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Journal of Clinical Microbiology, October 2004, p. 4697-4701, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4697-4701.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Phylogeny, Clinical Associations, and Diagnostic Utility of the Pilin Subunit Gene (sfpA) of Sorbitol-Fermenting, Enterohemorrhagic Escherichia coli O157:H–

Alexander W. Friedrich,* Katja V. Nierhoff, Martina Bielaszewska, Alexander Mellmann, and Helge Karch

Institut für Hygiene, Universitätsklinikum Münster, Münster, Germany

Received 19 March 2004/ Returned for modification 6 June 2004/ Accepted 8 June 2004


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ABSTRACT
 
The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H–. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H– strain 493/89, each of 107 SF EHEC O157:H– clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H– strains which contained eae, which encodes {gamma}-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H–. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H– strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H–.


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INTRODUCTION
 
Enterohemorrhagic Escherichia coli (EHEC) is defined as a subgroup of Shiga toxin (Stx)-producing E. coli (STEC) that causes diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) (33). The most common serotype of EHEC implicated worldwide is E. coli O157:H7 (33). It does not ferment sorbitol and lacks ß-D-glucuronidase activity. These phenotypes allow the use of a selective medium, such as sorbitol MacConkey (SMAC) agar (22), for its detection. A large number of STEC isolates that do ferment sorbitol are therefore not detected by diagnostic procedures appropriate for the detection of E. coli O157:H7, and many of these are human pathogens (12, 14, 18). Among these non-O157:H7 E. coli isolates, sorbitol-fermenting (SF) EHEC O157:H– strains are important causes of diarrhea and HUS in Germany (15, 17) and have caused large outbreaks (2, 21, 28). SF O157:H– strains have also been isolated in other European (1, 4, 18) and non-European countries (3), but they have not yet been found in North America (6, 14, 19).

Multilocus enzyme electrophoresis and multilocus sequence typing indicate that SF EHEC O157:H–, non-sorbitol-fermenting (NSF) EHEC O157:H7, and E. coli O55:H7 are closely related members of the EHEC 1 group (10, 27, 34). In a stepwise evolution model of EHEC O157, a nontoxigenic E. coli O55:H7 strain which already contained the locus of enterocyte effacement is proposed to be an ancestor of EHEC O157:H7 and SF EHEC O157:H– (10, 32). SF EHEC O157:H– possess a large plasmid (15), which we termed pSFO157 (9) to distinguish it from pO157, which is present in EHEC O157:H7. pSFO157 and pO157 share several determinants, such as the EHEC hly operon and the etp gene cluster, which encode EHEC hemolysin and a type II secretion system, respectively (9, 29, 30). Both plasmids also contain stcE, a C1 esterase inhibitor-specific metalloprotease (20). However, espP, which encodes a serine protease that cleaves factor V (8), and katP, the structural gene for a catalase typically found on pO157 (7), are absent from pSFO157 (9). Instead, the sfp gene cluster, which encodes novel pili, is integrated at those positions and is flanked by insertion elements (9). This cluster consists of six genes that mediate mannose-resistant hemagglutination and that allow pilus expression (9). Preliminary analysis in our laboratory with a limited number of E. coli strains suggests that the structural pilin gene sfpA is a characteristic feature of many SF STEC O157 strains (9). This prompted us to investigate the prevalence and distribution of sfpA in a large collection of diarrheagenic E. coli strains and to test its utility as a diagnostic target specific for SF EHEC O157:H– strains.


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MATERIALS AND METHODS
 
Bacterial strains. In total, 600 E. coli strains were investigated. The complete E. coli standard reference (ECOR) collection of strains comprised 72 wild-type E. coli strains from humans and 16 other mammalian species (25). Moreover, 78 E. coli strains comprising the complete diarrheagenic E. coli (DEC) collection were also tested. For further information about these strains, see http://www.shigatox.net/cgi-bin/deca. The ECOR and DEC collections were kindly provided by T. Whittam (National Food Safety & Toxicology Center, Michigan State University, East Lansing). A total of 450 clinical E. coli isolates originated in our laboratory, and their serotypes are shown in Table 1. Of these, 238 strains were isolated in previous studies (12, 13) or in routine diagnostic tests conducted between 1996 and 2001. The remaining strains were isolated in our laboratory between 2002 and 2003 by the procedures described below. Furthermore, 20 other bacterial species occurring in human stools were investigated. These included Clostridium difficile (ATCC 9689), Salmonella enterica serovar Typhimurium (ATCC 14028), Proteus mirabilis (ATCC 35659), Proteus vulgaris (ATCC 29905), Enterococcus faecalis (ATCC 19433), Enterococcus faecium (ATCC 19434), Enterobacter cloacae (ATCC 13047), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae subsp. pneumoniae (ATCC 13887), Streptococcus pyogenes (ATCC 16915), and the following clinical isolates from our laboratory: Klebsiella oxytoca, Serratia marcescens, Citrobacter freundii, Citrobacter koseri, Morganella morganii, Enterobacter aerogenes, Yersinia enterocolitica, Clostridium perfringens, and Aeromonas hydrophila.


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  		TABLE 1. Distribution of sfpA among E. coli strains tested

Stool analysis. Six hundred thirty-six stool samples were investigated at the Institute of Hygiene, University Hospital Münster, Münster, Germany, as part of clinically indicated routine diagnostic evaluations between January 2002 and December 2003. The majority of the samples were obtained from hospitalized children with HUS or diarrhea throughout Germany. HUS was defined as a case of microangiopathic hemolytic anemia (hematocrit, <30% with peripheral evidence of intravascular hemolysis), thrombocytopenia (platelet count, <150,000/mm3), and renal insufficiency (a serum creatinine concentration greater than the upper limit of the normal range for the patient's age) (35). Ninety-two of the samples from patients with diarrhea were prescreened in another laboratory by an Stx-specific enzyme immunoassay and sent to our laboratory to confirm the diagnosis of STEC infection. The HUS cases showed no obvious geographic or temporal linkage. The following procedure was used to screen for and isolate STEC: 1 g of stool was inoculated into 10 ml of GN broth Hajna (Difco Laboratories, Detroit, Mich.) for 6 h. E. coli O157 was enriched from 1 ml of this enrichment culture by immunomagnetic separation (IMS), as described previously (16), and subsequent culture of magnetically separated organisms on SMAC agar, cefixime-tellurite (CT)-SMAC agar (Oxoid, Basingstoke, United Kingdom) (36), and enterohemolysin agar (Heipha, Eppelheim, Germany) (16, 29). To find non-O157 STEC, 200 µl of the enrichment culture from GN broth was cultured on SMAC and enterohemolysin agars. The bacterial growth was harvested into 1 ml of saline, and ca. 106 cells were used in PCRs with primer pairs KS7 and KS8 (stxB1), LP43 and LP44 (stxA2), SK1 and SK2 (eae) (12), and O157-F and O157-R (rfbO157) (24). The same PCR approach was also applied to detect E. coli O157 in the IMS-enriched cultures from SMAC, CT-SMAC, and enterohemolysin agar plates. To identify STEC strains in PCR-positive samples, colony blot hybridization was performed with a digoxigenin-labeled probe for stxB1, stxA2, or eae (12, 31). In addition to this routine diagnostic protocol, the 636 stool specimens from patients with diarrhea or HUS were also analyzed for sfpA, as a marker for the sfp gene cluster (9), by PCR with primers sfpA-U (5'-AGCCAAGGCCAAGGGATTATTA-3') and sfpA-L (5'-TTAGCAACAGCAGTGAAGTCTC-3') (9). The PCR conditions included initial denaturation at 94°C for 5 min and 30 cycles of denaturation (94°C, 30 s), annealing (59°C, 60 s), and extension (72°C, 60 s), followed by a final extension at 72°C for 5 min (9). Strains producing an sfpA amplicon were isolated by colony blot hybridization with a digoxigenin-labeled probe for sfpA, as described previously (9).

fliC PCR-RFLP analysis and eae typing. The fliC PCR-restriction fragment length polymorphism (RFLP) procedure was performed as described by Fields et al. (11). The fliC gene was amplified with primers F-FLIC1 and R-FLIC2 (11) and digested with RsaI (Gibco BRL, Eggenstein, Germany). Restriction fragments were separated on a 2% (wt/vol) agarose gel and visualized by staining with ethidium bromide. eae typing was performed as described previously (37).

sfpA sequence analysis. The sfpA gene was amplified with primer pair sfpA-U and sfpA-L and was sequenced by using an automated ABI Prism 3100 Avant Genetic Analyzer and an ABI Prism BigDye Terminator Ready Reaction cycle sequencing kit (version 3.0; Perkin-Elmer Applied Biosystems, Darmstadt, Germany). Sequences were analyzed with DNASIS software (Hitachi Software, San Bruno, Calif.). Homology searches were performed with the GeneBlast program of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/).

Phenotypic methods. The isolates were serotyped with antisera against E. coli O antigens 1 to 181 and H antigens 1 to 56 by the microtiter method described by Prager et al. (26). Fermentation of sorbitol was detected on SMAC agar plates (22) after overnight incubation at 37°C. The biochemical profiles were determined with the API 20E system (bioMérieux, Lyon, France), a system for the identification of members of the family Enterobacteriaceae and other nonfastidious gram-negative rods.


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RESULTS
 
sfpA in clade EHEC 1. Clade EHEC 1 includes O157:H7, its nonmotile relative, O157:H–, and E. coli O55:H7, which is the proposed ancestor of EHEC O157. sfpA was absent from each of 63 NSF EHEC O157:H7 clinical isolates (Table 1). It was also absent from each of the 11 O157:H7 DEC strains (DEC3A to DEC3E and DEC4A to DEC4F) originating from humans, cattle, and food (http://www.shigatox.net/cgi-bin/deca). Also, two stx-negative E. coli O157:H7 strains from our strain collection tested negative for sfpA (Table 1). However, sfpA was detected in strain 493/89, an SF EHEC O157:H– strain of the DEC3F lineage (Table 1), and was present in each of 107 SF STEC O157:H– strains from patients with diarrhea or HUS (Table 1). Moreover, sfpA was found in each of 14 eae-positive SF E. coli O157:H– strains which did not contain stx genes (Table 1). We performed fliC RFLP analysis to characterize the fliC genes of the nonmotile SF E. coli O157 strains. This revealed that each of the 14 SF E. coli O157:H– strains contained fliC, which encodes the H7 antigen. In addition, eae, which encodes {gamma}-intimin, a typical trait of clade EHEC 1 strains, was found in each of these 14 strains. Notably, sfpA was absent from all 10 eae-positive clinical isolates of O55:H7 (Table 1), as well as from the 5 O55:H7 DEC strains comprising strains DEC5A to DEC5E (http://www.shigatox.net/cgi-bin/deca).

sfpA among STEC strains outside clade EHEC 1. Among the DEC strains outside clade EHEC 1, all stx-positive strains were negative for sfpA. In addition, all 217 non-O157 STEC strains isolated in our laboratory were negative for this gene (Table 1).

sfpA among non-STEC strains. Each of the 72 ECOR strains was negative by the sfpA PCR (Table 1). All stx-negative DEC strains and the non-STEC clinical isolates were also sfpA negative. These included seven eae-positive SF E. coli O157:H45 strains and SF isolates of serotypes O157:H19 and O157:H43 (Table 1 and strains DEC7A, DEC7C, and DEC7D).

sfpA among other bacterial species. None of the 20 other bacterial species found in human stools tested positive for sfpA. These species included Salmonella enterica serovar Typhimurium, Proteus mirabilis, Proteus vulgaris, Morganella morganii, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Citrobacter koseri, Serratia marcescens, Yersinia enterocolitica, Aeromonas hydrophila, Pseudomonas aeruginosa, Clostridium difficile, Clostridium perfringens, Enterococcus faecium, Enterococcus faecalis, and Streptococcus pyogenes.

Sequence analysis of sfpA PCR product. Sequence analysis of the sfpA PCR products from 10 representative SF E. coli O157:H– strains (5 stx-positive strains and 5 stx-negative strains) isolated over a period of 15 years demonstrated that the sfpA nucleotide sequences were identical to each other and to the sfpA sequence from SF STEC O157:H– strain 3072/96 (GenBank accession number AF228759) reported by Brunder et al. (9). This indicates that this gene is highly conserved among SF STEC O157:H– strains and is stable during storage in the laboratory.

sfpA PCR for screening for SF E. coli O157:H– strains. Six hundred thirty-six enriched primary cultures of stool specimens from HUS patients (n = 177) and patients with diarrhea (n = 459) were screened by PCRs for the stx, eae, rfbO157, and sfpA genes. To screen for E. coli O157, the O157 IMS enrichment procedure was applied before PCR. For the screening for non-O157 STEC, stool cultures were analyzed by the same procedure but without O157 IMS. The results are summarized in Table 2. A positive sfpA PCR result was always found in combination with positive rfbO157 and eae PCR results, and in 25 of 27 cases a positive sfpA PCR result was also found in combination with a positive stx PCR result. This could subsequently be confirmed by isolation. In contrast, a positive sfpA PCR result was not found for any of the other 609 stool samples, although 59 of those stool samples contained STEC O157:H7 (Table 2). The corresponding strains isolated from the stool samples are included in Table 1. On the basis of the apparent correlation between the sfpA PCR screening result and the isolation of E. coli O157 strains possessing the corresponding combination of stx, eae, rfbO157, and sfpA genes, a diagnostic scheme has been designed to identify SF STEC O157:H– strains in human stool samples (Fig. 1).


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  		TABLE 2. Results of PCR screening targeting stx, eae, rfbO157, and sfpA genes in 636 stool specimens from patients with diarrhea (n = 459) and HUS (n = 177)



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  		FIG. 1. Diagnostic scheme designed to screen for and to isolate established pathogens of E. coli O157 strains from human stools. PBS, phosphate-buffered saline.


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DISCUSSION
 
SF EHEC O157:H– strains pose a great diagnostic challenge because their ability to ferment sorbitol makes it impossible to distinguish them from physiological stool flora and most other diarrheagenic E. coli strains on SMAC agar (15, 16, 17). Moreover, both Stx-producing and stx-negative NSF and SF E. coli O157 strains can be isolated from clinical specimens (1, 15, 21, 31), demonstrating that Stx detection methods are insufficient for the detection of such organisms. Here we demonstrate that plasmid-encoded sfpA is a characteristic feature of all eae-positive SF E. coli O157:H– strains, whether or not they possess stx genes. Therefore, it represents a potential target for the identification of these strains.

sfpA was absent from each of the E. coli strains of the ECOR collection, which comprises a set of 72 wild-type E. coli isolates from humans and 16 other mammalian species originally obtained from a large collection of approximately 2,600 isolates (23, 25). The collection is thought to broadly represent genotypic variation in E. coli (25). In addition, sfpA was absent from all but 1 of the 78 strains from the DEC collection. The exception was DEC3F strain 493/89, which is an SF EHEC O157:H– strain previously isolated in our laboratory (15).

The PCR targeting sfpA, a locus specific to all eae-positive SF E. coli O157:H– strains, has three particular potential diagnostic utilities. First, it represents a fast, simple, and specific screening method that might identify cases early in an outbreak and that might allow the tracing of its spread and its transmission to humans. Once they are detected, SF EHEC O157 infections can trigger timely and appropriate epidemiological investigations and, subsequently, the implementation of public health measures that will allow interruption of the epidemiological chain and, thus, avert continued transmission. The large 1996-1997 and 2002-2003 EHEC O157:H– outbreaks in Germany (2, 28) have demonstrated drastically the need for clinical laboratories to be able to identify these pathogens. Second, because of the cumbersome laboratory procedures required for the detection of SF EHEC O157 strains, infections with SF EHEC O157 strains probably remain underdiagnosed. As a consequence, the geographic distribution of these pathogens and their prevalence in most countries around the world are unknown. This lack of knowledge regarding their epidemiology is in contrast to their considerable clinical significance. In this context it is therefore important that the sfpA PCR provides clinical laboratories worldwide with an easy and reliable diagnostic tool that can be used to screen for these pathogens in large-scale prospective epidemiological studies. Such studies would answer the question whether SF EHEC O157:H– strains are really limited only to certain countries or whether they also occur in other geographic areas and to determine their prevalence and clinical significance worldwide. Third, and most importantly, the epidemiology of SF EHEC O157 infections, including its reservoirs and source for humans, is poorly understood. Whereas cattle have been well established as a major reservoir of EHEC O157:H7, to our knowledge, only one SF EHEC O157:H– strain has been isolated to date from a cow (5), thorough attempts to find it notwithstanding (17). While these observations suggest that cattle and perhaps other animals can be reservoirs of SF EHEC O157:H–, the rare isolation of these pathogens from animals led to the assumption that SF EHEC O157:H– might be adapted to the human intestine and that humans could be their major reservoirs (17). Thus, the PCR strategy described in this study provides a suitable and reliable tool that can be used to better understand the epidemiology of SF EHEC O157 infections, especially in situations in which the stx2 gene is absent.

Recently, SF E. coli strains of serotype O157:H– that did not contain stx genes were isolated from patients with HUS or diarrhea in our laboratory (31), and stx-negative SF E. coli O157:H– strains were reported during a family outbreak in Austria (1). Here we describe 14 additional SF E. coli O157:H– strains which are eae positive and stx negative. However, the possession of sfpA by each of these strains suggests that they arose from the infecting SF EHEC O157:H– organisms by loss of their stx genes during infection, isolation, or subculture. Alternatively, the stx-negative SF E. coli O157:H– strains might be progenitors of SF EHEC O157:H– that could, in the future, become EHEC by transduction with stx-converting phages. Important from the diagnostic point of view is the fact that the stx-negative SF E. coli O157:H– strains would be overlooked in patients' stools not only on SMAC agar but also when diagnostic protocols that rely on the detection of stx genes or Stx production are used. Their isolation requires stx-independent recovery techniques, such as the detection of the eae, rfbO157, or sfpA gene. Among these, the sfpA PCR represents a specific, and apparently sensitive, approach to the identification of stx-negative SF E. coli O157:H– strains, which are potential pathogens for humans, and an approach that can be used to distinguish them from other E. coli O157 strains.

Several studies indicate that SF EHEC O157:H– strains are closely related to NSF EHEC O157:H7 strains (10, 27, 32). It was hypothesized that both groups have evolved from a common enteropathogenic E. coli (EPEC)-like O55:H7 ancestor (10, 27). Interestingly, sfpA was not present in any of the 10 clinical E. coli O55:H7 isolates or any of the 5 DEC O55:H7 strains analyzed here. Because all of the sfpA-harboring E. coli O157:H– strains possessed eae, we hypothesize that the sfp gene cluster has been acquired by the nonmotile SF E. coli O157 branch, presumably after the pathogenicity island locus of enterocyte effacement was acquired and motility was lost. The sfp fimbrial gene cluster, as we have shown, is inserted into a region corresponding to that where katP and espP reside in pO157 (9). This is in accord with earlier observations showing that the plasmids of SF EHEC O157:H– strains are distinct from those of NSF EHEC O157:H7 strains with regard to the katP and espP genes, which are present in NSF O157:H7 strains but not in SF EHEC O157:H– strains (5, 17). The remnants of different insertion sequences flanking the sfp cluster on the large plasmid of SF EHEC O157, as well as the katP and espP genes in pO157, lead us to propose that transposition processes engendered these differences. On the other hand, the existence of the RepFIB-like origin of replication downstream of the sfp cluster (9) indicates that a second plasmid is the source of the fimbrial gene cluster and that this plasmid underwent replicon fusion with a pO157-like precursor of pSFO157. However, the gene pool from which SF EHEC O157:H– acquired the sfp fimbrial genes remains unknown, and also, in this study we were not able to find sfpA genes in E. coli strains others than SF O157 and outside E. coli species. The question of why this gene cluster, despite its putative mobility, is so unique to the SF E. coli O157:H– strains within our collection of Enterobacteriaceae cannot be answered at present.

In conclusion, the sfpA PCR is a highly specific and sensitive technique for the detection of SF EHEC O157 in human stools. Therefore, it represents an easy and reliable approach potentially useful worldwide for investigation of the prevalence of SF EHEC O157:H– strains, their clinical significance, and the epidemiology of the diseases that they cause.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) Project Network of Competence Pathogenomics Alliance "Functional Genomic Research on Enterohaemorrhagic Escherichia coli" (BD number 119523).

We thank P. I. Tarr (Washington University School of Medicine, St. Louis, Mo.) for critical reading of the manuscript and fruitful discussions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Hygiene, Universitätsklinikum Münster, Robert-Koch Str. 41, 48149 Münster, Germany. Phone: 49-251-8355366. Fax: 49-251-8355341. E-mail: alexf{at}uni-muenster.de. Back


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Journal of Clinical Microbiology, October 2004, p. 4697-4701, Vol. 42, No. 10
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.10.4697-4701.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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