BRIEF REPORT

http://dx.doi.org/10.1016/j.ram.2016.04.009

Serotypes, virulence profiles and stx subtypes of Shigatoxigenic Escherichia coli isolated from chicken derived products

Serotipos, perfiles de virulencia y subtipos de stx en Escherichia coli productor de toxina Shiga aislados de productos de pollo

 

Mónica Z. Alonso ^a, Alejandra Krüger ^a, Marcelo E. Sanz ^a, Nora L. Padola ^a,*, Paula M.A. Lucchesi ^a

a. Laboratorio de Inmunoquímica y Biotecnología, Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, CICPBA, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Buenos Aires, Argentina

Received 16 December 2015; accepted 12 April 2016
Available online 22 September 2016

*Corresponding author.
E-mail address: nlpadola@vet.unicen.edu.ar (N.L. Padola).

0325-7541/© 2016 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

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Abstract

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 stx_2f 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.

Keywords

STEC; Chicken; Serotypes; Virulence factors; stx subtypes.

Resumen

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 stx_2f 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.

Palabras clave

STEC; Pollo; Serotipos; Factores de virulencia; Subtipos de stx.

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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:H7^2,13. 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 24 h¹¹. 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 stx_2f 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.

Table 1. Serotypes and virulence genes of STEC isolated from chicken products.

a - One O113:H21 isolate and one O171:H2 isolate were obtained from the same sample.
b - One O113:H21 isolate and one O130:H11 isolate were obtained from the same sample.

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 stx_2EDL933 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 stx_1EDL933 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 stx_2vhb subtype was the most prevalent, accounting for 43.5% of the isolates, followed by stx_2EDL933 (8 isolates, 33%), stx₂₀₁₁₈ (3 isolates, 12%) and stx_2vha (2 isolates, 8%). This data is important because stx_2vhb, stx_2EDL933 and stx_2vha have been frequently associated with HUS cases⁶. The stx_2O118 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 stx_2f-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 meat^2,13.

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 stx₁^5,10,11. 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 studies^9,10.

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 responsibilities

Protection of human and animal subjects

The authors declare that no experiments were performed on humans or animals for this study.

Confidentiality of data

The authors declare that no patient data appear in this article.

Right to privacy and informed consent

The authors declare that no patient data appear in this article.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgments

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).

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