Three-Decade Epidemiological Analysis of Escherichia coli O15:K52:H1

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JOURNAL OF CLINICAL MICROBIOLOGY, June 2009, p. 1857–1862 0095-1137/09/$08.00⫹0 doi:10.1128/JCM.00230-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 47, No. 6

Three-Decade Epidemiological Analysis of Escherichia coli O15:K52:H1䌤 Bente Olesen,1,2* Flemming Scheutz,2 Megan Menard,3 Marianne N. Skov,4 Hans Jørn Kolmos,4 Michael A. Kuskowski,3 and James R. Johnson3 Department of Clinical Microbiology, Hillerød Hospital, Helsevej 2, DK-3400 Hillerød, Denmark1; The International Escherichia and Klebsiella Centre (WHO), Statens Serum Institut, Copenhagen, Denmark2; Veterans Affairs Medical Center and Department of Medicine, University of Minnesota, Minneapolis, Minnesota3; and Department of Clinical Microbiology, Odense University Hospital, Sdr. Boulevard 29, DK-5000 Odense C, Denmark4 Received 3 February 2009/Returned for modification 14 March 2009/Accepted 25 March 2009

The successful Escherichia coli O15:K52:H1 clonal group provides a case study for the emergence of multiresistant clonal groups of Enterobacteriaceae generally. Accordingly, we tested the hypotheses that, over time, the O15:K52:H1 clonal group has become increasingly (i) virulent and (ii) resistant to antibiotics. One hundred archived international E. coli O15:K52:[H1] clinical isolates from 100 unique patients (1975 to 2006) were characterized for diverse phenotypic and molecular traits. All 100 isolates derived from phylogenetic group D and, presumptively, sequence type ST393. They uniformly carried the F16 papA allele and papG allele II (P fimbria structural subunit and adhesin variants), iha (adhesin-siderophore), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iutA (aerobactin receptor), and kpsM II (group 2 capsule); 85% to 89% of them contained a complete copy of the pap operon and ompT (outer membrane protease). Slight additional virulence profile variation was evident, particularly within a minor diarrhea-associated subset (biotype C). However, in contrast to the clonal group’s fairly stable virulence profiles over the past 30ⴙ years, during the same interval the clonal group members’ antimicrobial resistance profiles increased by a mean of 2.8 units per decade (P < 0.001). Moreover, the numbers of virulence genes and resistance markers were positively associated (P ⴝ 0.046), providing evidence against antimicrobial resistance and virulence being mutually exclusive in these strains. Thus, the O15:K52:H1 clonal group has become increasingly resistant to antimicrobials while maintaining (or expanding) its virulence potential, a particularly concerning trend if other emerging multiresistant enterobacterial clonal groups follow a similar pattern. Escherichia coli strains of serotype O15:K52:H1 first came to clinicians’ attention as significant pathogens in 1986 and 1987, during a year-long, community-wide outbreak of multiresistant infections in South London (23). The outbreak strain exhibited a 100-MDa plasmid and the signature resistance profile ACSSuTTp (resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim). Most patients had urinary tract infections, but some had septicemia associated with pneumonia, meningitis, or endocarditis, and three died. E. coli O15:K52:H1 was later documented as a significant extraintestinal pathogen elsewhere in Europe (19, 24) and also shown to be distributed widely outside Europe, where it exhibited previously unrecognized phenotypic and genotypic diversity (8). Subsequently, rapid and specific detection of the O15:K52:H1 clonal group by single-nucleotide polymorphism (SNP)-specific PCR was described (12). In a recent Italian study, the O15:K52:H1 clonal group accounted for 16 (11%) of 148 ciprofloxacin-resistant E. coli isolates from uncomplicated urinary tract infections in eight European countries (2003 to 2006), providing evidence of continued clinical importance and a seemingly new resistance phenotype for this clonal group (2).

The O15:K52:H1 clonal group can serve as a case study for other disseminated, multiresistant pathogens, whose mechanisms for emergence are important to understand for public health reasons. We hypothesized that this clonal group’s emergence might be explained by its members having become increasingly virulent and/or antimicrobial resistant over time. Accordingly, we took advantage of a large archival collection of E. coli strains and sought to clarify the epidemiology, phylogenetic background, virulence characteristics, and antimicrobial resistance patterns of E. coli O15:K52:H1 over the past approximately 3 decades. MATERIALS AND METHODS Clinical and epidemiological information. The International Escherichia and Klebsiella Centre (WHO) holds more than 60,000 E. coli isolates received from 85 different countries since 1951, obtained from both humans (50,000) and animals (10,000). The strains are highly selected, since the center participates in many national and international projects but has very few routine functions. The median number of isolates examined per year in the center is 1,330 (range, 677 to 2,671). To identify all available E. coli O15:K52:H1 isolates held by the International Escherichia and Klebsiella Centre, the center’s E. coli database (1951 to 2006) was searched for serogroup O15. The 839 E. coli O15 isolates were tested for motility, with motile isolates tested for H1 antigen expression by microtiter plate and tube agglutination (22). The 207 O15:H⫺ (i.e., nonmotile) and 156 O15:H1 isolates thus identified were tested for the K52 capsular antigen by Cetavlon precipitation, followed by countercurrent immunoelectrophoresis in K52 antiserum for Cetavlon-positive isolates. Among these two groups of isolates, 5 (2%) and 120 (77%), respectively, proved to be K52 antigen positive. The 125 total isolates of E. coli O15:K52:[H1] (brackets indicate the inclusion of nonmotile, i.e., H antigen-negative, variants) so identified were received from

* Corresponding author. Mailing address: Department of Clinical Microbiology, Hillerød Sygehus, Helsevej 2, DK-3400 Hillerød, Denmark. Phone: 45-4829-4379. Fax: 45-4829-4384. E-mail: benol@hih .regionh.dk. 䌤 Published ahead of print on 1 April 2009. 1857

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1975 to 2006. All were of human origin. The study was limited to (i) Danish isolates, regardless of previous publication status, and (ii) previously unpublished isolates from other countries, using one isolate per patient. Seven English, eight Spanish, and three American O15:K52:H1 isolates were excluded by virtue of previous publication (8, 24, 25), six isolates were duplicates from the same patients (all Danish), and one was subsequently lost (total number excluded, 25). Thus, 100 isolates from 100 different patients infected with E. coli O15:K52:[H1] from 1975 to 2006 were studied. Of these, 16 (all Danish) were described elsewhere (19), whereas 84 (38 Danish isolates and 46 isolates from other countries) had not been reported previously. Phenotypic and genotypic characteristics. Isolates were confirmed as E. coli by use of a Minibact E kit (SSI) (15) and a ␤-glucuronidase test. Biotyping was done according to the method of Kauffmann (14), using the following 12 carbohydrates: adonitol, dulcitol, sorbitol, raffinose, xylose, rhamnose, maltose, salicin, inositol, lactose, sucrose, and sorbose. Hemolysin production was determined on sheep’s blood agar plates (1). Aerobactin secretion was determined in a crossfeeding bioassay (30). Forty-one randomly selected isolates were examined for Shiga toxin production by the Vero cell assay (16). All isolates underwent EcoRI riboprinting (automated ribotyping), were manually ribotyped according to the method of Gerner-Smidt (4), and underwent pulsed-field gel electrophoresis analysis (20). Selected isolates underwent plasmid analysis by a modification (21) of the method of Kado and Liu (13). Plasmids in E. coli 39R861 (27) and V517 (17) were used as size references. Phylogenetic analysis and virulence genotyping. Isolates were assigned to major E. coli phylogenetic groups (A, B1, B2, and D) by use of a triplex PCRbased method (3) and were tested by PCR for an O15:K52:H1-specific SNP in fumC (12). They were tested for 35 virulence markers of extraintestinal pathogenic E. coli (ExPEC) and for the F16 papA allele (P fimbria structural subunit variant) by established PCR-based assays (6, 7, 9, 10). Testing was done in duplicate, using independently prepared boiled lysates of each isolate, together with appropriate positive and negative controls. Using DNA probe hybridization, the isolates were also examined for several diarrhea-associated genes, including those encoding the porcine (estAp) and human (estAh) variants of heat-stable enterotoxin; elt, encoding heat-labile toxin (26) (both characteristic of enterotoxigenic E. coli [ETEC]); eae (characteristic of enteropathogenic E. coli and attaching-and-effacing E. coli) (5); and astA (encoding enteroaggregative heatstable enterotoxin), which occurs in both diarrheagenic E. coli and ExPEC. The total number of unique markers detected was the virulence score. A dendrogram based on pairwise similarity relationships according to extended virulence profiles (i.e., the presence/absence of all markers tested) was inferred according to the unweighted-pair group method using average linkages. MLST. Multilocus sequence typing (MLST) was performed on 12 O15: K52:H1 isolates (selected for maximal diversity in locale, biotype, virulence profile, and year of receipt), using eight housekeeping genes. For adk, fumC, gyrB, icd, mdh, purA, and recA, procedures, primers, and sequence type (ST) assignments were as described at http://mlst.ucc.ie/mlst/dbs/Ecoli/documents /primersColi_html. For metG, the primers were metG F (5⬘-CACATCCAGGC TGATGTCTG-3⬘) and metG R (5⬘-GCAGATCAAAGAAGAAGTGT-3⬘). Full-length metG sequences were compared to an internal metG sequence database (11). Susceptibility testing. Susceptibility to 13 antimicrobial agents (ampicillin, ceftazidime, cefuroxime, piperacillin, streptomycin, gentamicin, tobramicin, amikacin, tetracycline, chloramphenicol, ciprofloxacin, trimethoprim, and sulfonamides) was assessed on 5% Danish blood agar (Statens Serum Institut), using Neo-Sensitabs according to the manufacturer’s instructions (Rosco). Isolates susceptible to all agents were defined as susceptible. Multiresistance was defined as resistance to five or more antimicrobial agents. The number of resistance markers was the resistance score. Antimicrobial agents were analyzed as independent, irrespective of possible commonality by class or resistance mechanism. Statistical methods. Comparisons were tested using Fisher’s exact test (twotailed) for proportions and the Mann-Whitney U test for scores. Assessments of multiple variables as predictors of an outcome variable were done by multivariable logistic regression analysis or multiple regression. Assessment for temporal trends in resistance or virulence scores was done by Poisson regression analysis. Correlations between variables were tested by using Spearman’s rho coefficient. Statistical significance was defined as P values of ⬍0.05.

RESULTS Clinical and epidemiological data. Of the 100 total E. coli O15:K52:[H1] study isolates (1975 to 2006), 57 were from the bloodstream, 27 were from urine, and 5 were from feces,

whereas 11 were of unknown sources. Fifty-four (54%) isolates originated from Denmark; the other 46 were from Sweden (n ⫽ 24), the United States (n ⫽ 15), Canada (n ⫽ 3), England (n ⫽ 2), Switzerland, and Germany (n ⫽ 1 each). The five earliest-received isolates, in chronological order, were from the United States (1975; two isolates), Sweden (1982), England (1986), Canada (1988), and Denmark (1988), with the 1975 U.S. isolates representing the earliest known examples of this serotype. Prevalence of E. coli O15:K52:[H1]. The study population included 34 isolates from two national studies of E. coli bacteremia in Denmark conducted in the 1990s, which comprised 377 (3) and 525 (B. Olesen and F. Scheutz, unpublished data) blood isolates. In these studies combined, the overall prevalence of serotype O15:K52:[H1] was 4% (34/902 isolates). In contrast, among Swedish E. coli urine isolates from several different prospective studies (1980 to 1993), 19/912 isolates (2%) were O15:K52:[H1] (P ⫽ 0.037). The prevalence of O15: K52:[H1] among all E. coli strains received at the WHO center was 0/19,338 isolates (0%) from 1951 to 1975 versus 125/35,276 isolates (0.4%) thereafter (P ⬍ 0.001), consistent with the clonal group having emerged significantly during this period. Phenotypic characteristics. Of the 100 O15:K52:[H1] isolates, 95 (95%) were H1 antigen positive, whereas 5 were nonmotile. Four biotypes were encountered, varying only in fermentation of raffinose, maltose, and lactose (among 12 carbohydrates). Biotype A (Raf⫺ Mal⫹ Lac⫹) was the most common biotype (88%) and the only one encountered until 1986. The prevalence of biotype A prior to 1986 was 28/28 isolates (100%), versus 60/72 isolates (83%) thereafter (P ⫽ 0.03), indicating a significant diversification of biotypes within the clonal group after 1986. Specifically, subsequent to 1986, two study isolates exhibited biotype B (Raf⫺ Mal⫺ Lac⫹), nine showed biotype C (Raf⫺ Mal⫹ Lac⫺), and one showed biotype D (Raf⫹ Mal⫹ Lac⫹). Biotype C (which differed from biotype A only by being Lac⫺) first appeared in 1991, exhibiting a prevalence of 0/42 isolates (0%) before 1991 versus 9/58 isolates (16%) thereafter (P ⫽ 0.01), consistent with significant emergence during the 1990s. Biotype C was also significantly associated with feces, accounting for 3/5 (60%) fecal isolates versus 6/84 (7%) known nonfecal isolates (P ⫽ 0.01). In contrast to these temporal and clinical associations, no geographical segregation of biotypes was evident. Regarding other phenotypic characteristics, all isolates but one produced aerobactin, whereas none produced either hemolysin or (among the 41 strains so tested) Shiga toxin. Molecular characteristics. All 100 isolates were from phylogenetic group D, exhibited the O15:K52:H1-specific fumC SNP (12), and belonged to the same ribotype. They were nontypeable by pulsed-field gel electrophoresis, probably due to autodigestion of DNA resulting in smeared gels, as described previously (24). To more stringently assess clonality, full MLST was done on 12 diverse O15:K52:[H1] study isolates. All 12 (including 2 from biotype C) exhibited identical sequences across the seven MLST loci and corresponded with ST393, which is within the ST31 clonal complex. All 12 isolates also exhibited an identical metG sequence to that of strain 2P9, a published O15:K52:H1 urosepsis isolate (29) from ST598. This ST, likewise, is within the ST31 clonal complex, differing from ST393 by a single SNP in fumC.

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FIG. 1. Virulence gene tree for 100 isolates of Escherichia coli O15:K52:[H1]. The following genes are included: astA (enteroaggregative heat-stable enterotoxin), cvaC (microcin V), estAp and estAh (characteristic of ETEC; heat-stable toxin), fyuA (yersiniabactin receptor), hlyF (hemolysin F), hra (heat-resistant agglutinin), iha (adhesin-siderophore), iroN (siderophore receptor), ireA (siderophore receptor), iss (increased serum survival), iutA (aerobactin receptor), kpsM II (group 2 capsule), ompT (outer membrane protease T), pap (P fimbria operon; presumed to be complete [based on detection of papAH, papC, papEG, and papG allele II], unless listed as “partial”), tsh (temperature-sensitive hemagglutinin), and traT (serum resistance-associated protein).

Virulence genotypes of all 100 isolates included the F16 papA allele, papG allele II (P adhesin variant), iha (adhesinsiderophore), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iutA (aerobactin receptor), and kpsM II (group 2 capsule) (Fig. 1). All but two isolates (i.e., 98%) exhibited sat (secreted autotransporter toxin), and 86% exhibited ompT (outer membrane protease T). Whereas 89 (89%) of the isolates exhibited a complete copy of the pap (P fimbria) operon, 11 (11%) lacked papEF (minor subunits); 1 also lacked papC (P fimbria assembly). Traits encountered less frequently (i.e., in 4% to 18% of isolates) included hra1 (heat-resistant agglutinin 1) and typically plasmid-associated traits, i.e., iroN (siderophore receptor), cvaC (microcin V), hlyF (hemolysin F), traT (serum resistance-associated protein), and iss (increased serum survival) (Fig. 1). Additionally, two biotype C isolates exhibited ETECassociated genes; one contained both the porcine (estAp) and human (estAh) variants of the heat-stable enterotoxin gene, and another contained estAp alone. Both were fecal isolates (from 1991 and 1999, respectively) from patients with diarrhea. One of these patients had traveled in India; no other pathogens were found in feces. No information was available regard-

ing the other patient. Thus, estAp was significantly more prevalent among biotype C isolates than among other isolates (2/9 versus 0/91 isolates; P ⫽ 0.015) and among fecal isolates than among urine and bloodstream isolates (2/5 versus 0/84 isolates; P ⫽ 0.005). Additionally, seven urine and blood isolates, representing biotypes A (n ⫽ 5) and C (n ⫽ 2), exhibited astA (enteroaggregative heat-stable enterotoxin), which occurs in both diarrheagenic E. coli and ExPEC. In a virulence factor-based tree, the 27 unique profiles (which yielded virulence scores ranging from 3.5 to 14) exhibited at least 89% similarity (Fig. 1). The most common profile, which included the pap operon (with the F16 papA allele and papG allele II), iha, sat, fyuA, iutA, kpsMT II, and ompT, accounted for 53% of study isolates, whereas the other 27 profiles accounted for only 1 to 9% of isolates each. Virulence scores associated with the majority virulence profile were slightly but significantly lower than those associated with other profiles (median difference, one gene; P ⫽ 0.002). Virulence profiles (not shown) and virulence scores (Table 1) were fairly stable throughout the study interval. All strains were negative for afa/dra (Dr family adhesins), bmaE (M fimbriae), cdtB (cytolethal distending toxin), clpG (fimbrial adhesin CS31A),

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TABLE 1. Virulence and resistance scores by time interval among 100 Escherichia coli isolates of serotype O15:K52:关H1兴 Mean, median (range) Time interval (n)

1975–1984 (27) 1985–1994 (46) 1995–2006a (27)

Virulence score

Resistance scoreb

8.2, 8 (6.75–13) 8.5, 8 (6.75–14) 8.3, 8 (3.5–11)

0.6, 0 (0–3) 1.4, 0 (0–6) 4.4, 4 (0–9)

a The years 2005 and 2006 were included with 1995 to 2004 as part of the most recent time interval (giving 12 years total rather than 10). b According to Poisson regression analysis, the resistance score increased by an average of 2.8 antibiotics (95% confidence interval, 2.2 to 3.6) every 10 years (P ⬍ 0.001).

cnf1 (cytotoxic necrotizing factor), eae (possessed by enteropathogenic E. coli and attaching and effacing E. coli), elt (possessed by ETEC), fliC (H7 flagellin), focG (F1C fimbriae), gafD (G fimbriae), hlyD (hemolysin), ibeA (invasion of brain endothelium), malX (pathogenicity island marker), papG alleles I and III (P adhesin variants), pic (serine protease autotransporter), sat (secreted autotransporter toxin), rfc (O4 lipopolysaccharide), sfa/focDE (S and F1C fimbriae), sfaS (S fimbriae), and usp (uropathogenic specific protein). Susceptibility testing. A minority (45%) of the isolates were susceptible to all 13 tested antimicrobial agents; in contrast, 55 (55%) exhibited resistance to 1 to 9 (median, 3) of the agents, and 16 (16%) exhibited multiresistance. Eight (89%) of nine biotype C isolates were multiresistant, versus eight (9%) of the other isolates (P ⬍ 0.001), and biotype C isolates exhibited significantly higher resistance scores than did other isolates (median, 7.0 versus 1.0; P ⬍ 0.001). Only the German isolate and three Danish isolates (total, 4%) exhibited the signature resistance profile of the London outbreak, i.e., ACSSuTTp, with or without additional resistance markers. Two of these isolates (both biotype C), but none of the other multiresistant strains, carried a 100-MDa plasmid. The prevalence of the ACSSuTTp profile among biotype C isolates was 3/9 (33%), versus 1/91 (1%) for other isolates (P ⫽ 0.004). Significant temporal trends were observed for resistance score and resistance to specific antibiotics. For example, resistance to streptomycin and the combination of sulfonamides and trimethoprim first appeared in 1986. Accordingly, the prevalence of streptomycin resistance was 0/28 isolates before 1986 versus 25/72 isolates (35%) thereafter (P ⬍ 0.001), whereas the prevalence of resistance to sulfonamides and trimethoprim was 0/28 isolates before 1986 versus 13/72 isolates (18%) thereafter (P ⫽ 0.02). Fluoroquinolone resistance first appeared in 1995, exhibiting a prevalence of 0/73 isolates before 1995 versus 8/27 isolates (30%) thereafter (P ⬍ 0.001). Likewise, multiresistance was observed in 4/73 (6%) pre-1995 isolates versus 12/27 (44%) later isolates (P ⬍ 0.001). According to Poisson regression analysis, the resistance score increased by an average of 2.8 antibiotics (95% confidence interval, 2.2 to 3.6) every 10 years (P ⬍ 0.001) (Table 1). The virulence score was significantly correlated with the resistance score (P ⫽ 0.046). Additionally, the majority virulence profile was associated with a significantly lower resistance score than those for the other virulence profiles (median score, 0 versus 2.0; P ⫽ 0.002). Accordingly, multivariable Poisson models were constructed to assess decade, biotype C,

and virulence genotype as correlates of resistance. When present in the model together, decade (P ⬍ 0.001), biotype C (P ⬍ 0.001), and virulence score (P ⫽ 0.009) all significantly predicted the resistance score, as did (in a separate model) decade (P ⬍ 0.001), biotype C (P ⬍ 0.001), and the majority virulence profile (negative predictor; P ⫽ 0.006). Virulence scores were slightly, although not significantly, greater (mean difference, 0.5 units) among the eight fluoroquinolone-resistant isolates than among the other 92 isolates. DISCUSSION We report the characteristics of 100 E. coli O15:K52:[H1] clinical isolates from 100 unique patients from six countries over an approximately 3-decade study interval. This study, which is the largest to date for this important pathogen, tested the hypotheses that the O15:K52:H1 clonal group has become increasingly virulent and has grown increasingly resistant to antibiotics over time as possible explanations for its emergence over the past 3 decades. Our findings suggest instead that the clonal group has actually maintained a fairly stable virulence profile over the past 30⫹ years, albeit with minor variation, which corresponded in some instances with biotype variants and altered pathogenic capabilities (e.g., for intestinal versus extraintestinal disease). This was counter to our first hypothesis of increasing virulence over time. However, supporting our second hypothesis, since the clonal group’s first appearance in 1975, its members’ antimicrobial resistance profiles have become progressively broader, now extending to fluoroquinolone resistance, with an average increase in the resistance score of 2.8 antibiotics per decade. Most study isolates (53%) shared a consensus virulence profile that included a complete copy of pap (including the F16 papA allele and papG allele II), iha, sat, fyuA, iutA, kpsMT II, and ompT. This profile has predominated within the clonal group since its first documented appearance in 1975, which suggests that this combination of traits may have conferred the pathogenic fitness required for the clonal group’s initial emergence. Interestingly, although the other observed virulence profiles typically included slightly more virulence genes than this majority profile, they possibly did not confer (or were not associated with) significantly enhanced fitness, since none exhibited ⬎9% prevalence within the population, and most occurred in only a single isolate each. However, given the relatively recent appearance of some of the minor virulence profile variants, it may be too early to draw conclusions regarding their comparative fitness based on observed prevalence levels. The clonal group’s broadening resistance profiles over time may have helped it to cope with intensifying selection pressure from widespread antimicrobial use, thereby facilitating its persistence and expansion. Unexpectedly, only 4% of isolates exhibited the signature ACSSuTTp resistance profile of the strains causing the South London outbreak in 1986 and 1987 (23). Perhaps the scarcity of the “classic” ACSSuTTp profile was because the associated plasmid has not become well established in Denmark and Sweden, the origins of 78% of study isolates. We found a significant positive association between the numbers of virulence genes and resistance markers. At least for this clonal group, this argues against the hypothesis that

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antimicrobial resistance and virulence are mutually exclusive, such that strains must give up virulence genes when they become resistant, as has been proposed for fluoroquinolone resistance (28). In this regard, the present fluoroquinoloneresistant isolates actually exhibited virulence scores approximately 0.5 unit greater than those of their fluoroquinolonesusceptible counterparts. Seemingly, members of the O15: K52:H1 clonal group continue to acquire new resistance capabilities yet retain their virulence genes and even acquire new ones. Contributing to the (albeit limited) observed diversity in virulence genotypes, certain isolates possessed diarrhea-associated genes. Specifically, two fecal isolates from patients with diarrhea exhibited genes normally found in ETEC, i.e., the porcine (estAp) and human (estAh) variants of the heat-stable enterotoxin gene. To our knowledge, these two genes have not previously been detected in typical ExPEC. This situation recalls the finding of (diarrheagenic) enteroaggregative E. coli within a lineage, ST394, that is closely related to “clonal group A” (ST69), which is another multiresistant ExPEC clonal group from phylogenetic group D that exhibits a consensus virulence profile similar to that of the O15:K52:H1 clonal group (29). However, in our study, the diarrhea-associated virulence traits were actually encountered among members of the ExPEC lineage per se, not in a separate but closely related lineage (as with clonal group A and ST394). The increasing diversity over time of biotypes within the O15:K52:H1 clonal group, although of uncertain clinical significance, warrants attention. Biotype C (Lac⫺) was particular interesting because of its statistical association with diarrhea, estAp, and fluoroquinolone resistance. Notably, in a recent Italian study of fluoroquinolone-resistant European cystitis isolates (2), the 16 identified O15:K52:H1 clonal group members were all non-lactose-fermenting, like the present biotype C isolates. Biotype C thus may represent an emerging subset within the O15:K52:H1 clonal group that exhibits enhanced fitness for multiple pathogenic niches. All 100 study isolates, which were selected without regard for host species, were from humans. Given the many animalsource E. coli isolates in the WHO center’s collection, this strict segregation by host group suggests strongly that the O15: K52:H1 clonal group is preferentially associated with humans over animals. Indeed, to our knowledge, E. coli O15:K52:H1 has never been isolated from either animals or meat products, although this could simply reflect limited sampling. A potential animal origin of certain human-associated multidrug-resistant uropathogenic E. coli strains has been proposed (18, 25) but remains unproven. The absence of animal isolates in the present population argues against a food animal reservoir for E. coli O15:K52:H1. In conclusion, this study demonstrates that over the more than 3 decades since its first known appearance, the O15: K52:H1 clonal group has maintained a fairly stable virulence profile. In contrast, its antimicrobial resistance profile has become progressively more extensive, coincident with the appearance of a distinctive minor biotype (biotype C) with abundant antimicrobial resistance markers and diarrhea-associated virulence genes. These findings suggest that the O15:K52:H1 clonal group could become even more extensively resistant to antimicrobials while maintaining, or even expanding, its viru-

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lence potential. This possibility, which conflicts with the established negative association between virulence and resistance in E. coli, is a very worrying prospect, particularly if other emerging multiresistant clonal groups of Enterobacteriaceae (e.g., the recently recognized O25:H4 ST131 clonal group [2]) follow a similar pattern. ACKNOWLEDGMENTS We thank Niels Frimodt-Møller (Statens Serum Institut) for antimicrobial susceptibility testing and Susanne Jespersen (Statens Serum Institut) for excellent technical assistance. Dave Prentiss (VA Medical Center) prepared the figures. This material is based upon work supported in part by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (J.R.J.). REFERENCES 1. Beutin, L., M. A. Montenegro, I. Ørskov, F. Ørskov, J. Prada, S. Zimmermann, and R. Stephan. 1989. Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J. Clin. Microbiol. 27:2559–2564. 2. Cagnacci, S., L. Gualco, E. Debbia, G. C. Schito, and A. Marchese. 2008. European emergence of ciprofloxacin-resistant Escherichia coli clonal groups O25:H4⫺ ST 131 and O15:K52:H1 causing community-acquired uncomplicated cystitis. J. Clin. Microbiol. 46:2605–2612. 3. Clermont, O., S. Bonarcorsi, and E. Bingen. 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66:4555–4558. 4. Gerner-Smidt, P. 1992. Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumanii complex. J. Clin. Microbiol. 30:2680–2685. 5. Jerse, A. E., J. Yu, B. D. Tall, and J. B. Kaper. 1990. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87:7839–7843. 6. Johnson, J. R., A. Gajewski, A. J. Lesse, and T. A. Russo. 2003. Extraintestinal pathogenic Escherichia coli as a cause of invasive non-urinary extraintestinal infections. J. Clin. Microbiol. 4:5798–5802. 7. Johnson, J. R., A. L. Stell, F. Scheutz, T. T. O’Bryan, T. A. Russo, U. B. Carlino, C. Fasching, J. Kavle, L. Van Dijk, and W. Gaastra. 2000. Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex polymerase chain reaction-based assay. Infect. Immun. 68:1587–1599. 8. Johnson, J. R., A. L. Stell, T. T. O’Bryan, N. Kuskowski, B. Nowicki, C. Johnson, J. N. Maslow, A. Kaul, J. Kavle, and G. Prats. 2002. Global molecular epidemiology of the O15:K52:H1 extraintestinal pathogenic Escherichia coli clonal group: evidence of distribution beyond Europe. J. Clin. Microbiol. 40:1913–1923. 9. Johnson, J. R., and A. L. Stell. 2000. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J. Infect. Dis. 181:261–272. 10. Johnson, J. R., and J. J. Brown. 1996. A novel multiply primed polymerase chain reaction assay for identification of variant papG genes encoding the Gal(alpha 1-4) Gal-binding PapG adhesins of Escherichia coli. J. Infect. Dis. 173:920–926. 11. Johnson, J. R., K. L. Owens, C. R. Clabots, S. J. Weissman, and S. B. Cannon. 2006. Phylogenetic relationships among clonal groups of extraintestinal pathogenic Escherichia coli as assessed by multi-locus sequence analysis. Microbes Infect. 8:1702–1713. 12. Johnson, J. R., K. Owens, M. Sabate, and G. Prats. 2004. Rapid and specific detection of the O15:K52:H1 clonal group of Escherichia coli by genespecific PCR. J. Clin. Microbiol. 42:3841–3843. 13. Kado, C. I., and S.-T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365–1373. 14. Kauffmann, F. 1966. The Bacteriology of Enterobacteriaceae, p. 1–400. Munksgaard, Copenhagen, Denmark. 15. Kjaeldgaard, P. B., B. Nissen, N. Lange, and H. Laursen. 1986. Evaluation of Minibact, a new system for rapid identification of Enterobacteriaceae. Comparison of Minibact, Micro-ID and API 20E with a conventional method as reference. APMIS 94:57–61. 16. Konowalchuk, J., J. I. Speirs, and S. Stavric. 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18:775–779. 17. Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. M. McCowen. 1978. A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1:417–420. 18. Manges, A. R., S. P. Smith, B. J. Lau, C. J. Nuval, J. N. Eisenberg, P. S. Dietrich, and L. W. Riley. 2007. Retail meat consumption and the acquisition of antimicrobial resistant Escherichia coli causing urinary tract infections: a case-control study. Foodborne Pathog. Dis. 4:419–431.

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