich, St Quentin Fallavier, France) at 37°C for 3 h in a final volume of 25 µl. Restriction fragments were separated by electrophoresis in 2% agarose gel stained by ethidium bromide. The expected fragment sizes were presented in table 2. We used a video gel analysis system (Photo-Capt, Bioblock scientific, Illkirch, France) to estimate the molecular weight.
Table 2
Expected fragment sizes after restriction by HincII of Lin PCR products
stx variant Expected fragments (bp) stx1 705, 158 and 32 stx2 555, 262, and 62 stx2c 555, 324 and 16
stx2e 555 and 340
3 - RESULTS
3.1 Detection of bacteria
A total of one hundred and fifty samples were analysed between Octo- ber 1995 and July 1996. All samples satisfied the regulation governing levels of faecal coliform bacteria and Salmonella (table 3), and all the samples would have been authorised for sale for human consumption. Results were conside- red as satisfactory if they were fewer than 300 cfu of faecal coliform bacte- ria/100 g and no Salmonella in 25 g, according to the legal requirements laid down by the French Ministry of Agriculture 8195, July 31, 1995. One hundred and thirty-nine samples had levels of faecal coliform bacteria contamination lower than the detection threshold (100 cfu/100 g). Only ten samples contained more than 100 cfu of faecal coliform bacteria/100 g and one had 276 cfu/100 g. Salmonella was not detected in any of the samples analysed.
One sample tested positive for E. coli, with 3 cfu/g accounting for 0.7% of the samples. Similarly, E. coli O157 was found in one sample (0.7%). This sample, positive for E. coli O157, was the most heavily contaminated by faecal coliform bacteria (276 cfu/100 g) although normal E. coli counts were below detection threshold (< 3 cfu/g).
Table 3
Results of analysis and satisfaction of the quality for human consumption criteria of the French regulations
cfu Number of samples Quality Statutory Fecal coliform bacteria < 100/100 g 139 (93.6%) Satisfactory
Analysis Fecal coliform bacteria ≥100/100 g and 11 (7.4%) Satisfactory
< 300/100 g
Salmonella 0/25 g 150 (100%) Satisfactory
Analysis not E. coli < 3/g 149 (99.3%) Satisfactory
required by law ≥3/g 1 (0.7%) Unsatisfactory
E. coli O157 Not detected 149 (99.3%) Satisfactory Positive 1 (0.7%) Unsatisfactory
The suspected strain was sent to the National Reference Centre (Institut Pasteur, Paris). It was confirmed to be E. coli O157 and H7 negative. It posses- sed the eae, EHEC-hlyA and stx genes.
3.2 Sub-typing of stx genes
The pattern of restriction by the enzyme HincII of the stx-Lin PCR products revealed several bands of about 700, 550, 260, 150 and 60 bp. According to table 2, we can conclude to the presence of stx1and stx2c.
4 - DISCUSSION
The shellfish satisfied the quality criteria of the French regulations. The rate of conformity to the regulations was good and comparable with that described by JAMET in 1992, about 2.4% of oyster samples did not conform the French regulations in 1990. We detected no Salmonella spp. and counts of faecal coli- form bacteria were low in all but one sample. The only strain of E. coli O157 detected was isolated from this sample which nevertheless satisfied legal requi- rements. The O157 strain possessed all the pathogenicity genes necessary to cause human disease. The prevalence in this study was low, 0.7%, but oysters are consumed raw, so the risk is not negligible. There are currently no regula- tions concerning the STEC contamination of shellfish. We suggest that no STEC should be allowed to be present. A recent requirement from the French Ministry of Agriculture and Fishing (DGAL/SDHA/N98-8137, August 19, 1998) modified the guidelines concerning counting of faecal indicator bacteria. It stated that for live shellfish E. coli should be counted rather than faecal coliform bacteria according to the recommendation of European directive 91/492/CEE which defined E. coli as a faecal coliform bacterium that produces indol from trypto- phan at 44°C in 24 h. The new quality threshold for E. coli is defined as 230 cfu/100 g of tissue. According to these guidelines, the sample contaminated by E. coli O157 with a faecal coliform bacteria count (E. coli for the new circular) of 276 cfu/100 g would be classified as unsatisfactory.
5 - CONCLUSION
One strain of Shiga toxin-producing E. coli O157 was detected in one of 150 oyster samples analysed (0.7%). This E. coli O157 strain possessed all the three pathogenicity genes of enterohaemorrhagic E. coli. All legal requirements were satisfied (< 300 cfu of faecal coliform bacteria/100 g and no Salmonella in 25 g), so these oysters would be passed fit for human consumption. Although, the change in regulations for live shellfish in 1998 would result in this sample being classified as unsatisfactory due to high level of faecal coliform bacteria (E. coli),
there are no specific legal requirements relating to STEC. The investigation of shellfish contamination by STEC would be of value for guaranteeing public health and an incitement for change legal requirements.
ACKNOWLEDGEMENTS
This work was supported by the County Council of Calvados (France) and the Veterinary Service of Calvados (control administration). We would like to thank F. Grimont, Institut Pasteur of Paris, for confirmation of STEC serotyping.
We appreciate the assistance of S. Dumontier, V. Bouchart and G. Fortier in the preparation of this manuscript.
Receveid 11 January 2000, accepted 22 May 2000.
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