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Journal of Clinical Microbiology, January 2003, p. 218-226, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.218-226.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Extended Virulence Genotype of Pathogenic Escherichia coli Isolates Carrying the afa-8 Operon: Evidence of Similarities between Isolates from Humans and Animals with Extraintestinal Infections

Jean Pierre Girardeau,1* Lila Lalioui,2 A. Mohamed Ou Said,1 Christophe De Champs,3 and Chantal Le Bouguénec2

Laboratoire de Microbiologie, Centre de Recherche, INRA de Clermont-Ferrand-Theix, 63122 St Genès Champanelle,1 Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, 75724 Paris Cedex 15,2 Laboratoire de Bactériologie, Faculté de Médecine, 63001 Clermont-Ferrand Cedex, France3

Received 6 May 2002/ Returned for modification 18 June 2002/ Accepted 24 October 2002


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ABSTRACT
 
The afimbrial AfaE-VIII adhesin is common among Escherichia coli isolates from calves with intestinal and/or extraintestinal infections and from humans with sepsis or pyelonephritis. The virulence genotypes of 77 Escherichia coli afa-8 isolates from farm animals and humans were compared to determine whether any trait of commonality exists between isolates of the different host species. Over half of the extraintestinal afa-8 isolates were associated with pap and f17Ac adhesin genes and contained virulence genes (pap, hly, and cnf1) which are characteristic of human extraintestinal pathogenic E. coli (ExPEC). PapG, which occurs as three known variants (variants I to III), is encoded by the corresponding three alleles of papG. Among the pap-positive strains, new papG variants (papGrs) that differed from the isolates with genes for the three adhesin classes predominated over isolates with papG allele III, which in turn were more prevalent than those with allele II. The data showed the substantial prevalence of the enteroaggregative E. coli heat-stable enterotoxin gene (east1) among afa-8 isolates. Most of the afa-8 isolates harbored the high-pathogenicity island (HPI) present in pathogenic Yersinia; however, two-thirds of the HPI-positive strains shared a truncated HPI integrase gene. The presence of ExPEC-associated virulence factors (VFs) in extraintestinal isolates that carry genes typical of enteric strains and that express O antigens associated with intestinal E. coli is consistent with transfer of VFs and O-antigen determinants between ExPEC and enteric strains. The similarities between animal and human ExPEC strains support the hypothesis of overlapping populations, with members of certain clones or clonal groups including animal and human strains. The presence of multiple-antibiotic-resistant bovine afa-8 strains among such clones may represent a potential public health risk.


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INTRODUCTION
 
The fimbrial and afimbrial adhesins of the Afa family mediate the adherence of uropathogenic and diarrhea-associated Escherichia coli to various host tissues. Among the Afa-related surface antigens, the Afa(Dr+) adhesins (AfaE-I, AfaE-III, Dr, and F1845) bind to epithelial cells via recognition of the decay-accelerating factor (DAF; also referred to as CD55) carrying the Dr blood group antigen (15, 33, 36, 37, 44). We recently described a new afa operon (afa-8) encoding afimbrial adhesin AfaE-VIII which does not recognize DAF molecules (Afa(Dr-) adhesin). The afa-8 operon can be either chromosome or plasmid borne, suggesting that it may be carried by a mobile element, facilitating its dissemination (16, 34). It therefore appears that in some strains the afa-8 operon is carried by a 61-kb pathogenicity island (PAI) inserted into pheV and/or pheR tRNA-encoding genes. Partial characterization of the afa-8-containing PAI indicated that this PAI carries the afa-8 operon as the only known virulence determinant (35).

The afa-8 operon is common among pathogenic E. coli strains isolated from animals and humans (16, 34, 52). It was frequently found in animal and human isolates producing CNF toxins, but it has also been detected in CNF-negative strains isolated from calves and piglets (16, 41). Bloodstream infections in which the bacteria are derived from the intestinal flora by bacterial translocation are common in patients with cancer. Preliminary results showed that the afa-8 operon is found in CNF1-producing strains associated with this type of bacteremia (22, 38, 42). Therefore, afa-8 is probably involved in the development of extraintestinal infections associated with primary colonization of the intestine. However, afa-8 has never been detected in diarrhea-associated human isolates (38). In addition to CNF1, certain afa-8-positive strains carry virulence factor (VF) genes including pap, sfa, f17A, and clpG (P, S, F17, and CS31A adhesins, respectively) and hlyA (hemolysin), which are frequently detected in extraintestinal pathogenic E. coli (ExPEC) (1, 17, 36, 41, 42).

ExPEC strains are the major cause of neonatal meningitis, gram-negative organism-associated bacteremia, pyelonephritis, cystitis, and prostatitis. Most ExPEC strains are derived from phylogenetic group B2 (and, to a lesser extent, from group D) and belong to restricted O serogroups (serogroups O1, O2, O4, O6, O18, O75, and O83) (25, 26, 47). The virulence potential of ExPEC is determined largely by the presence of specialized VFs, which can be located on large plasmids or on the chromosomes of pathogenic strains. Recognized VFs of ExPEC, which are infrequent among commensal strains, include adhesins (P, S, and F1C fimbriae and Afa-related adhesins), toxins (hemolysin and cytotoxic-necrotizing factor 1), siderophores (aerobactin and yersiniabactin), capsules (K1, K5, and K12), invasins (Ibe10 and afaD genes), and factors contributing to serum resistance (5, 22, 28, 55). Most VF genes are located on PAIs, which are large segments of chromosomal DNA inserted within or near tRNA genes. These PAIs may provide a mechanism of horizontal transfer of genes between lineages within E. coli and even between species (6, 7, 19, 20). A high prevalence of VFs typical of human ExPEC among animal isolates allows one to speculate about cross-infection between different host species (40, 42). This idea was supported by phylogenetic studies, which have demonstrated clonal overlap between pathogenic isolates from humans and animals (poultry, calves, piglets, and dogs) belonging to serogroups O4, O6, and O78 that contain CNF1, hemolysin, and other putative VFs (9, 10, 25, 29, 30).

In the present study, to determine whether any trait of commonality exists between animal and human afa-8 isolates, 32 human and 45 animal pathogenic isolates were characterized with respect to the prevalence of a broad range of VFs. We also compared the susceptibilities of the isolates to antimicrobial agents in relation to the epidemiological source.


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MATERIALS AND METHODS
 
Bacterial isolates. The population studied included 77 E. coli isolates from humans (n = 32) with bacteremia or urinary tract infections (UTIs; with or without extraurinary tract involvement) and from calves (n = 42) and piglets (n = 3) with intestinal or extraintestinal colibacillosis. The human, bovine, and porcine isolates were partially characterized previously (13, 22, 42). All the strains carried sequences of the afa-8 operon, as evidenced by detection of the afaD8 and afaE8 genes by PCR or colony hybridization assays as described in a previous report (38).

Human isolates included afa-8 strains of different origins. Fourteen E. coli strains were isolated between 1992 and 1993 from the blood of cancer patients who were hospitalized with bacteremia at the Institut Gustave Roussy (a French cancer reference center). This collection was obtained from A. Andremont (Groupe Hospitalier Bichat-Claude Bernard, Paris, France). Fifteen strains were isolated from the urine of patients with pyelonephritis who were admitted to five different hospitals over the period from 1985 to 1989. This collection was obtained from M. Archambaud (Centre Hospitalier Universitaire Rangueil, Toulouse, France). Three afa-8 strains isolated in 1999 from patients hospitalized with UTIs were kindly provided by F. Bourlioux (EPS Perray-Vaucluse, Epinay sur Orge, France).

Forty-two E. coli strains obtained from intestinal or extraintestinal sites in calves with signs and lesions typical of septicemia (n = 16) or enteritis (n = 26) in European countries (Belgium, France, Spain) were also studied. All these strains were obtained from J. G. Mainil (Faculté de Médecine Vétérinaire, Université de Liège, Liège, Belgium) and from M. Contrepois (Laboratoire de Microbiologie, Centre de Clermont-Ferrand/Theix, Institut National de la Recherche Agronomique, St Genès Champanelle, France). Three strains isolated from extraintestinal sites of piglets with septicemia were provided by J. Fairbrother (Faculty of Veterinary Medicine, University of Montreal, Montreal, Quebec, Canada).

Uropathogenic E. coli strain J96 was used as a reference strain for papG alleles I and III, and IA2 was used as a reference strain for papG allele II and for other VF genes (papAH, papC, papEF, sfa/foc, hlyA, cnf1). CFT073 was used as a reference strain for PAI ICFT073 markers malX, cysB, and modB. Bovine septicemia-associated strain 31A was used as a reference strain for the f17Ac (also called 20K or gafD), papGrs (a new papG variant), clpG (major subunit of the CS31A adhesin), east1 (enteroaggregative E. coli heat-stable enterotoxin 1), and iutA (aerobactin) genes.

Bacterial cultures, DNA extraction, and serotyping. E. coli strains were grown in Luria-Bertani broth at 37°C overnight. Genomic DNA was extracted by rapid lysis, as described previously (3), and stored at -20°C for further analysis.

Fifty-two strains were O serotyped by the International Escherichia and Klebsiella Center, Statens Serum Institut, Copenhagen, Denmark.

Genotyping of VFs. Isolates were tested by multiplex PCR assays (28) for putative VF genes of extraintestinal pathogenic E. coli, including pap-encoding sequences (papAH, papC, papEF, and the three papG alleles), sfa/foc (central region of the sfa/foc operon), hlyA (hemolysin), cnf1 (cytotoxic necrotizing factor type 1), cnf2 (cytotoxic necrotizing factor type 2), iutA (aerobactin), kpsMT II (group II capsule synthesis), iha (nonhemagglutinating adhesin-associated PAI ICFT073), and iroN (catechol siderophore receptor-associated PAICP9). Hemolysin was detected both by PCR and by detection of hemolysis on sheep blood agar.

As reported previously (4), bovine septicemia-associated strain 31A was positive for the different pap elements but negative for all three recognized papG adhesin classes. However, hybridization experiments revealed that strain 31A carried a complete pap operon, suggesting the presence of a new papG variant (referred to as papGrs). Detection of such a papG-related sequence was done by a PCR assay with a new pair of primers: papG4 (3'-CCTGTCAGGCTGTAATGATGCT-5') and papG-C (3'-CAAGACACAGAAAGAGTCTGAGCC-5'). PCR was carried out as described previously (4) at an annealing temperature of 49°C. When used in combination, primers papG4 and papG-C revealed a product of 708 bp for pap-positive strains that were negative for the three recognized adhesin classes. Slight differences in the sizes of the PCR products suggest that different variants of papGrs may exist.

To detect the F17-related fimbrial adhesins and to identify the four alleles of the major structural subunit (F17A), strains were tested for the f17Aa, f17Ab, f17Ac, and f17Ad genes as described by Bertin et al. (2). Genes clpG and east1 were detected by PCR as described previously (3). Production of F17 and CS31A adhesins was detected by both PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of protein extracts. Concordant results were obtained by the two methods (data not shown). Investigation of the location of the afa-8 operon by hybridization between the afaD8 and afaE8 amplification products, which were used as probes, and plasmid DNA from the isolates was done as described previously (35).

The genes malX, cysB, and modD were used as markers for PAI ICFT073 (18, 31). The primers used for PCR and the sizes of the amplified fragments were as follows: for malX, primers malX-L (5'-GCGATCGGCCAACCTGTTCT-3') and malX-R (5'-CGGTTCGGCTGTGATTGGTG-3'), 429 bp; for cysB, primers cysB-L (5'-GGATAACCAATAGCAGAACAA-3') and cysB-R (5'-AGTTATTGACATCGCATGGT-3'), 682 bp; and for modD, primers modD-L (5'-AGCTGAAGTACGTCTGGTTG-3') and modD-R (5'-TTCTGTCGCTTGTAAGATGT-3'), 510 bp. PCR was carried out at an annealing temperature of 52°C. The espB gene from the LEE locus carried by enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) and the stx (Shiga-like toxin) virulence gene of Shiga toxin-producing E. coli (STEC) were detected by a PCR assay as described previously (43).

Detection of Yersinia HPI and determination of integration site of HPI in afa-8 strains. PCR assays were performed as described previously (49) to detect marker genes (irp1, irp2, and fyuA) for the high-pathogenicity island (HPI) of Yersinia. To examine the linkage of HPI to the asnT tRNA gene, the primer pair Ec-chrom and HPI-5' end generated by Schubert et al. (49) was used. As most of the HPI-positive strains did not generate any PCR fragments with this primer pair, a new primer, P4del (5'-GAACACATGCAGCATCAGCAG-3'), derived from the 5' end of the HPI integrase gene, was defined. By using the primer pair Ec-chrom and P4del, PCR amplicons were obtained with all of the HPI-positive strains. To examine the HPI integration site and the HPI integrase gene, the PCR fragments obtained with primer pair Ec-chrom and P4del were sequenced for representative HPI-positive strains. Automated DNA sequencing was performed on double-stranded DNA templates with the Prism Ready Reaction dye dideoxy terminator cycle-sequencing kit (Applied Biosystems Inc.) according to the instructions of the manufacturer. Electrophoresis of the sequencing products was performed with an automated sequencer (model 373A; Applied Biosystems Inc.).

Identification of DNA polymorphisms in the mutS-rpoS region. In comparison to E. coli MG1655, previous studies (14, 21, 39) revealed that EPEC, EHEC, and E. coli group B2 strains harbor specific DNA insertions within the mutS-rpoS intergenic region. A PCR assay was developed to determine the nature of the mutS-rpoS intergenic region of the afa-8 isolates. The production of two amplicons of 798 bp (primers O454 and yclC) and 608 bp (primers slyA and rpoS) indicated the presence of the inserted sequence of EPEC 1, EPEC 2, and EHEC 2 groups within the mutS-rpoS intergenic region. The production of two amplicons of 1,072 bp (primers O218 and yclC) and 608 bp (primers slyA and rpoS) indicated the presence of the inserted sequence of O157:H7 and EHEC 1 strains within the mutS-rpoS intergenic region. The production of two amplicons of 747 bp (primers O454 and O347) and 473 bp (primers O183 and rpoS) indicated the presence of the inserted sequence of the phylogenetic group B2 strains within the mutS-rpoS intergenic region. The production of a 650-bp amplicon (primers O454 and rpoS) indicated the presence of the mutS-rpoS intergenic region in E. coli K-12. The cycling conditions were as follows: denaturation for 60 s at 95°C, annealing for 90 s at 52°C, and extension for 90 s at 72°C (30 cycles). The primers used for the PCR analysis were as follows: O454 (3'-GATTGACCTGCCTCTGTTAC-5'), yclC (3'-GAATATGTCCGTGCTGGAA-5'), slyA (3'-GATAGAAGCAGCTGTCAGCA-5'), rpoS (3'-GATATGAAGCAGAGCATC-5'), O218 (3'-CGTTGGTGATATTCATGGTG-5'), O347 (3'-CGCTGAAGGTTCAACATAAC-5'), and O183 (3'-TTGCAGATAGAT-CAGCGTAAC-5').

Serum bactericidal assay. Survival of the bacteria in 90% fresh nonimmune human serum was examined by the bactericidal assay described by Taylor and Kroll (53). Serum-resistant strain 31A (an E. coli strain causing septicemia) and serum-sensitive strain HB101 were used as positive and negative controls, respectively.

Antibiotic susceptibility. The susceptibilities of the isolates to 19 antimicrobial agents were determined in a semisolid medium by use of a commercially available microdilution system (ATB G-strips; BioMerieux, Lyon, France), as recommended by the manufacturer. The following antibiotics and amounts (in micrograms per milliliter of semisolid medium) were used per assay: amoxicillin (16 µg/ml), ticarcillin (16 µg/ml), piperacillin (8 µg/ml), piperacillin-tazobactam (8 and 4 µg/ml, respectively), imipenem (4 µg/ml), cephalothin (8 µg/ml), cefoxitin (8 µg/ml), cefotaxime (4 and 32 µg/ml), ceftazidime (1 and 4 µg/ml), cefepime (4 µg/ml), cefpirome (4 µg/ml), tobramycin (4 µg), amikacin (8 µg/ml), gentamicin (4 µg/ml), netilmicin (4 µg/ml), sulfamethoxazole-trimethoprim (2 and 38 µg/ml, respectively), nalidixic acid (8 µg/ml), pefloxacin (1 and 4 µg/ml, respectively), and ciprofloxacin (1 and 2 µg/ml).

Statistical analysis. The data obtained for the different groups were compared by using the chi-square test and Fisher's exact test. Correlations between traits were assessed by using Pearson's correlation. Comparison of the prevalences of different traits within the same population were made by McNemar's test. The threshold for statistical significance was a P value <0.05. An aggregate VF score was calculated for each strain by summing the number of nonredundant VFs present in the same strain, and VF scores were compared as described by Johnson et al. (23).

Distance matrices based on VF genotype and phenotype data (including biotype and serum resistance) were calculated for all pairwise combinations and were used to infer a similarity dendrogram by the unweighted pair group method with averaging (UPGMA) (50) by use of the numerical taxonomy and multivariate analysis system (SPSS software for Windows, version 10.1; SPSS, Paris, France).


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RESULTS
 
Prevalence of VFs. All of the afa-8 strains contained at least one of the VF genes studied, which ranged in prevalence from 7% (kpsMT II) to 80% (iutA) (Table 1). Of the adhesin-encoding genes studied, pap and f17Ac were more prevalent than clpG, iha, and sfa/foc (for all comparisons, P < 0.05 by McNemar's test). Over half of the isolates contained pap elements (papAHCEF) and various papG alleles. The newly identified variants (papGrs) predominated over those with allele III, which were in turn more prevalent than those with allele II (for all comparisons, P < 0.05 by McNemar's test). papG allele I was not encountered among the afa-8 isolates. Of the siderophore-encoding genes studied, iutA and fyuA were more prevalent than iroN. Of note was the substantial prevalence of the enteroaggregative E. coli heat-stable enterotoxin gene (east1) and the low prevalence of the PAI ICFT073 marker genes.


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  		TABLE 1. Distribution of VFs in the 77 E. coli afa-8 strains by epidemiological source

Serotyping. The O serogroups of 52 isolates were determined. Although over half of the afa-8 strains carried ExPEC-associated virulence genes, serotyping showed the paucity of ExPEC-associated O antigens among these strains (only six isolates had the O4, O83, and O78 antigens). The most frequently observed serogroups were O11 (21 isolates), O101 (9 isolates), and O8 (6 isolates). Two isolates each were serogroup O118, O86, and O9; and four strains were nontypeable.

Antibiotic susceptibility. Most of the isolates (88%) were resistant to at least two antimicrobial agents; only five strains were sensitive to all of the antimicrobial agents tested (Table 2). Only one bovine isolate was resistant to imipenem and ciprofloxacin. A high prevalence of resistance (>50% of isolates) to piperacillin, ticarcillin, and trimethoprim-sulfamethoxazole was observed. Compared with all other epidemiological sources, antimicrobial resistance was highest among animal bacteremia isolates (antimicrobial resistance score, 9.0 versus 5.9).


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  		TABLE 2. Antibiotic resistance of the E. coli afa-8 strains by epidemiological source

Bactericidal effect of normal human serum. Among the 77 isolates, 52 (67%) were resistant to killing by 90% human serum (Table 1). Compared to the isolates associated with intestinal infections, extraintestinal isolates exhibited a significantly higher prevalence of resistance to serum (P < 0.05). Several VFs were associated with increased serum resistance, including pap genes (P = 0.02), hlyA (P = 0.01), and HPI (P = 0.03). In contrast, serum resistance was negatively associated with cnf2 (P < 0.01) and iha (P = 0.005).

HPI of Yersinia. Among the afa-8 isolates, HPI was the second most frequent VF overall (Table 1). Compared to the diarrhea-associated isolates, the extraintestinal isolates had a higher prevalence of HPI (77 versus 51%; P = 0.02). The products obtained by PCR with the Ec-chrom and P4del primer pair with 19 (36%) of the 52 HPI-positive strains had the predicted lengths (1,360 bp). Sequencing of this 1,360-bp amplicon showed that the HPIs of these strains were directly linked to the asnT tRNA gene and had an intact HPI integrase gene. Interestingly, the PCR products obtained with the remaining 33 (64%) HPI-positive strains were about 350 bp smaller than those obtained with the other strains (1,010 bp instead of 1,360 bp), indicating a deletion in the HPI integrase gene. Sequencing of the 1,010-bp amplicon from four different strains (data not shown) revealed a 347-bp deletion, resulting in a truncated HPI integrase gene.

Plasmid-borne afa-8 operon. The location of the afa-8 operon was investigated by hybridization between afaD8 and afaE8 amplification products, which were used as probes, and plasmid DNA from the 77 isolates. Hybridization with plasmid DNA was detected for 16 of the 77 (20%) isolates. Several VFs displayed specific associations with either the chromosome- or the plasmid-borne afa-8 operon. Although clpG, papG allele III, hly, and east1 were significantly associated with the chromosomal location of the afa-8 operon (for all comparisons, P < 0.03), for the bovine isolates, cnf2 and papG allele II were significantly associated with a plasmid location of the afa-8 operon (for all comparisons, P < 0.01) (Table 1).

Polymorphism in the mutS-rpoS region. Among the 77 afa-8 strains, 22 (29%) differed from E. coli K-12 in the mutS-rpoS intergenic region (Table 1). A first group (10 isolates) comprising essentially animal isolates (including 6 of the 7 STEC isolates) harbored the mutS-rpoS intergenic region of EPEC group 1, EPEC group 2, and EHEC group 2 strains. A second group (seven isolates) comprising members of different host groups (five human extraintestinal isolates and two bovine diarrhea-associated isolates) harbored the mutS-rpoS intergenic region of E. coli O157:H7 and EHEC group 1 strains. A third group (five isolates), which included exclusively the O4 and O83 human isolates, harbored the mutS-rpoS intergenic region of the phylogenetic group B2 strains.

Cluster analysis of VF profiles and O antigens. To determine whether the isolates segregate according to their VF serotype profiles, similarity relationships among the isolates were assessed by UPGMA. The dendrogram (data not shown) resolved the population into five main clusters. All strains containing traits of ExPEC were segregated into clusters I, III, and V. The remaining isolates segregated into clusters II and IV according to their diarrhea-associated VF profiles (Table 3).


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  		TABLE 3. Characteristics of the 77 E. coli isolates from humans and farm animals with intestinal or extraintestinal infections

Cluster I was the largest and accounted for 52% of the afa-8 isolates. Within this cluster, five subclusters with different O antigens and VF profiles were resolved. Of particular interest were the seven bovine and the three human isolates that constituted subcluster ID. With one exception, all isolates expressed the O11 antigen and displayed the profile of uropathogenic E. coli strains, including the pap operon with papG allele III, fyuA, iutA, cnf1, hlyA, and serum resistance. Interestingly, all isolates in this subcluster shared, in addition to ExPEC-associated traits, the two diarrhea-associated traits clpG and east1. Cluster II comprised 17 bovine isolates. None of these strains contained ExPEC-associated traits, but most of the isolates bearing the f17Ab and cnf2 genes and the plasmid-borne afa-8 operon were concentrated in this cluster. Cluster III, comprising exclusively human extraintestinal isolates, had the highest aggregate VF score (12.4 versus 6.0; P = 0.01). Isolates of this cluster exhibited ExPEC-associated VF serotype profiles and shared the mutS-rpoS intergenic region of group B2 strains. The markers PAI ICFT073 (malX and cysB) and sfa/foc were found exclusively within this cluster. In addition to ExPEC-associated traits, all isolates in this cluster expressed bovine intestinal adhesin F17c. In cluster IV, seven diarrhea-associated bovine isolates displayed the VF profile of STEC. All strains in this cluster shared the mutS-rpoS intergenic region of EPEC or EHEC strains. In cluster V, seven human isolates associated with extraintestinal infections exhibited VF profiles that differed from modal VF profiles for both intestinal and extraintestinal strains in that they had a combination of the VFs of the two groups (kpsMT II, east1, modD, and serum resistance). All isolates that constituted this cluster shared the mutS-rpoS intergenic region of EPEC or EHEC strains.

Distribution of VFs by epidemiological source. The distributions of the VFs among the 77 afa-8 strains were analyzed in relation to the epidemiological sources of the isolates (Table 1), with strains stratified into five groups (human pyelonephritis [n = 18], human bacteremia [n = 14], animal bacteremia [n = 19], non-STEC animal enteritis [n = 19], and STEC [n = 7]). No statistically significant difference (P < 0.01) between human and animal extraintestinal isolates with respect to ExPEC-associated traits (including the uniform presence of pap, F17Ac, hly, cnf1, fyuA, and serum resistance) was observed. However, a significant difference with respect to the prevalence of individual VFs was observed, including sfa/foc, kpsMT II, iroN, PAI ICFT073, and the mutS-rpoS intergenic region of the group B2 strains (which were more prevalent in human isolates than in animal isolates), east1 (which was more prevalent in bacteremia-associated isolates than in isolates of any of the other groups), and pap, clpG, iroN, fyuA, and serum resistance (which were more prevalent in bacteremia-associated isolates than in enteritis-associated isolates). When the prevalence of individual traits was examined, the 26 diarrhea-associated bovine isolates differed from the extraintestinal isolates with respect to the lack of ExPEC-associated traits. Most of the cnf2 genes and the plasmid-borne afa-8 operon were found among diarrhea-associated bovine isolates.


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DISCUSSION
 
Previous studies have reported the association of the afa-8 operon with CNF1- or CNF2-producing E. coli strains isolated from intestinal or extraintestinal infections in humans and animals (16, 38, 41). The present study provides novel epidemiological information on the distributions of the various VFs of AfaE-VIII-producing E coli strains.

Although the enteroaggregative E. coli heat-stable enterotoxin 1 gene (east1) was common in E. coli diarrheagenic isolates from humans and animals and EAST1 has primarily been regarded as an enteric VF (48, 54), this study revealed the substantial prevalence of the east1 gene among extraintestinal afa-8 isolates. Little is known about the pathogenic significance of EAST1 in diarrhea, but previous reports (3, 54) indicated that it may be found in association with different diarrheagenic VFs, including adhesins (CS31A, CFA/II, K88) and toxins (heat-stable toxin STa, heat-labile toxin). Consistent with these previous findings, a significant association between east1 and clpG (the major subunit of CS31A) was observed among extraintestinal afa-8 isolates. The recent finding that fecal strains with ExPEC-associated traits were isolated from dogs with diarrhea (51) raises the question whether these isolates are capable of causing intestinal as well as extraintestinal infections. In our opinion, the different findings from this study yield further evidence for the involvement of diarrheagenic isolates in the development of extraintestinal infections: (i) cluster 1D isolates from calves with diarrhea were indistinguishable in terms of their O types and VF profiles from isolates from calves with extraintestinal infections; (ii) among certain extraintestinal isolates, enteritis-associated traits (f17Ac, clpG, east1) were found to be associated with ExPEC-associated traits (papG allele III, hlyA, cnf1); and (iii) many extraintestinal afa-8 isolates with ExPEC-associated traits exhibited the O8, O9, O11, and O101 antigens, which are commonly found in fecal or diarrheagenic isolates (11, 13, 55).

The HPI of Yersinia, which carries the yersiniabactin siderophore system, was common in afa-8 isolates. Consistent with previous reports involving other human clinical isolates (12, 28, 49), HPI was significantly more prevalent among extraintestinal afa-8 isolates than among diarrhea-associated afa-8 isolates. Among the HPI-positive strains, those which carried a 347-bp deletion in their HPI integrase genes predominated over strains with intact HPI integrase genes. Karch et al. (32) previously identified an identical deletion in the integrase gene of the HPI carried by Shiga toxin-producing E. coli serotype O26 strains. The presence of this deletion in the HPI integrase gene carried by all strains in subcluster ID suggests the fixation of HPI in the genomes of these strains.

Recent studies revealed a pathotype-associated polymorphism in the mutS-rpoS region of the E. coli chromosome (14, 21, 39). PCR analysis showed that 29% of the afa-8 isolates differed from E. coli K-12 in the mutS-rpoS intergenic region. The five O4 and O83 human isolates included in cluster III harbored the DNA insertion characteristic of group B2 strains, suggesting that these strains belong to phylogenetic group B2. Among the strains in cluster IV, the concordant distribution of STEC-associated traits and the mutS-rpoS intergenic region of EPEC or EHEC strains suggests that these afa-8 isolates are true STEC strains. Of particular interest was the concentration of the DNA segment specific to EPEC or EHEC strains within human pyelonephritis-associated isolates that lack modal ExPEC-associated traits (cluster V). The DNA sequence conserved in the mutS-rpoS intergenic region of EPEC and EHEC isolates encodes the Salmonella enterica transcriptional regulator (SlyA). A function of SlyA in pathogenesis is suggested by the results for Salmonella, in which SlyA has been shown to play a role in bacterial survival in the intracellular environment of host macrophages and in the invasion of M cells (8). This finding raises the question whether SlyA has a function in the pathogenesis of certain E. coli extraintestinal isolates that lack ExPEC-associated traits.

Two-thirds of the extraintestinal afa-8 isolates contained pap elements and various papG alleles. However, half of the pap-positive strains carried a papG variant which differd from the three recognized adhesin classes. Recently, Bertin et al. (4) demonstrated that bovine septicemia-associated strain 31A carried a complete pap operon with a structural subunit (papA) which is highly homologous to F11 and an unknown papG allele (papGrs). The present study indicates that among afa-8 isolates, papGrs predominates over papG alleles II and III. Strains containing papG allele III differed significantly from those containing papGrs and papG allele II with respect to the association with hlyA and cnf1. The observed association of papG allele II with intestinal adhesins CS31A and F17 is a novel finding of this study. Consistent with recent findings for avian pathogenic E. coli strains (52), this study revealed a high prevalence of intestinal adhesin F17c among human and bovine extraintestinal afa-8 isolates. In addition to afa-8, a significant proportion (22%) of the extraintestinal isolates displayed the associated set of adhesin genes, pap, clpG, and f17Ac.

The hypothesis that humans may acquire ExPEC strains from their domestic animals has been suggested by the similarities observed among isolates recovered from dogs, cats, and humans with UTIs. Recently, Johnson et al. (25) provided evidence of a commonality between canine and human ExPEC strains and suggested that canine feces represent a potential reservoir of E. coli with infectious potential for humans. Our finding that bovine and human isolates in subcluster ID were indistinguishable with respect to their O-type and VF profiles also supports the hypothesis of overlapping populations. With respect to their VF profiles, the two human O4 isolates appeared to be highly similar to an isolate taken from a dog with an UTI (strain 840383) described by Johnson et al. (24). Given these similarities, it is possible that certain clones within the afa-8 population are also pathogenic for humans and dogs.

The AfaE-VIII adhesin is very similar to the M adhesin, an afimbrial adhesin encoded by the bma gene cluster (46). The prevalence of the M adhesin in pathogenic E. coli strains from humans and dogs has been estimated to be on the order of 1% (24), whereas the prevalence of the afa-8 operon in pathogenic E. coli isolates from humans and farm animals has been estimated to be significantly higher (5 to 25%) (16, 39, 52). Since the prevalence of afa-8 and bma was assessed by PCR assays with different primer sets, differences in the prevalence of afa-8 and bma need to be evaluated in terms of the primers used.

A large number of extraintestinal afa-8 isolates were resistant to several of the antimicrobial agents frequently used in human medicine. afa-8 isolates from farm animals were significantly more likely to be multidrug resistant than human isolates. Similar results have been observed with other pathogenic E. coli isolates and may reflect the selection of a resistant population due to the use of antibiotic additives in animal feed and the extensive use of antibiotics in veterinary medicine. Previous studies have reported that pathogenic isolates that produce VFs are more sensitive to antimicrobial agents than those without VFs (27, 28, 45). In contrast, the antimicrobial resistance of afa-8 strains was not correlated with the prevalence of the VFs.

Taken together, our findings suggest that extraintestinal afa-8 isolates represent distinct clonal groups that differ from other ExPEC isolates. The zoonotic potential of certain clones and the high prevalence of multiple antibiotic resistance among bovine afa-8 isolates suggest that such strains present a risk to public health.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Microbiologie, INRA, Centre de Clermont-Theix, 63122 St Genès Champanelle, France. Phone: 33 04 73 62 42 42. Fax: 33 04 73 62 45 81. E-mail: girard{at}clermont.inra.fr. Back


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Journal of Clinical Microbiology, January 2003, p. 218-226, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.218-226.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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