Shigatoxigenic Escherichia coli (STEC) is a foodborne pathogen that causes hemolytic uremic syndrome (HUS) and the consumption of chicken products has been related to some HUS cases. We performed a non-selective isolation and characterization of STEC strains from retail chicken products. STEC isolates were characterized according to the presence of stx1, stx2, eae, saa and ehxA; stx subtypes and serotypes. Most of them carried stx2, showing subtypes associated with severe human disease. Although reported in other avian species, the stx2f subtype was not detected. The isolates corresponded to different serotypes and some of them, such as O22:H8, O113:H21, O130:H11, O171:H2 and O178:H19, have also been identified among STEC isolated from patients suffering from diarrhea, hemorrhagic colitis, HUS, as well as from cattle. Considering the virulence profiles and serotypes identified, our results indicate that raw chicken products, especially hamburgers sold at butcheries, can be vehicles for high-risk STEC strains.
Escherichia coli productor de toxina de Shiga (STEC) es un patógeno transmitido por alimentos que causa el síndrome urémico hemolítico (SUH). Algunos casos de SUH están relacionados con el consumo de productos de pollo. Se realizó el aislamiento no selectivo y la caracterización de cepas STEC provenientes de productos de pollo atendiendo a la presencia de stx1, stx2, eae, saa y ehxA, subtipos de stx y serotipos. La mayoría de los aislamientos portaba stx2 y subtipos de stx asociados con enfermedades graves en humanos. Aunque se ha detectado en otras especies aviares, el subtipo stx2f no se encontró. Se detectaron diferentes serotipos, entre ellos O22:H8, O113:H21, O130:H11, O171:H2 y O178:H19, también identificados como STEC aislados de pacientes con diarrea, colitis hemorrágica y SUH, y de ganado bovino. Teniendo en cuenta los perfiles de virulencia y los serotipos identificados, nuestros resultados indican que los productos de pollo crudos, especialmente las hamburguesas que se venden en las carnicerías, pueden ser vehículos de cepas STEC de alto riesgo.
Shigatoxigenic Escherichia coli (STEC) is a foodborne pathogen of public health importance that causes diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. The main virulence factors of STEC are Shiga toxins (Stx1 and Stx2), which inhibit protein synthesis by inactivating ribosome function8. The Stx1 group is more homogenous than Stx2 since it includes only three subtypes. In contrast, a great number of subtypes have been identified for Stx2. The stx subtypes have been differently associated with HUS6. In addition to Shiga toxins, STEC can synthesize the adhesin intimin (encoded by eae), an enterohemolysin (EhxA), and an autoagglutinating protein (Saa) in some eae-negative strains, among other virulence factors8.
Different STEC serogroups have been identified in strains isolated from humans suffering from gastrointestinal disease. Five STEC serogroups (O26, O103, O111, O145, O157) are considered to be the “top five” serogroups most frequently associated with severe human disease in the European Union, and two others (O45 and O121) are also regarded as the most pathogenic ones in the USA. The serotype most frequently associated with outbreaks and sporadic cases of severe disease is O157:H7; however, more than 50% of all STEC infections are attributed to non-O157 strains3.
STEC transmission occurs through the consumption of contaminated food or water, direct contact with animals or their environments, and person-person contact8. With regard to food, the consumption of chicken products has been related to HUS cases, but most of the studies performed on this kind of products have been focused only on the detection of STEC O157:H72,13. Therefore, the aim of this study was to perform a non-selective isolation and characterization of STEC strains from retail chicken products.
Samples analyzed in the present study corresponded to 10 giblets and 54 chicken hamburgers previously identified as stx-positive by Alonso et al.1 in a screening of 300 giblets and 300 chicken hamburgers. Peptone water cultures were stored at −70°C with 20% (v/v) glycerol. To isolate the STEC strains, an aliquot of each stx-positive culture, was streaked on MacConkey agar plates and incubated at 37°C for 24h11. Individual colonies were analyzed by a multiplex PCR to detect stx1, stx2, eae, saa and ehxA genes with the PCR protocol and primers described by Paton and Paton12. Amplification products were electrophoresed in 2% agarose gels and stained with ethidium bromide. Only one colony was further characterized except when colonies with different virulence profiles were detected by this multiplex PCR.
As several samples were contaminated with Proteus, subsequent cultures were streaked repeatedly on cysteine lactose electrolyte deficient agar (CLED) to obtain pure colonies of E. coli. Afterwards, the absence of Proteus was verified by culture on a non-selective medium such as trypticase soy agar (TSA).
The O-antigens were determined by the microagglutination technique, and H antigens were determined by the tube agglutination technique using antisera provided by the Laboratorio de Referencia de E. coli (LREC) (Lugo, Spain) as described by Fernández et al.5.
To subtype stx1 and stx2 genes, PCR-restriction fragment length polymorphism (RFLP) assays were used9. In addition, a monoplex PCR described by Schmidt et al.14 was used to detect the stx2f subtype.
Twenty-three STEC isolates were recovered from 54 stx-positive cultures of chicken hamburgers and only one isolate was obtained from 10 stx-positive giblet samples (Table 1). It was not possible to obtain any STEC isolate from some stx-positive samples although up to 200 colonies from those samples were analyzed.
Serotypes and virulence genes of STEC isolated from chicken products.
| Number of isolates | Sample | Origin | Serotype | Virulence genes |
|---|---|---|---|---|
| 1 | Hamburger | Butchery | O22:H8 | stx2EDL933 |
| 1 | Hamburger | Butchery | O91:H14 | stx1EDL933ehxA |
| 1 | Hamburger | Butchery | O91:H40 | stx2EDL933ehxA |
| 3 | Hamburger | Butchery | O113:H21a,b | stx2EDL933ehxA saa |
| 2 | Hamburger | Butchery | O117:H7 | stx2vhb |
| 2 | Hamburger | Butchery | O130:H11b | stx1EDL933stx2vhbehxA saa |
| 1 | Hamburger | Butchery | O130:H11 | stx1EDL933stx2EDL933ehxA saa |
| 1 | Hamburger | Butchery | O153:H28 | stx2vhb |
| 1 | Hamburger | Butchery | O160:H40 | stx2EDL933 |
| 1 | Hamburger | Butchery | O171:H2 | stx2O118 |
| 1 | Hamburger | Butchery | O171:H2a | stx2vha |
| 1 | Hamburger | Butchery | O178:H19 | stx2vha |
| 1 | Hamburger | Butchery | ONT:H2 | stx2O118 |
| 1 | Giblet | Butchery | ONT:H8 | stx2EDL933ehxA saa |
| 2 | Hamburger | Butchery | ONT:H40 | stx2vhb |
| 1 | Hamburger | Poultry shop | ONT:H- | stx2O118 |
| 3 | Hamburger | Butchery | ONT:H- | stx2vhb |
Isolates carrying only the stx2 gene predominated over the strains carrying both stx1 and stx2 or only stx1, a similar trend to studies from other countries that detected stx2 and not stx1 in STEC isolated from chicken meat. This finding is important considering that Stx2 is more cytotoxic than Stx18, and is associated with high virulence in humans6.
None of the STEC isolates carried the eae gene but some of them harbored the saa gene. STEC isolates that were saa-positive and eae-negative, belonging to serotypes O91:H21 and O113:H21 have been isolated from human patients with HUS8. Noticeably, in the present study O113:H21 isolates positive for saa were found in 3 samples and also harbored the stx2EDL933 subtype which has been associated with severe human disease.
Five virulence profiles could be determined by the multiplex PCR described by Paton and Paton12, with stx2 being the predominant profile (62.5%), followed by stx2ehxA saa and stx1stx2ehxA saa (17 and 12.5%, respectively). Furthermore, when the stx subtypes were also considered, 9 virulence profiles could be determined (Table 1).
With regard to STEC from chicken and derived products, there are few studies which identified the stx subtypes, and furthermore, these studies were focused exclusively on the characterization of O157:H7 strains2. In the present study, all stx1-positive isolates possessed the stx1EDL933 subtype, which has been associated with HUS cases and predominates in stx1-positive isolates from cattle and meat products9. As far as we know, this is the first report about stx1 subtypes in chicken samples. For the stx2 gene, different subtypes were detected, but no more than one stx2 subtype was identified within the same isolate. The stx2vhb subtype was the most prevalent, accounting for 43.5% of the isolates, followed by stx2EDL933 (8 isolates, 33%), stx20118 (3 isolates, 12%) and stx2vha (2 isolates, 8%). This data is important because stx2vhb, stx2EDL933 and stx2vha have been frequently associated with HUS cases6. The stx2O118 subtype predominates in STEC strains from sheep, is rarely found in cattle, and, in contrast to our results, it is usually found associated with other stx subtypes in strains isolated from cattle and foods9.
Although stx2f-positive strains have been isolated from avian species (pigeons), and Etoh et al.4 reported the isolation of a STEC strain harboring this subtype from a patient that had eaten raw chicken, we did not detect this subtype in any of the chicken samples.
STEC isolates belonging to O157:H7 were not detected in any of the samples, in agreement with the results obtained by other researchers who did not find this serotype in raw chicken meat and carcasses, even though they used selective methods for the isolation15. Indeed, there are only few studies that report the presence of STEC O157:H7 in chicken meat2,13.
Several non-O157 serotypes were isolated from chicken products in the present study (Table 1). Some of the serotypes, such as O22:H8, O113:H21, O130:H11, O171:H2 and O178:H19, have also been isolated from patients suffering from diarrhea, HC or HUS, highlighting the importance of these findings7. Two hamburgers presented STEC isolates belonging to two different serotypes (O113:H21 and O171:H2 in one sample, and O113:H21 and O130:H11 in the other).
A comparison was made between STEC isolated in the present study and STEC isolates from cattle and derived products in Argentina. Noticeably, some serotypes such as O91:H14, O117:H7, O113:H21, O130:H11, O171:H2 and O178:H19 were present in both groups of strains, and also stx2 predominated over stx15,10,11. Furthermore, some of the isolates belonging to serotypes such as O113:H21, O117:H7, O171:H2 and O178:H19 harbored the same virulence genotype and stx subtype as the isolates obtained from cattle, ground beef and evisceration tray samples in other studies9,10.
In conclusion, the characterization of the STEC isolates in terms to serotype, virulence profile and stx subtype performed in the present study shows that chicken hamburgers can carry STEC strains that are potentially pathogenic to humans. Moreover, most of the isolates obtained from hamburgers presented the same serotype and genotype as the STEC strains recovered from cattle and derived meat products in our country.
Ethical responsibilitiesProtection of human and animal subjectsThe authors declare that no experiments were performed on humans or animals for this study.
Confidentiality of dataThe authors declare that no patient data appear in this article.
Right to privacy and informed consentThe authors declare that no patient data appear in this article.
Conflict of interestThe authors declare that they have no conflicts of interest.
Authors thank María Rosa Ortiz for her technical assistance. This work was supported by grants from SECAT-UNICEN, CONICET and FONCYT. M. Z. A. was a holder of a fellowship from CONICET. P.M.A.L. and A. K. are members of the Research Career of CONICET. N.L. P. is member of the Scientific Research Commission Prov. Buenos Aires (CIC).
