Detection and Identification by PCR of a Highly Virulent Phylogenetic Subgroup among Extraintestinal Pathogenic Escherichia coli B2 Strains

Closely related Escherichia coli B2 strains O1:K1, O2:K1, O18:K1,and O45:K1 constitute a major subgroup causing extraintestinalinfections. A DNA pathoarray analysis was used to develop aPCR specific for this subgroup that was included in the multiplexphylogenetic-grouping PCR method. Our PCR may serve to identifythis virulent subgroup among different ecological niches.

INTRODUCTION

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Introduction

Escherichia coli is the main bacterial constituent of mammalianand avian gut aerobic microflora and a major cause of extra-intestinalinfections. E. coli strains causing extraintestinal infections(extraintestinal pathogenic E. coli [ExPEC]) belong mainly tothe phylogenetic group B2 (4, 5, 17). Among the B2 ExPEC group,strains harboring serotypes O2:K1, O18:K1, and O1:K1 have beenshown to predominate (2, 21, 22) and to be closely related,according to several methods, including, recently, multilocussequence typing (MLST) (1, 32), and they therefore representa major human ExPEC phylogenetic subgroup. This subgroup includes,notably, the worldwide O18:K1:H7 strains causing cystitis andneonatal meningitis (18). We have previously shown that B2 strainsbelonging to these serotypes share the same ribotype that wedesignated B2₁ (7). The B2₁ ribotype also contains the neonatalmeningitis O45:K1:H7 serotype strains that predominate in Franceand that are also present elsewhere in Europe (14, 29). Finally,analyzing several clinical collections of E. coli strains, wefound that B2₁ was the ribotype most frequently encounteredamong urosepsis strains infecting non-host-compromised adultsand young infants as well as among meningitis strains (5, 9).

Serogroup strains O2:K1, O18:K1, and O1:K1, similar to thoseof human ExPEC, also cause invasive diseases in animals, notablyfatal avian septicemia (27, 33). Moreover, O45:K1:H7 strainsresembling human neonatal meningitis strains have been sporadicallydescribed in cases of severe avian dermatitis (29). Thus, ithas been suggested that poultry may be a vehicle for human ExPECinfection (27, 29).

All these observations imply that ribotype B2₁ strains mightprovide a key to understanding the pathogenetic mechanisms ofinvasive E. coli infections. However, ribotyping and MLST arecostly and time consuming and are not suited to large-scalestudies of the prevalence of this subgroup among human and animalExPEC or to its detection within complex microflora.

In this study, we first applied MLST to published collectionsof B2₁ strains (O1:K1, O2:K1, O18:K1, and O45:K1) in order todetermine the sequence type(s) (ST) to which ribotype B2₁ corresponds.We then used subtractive DNA pathoarray analysis to developa PCR-based tool for the rapid identification of this highlyvirulent clonal group. Finally, we tested the capacity of thisPCR method to detect these strains directly in human stools.

Highly virulent strains harboring ribotype B2₁ share a unique ST.

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Highly virulent strains...

To establish the correspondence between ribotype B2₁ and MLSTdata, we selected 23 and 16 E. coli strains of ribotype B2₁and non-B2₁, respectively, from clinical and reference collections(Table 1) (2, 6, 7, 9, 24, 31). MLST was performed as describedby Whittam et al. at the EcMLST website, using seven housekeepinggenes (http://www.shigatox.net) (26).

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TABLE 1. E. coli strains belonging to ribotype B2₁ or to other ribotypes used for MLST and DNA pathoarray analyses

All B2₁ strains had identical DNA sequences in the seven genesstudied, corresponding to ST 29 of the EcMLST database, an STpreviously attributed to only two strains, including the E.coli O18:K1:H7 neonatal meningitis (ECNM) strain RS218. Thisshowed that the "O1, O2, O18:K1" clonal group (3, 30, 32) alsoencompasses a fourth serotype, 045:K1. This clonal group isdesignated B2₁/ST29 throughout this report.

DNA pathoarray-based identification of an svg open reading frame specific for the B2₁/ST29 clonal group.

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Dna pathoarray-based...

Open reading frames (ORFs) specific for O18:K1, without homologson the E. coli K-12 MG1655 chromosome, were amplified by PCRwith primers based on the incomplete chromosomal sequence ofthe O18:K1 ECNM strain RS218 (www.genome.wisc.edu) (2, 6, 28),because the UTI89 (uropathogenic E. coli isolate O18:K1:H7)sequence (11) was not yet available. DNA fragments specificfor strain RS218 were sought in silico by using sequences ofclones (GenBank accession numbers AF222070 to AF222307) generatedby subtractive hybridization between O18:K1:H7 ECNM and nonpathogenicE. coli strains (10). Specific ORFs were amplified in sectionsof about 500 bp. The amplicons of 300 known or putative ORFsgenerated by PCR from chromosomal DNA were spotted in duplicateby a robot (Eurogentec, Belgium) on nylon membranes which werethen hybridized and analyzed as previously described (25).

On our DNA array, only one ORF, of unknown function and codingfor a hypothetical protein 277 amino acids long with no significanthomology in the databases (now corresponding to GenBank accessionnumber ABE08649 for E. coli strain UTI89) (11), hybridized withall the B2₁ strains and with none of the strains belonging toother groups and subgroups. We then assessed the specificityof this ORF, which we designated specific for virulent subgroup(svg) ORF, by using a PCR method. A pair of primers (svg.1,5'-TCCGGCTGATTACAAACCAAC-3'; and svg.2, 5'-CTGCACGAGGTTGTAGTCCTG-3')were designed to amplify a 434-bp fragment of the svg ORF. ThisPCR was then included, together with uidA (a ß-glucuronidasegene) as a control amplification (16), in our triplex PCR methodfor phylogenetic group affiliation (12), with a modified protocol.Briefly, PCR was carried out in a 50-µl volume with 25µl of 2x QIAGEN Multiple PCR Master Mix (QIAGEN, Courtaboeuf,France), 5 µl of 5x Q-solution, 1 µM of each primer,and 5 µl of bacterial lysate. PCR was performed as follows:DNA denaturation and polymerase activation for 15 min at 95°C;30 cycles of 30 s at 94°C, 90 s at 55°C, and 90 s at72°C; and a final extension step for 10 min at 72°C.Samples were electrophoresed as previously described (8) (Fig.1A). This PCR screening test was then applied to 340 E. colistrains (including 97 strains of ribotype B2₁) belonging topreviously published collections (5, 7, 9) (Table 2). In addition,13 avian pathogenic E. coli (APEC) strains were assayed by PCR.Eight of them were B2 strains belonging to ribotype B2₁: oneO1:K1 strain (LDA 6042253), three O2:K1 strains (LDA 5063391,LDA 5067912, and LDA 6081105), and four O45:K1 strains (BEN1068,BEN1082, BEN1090, and BEN1354). The five other APEC strainswere group D strains belonging to serogroup O1 (LDA 6072791)and to serogroup O45 (BEN0058, BEN0214, BEN0289, and BEN0456).Strains "LDA" and strains "BEN" were kindly provided by HervéMorvan (Laboratoire de développement et d'analyses desCôtes d'Armor, Ploufragan, France) and Maryvonne Moulin-Schouleur(INRA, Centre de Tours, UR1282 IASP, Pathogenie Bacterienne,Nouzilly, France), respectively. The results showed perfectspecificity and sensitivity for the svg PCR for the identificationof B2₁/ST29 strains (Table 2).

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FIG. 1. (A) Pentaplex PCR combining the amplification of a 434-bp fragment of the svg ORF specific for highly virulent B2₁/ST29 E. coli strains; uidA (ß-glucuronidase gene) was used as a control (16), and chuA (outer membrane hemin receptor gene) and yjaA (unknown function) and DNA fragment TspE4.C2 were used for phylogenetic group affiliation (12). M, molecular weight marker (100-bp DNA ladder; New England BioLabs); lanes 1, 2 and 3, strains belonging to phylogenetic groups A, B1, and D, respectively; lane 4, reference strain belonging to the phylogenetic group B2 but not to B2₁/ST29 (strain CFT073); lane 5, meningitis strain belonging to B2₁/ST29 (strain S88). (B) Biplex PCR combining the amplification of a 434-bp fragment of ORF svg (upper band) together with uidA (ß-glucuronidase gene, lower band), used as a control (16), for the detection of highly virulent B2₁/ST29 E. coli strains directly in stool samples. Lanes 1 and 3, stool samples harboring B2₁/ST29 E. coli strains (svg-positive PCR); lane 2 and 4, stool samples harboring E. coli strains not belonging to B2₁/ST29 subgroup; lane 5, negative control.

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TABLE 2. Results of svg ORF PCR specific for highly virulent B2₁/ST29 E. coli strains, applied to E. coli collections and other Enterobacteriaceae species

svg amplification for B2₁/ST29 strain identification in a complex microflora.

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svg amplification for B21/ST29...

We then tested the capacity of the svg ORF simplex PCR to detectB2₁/ST29 strains in a complex microflora. Specificity was firsttested with 52 strains belonging to different Enterobacteriaceaespecies and with 30 stool samples that were culture negativefor E. coli, as described below (Table 2). None of these strainsor stool samples was positive, suggesting that the svg ORF isrestricted to E. coli. We then applied the new method to 92stool samples from healthy children who were culture positivefor E. coli on two different types of samples: (i) 10 coloniesof E. coli strains obtained by stool cultured on Uriselect 4chromogenic medium (Bio-Rad, Marnes-la-Coquette, France) and(ii) whole-stool samples, as previously described (23). Briefly,about 100 µg of stool was mixed with 9 ml of peptone waterand incubated at 37°C for 4 h. After centrifugation (1500rpm for 10 min), the supernatant was boiled and 5 µl wasused as template DNA for the PCR. Amplification of the geneuidA was used to check for PCR inhibitors (Fig. 1B). The svgPCR was positive for 10% of cases (9/92) of the predominantisolates and 14% (13/92) of the stool samples. Four specimenswere thus PCR positive only for stool samples, suggesting thatB2₁/ST29 strains may be a subdominant E. coli population inthe microflora of some healthy children.

Serotype strains O1:K1, O2:K1, and O18:K1 encountered in humanextraintestinal infections were recently shown to cluster withina single sequence type (ST95), based on the MLST method describedby Achtman et al. (www.mlst.net) (19, 30, 32). However, severalother sets of MLST target genes have been published, and noreference set of genes has yet been established (15, 26). Inour study, using the set of genes cited by Whittam et al., weconfirmed that strains O1:K1, O2:K1, and O18:K1 (all ribotypeB2₁ in our study) clustered in a single ST (EcMLST, ST29) andthat this clonal group includes meningitis strains of serotypeO45:K1. Considering that (i) ST29 corresponds perfectly to ribotypeB2₁, the leading ribotype causing septicemia in non-host-compromisedhumans (5, 7, 9) and (ii) since serotype strains O1:K1, O2:K1,O18:K1, and O45:K1 are major causes of fatal bacteremia in birds(29, 33), it would be useful to be able to detect this highlyvirulent ExPEC subgroup both rapidly and simply. By using DNAarray technology, we developed a PCR test for inclusion in ourmultiplex phylogenetic grouping PCR (12), one of the most widelyused methods for determining the main phylogenetic groups (A,B1, B2, and D). The resulting pentaplex PCR test will be helpfulfor determining in a unique reaction both the main phylogeneticgroup of a strain and its affiliation with the highly virulentsubgroup B2₁/ST29.

We also showed that an svg PCR applied to stool samples is moresensitive than culture for B2₁/ST29 E. coli strains. These resultsare consistent with previous reports suggesting that E. colistrains harboring virulence factors may represent a minor subpopulationof the fecal microflora that might not be detected by colonysampling (20). To our knowledge, this is the first PCR-basedapproach to permit the detection of E. coli strains belongingto a particular phylogenetic subgroup directly within a complexbacterial population. In addition to its value as an epidemiologicaltool, our svg PCR method may prove useful in several fieldsof human and veterinary medicine. For example, it could servefor early identification (and treatment) of human neonates colonizedby such E. coli strains and to screen avian hosts in industrialanimal husbandry. Finally, this method could serve as a cost-effectivefirst-line screening test in an E. coli serogrouping assay,as B2₁/ST29 strains belong mainly to only four serogroups.

ACKNOWLEDGMENTS

We thank Colin Tinsley for helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Service de Microbiologie, Hôpital Robert Debré, 48 Bd Sérurier, 75395 Paris cedex 19, France. Phone: 33 1 40 03 23 40. Fax: 33 1 40 03 24 50. E-mail: edouard.bingen{at}rdb.ap-hop-paris.fr.

Published ahead of print on 9 February 2007.

REFERENCES

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