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Journal of Clinical Microbiology, October 2006, p. 3703-3711, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00429-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Virulence Genes Linked with Diarrhea Due to Atypical Enteropathogenic Escherichia coli by DNA Microarray Analysis and PCR{dagger}

Jan Egil Afset,1* Guillaume Bruant,2 Roland Brousseau,3 Josée Harel,2 Endre Anderssen,4 Lars Bevanger,1,5 and Kåre Bergh1,5

Department of Laboratory Medicine, Children's and Women's Health, Faculty of Medicine,1 Department of Chemistry, Faculty of Natural Sciences and Technology, Norwegian University of Science and Technology,4 Department of Medical Microbiology, St. Olavs University Hospital, Trondheim, Norway,5 Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C2,2 Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada3

Received 27 February 2006/ Returned for modification 24 April 2006/ Accepted 17 July 2006


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ABSTRACT
 
The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n = 37) and without (n = 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P = 0.0008). Other OI-122 genes (set/ent, nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P = 0.0004). Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea.


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INTRODUCTION
 
Enteropathogenic Escherichia coli (EPEC) is characterized by its ability to cause attaching and effacing (A/E) lesions in the intestinal epithelium. Typical features of A/E lesions are intimate attachment of the bacterium to the intestinal epithelial surface, effacement of microvilli, formation of pedestal-like structures, and reorganization of actin filaments in the intestinal cell just beneath the site of bacterial attachment (43). The genes necessary for A/E lesion formation are located on a pathogenicity island on the E. coli chromosome called locus of enterocyte effacement (LEE). EPEC is differentiated from enterohemorrhagic E. coli (EHEC), which also causes A/E lesions, by the ability of the latter to express Shiga toxins. EPEC strains are classified as typical if they possess the E. coli adherence plasmid (EAF) with bfp genes encoding bundle-forming pili (27). Typical EPEC strains produce a characteristic adherence pattern called localized adherence in tissue culture (43). A/E E. coli strains which do not possess the EAF plasmid or bfp genes are classified as atypical EPEC (27).

Whereas typical EPEC is well recognized as a leading cause of severe pediatric diarrhea in developing countries (39), the role of atypical EPEC in childhood diarrhea has been controversial (58). Atypical EPEC has been shown to be prevalent among children in both developing (15, 18, 59) and developed countries (2, 7, 11, 32, 45), but most studies have not been able to demonstrate a significant association with diarrhea for this class of bacteria (16, 17, 19, 32, 37, 44, 45, 47, 49). However, such an association has been reported in some studies (2, 11, 50, 60), and atypical EPEC has also been the cause of diarrhea outbreaks (22, 23, 61, 63). In addition, EAF plasmid-cured and bfpA mutant strains of typical EPEC caused diarrhea in healthy adult volunteers, although to a lesser extent than the original EPEC strains (8, 33).

Molecular characterization has shown a great diversity of virulence factors among different strains of atypical EPEC (6, 15, 60). It is therefore possible that different strains within the group of atypical EPEC have different pathogenic potentials. Such a difference may have its basis in the variability in the LEE pathogenicity island or in virulence factors encoded by genes located in some other part of the genome. After the sequencing of the genomes of two O157:H7 EHEC outbreak strains (EDL933 and Sakai) was completed, the search for potential virulence genes was made easier. By comparing the genomes of the two O157:H7 EHEC strains with that of the nonpathogenic E. coli K-12 strain, many regions unique to these strains were identified, and several of these regions fulfilled criteria as pathogenicity islands (21, 46). This has led to the identification of a number of new potential virulence genes within the E. coli chromosome which may be associated with diarrhea (12).

Microarrays are excellent tools for the simultaneous detection of high numbers of genes. Recently, an oligonucleotide microarray was developed for the detection of E. coli virulence genes (9). This microarray, which contained probes specific for a high number of virulence genes and virulence markers from all known pathotypes of E. coli, was validated as a powerful molecular tool with a collection of well-characterized and reference E. coli strains. Based on the hypothesis that pathogenicity may depend on the presence of specific virulence factors, the aim of the present study was to search for genes linked with diarrheal disease in atypical EPEC strains from a case-control study using this oligonucleotide microarray and PCR.


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MATERIALS AND METHODS
 
Bacterial strains. Atypical EPEC strains were isolated from fecal specimens from children less than 5 years old participating in a case-control study conducted during the period 2002 to 2003 in the County of Sør-Trøndelag, Norway (2). The cases (n = 251) were children with community-acquired diarrhea who attended a physician and had a fecal specimen taken for investigation of causative agent. Healthy controls (n = 210) were recruited at Maternal and Child Health Centers. EPEC identification was based on PCR amplification of the eae, stx1/stx2, and bfpA genes and verification of eae-positive isolates as E. coli by biochemical testing. A total of 57 isolates identified as atypical EPEC, 37 from cases and 20 from controls, were stored (1 isolate per subject) at –70°C until further study by microarray analysis and PCR. Specimens from cases were also tested for other bacterial and viral diarrheagenic agents. Information on duration of disease was recorded, and persistent colonization with EPEC was tested in follow-up specimens.

Virulence oligonucleotide microarray. A DNA microarray recently developed by Bruant et al. (9), was used for the characterization of the collection of atypical EPEC strains. The version of this microarray used in the present study was composed of 242 70-mer oligonucleotide probes specific for 182 virulence genes or markers found in various intestinal and extraintestinal E. coli strains of all known pathotypes. Probes were specific for genes encoding adhesins; toxins; bacteriocins; antiaggregative factors; autotransporters; capsular, flagellar, and somatic antigens; hemolysins; invasins; iron acquisitions system or transport proteins; and outer membrane proteins, as well as other genes recently shown to be associated with virulence in E. coli (see Table S1 in the supplemental material). The oligonucleotide microarray also allowed the detection of genetic variants of particular genes, such as the intimin-encoding gene eae (variants eae-alpha, eae-alpha2, eae-beta, eae-beta2, eae-delta, eae-epsilon, eae-epsilon2, eae-eta, eae-gamma, eae-iota, eae-iota2, eae-lambda, eae-mu, eae-nu, eae-pi, eae-xi, and eae-zeta), espA (variants espA1, espA2, and espA3), espB (variants espB1, espB2, and espB3), and tir (variants tir-1, tir-2, and tir-3) from the LEE. Oligonucleotides specific for three variants of the long polar fimbria (LPF)-encoding gene lpfA are also included. These are based on sequences from the lpfA genes of EPEC strains of serogroup O113 (lpfAO113), O-island 141 of serogroup O157:H7 (lpfA1), and rabbit EPEC (lpfAR141). The last is identical to the lpfA sequence of serogroup O26. Compared to the recently published version (9), the microarray used in the present study did not include nine probes specific for seven genes (cif, epeA, focG, nleA/espI, sfaD, sopA, and tspE4.C2), 12 probes specific for variants of virulence genes already represented on the array, and duplicate probes of control genes (see Fig. S1 in the supplemental material).

DNA labeling and hybridization of labeled DNA on the microarray. E. coli genomic DNA was fluorescently labeled with Cy5, as described previously (9), with a random-priming protocol derived from the Invitrogen's Bioprime DNA-labeling system (Invitrogen Life Technologies, Burlington, Ontario, Canada).

Hybridization of labeled DNA was performed as described previously (9). Briefly, the microarrays were prehybridized at 50°C for 1 h in a slide hybridization chamber (Corning, Canada) with 30 µl of prewarmed (37°C) DIG Easy Hyb buffer (Roche Diagnostics, Laval, Quebec, Canada) containing 0.5% (vol/vol) purified bovine serum albumin (10 mg/ml) (New England BioLabs, Pickering, Ontario, Canada). After prehybridization, 500 ng of labeled DNA was resuspended in 6 µl of prewarmed DIG Easy Hyb buffer and denatured by heating it for 5 min at 95°C. Microarrays were then hybridized overnight at 50°C in a slide hybridization chamber. After hybridization, four stringency washes (three in 0.1x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]-0.1% sodium dodecyl sulfate and one in 0.1x SSC) were performed at 37°C for 5 min and under agitation. The slide was then scanned at a resolution of 5 µm at 90% laser power, with a ScanArray Lite fluorescent microarray analysis system (Canberra-Packard Canada, Montreal, Quebec). All hybridization experiments were performed in duplicate with DNA obtained from two separate bacterial cultures.

Detection of putative virulence genes by PCR. PCR was employed for the detection of seven putative virulence genes (nleA/espI, cif, nleB, nleC, nleD, and nleE) which were not included in the microarray version used in this study. Bacterial DNA samples were obtained by boiling them for 10 min and centrifuging them or by using the MagAttract DNA mini M48 kit (QIAGEN, Hilden, Germany). PCR for the genes nleA/espI and cif was done as previously described (34, 38). Oligonucleotide primers for the EHEC O157:H7 EDL933 genes nleB(Z4328), nleC(Z0986), nleD(Z0990), nleE(Z4329), and nleF(Z6020) were designed using the Primer3 software (48). These genes are homologous to Citrobacter rodentium genes encoding effector proteins which are secreted through the LEE-encoded type III secretion system (13). The primer sequences and cycling conditions used in the PCR analysis are listed in Table S2 in the supplemental material. PCR amplification was performed in a total volume of 50 µl, containing 50 µM (each) dATP, dCTP, dGTP, and dTTP; 0.5 µM each primer; 10x PCR buffer (Applied Biosystems, Branchburg, N.J.); 1.5 mM MgCl2; 1 U AmpliTaq Gold polymerase (Applied Biosystems); and 2 µl bacterial DNA extract as the template. The polymerase enzyme was activated by heating it at 94°C for 15 min. After the final cycle, the mixture was held at 72°C for 7 min before cooling at 10°C. The amplified products were analyzed by 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. The EDL933 strain (CCUG29197) and the REPEC E22 strain (kindly provided by E. Oswald) were used as positive controls for the genes nleA/espI, nleB, nleC, nleD, nleE, and nleF and the genes cif and efa1/lifA, respectively.

PCR was also employed to control the specificity of newly designed probes (bfpA), to differentiate between truncated and nontruncated genes (astA, efa1/lifA, cif), to ensure that gene variants were not missed (lpfA, paa), and to control the result for unexpected genes in atypical EPEC strains (malX, etpD, stxB1, and usp). The PCR analyses were performed as previously described (9), with published primers for the genes astA (62), bfpA (20), cif (34), efa1/lifA (3), lpfAO113 (14), lpfA1 (53), lpfAR141 (41), malX (24), etpD, paa, stxB1, and usp (5) (Table S2 in the supplemental material). The PCR results were based on concordant results when bacterial strains were tested in duplicate.

Statistical analysis. Of the 189 virulence genes or gene markers targeted in this study, 53 contained sufficient variation, present and absent in more than five strains, to be useful in further analysis. Genes associated with diarrhea were determined using Fisher's exact test (P < 0.05). As the numbers of genes and cases were comparable in this study, multiple testing was not expected to be as much of a problem as in other microarray studies. Nevertheless, a permutation test was conducted and a P value of 0.05 was found to correspond to a false-discovery rate of 0.07. Further correction for multiple testing was therefore deemed unnecessary.


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RESULTS
 
As expected, duplicate microarray hybridization experiments gave identical results for each of the atypical EPEC strains tested in the study. The microarray results were confirmed by PCR for the bfpA, etpD, lpfAO113, lpfA1, lpfAR141, malX, paa, and usp probes. For astA probe-positive strains, PCR had to be done to distinguish complete astA sequences from truncated ones, as mentioned by Bruant et al. (9). The only discrepancy was for the stxB1 gene, encoding the B subunit of EHEC Shiga toxin 1, which was not detected by the PCR method in any of the 16 strains which had hybridized with this probe in the microarray experiments. The results of the PCR analyses for the recently described virulence genes nleA/espI, cif, nleB, nleC, nleD, nleE, and nleF were analyzed together with the microarray data.

A total of 95 putative virulence genes or gene variants were detected in the 57 atypical EPEC strains (Table 1). About one-third of these genes were observed in five strains or fewer. The majority of virulence genes detected were either EPEC- or EHEC-associated genes or recognized to be common in different pathotypes of E. coli. Among genes linked to extraintestinal E. coli disease, only the ibeA (23 strains); the fyuA, irp-1, and irp-2 (12 strains each); and the usp and malX (9 strains each) genes were present in more than five strains. Genes of diarrheagenic E. coli pathotypes other than A/E E. coli were each seen in only one or two strains each.


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  		TABLE 1. Virulence genes and markers detected in 57 atypical EPEC strains isolated from children with and without diarrhea

Genes linked with diarrhea. Twelve genes were found to be statistically associated with diarrhea in this study (Table 2). The association was positive for eight and negative for four of these genes. The set/ent, nleB, and nleE genes were always found together in 23 of the strains, consistent with a location on a pathogenicity island, as previously reported for typical EPEC and EHEC strains (26, 36). The efa1/lifA gene, which also belongs to this pathogenicity island, named OI-122 in the EHEC reference strain EDL933, was present in 17 of the same strains. All efa1/lifA-positive strains were tested for completeness of the gene using PCR with primers specific for three different parts of the gene. All but one of the strains were positive with all primer sets, whereas only the 5'-end part of the gene was detected in the last strain.


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  		TABLE 2. Genes significantly associated with diarrhea in atypical EPEC strains from Norwegian children less than 5 years old with and without diarrhea

Of the three lpfA gene variants, only lpfAO113 was significantly associated with diarrhea when the results with the three different lpfA probes were analyzed separately (Table 2). However, when the analysis was done for all three lpfA variants together, the association with diarrheal disease was highly significant (P = 0.0008). None of the atypical EPEC strains hybridized with more than one of the three lpfA probes. The lpfA1 gene variant (n = 5) was found exclusively in children with diarrhea, whereas the eight strains with lpfAR141 were found almost equally frequently in children with (6/37) and without (2/20) diarrhea. Among the 26 lpfA-positive strains, 21 also contained the set/ent, nleB, and nleE genes and 17 contained the efa1/lifA gene (Table 3).


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  		TABLE 3. Virulence profiles of atypical EPEC strains isolated from Norwegian children less than 5 years of agec

In an alternative analysis, eight atypical EPEC strains from children with diarrhea who also had other concurrent diarrheagenic agents were excluded from the analysis. However, the results from this analysis compared to those from the previous analysis were principally unchanged, except for the genes ureD, ehxA, and b1121, for all of which a P value of 0.07 was found. The lack of significance for these genes in the alternative analysis may reflect the effect of the simultaneous enteropathogenic agents but will also in part be the result of the reduction in statistical power due to the lower number of strains in the analysis. In this analysis, borderline significance (P = 0.05) was detected for two additional genes, the outer membrane protein gene ompT and the intimin gene variant eae-gamma.

Genes from the LEE. None of the LEE gene variants were found to be significantly associated with diarrheal disease, except for the intimin gene eae-gamma variant that had borderline significance in the alternative analysis (P = 0.05) as mentioned above. Four main variants of the eae gene were most common. The alpha2, beta1, beta2, and gamma variants were detected in 12, 13, 9, and 12 strains, respectively (Table 1). eae variants seen more rarely were the alpha1, delta, epsilon1, and iota1 variants. Two strains did not hybridize with any of the 17 eae variant probes present on the array, although they were positive for the common eae-specific probe. For the espA and espB genes, the espA1 and espB2 variants dominated, found in 40 and 36 strains, respectively. The most common tir variant, tir-3, was seen in 24 strains.

Other EPEC- and EHEC-related genes. Several other genes which have previously been reported as putative virulence factors in EPEC were detected but were not statistically associated with diarrheal disease in this study (Table 1). The genes nleA/espI, nleC, nleD, nleF, and espC were present in 24, 50, 34, 40, and 8 strains, respectively. The cycle-inhibiting factor gene cif was detected in all together 29 strains, with a truncated gene in three of these strains. Several of the atypical EPEC strains contained genes associated with the EHEC pathotype. In addition to the already mentioned genes stxB1 (16 strains), ehxA (8 strains), and ureD (7 strains), the etpD and rtx genes were found in 7 and 6 strains, respectively. Other EHEC-associated genes were seen in five strains or fewer. Except for the ehxA and ureD genes, none of the EHEC genes were found to be associated with diarrheal disease.

Virulence groups. All except one of the atypical EPEC strains in this study could be classified into two main virulence groups: group I strains were defined by the presence of OI-122 genes and/or lpfA genes as well as the absence of the yjaA gene, while group II strains were classified by their content of the yjaA gene and the absence of OI-122 and lpfA genes (Table 3). Group I strains were further divided into the subgroups Ia and Ib depending on whether or not they contained the gene with the strongest association with diarrhea, efa1/lifA. One strain containing both OI-122 and the yjaA genes could not be allocated to any of these virulence groups.

The main virulence groups were compared with respect to clinical and various other relevant features to assess the impact of the combination of virulence genes in each strain as an alternative to an analysis of each gene separately (Table 4). There was no difference between the virulence groups with respect to demographic variables or in the quantities of atypical EPEC organisms in the stool specimens. However, the association between virulence group I strains and diarrhea was highly significant (P < 0.001). There was also significant difference between the virulence groups in the durations of colonization (P = 0.005). Among children with diarrhea, a nonsignificant tendency was seen towards persistence of diarrhea in children infected with virulence group II strains compared with children with strains belonging to virulence group I.


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  		TABLE 4. Comparison of various features of the main virulence groups of atypical EPEC strains isolated from Norwegian children less than 5 years old with and without diarrhea


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DISCUSSION
 
The results of the present study clearly indicate that several atypical EPEC genes are statistically associated with diarrheal disease (Table 2). This finding is in contrast to the majority of previous epidemiological studies, which have not been able to find any significant difference between strains from children with and without diarrhea (16, 17, 19, 32, 37, 44, 45, 47, 49). The basis for this detection was the ability to simultaneously analyze a high number of putative virulence genes, which would have been very laborious and in practice impossible without the use of microarray technology.

The statistical association with diarrhea for the efa1/lifA gene demonstrated in this study (Table 2) is consistent with results from experimental studies. The efa1/lifA gene encodes a protein, lymphostatin, which has been shown to inhibit lymphocyte proliferation and proinflammatory cytokines (1, 30) and has also been reported to have adhesive properties (42). The nleB gene was also recently shown to be involved in virulence, while no such effect was seen for the nleE gene (29). The function of the set/ent gene, which encodes a putative enterotoxin (40), is still unknown. Among the 23 strains with OI-122 genes in this study, the efa1/lifA gene was present in 17 and absent in 6 strains. This fact demonstrates the existence of two main variants of OI-122 in atypical EPEC strains in this region of Norway. Interestingly, Karmali et al. have reported that EHEC strains with a complete OI-122 gene (including the pagC gene not tested in this study) are associated with increased epidemic potential and severity of disease (28). Whether the same applies to atypical EPEC strains has yet to be investigated. The virulence potential of truncated efa1/lifA genes similar to those of one of the atypical EPEC strains in this study is not clear (1, 52).

For the lpfA genes, the statistical analysis was done for each gene variant separately and for all three variants together. lpfA genes were statistically associated with diarrhea, both for lpfAO113 variants when analyzed separately and all lpfA variants analyzed together. The lpf family of genes has been reported to be involved in the adherence to epithelial cells. However, conflicting results regarding the function of different gene variants have been reported (14, 41, 55, 56). Recently, Tatsuno et al. could find no involvement of lpf genes in the adherence of EPEC (54). The apparent conflict between the results of the present study and those reported by Tatsuno et al. may be caused by differences in the sequences or the expression levels of the lpf genes in atypical EPEC strains and the reference strain used in that study. It is also possible that the significant result for the lpfA gene in the present study may be due to other factors present in the same bacterial strains. The results from this study of atypical EPEC strains collected in an epidemiological study should therefore be confirmed experimentally.

The paa gene has been found to correlate with the A/E phenotype of the swine pathogen PEPEC O45 but has so far not been associated with human disease (4). Based of the results of the current study, however, it appears that the paa gene may play a role in the virulence of atypical EPEC strains (Table 2).

Interestingly, for some of the genes tested in this study, the statistical association with diarrheal disease was negative (Table 2). Especially for the yjaA gene, the negative association was strong. This gene, which has no known function, has been used for the determination of the E. coli phylogenetic group (10). The fact that the yjaA gene may be a marker for an atypical EPEC phylogenetic group with low diarrheagenic potential may be the reason behind its negative association with diarrhea observed in this study. Another possibility could be that the protein encoded by the yjaA gene may confer protection against disease to the host, with the consequence of increased virulence in strains where it is absent. Such a negative association between a gene and virulence has been reported for other pathotypes of E. coli as well as for other bacterial species (35, 51). Another gene negatively associated with diarrhea in this study was the enteroaggregative E. coli toxin gene astA. This gene has previously been reported to be associated with diarrhea in subjects with an atypical EPEC strain (15). The reason behind the discrepancy between the results of these two studies is not clear but may be explained by different populations of atypical EPEC strains in the two geographic areas.

Different variants of the LEE genes have been associated with various characteristics of colonization and tissue trophism (57). Such differences might influence the ability of the bacteria to induce diarrhea. In this study, however, except for the borderline significance for the eae-gamma variant, none of the LEE gene variants were significantly associated with diarrhea. On the other hand, there appeared to be an association between the two virulence groups with respect to eae variants. Virulence group I strains contained mainly beta1 and gamma variants (23/27 strains), while alpha2 and beta2 variants of the eae gene predominated among virulence group II strains (21/29 strains). Based on these findings, it appears relevant to differentiate between the two beta variants, since they are found in different virulence groups.

The large number of virulence profiles among the strains in this study (Table 3) indicate that atypical EPEC is a highly diverse group of bacteria, a finding which is in agreement with what has been reported from other geographic areas (15, 60). Interestingly, the atypical EPEC strains of virulence group I with the OI-122 and lpfA genes were more diverse; there were few strains of each virulence profile, unlike with virulence group II strains, more than half of which belonged to one of two virulence profiles.

When studying the pathogenicity of atypical EPEC strains, it may be as relevant to study the virulence profiles of the strains as to analyze each gene separately. The combination of virulence factors may influence the pathogenic potential of the strains. The diarrheagenic potential of virulence groups I and II does not appear to depend on the quantity of bacteria in the intestine, at least as estimated from specimens taken several days after initial symptoms, as was done here (Table 4). Such an association might be expected since both the efa1/lifA- and the lpf-encoded proteins have been reported to influence initial events of infection (25, 31). On the other hand, distinct differences were observed in colonization and diarrhea when the two virulence groups were compared. Whereas the persistence of bacteria in stools was significantly more common for virulence group II than for group I bacteria, only the latter group was statistically associated with diarrheal disease. These results seem to be in contrast to those with EHEC O157, in which LPF (present in virulence group I strains in the present study) increased the persistence of bacteria in sheep and pigs (25). The result of this analysis, however, may be uncertain due to the high number of subjects without a recording of colonization at the 1-month follow-up.

Whether the virulence profiles and pathogenic potential of atypical EPEC strains observed in this study are comparable to those of other parts of Norway or other geographic areas of the world is not known. Recently, Dulguer et al. reported that there was no difference in the prevalences of the genes efa1/lifA, lpfDR141, and nleA/espI in atypical EPEC strains from children with and without diarrhea in Brazil (M. V. Dulguer, S. H. Fabbricotti, and I. C. A. Scaletsky, Abstr. 105th Gen. Meeting Am. Soc. Microbiol., abstr. D-102/195, 2005). The discrepancy between these findings and those of the present study might be explained by different populations of atypical EPEC strains, by different panoramas of other infectious agents in the two geographical regions, and possibly by differences in the child populations. It may also at least in part be caused by the use of different methods for gene detection. It will be of great interest to clarify if the genes associated with diarrhea as well as the virulence profiles observed in the present study are representative for atypical EPEC strains in other parts of the world.

In conclusion, by the use of a DNA microarray to simultaneously analyze a high number of putative virulence genes in atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Among these, the OI-122 gene efa1/lifA was most strongly associated with disease. Interestingly, the phylogenetic marker gene yjaA was negatively associated with diarrheal disease. Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 and lpfA negative, yjaA positive).


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ACKNOWLEDGMENTS
 
The work was in part supported by a grant M11217.11 to J.E.A. from SINTEF Health Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medical Microbiology, St. Olavs University Hospital, N-7006 Trondheim, Norway. Phone: (47) 725 73319. Fax: (47) 725 76417. E-mail: jan.afset{at}ntnu.no. Back

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/. Back


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Journal of Clinical Microbiology, October 2006, p. 3703-3711, Vol. 44, No. 10
0095-1137/06/$08.00+0     doi:10.1128/JCM.00429-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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