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Journal of Clinical Microbiology, October 2005, p. 5362-5365, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5362-5365.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Single Multiplex PCR Assay To Identify Simultaneously the Six Categories of Diarrheagenic Escherichia coli Associated with Enteric Infections

Maricel Vidal,1 Eileen Kruger,1 Claudia Durán,1 Rosanna Lagos,2 Myron Levine,3 Valeria Prado,1 Cecilia Toro,1 and Roberto Vidal1*

Programa de Microbiología, ICBM, Facultad de Medicina, Universidad de Chile, Santiago, Chile,1 Centro para Vacunas en Desarrollo, Santiago, Chile,2 Center for Vaccine Development, University of Maryland, Baltimore, Maryland3

Received 30 March 2005/ Returned for modification 25 May 2005/ Accepted 12 July 2005


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ABSTRACT
 
We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx1, stx2, and eae), enteropathogenic (eae and bfp), enterotoxigenic (stII and lt), enteroinvasive (virF and ipaH), enteroaggregative (aafII), and diffuse adherent (daaE) Escherichia coli in stool samples.


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TEXT
 
Most Escherichia coli strains are commensal; however, there are several highly adapted clones that have the capacity to cause human illness. Strains that cause enteric infections are designated diarrheagenic E. coli, a group that includes emergent pathogens with public health relevance worldwide (13). Six categories of diarrheagenic E. coli that differ in their virulence factors have been described (13). The most commonly reported diarrheagenic E. coli strains in Chile are enterotoxigenic E. coli (ETEC), which produces one or more enterotoxins that are heat labile LT (LT-1 and LT-2) or heat stable ST (STa and STb) (11); enteropathogenic E. coli (EPEC), which harbors a pathogenicity island that encodes a series of proteins involved in the attaching and effacement lesions of the intestinal microvilli of the host cell (8); and the presence of the large EPEC adherence factor (EAF) plasmid, on which also the cluster of genes encoding bundle-forming pili (bfp) is present (9). Based on these, EPEC strains are classified as typical when they possess the EAF plasmid, whereas atypical EPEC strains do not possess the EAF plasmid (18); Shiga toxin-producing E. coli (STEC) is characterized by the production of two potent cytotoxins denominated Shiga-like toxins 1 and 2 (Stx1 and Stx2) (17) and in some strains the presence of the LEE locus related to the attaching and effacement lesion (7, 16). The three other categories seem to be less prevalent. Enteroinvasive E. coli (EIEC) has biochemical, physiological, and genetic properties similar to those of Shigella, invading the epithelial cells of the colon, where it proliferates and causes necrosis of the tissue. The genes related to invasion are located in a virulence plasmid (pInv) of 140 MDa that encodes a type III secretion system (1, 12). Enteroaggregative E. coli (EAggEC), first discovered by studies of adherence to HEp-2 cells, displays a pattern of adherence characterized by self-agglutination that is denominated aggregative adherence (AA). Fimbrial structures denominated AA fimbriae I and II (AAF-I and -II) have been associated with adhesion to HEp-2 cells and human erythrocytes (5). The AAF-II fimbriae (coded in the pAA plasmid) seem to be more prevalent and are related to the capacity for adherence of EAggEC to the intestinal surface (14). The most recently characterized category corresponds to diffuse adherent E. coli (DAEC), strains that are capable of adhering to HEp-2 cells in a nonlocalized pattern. A surface fimbria (denominated F1845) has been proposed as a putative virulence factor that could be mediating this adherence phenotype (2). The "gold standard" method for detection of DAEC strains is based on the diffuse adherence phenotype in tissue cultures or by detection of the gene daa that is necessary for the expression of the F1845 fimbriae (4, 20).

Identification of different diarrheagenic E. coli pathotypes is not routinely performed because it is cumbersome and techniques are not readily available. Diagnosis is currently recommended for cases of persistent diarrhea, especially among tourists, children with severe diarrhea unresponsive to treatment, and immunodeficient patients with moderate to severe diarrhea, and in epidemic outbreaks of gastroenteritis (13).

Considering the epidemiological impact of diarrheagenic E. coli worldwide, especially ETEC, STEC, and EPEC, we previously designed a multiplex PCR to detect these three enteropathogens that proved to be sensitive and specific (19). In the present study we incorporated into this one-step multiplex PCR the detection of the remaining categories of diarrheagenic E. coli, EIEC, EAggEC, and diffuse adherence E. coli.

A total of 509 stool samples were obtained from Chilean children younger than 9 years of age with acute diarrhea attending different outpatient clinics in Santiago between April 2004 and January 2005. Study protocols of acute diarrhea that considered evaluation of one stool sample per diarrhea episode were approved by the Institutional Review Board of the Faculty of Medicine, University of Chile, and the Ethics Committee of the Servicio de Salud Metropolitano Norte.

Diarrheagenic E. coli reference strains 933J (stx1 stx2 eae), C600J (stx1), C600W (stx2), 2348/69 (eae), H10407 (st lt), STEC O159 (st), STEC O8 (lt), STEC O6 (lt), EI-34 (ipaH virF), F-1845 (daaE), and O42 (aafII) were used as positive controls. To determine the specificity of the primers, other members of the Enterobacteriaceae family, e.g., Shigella sonnei, Shigella flexneri, Enterobacter sp., Proteus mirabilis, Klebsiella oxytoca, Salmonella group B, Salmonella group D, Salmonella enterica serovar Typhi, "normal" colonic flora Escherichia coli HS, and nonenteropathogenic Escherichia coli 60120, were included as negative controls (Table 1) (10).


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  		TABLE 1. Bacterial strains tested by multiplex PCR, including reference diarrheagenic E. coli

PCR primers specific for stx1 and stx2 were previously described by Cebula et al. (3) and those for eae, bfp, stII, and lt were described by Vidal et al. (19). Primers for virF, ipaH, daaE, and aafII region 2 were designed from sequences available in the GenBank database using OMIGA 2.0 software for alignment and the Primer 3 program for primer design. Sequences, sizes of PCR products, and references are shown in Table 2.


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  		TABLE 2. Primers used in the multiplex PCR for amplification of diarrheagenic E. coli genesa

A pool of five E. coli colonies from cultures of reference strains and stool samples were analyzed by multiplex PCR for detection of virulence genes (stx1, stx2, eae, bfp, stII, lt, virF, ipaH, daaE, and aafII). When multiplex PCR was positive for the pool, each separate isolate was tested by multiplex PCR and then biochemically identified.

The multiplex PCR assay was performed as follows. Each 50 µl of reaction mixture contained 1 mM deoxynucleoside triphosphates, 2 pmol of each primer, 1.5 mM MgCl2, 1x reaction buffer (10 mM Tris-HCl, 50 mM KCl), 0.2 µl of Taq DNA polymerase, and 3 µl of template DNA. The crude cell lysate used as template DNA was prepared by boiling five colonies of E. coli in 0.5% Triton X-100 for 20 min. The hot start technique was used to prevent nonspecific amplification: 40 µl of the reaction mixture was preheated to 94°C for 5 min before Taq DNA polymerase (2 U in a 10-µl reaction mixture) was added. Samples were amplified for 35 cycles, with each cycle consisting of 1.5 min at 94°C for denaturation, 1.5 min at 60°C for primer annealing, and 1.5 min at 72°C for strand elongation. PCR products were visualized following electrophoresis through 1.5% agarose gels stained with ethidium bromide, and the amplicons were identified based only on the size of the amplified product.

Specificity of the multiplex PCR was tested with reference strains (Table 1). The different sizes of the amplification products for the stx1, stx2, eae, bfp, stII, lt, virF, ipaH, daaE, and aafII genes are shown in Table 2. EPEC and STEC strains detected by multiplex PCR were serotyped by an agglutination test using a commercial antiserum (PROBAC, Sao Paulo, Brazil).

The multiplex PCR assay designed in this study incorporated 20 primers for the amplification of 10 virulence genes (Table 2). The assay proved to be specific for the different categories of diarrheagenic E. coli when applied to prototype reference strains. Also, the laboratory protocol design allowed us to detect some of the most frequent categories and serogroups of diarrheagenic E. coli isolated from stool samples in Chilean children with acute diarrhea (Tables 1 and 3; Fig. 1) (15). All stool samples were cultured on MacConkey, SS, and XLD agar (Oxoid) for isolation of Escherichia coli, Salmonella spp., and Shigella spp., and Campylobacter spp. and Yersinia enterocolitica were cultured on campylobacter blood-free selective and yersinia-selective agar (Oxoid), respectively. In the series of children with acute diarrhea, we observed mixed infections by different categories of diarrheagenic E. coli in only one patient (Fig. 1), and no other bacterial enteropathogens were isolated as mixed infection.


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  		TABLE 3. Detection by multiplex PCR of different categories of diarrheagenic Escherichia coli in 2,545 colonies obtained from 509 stool samples from Chilean children with acute diarrhea



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  		FIG. 1. Multiplex PCR analysis of stool samples from Chilean children with acute diarrhea and selected reference strains. Bands corresponding to stx2, stx1, eae, bfp, st, lt, aafII, daaE, virF, and ipaH are indicated. Lanes 1 and 20, 100-bp size ladder; lanes 2, 4, and 5, colonies in patient B.R.G. 1001 with mixed infection (DAEC daaE and ETEC lt genes, respectively); lanes 7 and 19, reagent control; lanes 10 to 12, patient G.M.M. 956 with EAggEC (three colonies); lanes 3, 8, and 9, commensal E. coli colonies without virulence factor in both studied patients; lane 13, enterohemorrhagic E. coli 933J (stx1 stx2 eae); lane 14, enteropathogenic E. coli 2348/69 (eae bfp); lane 15, enterotoxigenic E. coli H10407 (lt st); lane 16, enteroaggregative E. coli O42 (aafII); lane 17, diffuse adherent E. coli F-1845 (daaE); lane 18, enteroinvasive E. coli EI-34 (virF ipaH).

With the exception of enteroinvasive E. coli, which was not detected in the 509 stool samples studied, we were able to differentiate five categories of diarrheagenic E. coli, including the less common DAEC and EAggEC and a variety of different serogroups of STEC and EPEC; results were comparable to those reported by Cebula et al. (3) and Vidal et al. (19) (Table 3). Eight STEC strains from patients with sporadic diarrhea were detected. Two of them had the stx2 gene, the predominant toxin phenotype pattern described in countries of the Northern hemisphere (6). However, six strains were non-O157:H7 and harbored stx1; these results are comparable with the toxigenic pattern of STEC strains observed in other studies in Chile (15).

The most frequent category of diarrheagenic E. coli detected was EPEC (54/509) (Table 3). To discriminate between typical and atypical EPEC, primers previously described for bfp gene detection were included in multiplex PCR (19). In this sample of children with acute diarrhea, 14 out of 54 EPEC strains were typical (Table 3).

The main challenge of designing a multiple PCR assay is the possibility for primer dimers and nonspecific products. So, it is necessary to design primers with close annealing temperatures, to begin the program with a hot start, and to use reference strains to determine reaction specificity (Table 1). The multiplex PCR is a rapid method for detecting multiple targets in a single reaction and in a short time.

Our results confirm that it is possible and feasible to perform a simultaneous amplification of the virulence genes from all categories of diarrheagenic E. coli (STEC, ETEC, typical or atypical EPEC, EIEC, DAEC, and EAggEC) and that this technique can be applied for the etiologic diagnosis of patients with sporadic diarrhea.This multiplex PCR showed high specificity for diarrheagenic E. coli, becoming a novel diagnostic tool for future epidemiological studies.


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ACKNOWLEDGMENTS
 
We thank Miguel O'Ryan and Gonzalo Osorio for careful review of the manuscript and helpful discussions.

This work was supported by DID grant 12-02/4-2.


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FOOTNOTES
 
* Corresponding author. Mailing address: Programa de Microbiología, Facultad de Medicina, Universidad de Chile, Av. Independencia 1027, Santiago, Chile. Phone: 56-2-678 6641. Fax: 56-2-735 5855. E-mail: rvidal{at}med.uchile.cl. Back


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Journal of Clinical Microbiology, October 2005, p. 5362-5365, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5362-5365.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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