Received 3 September 2007/Returned for modification 26 October 2007/Accepted 30 April 2008

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1 JOURNAL OF CLINICAL MICROBIOLOGY, July 2008, p Vol. 46, No /08/$ doi: /jcm Copyright 2008, American Society for Microbiology. All Rights Reserved. Phylogenetic Backgrounds and Virulence Profiles of Atypical Enteropathogenic Escherichia coli Strains from a Case-Control Study Using Multilocus Sequence Typing and DNA Microarray Analysis Jan Egil Afset, 1,5 * Endre Anderssen, 2 Guillaume Bruant, Josée Harel, 3 Lothar Wieler, 4 and Kåre Bergh 1,5 Department of Laboratory Medicine, Children s and Women s Health, 1 and Department of Cancer Research and Molecular Medicine, 2 Faculty of Medicine, Norwegian University of Science and Technology, and Department of Medical Microbiology, St. Olavs University Hospital, 5 Trondheim, Norway; Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté demédecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C2, Canada 3 ; and Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, P.O. Box , D Berlin, Germany 4 Received 3 September 2007/Returned for modification 26 October 2007/Accepted 30 April 2008 Atypical enteropathogenetic Escherichia coli (EPEC) strains are frequently detected in children with diarrhea but are also a common finding in healthy children. The aim of this study was to compare the phylogenetic ancestry and virulence characteristics of atypical (eae positive, stx and bfpa negative) EPEC strains from Norwegian children with (n 37) or without (n 19) diarrhea and to search for an association between phylogenetic ancestry and diarrhea. The strains were classified in phylogenetic groups by phylogenetic marker genes and in sequence types (STs) by multilocus sequence typing. Phylogenetic ancestry was compared to virulence characteristics based on DNA microarray analysis. Serotyping and pulsed-field gel electrophoresis (PFGE) were also performed. All four phylogenetic groups, 26 different STs, and 20 different clonal groups were represented among the 56 atypical EPEC strains. The strains were separated into three clusters by overall virulence gene profile; one large cluster with A, B1, and D strains and two clusters with group B2 strains. There was considerable heterogeneity in the PFGE profiles and serotypes, and almost half of the strains were O nontypeable. The efa1/lifa gene, previously shown to be statistically linked with diarrhea in this strain collection (J. E. Afset et al., J. Clin. Microbiol. 44: , 2006), was present in 8 of 26 STs. The two phylogenetic groups B1 and D were weakly associated with diarrhea (P 0.06 and P 0.09, respectively). In contrast, group B2 was isolated most frequently from healthy controls (P 0.05). In conclusion, the atypical EPEC strains were heterogeneous both phylogenetically and by virulence profile. Phylogenetic ancestry was less useful as a predictor of diarrhea than were specific virulence genes. Escherichia coli is a commensal of the intestinal tract but also one of the most common causes of diarrhea in humans (34, 40). Several chromosomal and plasmid-borne virulence factors have been linked with the ability of certain E. coli strains to cause diarrhea. Based on the content of such factors, several different E. coli pathotypes have been defined (30). A key characteristic of two of these pathotypes, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), is a chromosomally located pathogenicity island named the locus of enterocyte effacement (LEE). Genes located on the LEE encode factors enabling the bacterium to adhere intimately to intestinal epithelial cells and to cause attaching-and-effacing (A/E) lesions (36, 37). EPEC is differentiated from EHEC by the possession of the latter of Shiga toxin genes and is further characterized as typical or atypical depending on whether or not it carries the virulence plasmid E. coli adherence factor (EAF) encoding bundle-forming pili (29). The E. coli species was subdivided into four phylogenetic * Corresponding author. Mailing address: Department of Medical Microbiology, St. Olavs Hospital, N-7006 Trondheim, Norway. Phone: (47) Fax: (47) jan.afset@ntnu.no. Published ahead of print on 7 May groups A, B1, B2, and D- based on multilocus enzyme electrophoresis (52). It has not been possible to establish reliable ancestral relationships between the phylogenetic groups due either to different speed of evolution among the groups or to frequent recombination (27, 61). Typical EPEC strains from different parts of the world have been shown to belong to four main clonal groups (33). In contrast, it has been argued that atypical EPEC strains may comprise a heterogeneous group of strains from different pathotypes that have acquired the LEE by horizontal transfer or are typical EPEC that have lost the EAF plasmid (58). Some atypical EPEC strains have shown a higher degree of genetic similarity to EHEC (O157:H7) than to typical EPEC (55). It has also been reported that strains classified as atypical EPEC may have very heterogeneous virulence profiles (2, 58). Whereas typical EPEC is well recognized as a leading cause of severe childhood diarrhea in low-income countries (39), the role of atypical EPEC as a diarrheagenic agent has been controversial. Atypical EPEC has been shown to be prevalent both in children with diarrhea and in healthy children (14, 16, 20, 23, 31, 38, 40 44, 47, 50, 57). However, several investigators have reported a statistical association between atypical EPEC and diarrhea (4, 15, 41, 49, 51, 58). 2280

2 VOL. 46, 2008 CHARACTERIZATION OF ATYPICAL EPEC 2281 In a recent case-control study from Norway, we found a high prevalence of atypical EPEC in children with or without diarrhea (1). When the strains were analyzed with respect to virulence gene content using DNA microarray and PCR, several genes were significantly associated with diarrhea (2). Among these, the association was highly significant for genes belonging to the pathogenicity island OI-122 (efa1/lifa, nleb, nlee, and set/ent), and for long polar fimbriae (LPF) when three variants of the lpfa gene were analyzed together. In contrast, the yjaa gene, which is often used as a phylogenetic marker, was negatively associated with diarrhea. This finding indicated a possible association between pathogenic potential of the atypical EPEC strains and their phylogenetic relationships. Multilocus sequence typing (MLST), by which a set of multiple housekeeping gene loci are compared using sequence analysis, is a powerful tool for long-term epidemiological and phylogenetic analysis (35, 56). In the present study we used MLST to characterize the phylogenetic relationships between the atypical EPEC strains from the above-mentioned casecontrol study. In addition, we classified the strains in phylogenetic groups using PCR and compared the phylogenetic ancestry of the strains with their virulence characteristics based on microarray analysis and with the results of serotyping and pulsed-field gel electrophoresis (PFGE). Finally, we searched for a possible association between phylogenetic ancestry and diarrhea. MATERIALS AND METHODS Bacterial strains. Atypical EPEC strains, defined as eae-positive E. coli without stx 1, stx 2, and bfpa genes, were isolated from fecal specimens from children younger than 5 years old in a case-control study conducted during the period from 2002 to 2003 in the county of Sør-Trøndelag, Norway (1). Thirty-seven of the strains were isolated from 251 children from whom a fecal specimen had been taken due to community-acquired diarrhea, and 19 strains were from 210 healthy children recruited at Maternal and Child Health Centers. Three eaepositive strains from the case-control study were classified as typical EPEC (bfpa positive, two strains) or EHEC (stx 2f positive, one strain) and were therefore not included in the present investigation. Fifteen strains, which had hybridized with the stxb 1 probe in DNA microarray analysis (2), were classified as atypical EPEC on basis of the following: they did not hybridize with the corresponding stxa 1 probe, they were negative in the PCR analysis with stxb 1 sequence-specific primers (5, 10), and they did not produce Shiga toxins (Premier EHEC; Meridian Bioscience) (unpublished results). We therefore concluded that the gene sequences detected by the stxb 1 hybridization probe did not represent a complete stxb gene. The 56 atypical EPEC isolates were stored at 70 C until further analysis. Phylogenetic group determination. Each atypical EPEC strain was assigned to one of the E. coli phylogenetic groups according to the presence or absence of the genes chua, yjaa, and tspe4c2 as proposed by Clermont et al. (13). MLST. For each strain the seven housekeeping genes adk, fumc, gyrb, icd, mdh, pura, and reca were amplified and sequenced according to the protocol of the Escherichia coli MLST database ( After overnight culture, bacterial DNA for PCR was obtained by heat lysis. Amplification was carried out with primers as previously published (61) in a total volume of 50 l with 50 M concentrations (each) of datp, dctp, dgtp, and dttp; 0.5 M concentrations of each primer (MedProbe, Oslo, Norway); 10 PCR buffer (Applied Biosystems, Branchburg, NL); 1.5 mm MgCl 2 ;1UofAmpliTaq Gold polymerase (Applied Biosystems); and 2 l of bacterial DNA extract as a template. The reaction conditions used were 15 min at 95 C, followed by 30 cycles of 1 min at 95 C, 1 min at the annealing temperature specified for each gene (see reference 61 or the E. coli MLST website), and a 2-min extension at 72 C, followed finally by 5 min at 72 C in a thermocycler, either an MJ Research PTC-200 (Bio-Rad, Hercules, CA) or a GeneAmp PCR System 9700 (Applied Biosystems). The PCR products were purified for sequencing with a QIAquick PCR purification kit (Qiagen, Valencia, CA). Both DNA strands were sequenced with the PCR primer set, with primers published by Tartof et al. (54), or with primers designed in the present study gyrb(trh)f, 5 -AAGTGATCATGACC GTTCTG-3 ; icd(trh)f, 5 -GATGGAATCGGTGTAGATGT-3 ; icd(trh)r 5 - GTAGCCCCAGTCTTTAAACG-3 ; and pura(trh)r, 5 -TGCTTGCAGAGG AACTCGC-3. Sequencing was performed by using either the CEQ DTCS- Quick Star kit (Beckman Coulter, Fullerton, CA) or a BigDye terminator cycle sequencing kit (v3.1; Applied Biosystems) with subsequent capillary electrophoresis, respectively, on a Beckman Coulter CEQ 8800 or an ABI 3130x genetic analyzer according to the protocol of the manufacturers. Sequence analysis. Raw sequence traces were reviewed by visual inspection using Sequencher software version 4.2 (Gene Code Corp.). Forward and reverse sequences were aligned, and consensus sequences corresponding to the allele templates were compared to known variants of the corresponding gene at the E. coli MLST database. Ambiguities in consensus sequences were resolved by resequencing. Each of the seven gene loci was assigned an allele number by submission of the sequences to the E. coli MLST database. Allelic sequences previously not reported were given new allele numbers by the curator of the database after independent review of sequence traces. Each isolate was assigned a sequence type (ST) according to its allelic profile. Phylogenetic analysis. For each bacterial strain the sequences from all seven gene loci were concatenated for phylogenetic analysis. Concatenated sequences were then aligned by using the CLUSTAL W algorithm of the MEGA3 software (32). A rooted neighbor-joining tree was constructed by using a Kimura twoparameter model of nucleotide substitution and the phylogenetically divergent E. coli strain Z205 from the E. coli MLST database as an outgroup (61). Tree stability was assessed by bootstrap analysis with 1,000 iterations. Using SplitsTree 4 software (26), phylogenetic network analysis was done with the neighbor-net algorithm and untransformed distances (p distance). The SplitsTree w recombination test was applied to concatenated sequences and for each of the seven gene loci individually to distinguish recombination from recurrent mutation. The phylogenetic relationships between different STs were analyzed by using the MSTree application of Bionumerics version 4.6 (Applied Maths, Sint-Martens-Latem, Belgium) to identify closely related genotypes. In this analysis atypical EPEC strains are compared based on similarity in allelic profiles. Strains of different STs sharing six of seven alleles were interpreted as belonging to the same clonal lineage but were assigned to an ST complex by the curator of E. coli MLST database only when that lineage included at least three different STs. The phylogenetic diversity of atypical EPEC strains isolated from children with diarrhea and healthy children was compared by using Simpson s index of diversity: D 1 [ n(n 1)/N(N 1)], where n is the number of subjects with atypical EPEC strains belonging to each ST, and N is the total number of subjects in each of the two groups (25, 53). Virulence gene profile. The atypical EPEC strains were compared with respect to virulence gene content based on the previously reported results of DNA microarray experiments and PCR (2). The microarray used in that study, derived from an E. coli virulence and antimicrobial resistance microarray (8), was composed of 70-mer oligonucleotide probes specific for 182 virulence genes or markers found in various intestinal and extraintestinal E. coli strains of all known pathotypes. The oligonucleotide microarray allowed the detection of genetic variants of the eae gene (17 variants), and three variants each of the espa, espb, and tir genes, all located on the LEE pathogenicity island. Oligonucleotides specific for three variants [lpfa(o113), lpfa1, and lpfa(r141)] of the LPFencoding gene lpfa were also included. The overall virulence profiles of the atypical EPEC strains were compared by principal-component analysis (R, version 2.6 [ of the 95 virulence genes detected in one or more of the strains (2). The strains were assigned to virulence clusters based on their distribution in the principal component analysis. PFGE. Macrorestriction analysis (PFGE) of chromosomal DNA was done using XbaI with the following electrophoretic conditions: 14 C, linear ramp of 5 to 60 s over 24 h, 120 switch angle, and a gradient of 6.0 V cm 1. Analysis was done by using Bionumerics software. Similarities of fragments between strains were compared by using a Dice coefficient at 1.0% tolerance and 0.5% optimization, and a dendrogram was constructed with the UPGMA (for unweighted pair-group method with arithmetic averages) clustering method. Significant clusters were determined by calculating the cutoff value that produced the highest point-bisectional correlation (Bionumerics manual, version 4.6). Serotyping. Serotyping of somatic (O) antigens (serogroups O1 to O177) and flagellar (H) antigens was done by using standard methods (24) at the Escherichia, Shigella, Yersinia, and Vibrio Reference Unit, Laboratory for Enteric Pathogens at the Health Protection Agency (United Kingdom). Statistical analyses. Fisher s exact test was used for the statistical analyses of categorical variables, and the Mann-Whitney U-test was applied to the analysis of differences between quantitative data which were not normally distributed. P values 0.05 were considered significant.

3 2282 AFSET ET AL. J. CLIN. MICROBIOL. TABLE 1. Comparison of phylogenetic classification, virulence profile, serotype, PFGE clustering, and clinical information for each of the 56 atypical EPEC strains identified in a case-control study among children 5 years old in Norway Strain ID Phylogenetic classification Phylogenetic group ST ST cplx a Combinations of LEE genes b OI-122 (efa1/lifa) c Virulence profile lpfa genes d EHEC-related gene variants e Virulence cluster f Serotype g PFGE cluster h Diarrhea Trh2 B G B2-A O?:H? I Trh3 B G B2-A O?:H? I Trh4 B G B2-A O?:H? II Trh5 B A B2-B O?:H? IV Trh7 A D A-B1-D O145:H40 Trh8 B A B2-B O?:H? IV Trh9 B A B2-B O132:H34 III Trh10 A D A-B1-D O51:H10 Trh11 A E A-B1-D O157:H16 IV Trh12 A B ( ) A-B1-D O117:H40 IV Trh13 B D A-B1-D O51:H IV Trh14 B A B2-B O125ac:H? IV Trh15 B A B2-B O132:H34 III Trh16 A E A-B1-D O157:H16 IV Trh17 B G B2-A O139:H? I Trh18 B G B2-A O?:H? I Trh19 B A B2-B O132:H34 III Trh20 B B ( ) A-B1-D O?:H21 IV Trh21 B A B2-B O132:H34 III Trh22 B A B2-B O132:H34 III Trh23 B D A-B1-D O?:H21 IV Trh24 B D A-B1-D O45:H IV Trh25 A 342 D A-B1-D O?:H51 IV Trh26 D 586 D A-B1-D O?:H51 IV Trh27 B G B2-A O?:H? I Trh28 A 342 D A-B1-D O177:H IV Trh29 A 342 D A-B1-D O177:H25 IV Trh30 D K A-B1-D O145:H III Trh31 D J A-B1-D O157:H III Trh32 B1 588 B ( ) A-B1-D O?:H IV Trh33 A H A-B1-D O?:H? II Trh34 B2 589 F B2-B O51:H49 IV Trh35 B A B2-B O132:H34 III Trh36 B A B2-B O136:H? IV Trh37 A 342 D A-B1-D O177:H? IV Trh38 B D A-B1-D O?:H11 IV Trh39 B1 327 B A-B1-D O?:H8 I Trh40 B I A-B1-D O?:H8 IV Trh41 D K A-B1-D O55:H7 V Trh42 B2 35 * C B2-B O145:H34 III Trh43 B1 327 B A-B1-D O?:H8 I Trh44 B B ( ) A-B1-D O?:H27 IV Trh45 B D A-B1-D O51:H IV Trh46 B2 526 * C B2-B O145:H25 III Trh47 B1 333 B ( ) A-B1-D O?:H IV Trh48 B1 154 E A-B1-D O152:H38 IV Trh49 B G B2-A O133:H38 I Trh50 B G B2-A O?:H? I Trh51 B A B2-B Orough:H? IV Trh52 B G B2-A O?:H7 I Trh53 D J A-B1-D O55:H7 V Trh54 B1 442 B ( ) A-B1-D O146:H27 IV Trh55 B A B2-B O?:H40 III Trh56 B1 327 B A-B1-D O?:H8 IV Trh57 B1 327 B A-B1-D O?:H8 IV Trh58 D J A-B1-D O55:H7 V a ST cplx, ST complex, based on the identity in at least six of the seven gene loci analyzed, between strains of at least three different STs. *, STs with identity in six of seven gene loci but which have not been assigned to an ST complex at the E. coli MLST database. b Classified by the combination of the variants of the genes espa, espb, tir, and eae detected in each strain. See Table 3 for details. c Based, in addition to the efa1/lifa gene, on the genes nleb, nlee, and set/ent. d Based on the gene variants lpfa1, lpfa(o113), and lpfa(r141). e Classified by the EHEC plasmid genes katp, espp, etpd, ehxa, and L7095 and the chromosomal ured gene. f Based on principal component analysis of 94 putative virulence genes or gene variants present in one or more of the strains. g O?, O serogroup not identified; H?, motile, but H-type not identified. h PFGE clusters were defined by the cluster cutoff method of the Bionumerics software., Two ST10 strains were not typeable with the method used.

4 VOL. 46, 2008 CHARACTERIZATION OF ATYPICAL EPEC 2283 Downloaded from FIG. 1. Phylogenetic relationships between 56 atypical EPEC strains from Norwegian children. A rooted phylogenetic tree was constructed by the neighbor-joining algorithm based on the Kimura two-parameter model of nucleotide substitution. The ST with the number of isolates (in brackets) is given at each branch tip. Bootstrap values greater than 50% based on 1,000 replications are shown at the internal nodes. Clonally related STs are shown by shaded boxes. The classification in phylogenetic groups, distribution of LEE gene variants, the presence of OI-122, lpfa, and EHEC-related genes, and the sources of the strains are shown for each ST. For combinations of LEE gene variants, see Table 3. RESULTS Phylogenetic group determination. Ten of the atypical EPEC strains belonged to phylogenetic group A, 16 strains belonged to group B1, 24 strains belonged to group B2, and 6 strains belonged to group D when classified according to the scheme proposed by Clermont et al. (13) (Table 1). MLST analysis. Sequencing of each of the seven MLST gene loci generated acceptable tracings in all 56 atypical EPEC strains tested. Seven new alleles were detected, and altogether 26 different STs were identified (Table 1). Fifteen strains with allele profiles not previously reported were assigned to 10 new STs. Phylogenetic analysis of concatenated sequences of the seven MLST loci showed that STs within the phylogenetic group B2 were grouped together and were clearly distinct from STs of the three other phylogenetic groups (Fig. 1). SplitsTree analysis revealed several parallel paths indicating phylogenetic incompatibility in the divergence of atypical EPEC clones (Fig. 2). By using the w test, statistical significant evidence of recombination was demonstrated (P ). When each gene was analyzed separately, evidence for recombination was found for the genes fumc (P 0.002) and gyrb (P 0.025) but not for the five other genes. Ten STs had at least six alleles similar to other STs in the study. Eight of these STs belonged to three different ST complexes, while two STs (ST35 and ST526) were not assigned to an ST complex due to the lack of a third closely related ST (Table 1 and Fig. 1). Clonal complex 582 (ST582, ST584, and ST585) has not been described previously. Altogether, 20 different clonal lineages were represented among the 56 atypical EPEC strains in the study. Phylogenetic background compared to virulence characteristics. The atypical EPEC strains were assigned to three different clusters by principal-component analysis of the 94 virulence genes or markers identified in the study (Fig. 3). Two of the clusters were highly compact, indicating a high degree of similarity in virulence profiles between the strains. Both of these clusters consisted exclusively of phylogenetic group B2 strains and were labeled clusters B2-A and B2-B. The third cluster was much wider, indicating more heterogeneity in vir- on August 19, 2018 by guest

5 2284 AFSET ET AL. J. CLIN. MICROBIOL. FIG. 2. Phylogenetic relationships between 56 atypical EPEC strains from Norwegian children are presented as a phylogenetic splits network based on the neighbor-net algorithm using a p distance matrix. STs are indicated at the branch tips. The sources of the strains are listed in parentheses (patients/controls). STs belonging to the same phylogenetic group are enclosed by an ellipse. ulence genes between these strains. This cluster included all of the phylogenetic group A, B1, and D strains and was accordingly labeled cluster A-B1-D. Phylogenetic group B and especially group A strains were widely dispersed within this cluster. Phylogenetic group D strains, in contrast, were located at the periphery and may represent a separate cluster. Thirty of the genes or gene variants observed in at least five bacterial strains were restricted to strains of either phylogenetic group B2 or one or more of the phylogenetic groups A, B1, and D (Table 2). Among genes present only in phylogenetic group B2 strains, all but one gene were observed in only one of the B2 virulence clusters, either B2-A or B2-B. In contrast, 18 of the 23 genes detected exclusively in strains belonging to the virulence cluster A-B1-D were present in more than one of the three phylogenetic groups of this cluster. Four genes were found only in strains belonging to phylogenetic group D, and one gene variant [lpfa(r141)] was detected only in phylogenetic group B1 strains. The atypical EPEC strains were also analyzed with respect to their total content of the virulence genes and markers analyzed in the study. There were considerable differences in the number of virulence genes between the strains, ranging from 22 of 46 genes per strain (median, 29 genes). Strains belonging to phylogenetic group D contained significantly more virulence genes (median, 37 genes; range, 34 to 42 genes) than strains belonging to the phylogenetic groups A, B1, and B2 (P 0.02, 0.01, and 0.001, respectively). The differences between the three other groups were not significant. Ten combinations of different variants of the LEE genes espa, espb, tir, and eae were present among the 56 atypical EPEC strains (Table 3). Each of the four phylogenetic groups, and even some of the STs, included strains with different combinations of LEE genes (Fig. 1). Some of these combinations were observed only within one phylogenetic group, but two of the most frequent combinations were detected in strains from more than one group. Although most of the eae variants were associated with only one specific combination of the espa, espb, and tir gene variants, the gamma and the iota variants were linked with two different combinations of the other LEE genes (Table 3). FIG. 3. Principal component analysis of the distribution of 94 putative virulence genes or gene variants in 56 atypical EPEC strains isolated from Norwegian children. Scores (A) and loadings (B) for the first two principal components; the phylogenetic group of each strain is indicated by the design of the marker, while the color shows whether the strain was isolated from children with (red) or without (blue) diarrhea. The loadings show genes previously shown to be statistically linked with diarrhea in red (2).

6 VOL. 46, 2008 CHARACTERIZATION OF ATYPICAL EPEC 2285 TABLE 2. Genes or gene variants restricted to one virulence cluster or phylogenetic group in 56 atypical EPEC strains Gene or gene variant a Function No. of strains (cases/controls) No. of strains in each phylogenetic group/virulence cluster b B2-A* B2-B* A B1 D agn43 Antigen 43 precursor 8/ cdtb-1 Cytolethal distending toxin type I, subunit B 3/4 7 cdtb-4 Cytolethal distending toxin type IV, subunit B 2/3 5 eae-alpha2 Intimin, alpha2 variant, LEE encoded 5/7 12 eae-beta Intimin, beta variant, LEE encoded, 9/ eae-beta2 Intimin, beta2 variant, LEE encoded 4/5 9 eae-gamma Intimin, gamma variant, LEE encoded 11/ efa1/lifa EHEC factor for adherence/lymphocyte activation inhibitor (OI-122 gene) 17/ ehxa EHEC hemolysin gene 8/ espa-2 Variant of the EspA protein secreted by the type III secretion system of the LEE, 5/0 5 involved in the formation of surface appendages espb-1 Variant of the EspB protein secreted by type III secretion system of the LEE 5/0 5 espb-3 Variant of the EspB protein secreted by type III secretion system of the LEE 10/ espc Enterotoxin EspC 8/7 15 etpd Type II secretion pathway, plasmid po157 (etp: EHEC type II pathway EtpD) 6/ flma54 E. coli flagellin, major subunit, flic variant 12/ ibea Invasion protein, E. coli invasion of the blood-brain barrier 11/ lpfa(o113) Variant of the major fimbrial subunit of LPF 12/ lpfa1 Variant of the major fimbrial subunit of LPF 5/0 5 lpfa(r141) Variant of the major fimbrial subunit of LPF 6/1 7 malx Maltose- and glucose-specific IIABC component, pathogenicity island associated 4/5 9 nlea/espi Non-LEE encoded effector A (type III secreted effector) 19/ nleb Non-LEE encoded effector B (type III secreted effector) (OI-122 gene) 21/ nlee Non-LEE encoded effector E (type III secreted effector) (OI-122 gene) 21/ rtx Putative RTX family exoprotein 6/0 6 set/ent Probable enterotoxin, similar to ShET2 enterotoxin (S. flexneri; OI-122 gene) 21/ stxb 1 Shiga-like toxin I, subunit B 10/ tir-1 Variant of the LEE-encoded translocated intimin receptor 10/ tlra Tellurium resistance protein A 7/ tsh Temperature-sensitive hemagglutinin of avian E. coli, autotransporter 6/0 5 1 ured Urease-associated protein UreD 7/ usp Uropathogenic specific protein (putative virulence island of UPEC) 4/5 9 a Only genes present in at least five strains are included in this analysis. b *, Strains belonging to phylogenetic group B2 are separated into two clusters (B2-A and B2-B) by principal component analysis of virulence genes (see Fig. 3);, strains belonging to the phylogenetic groups A, B1, and D are located in the same cluster (A-B1-D) based on principal component analysis of virulence genes (see Fig. 3). Genes located on the pathogenicity island OI-122 (nleb, nlee, set/ent, and efa1/lifa) were detected in strains belonging to 12 different STs (Fig. 1). Such genes were present in strains belonging to the three phylogenetic groups A, B1, and D but not group B2. The presence of the OI-122 genes was associated with four different combinations of LEE gene variants (Table 3). Incomplete OI-122s lacking the efa1/lifa gene always corresponded with one specific combination of LEE gene variants. The three variants of the lpfa gene were detected in 14 different STs within the phylogenetic groups A, B1, and D (Fig. 1). Genes linked to the EHEC pathotype, the po157 plasmid genes katp, espp, etpd, ehxa, and L7095 and the EHEC urease gene ured were detected in 14 (25%) strains. These genes were present in strains of nine different STs within the phylogenetic groups A, B1, and D (Fig. 1). Eight different combinations between the EHEC-related genes were observed (data not shown). The 15 strains that contained an incomplete stxb 1 sequence belonged to seven different STs within the phylogenetic groups A, B1, and D. PFGE. When the 56 atypical EPEC strains were analyzed by PFGE, all but two ST10 strains were typeable. Among the 54 typeable strains, the majority displayed unique genotypic patterns (Fig. 4). Except for two strains with identical PFGE restriction patterns isolated from siblings, there was no known epidemiological connection between other strains with closely related PFGE profiles. Five distinct clusters were identified by using the cluster cutoff method, while 42 different clusters identified if a similarity cutoff value of 90% was applied. Serotypes. An O serogroup was identified in 31 (55.3%) of the 56 atypical EPEC strains (Table 1). The remaining strains were either nontypeable with the O antisera used (24 strains) or rough (1 strain). Four strains belonged to classical EPEC serogroups (39). The other typeable strains belonged to 12 different serogroups. Five O serogroups were detected in more than one strain; O132 (six strains), O145 (four strains), O55 and O157 (three strains each), and O177 (two strains). Eleven different H-types were detected among the atypical EPEC strains. Most common were the flagellar types H34 (seven strains), H8 (five strains), and H7 (four strains). Seven strains were nonmotile (H ), and the H-type was not identified in 14 strains. Phylogenetic ancestry and diarrhea. An association between phylogenetic decent and diarrhea could indicate the presence of virulence traits linked to phylogenetic ancestry. In the

7 2286 AFSET ET AL. J. CLIN. MICROBIOL. TABLE 3. Distribution of different variants of four LEE genes compared to phylogenetic group and the presence of OI-122 genes in 56 atypical EPEC strains isolated from children 5 years old from Norway with or without diarrhea a Combination of LEE genes LEE gene variants b present study none of the phylogenetic groups A, B1, and D were significantly associated with diarrhea (Table 4). However, a trend toward significance was observed for the groups B1 (P 0.06) and D (P 0.09). In contrast, strains belonging to phylogenetic group B2 were more common in healthy controls than in children with diarrhea. Phylogenetic group A strains were equally common in patients and controls. Among STs that included three or more strains, three STs were detected only in children with diarrhea, one ST was observed only in healthy children, and four STs were present in strains from both healthy and ill children (Fig. 1). The STs that were detected exclusively in strains from children with diarrhea all contained the virulence gene efa1/lifa gene. There was a higher degree of diversity in phylogenetic ancestry between strains isolated from children with diarrhea than in strains from healthy children. All but one of the 20 different clonal lineages detected in the study were present among the 37 strains from symptomatic children (Simpson s diversity index of 0.95). In comparison, no more than six clonal lineages were present among the 19 strains from healthy children (Simpson s diversity index of 0.83). DISCUSSION In this study we show that atypical EPEC strains from Norwegian children with or without diarrhea are very heterogeneous with respect to phylogenetic ancestry. The 56 strains belonged to all four phylogenetic groups and to 26 different STs within 20 clonal groups (Table 1 and Fig. 1). This finding of phylogenetic heterogeneity is consistent with analyses from the E. coli MLST database, where EPEC strains, probably mainly atypical EPEC (unpublished data), were found within many different STs and clonal complexes (61). In contrast, Lacher et al. have shown that typical EPEC strains are more homogeneous and mainly belong to four clonal groups (33). However, both in the typical EPEC strains in that study and the atypical strains in the present study there was significant evidence of recombination. Comparison between phylogenetic background and virulence characteristics has usually been done only for a limited number of virulence factors (15, 22, 42, 45, 48, 58) or for strains No. of strains (cases/controls) Phylogenetic group c No. of strains with OI-122 genes (efa1/lifa) A espa1 espb2 tir-2 eae-alpha2 5/7 B2 B espa1 espb2 tir-3 eae-gamma 8/2 B1 (9), A (1) 10 (4) C espa1 espb2 tir-3 eae-iota 2/0 B2 D espa1 espb3 tir-1 eae-beta 9/4 D (1), B1 (5), A (6) 8 (8) E espa1 espb3 tir-1 eae-epsilon 1/2 B1 F espa3 espb2 tir-3 eae-alpha 1/0 B2 G espa3 espb2 tir-3 eae-beta2 4/5 B2 H espa3 espb2 tir-3 eae-delta 1/0 A I espa3 espb2 tir-3 eae-iota 1/0 B1 J espa1/2 espb1 tir-2 eae-gamma 3/0 D 3 (3) K espa1/2 espb1 tir-2 eae nt 2/0 D 2 (2) a For hybridization probe sequences, see reference 2. b For descriptions of the genes, see Table 2. nt, nontypeable. c In cases of more than one group, the number of results for each group is given in parentheses. belonging to specific EPEC serotypes (6). The use of data from DNA microarray experiments in the present study permitted an extensive characterization of the atypical EPEC strains with respect to overall virulence gene content, as well as a comparison between virulence profile and phylogenetic ancestry (Fig. 3). The main division between group B2 strains and the three other groups A, B1, and D observed in the phylogenetic analysis (Fig. 1) was supported by differences in virulence profiles (Fig. 3). There was also agreement between the two different types of analyses in the separation of phylogenetic group B2 strains in two clusters, while phylogenetic group A and B1 strains were not reliably differentiated by any of the two methods. Group D strains, on the other hand, were narrowly scattered at the periphery of this cluster (Fig. 3) and were also shown to contain significantly more virulence genes than strains belonging to the other virulence groups. The link observed in the present study between phylogenetic ancestry and virulence profile (Table 2 and Fig. 3) may be explained by the requirement of a specific genetic background for the acquisition of certain virulence factors (17). One possible reason for poor separation between strains belonging to different groups in the phylogenetic analysis might be the presence of hybrid strains carrying ancestry from more than one source (61). Such hybrid strains were not identified by the PCR method used for the analysis of phylogenetic groups in the present study. However, at least four of the STs identified in the study (ST 28, 32, 154, and 206) were previously observed to contain phylogenetic ancestry from more than one source (61). Typing of LEE genes may be of importance in the characterization of A/E pathogens since different variants of these genes have been associated with tropism to different locations in the human intestine (19, 46) and may be used for epidemiological characterization of LEE-containing strains. The LEE genes espa, espb, tir, and eae are all genes that may be exposed to high selection pressure since they encode adhesins and effector proteins that interact directly with the host. In the present study 11 different combinations of these four LEE gene variants were identified. In contrast to previous reports (12, 21), several of the espa, espb, tir, and eae variants could be

8 FIG. 4. PFGE cluster analysis compared to phylogenetic ancestry, virulence profile, phylogenetic group, and source of atypical EPEC strains from Norwegian children with or without diarrhea. Clusters identified by the cluster cutoff method (see the text) are visualized by dense lines in the dendrogram. Two strains (Trh7 and Trh10) were not typeable by the PFGE method used. The STs 35 and 526 share six of seven alleles but have not been assigned to a ST complex. See Table 1 for more information. 2287

9 2288 AFSET ET AL. J. CLIN. MICROBIOL. TABLE 4. Distribution in phylogenetic groups of 56 atypical EPEC strains from children with or without diarrhea with respect to the source of the strains Phylogenetic group Patients (n 37) No. of strains Controls (n 19) A B B D observed in combination with more than just one variant of the other LEE genes. This finding is consistent, as recently suggested, with horizontal exchange between different strains not only of entire LEE sequences but also of smaller gene elements within the LEE (11). The link between certain combinations of LEE gene variants and OI-122 genes shown in the present study (Table 3) may be due to close proximity of the genomic islands in the chromosome of these atypical EPEC strains, a finding similar to what has been shown for O103:H2 EHEC strains (28). The finding of genes usually linked to the EHEC pathotype in a considerable proportion of the atypical EPEC strains (Table 1) is consistent with evidence from epidemiological and experimental studies showing that atypical EPEC may convert to, or be a conversion from, the EHEC pathotype through the acquisition or loss of stx genes (7, 58, 61). Such a relationship is further supported by the presence of STs belonging to the phylogenetic lineages EHEC1 (ST335 and ST587) and EHEC2 (ST29) (59; among the atypical EPEC strains. The variability in the content of plasmid genes between different strains is in agreement with reports of extensive heterogeneity of large plasmids in STEC (9) and A/E E. coli of animal origin (3). The main impression from the results of the PFGE analysis is that of extensive heterogeneity between the atypical EPEC strains in the study. Differences in the PFGE banding pattern of several fragments were observed even between strains belonging to the same clonal lineage or virulence group (Fig. 4). The PFGE results also confirm a pattern of endemic infection among children during the study period and demonstrate, as expected, that the PFGE method has greater discriminatory power than MLST in differentiating between epidemiologically unrelated atypical EPEC strains. The finding that an O serogroup was identified in less than half of the strains in the study (Table 1) supports the view that O serogrouping is not useful in the diagnosis of atypical EPEC infections, at least with endemic atypical EPEC strains as reported here. This view is also supported by the observation that OI-122-positive atypical EPEC strains, which we have previously shown to be significantly associated with diarrhea (2), belonged to many different serogroups or were nontypeable (Table 1). Phylogenetic ancestry was a less useful indicator of diarrheagenic potential in this collection of atypical EPEC strains (Table 4) than specific virulence genes reported previously (2). This is most likely explained by the considerable heterogeneity in virulence factors within each of the phylogenetic groups P (Fig. 3 and Table 2). The OI-122 gene efa1/lifa most strongly associated with diarrhea in our previous study was present only in some of the strains within the phylogenetic groups A and B1 (Fig. 1). On the other hand, the negative statistical association with diarrhea shown for the phylogenetic group B2 seems to indicate a link between phylogenetic descent and lack of diarrheagenic potential. The lack of significant association observed between phylogenetic groups and diarrhea in the present study may in part be explained by the limited number of strains analyzed. In addition, the high number of STs among the strains made a statistical analysis for the relationship with diarrhea for each ST impossible. A considerably larger study than the present will be needed to clarify this issue further. However, the finding that some STs, present in more than two strains, were represented in only one of the groups of children (patients or controls) may indicate a difference in virulence potential between these STs (Fig. 1). Interestingly, the three STs that were detected exclusively in strains from children with diarrhea all contained the efa1/lifa gene. A limitation of the comparison between phylogeny and virulence genes used in the present study is that only a small part of the genome of each strain was included in the analysis. It is therefore possible that as-yet-unrecognized virulence genes were missed. An alternative could be to do a comparison at the whole-genome level, for instance, by comparative genomic (18) or subtractive (60) hybridization. However, the strength of the present study is the analysis of virulence genes of all pathotypes. In contrast, an analysis at the whole-genome level is usually based on hybridization of the test strain against a sequenced reference strain. In such an analysis, genes which are not present in the genome of the reference strains will not be detected. In conclusion, we have shown that atypical EPEC strains from Norwegian children with or without diarrhea belonged to all four phylogenetic groups, to 26 different STs, and to 20 different clonal groups. The strains were separated into three clusters by overall virulence gene profile. One large cluster included all phylogenetic group A, B1, and D strains, and two clusters consisted exclusively of strains belonging to group B2. Almost one-third of the virulence genes were detected in only one virulence cluster or phylogenetic group. There was considerable heterogeneity in PFGE profiles and serotypes, and almost half of the strains were O nontypeable. The efa1/lifa and other OI-122 genes were detected in many different STs, but only within the phylogenetic groups A, B1, and D, and were linked with certain combinations of LEE gene variants. EHEC pathotype-related genes were present in one-fourth of the strains. There was borderline significant association with diarrhea for the phylogenetic groups B1 and D, but phylogenetic ancestry was less useful as a predictor of diarrhea than the specific virulence genes shown previously. ACKNOWLEDGMENTS We thank Kirsti Løseth, Hilde Lysvand, Anne Nor, Sidsel Krokhaug, and Torunn Rønning for excellent technical assistance and Mark Achtman, curator of the Escherichia coli MLST database, for updated information on ST complexes. J.E.A. was supported by a Ph.D. grant from the Central Norway Regional Health Authority.

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