Molecular evolution of large virulence plasmid in Shigella clones and enteroinvasive Escherichia coli.

Three genes, ipgD, mxiC, and mxiA, all in the invasion region of the Shigella virulence plasmid, were sequenced from strains representing a range of Shigella serotypes and from two enteroinvasive Escherichia coli (EIEC) isolates. The plasmids can be classified into two relatively homogeneous sequence forms which are quite distinct. pINV A plasmids are found in Shigella flexneri strains F6 and F6A, S. boydii strains B1, B4, B9, B10, B14, and B15, S. dysenteriae strains D3, D4, D6, D8, D9, D10, and D13, and the two EIEC strains (M519 and M520). pINV B plasmids are present in S. flexneri strains F1A, F2A, F3A, F3C, F4A, and FY, two S. boydii strains (B11 and B12), and S. sonnei. The D1 pINV plasmid is a recombinant with ipgD gene more closely related to those of pINV A but with mxiA and mxiC genes more closely related to those of pINV B. The phylogenetic relationships of the plasmid and those of the chromosomal genes of Shigella strains are largely consistent. The cluster 1 and cluster 3 strains tested (G.M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000) have pINV A and pINV B plasmids, respectively. However, of the three cluster 2 strains (B9, B11, and B15), B9 and B15 have pINV A while B11 has a pINV B plasmid. Those Shigella (D8 and D10 and S. sonnei) and EIEC strains which do not group with the main body of Shigella strains based on chromosomal genes were found to have plasmids belonging to one or the other of the two types and must have acquired these by lateral transfer.     

Comparison of two major forms of the Shigella virulence plasmid pINV: positive selection is a major force driving the divergence

All Shigella and enteroinvasive Escherichia coli (EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneri serotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneri serotype 6 other than insertion sequence or related sequences and compared it with the pINV B form. More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions. This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative. There are also genes absent in the S. flexneri serotype 6 plasmid, including the sepA gene encoding serine protease, the major secreted protein of S. flexneri serotype 2a, and the stbAB genes, which encode one of the two partition systems found in S. flexneri serotype 5. The incompatibility of the two pINV forms appears to be due to either small differences in the mvpAT postsegregational killing system or the presence of an unknown system in pINVA.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies.

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups.

Shigella species and enteroinvasive Escherichia coli strains carry a large (120- to 140-megadalton) plasmid called pINV, which contains genes essential for the invasiveness of these pathogens. Hybridization with specific probes derived from the RepFIC and RepFIB replicons of the IncF1 Ent plasmid P307 showed that pINVs present in 35 clinical isolates are homologous with RepFIC but not RepFIB, regardless of the serogroup of the Shigella or E. coli strain. RepFIC of P307, in turn, is very similar to RepFIIA replicons of IncFII R plasmids. These and other related replicons constitute the RepFIIA family. With one pINV, pWR110, a plasmid of Shigella flexneri 5, we demonstrated the existence of a functional replicon, RepINV, with a restriction map similar to that of RepFIIA of plasmid R1. We isolated the putative inc RNA coding region of RepINV, which is a major determinant of incompatibility. The nucleotide sequence of the RepINV-inc RNA-coding region was determined and compared with the corresponding sequences of RepFIC and RepFIIA. The differences were small, but apparently were sufficient to affect the target specificity of the inc RNAs, thus rendering the replicons compatible with each other. We conclude that pINVs present in Shigella spp. and enteroinvasive E. coli constitute a homogeneous group, containing one basic replicon that belongs to the RepFIIA family of replicons.     

Sequence analysis of four Shigella boydii O-antigen loci: implication for Escherichia coli and Shigella relationships

Shigella strains are in reality clones of Escherichia coli and are believed to have emerged relatively recently (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567-10572, 2000). There are 33 O-antigen forms in these Shigella clones, of which 12 are identical to O antigens of other E. coli strains. We sequenced O-antigen gene clusters from Shigella boydii serotypes 4, 5, 6, and 9 and also studied the O53- and O79-antigen gene clusters of E. coli, encoding O antigens identical to those of S. boydii serotype 4 and S. boydii serotype 5, respectively. In both cases the S. boydii and E. coli O-antigen gene clusters have the same genes and organization. The clusters of both S. boydii 6 and S. boydii 9 O antigens have atypical features, with a functional insertion sequence and a wzx gene located in the orientation opposite to that of all other genes in S. boydii serotype 9 and an rmlC gene located away from other rml genes in S. boydii serotype 6. Sequences of O-antigen gene clusters from another three Shigella clones have been published, and two of them also have abnormal structures, with either the entire cluster or one gene being located on a plasmid in Shigella sonnei or Shigella dysenteriae, respectively. It appears that a high proportion of clusters coding for O antigens specific to Shigella clones have atypical features, perhaps indicating recent formation of these gene clusters.     

Virulence plasmids of enteroinvasive Escherichia coli and Shigella flexneri integrate into a specific site on the host chromosome: integration greatly reduces expression of plasmid-carried virulence genes.

The ability of enteroinvasive Escherichia coli and Shigella flexneri to cause disease depends on the presence of a large virulence plasmid (pINV). In this report we show that pHN280, the pINV of the O135:K-:H- enteroivasive strain E. coli HN280, and pWR100, the pINV of S. flexneri serotype 5 strain M90T, are able to integrate into a specific site on the host chromosome. pINV-integrated HN280 and M90T strains required methionine (Met-) to grow in minimal medium, were noninvasive, did not produce contact-mediated hemolysin, and had lost the ability to bind Congo red (Crb-) at 37 degrees C. Immunoblots of whole bacterial extracts from pHN280-integrated HN280 derivatives revealed that integration severely reduced the expression of ipa and virG (icsA) plasmid genes. Met- HN280 and M90T derivative strains spontaneously generated Met+ revertants that either contained excised forms of pINV or had lost pINV. Restriction analysis of excised pINVs showed that they either were virtually identical to parental pINVs (precise excision) or had suffered some deletion (imprecise excision). Precisely excised pINVs expressed the full pattern of virulence, whereas imprecisely excised pINVs were always Crb- and noninvasive. The revertion to Met+ was shown to be recA dependent, indicating that homologous plasmid and chromosomal DNA sequences are involved in the integration-excision process. The maintainance of pINV through integration and downregulation of its virulence genes may represent an advantageous mechanism for enteroinvasive bacteria, particularly when they are outside host cells and/or have to face adverse environmental conditions.     

Structure of the Shigella dysenteriae 7 O antigen gene cluster and identification of its antigen specific genes

Shigella strains are human pathogens. The O antigen gene cluster of Shigella dysenteriae O7 was sequenced and analyzed. It contains genes for synthesis of nucleotide sugars including UDP-2-acetamido-2-deoxy-D-galacturonamide, UDP-2-acetamido-2-deoxy-D-galacturonic acid and dTDP-4-amino-4,6-dideoxy-D-glucose. Also found in the gene cluster are genes encoding O unit flippase, O antigen polymerase and sugar transferases. The Escherichia coli O121 O antigen, which is present in an important Shiga toxin-producing strain, has the same structure as that of S. dysenteriae O7, and we found that the gene clusters also had the same genes and organization. Four genes specific to S. dysenteriae O7 and E. coli O121 were identified by PCR screening against representatives of 186 E. coli (including Shigella) O serotypes. E. coli O121 and S. dysenteriae O7 isolates can be distinguished by PCR of the H antigen fliC gene.     

Distribution and structural variation of the she pathogenicity island in enteric bacterial pathogens

Shigella flexneri serotype 2a carries a chromosomal pathogenicity island (PAI), termed the she PAI, that has been implicated in the pathogenesis of diarrhoeal disease. The complete nucleotide sequence and genetic organisation of the she PAI of S. flexneri 2a strain YSH6000T was determined recently. In the current study the distribution and structure of the she PAI was investigated by PCR and Southern analysis in 65 isolates of enteric pathogens including Shigella spp., enterohaemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Yersinia enterocolitica and Salmonella enterica serovar Typhimurium. The study showed that the she PAI has undergone a variety of structural changes, defined by the presence or absence of specific marker genes in the PAI. The she PAI or structural variants of this element were found in all species of Shigella as well as in EIEC, EHEC and EPEC. No evidence of the PAI was found in Y. enterocolitica or Sal. Typhimurium. The structural form of the she PAI that exists in strain YSH6000T was present in all strains of S. flexneri serotype 2a and in some strains of S. flexneri serotypes 2b and 3c. Variants of the PAI that were missing one or more marker regions were found in all species of Shigella and in pathogenic strains of E. coli. In all strains, the PAIs have inserted into either pheV or a phe tRNA gene in another location on the chromosome. It was concluded that the she PAI is one of several closely related genetic elements that have disseminated throughout Shigella and pathogenic strains of E. coli and diverged into distinct stuctural forms.     

Invasiveness of enterobacteria related to the presence of high molecular weight plasmids

A study of 41 strains of Shigella, Escherichia coli, and Salmonella was performed. The presence of high-molecular-weight plasmids, invasiveness in HEp-2 cells and the ability to produce a positive Serény test were tested. Five of the seven strains of Salmonella typhimurium harboured a 62 Md plasmid and invaded HEp-2 cells. Two strains of S. typhimurium and a S. paratyphi-B strain lacked plasmids but were still invasive. Among the 27 strains of Shigella and enteroinvasive E. coli (EIEC) 25 strains harboured a high molecular weight plasmid. 27 of the Shigella/EIEC strains invaded HEp-2 cells and 25 produced a positive Serény test. One strain of Shigella sonnei was invasive in HEp-2 cells and gave a positive Serény test, but plasmids were not demonstrated. Of the eight non-enteropathogenic E. coli 5 strains harboured plasmids of 100 to 140 Md size; only one of the strains invaded HEp-2 cells; none of the strains produced a positive Serény test. The study shows that tests for pathogenicity (Serény test, HEp-2 cell test) are usually positive in the Shigella and EIEC group of bacteria; these bacteria also usually carry a high molecular weight plasmid. However, among non-enteropathogenic bacteria plasmids of 100 to 140 Md size can be observed without any correlation to invasive properties. Genetic information from gene loci located to plasmids and chromosomes is required to give a positive Serény test. The presence of high molecular weight plasmids does not seem to be necessary for expression of in vitro invasiveness of S. typhimurium.     

Development and testing of invasion-associated DNA probes for detection of Shigella spp. and enteroinvasive Escherichia coli.

Genetic determinants of the invasive phenotype of Shigella spp. and enteroinvasive Escherichia coli (EIEC), two common agents of bacillary dysentery, are encoded on large (180- to 210 kilobase), nonconjugative plasmids. Several plasmid-encoded antigens have been implicated as important bacterial ligands that mediate the attachment and invasion of colonic epithelial cells by the bacteria. Selected invasion plasmid antigen (ipa) genes have recently been cloned from Shigella flexneri serotype 5 into the lambda gt11 expression vector. Portions of three ipa genes (ipaB, ipaC, and ipaD) were tested as DNA probes for diagnostic detection of bacillary dysentery. Under stringent DNA hybridization conditions, all three DNA sequences hybridized to a single 4.6-kilobase HindIII fragment of the invasion plasmids of representative virulent Shigella spp. and EIEC strains. No hybridization was detected in isogenic, noninvasive Shigella mutants which had lost the invasion plasmid or had deleted the ipa gene region. Furthermore, these probes did not react with over 300 other enteric and nonenteric gram-negative bacteria tested, including Salmonella, Yersinia, Edwardsiella, Campylobacter, Vibrio, Klebsiella, Aeromonas, Enterobacter, Rickettsia, and Citrobacter spp. and various pathogenic E. coli strains. The use of unique invasion-essential gene segments as probes for the specific detection of invasive dysentery organisms should benefit both epidemiologic and diagnostic analyses of Shigella spp. and EIEC.     

Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic Escherichia coli and Shigella spp

We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E. coli (EAEC) isolates, enterotoxigenic E. coli (ETEC) isolates, enteroinvasive E. coli (EIEC) isolates, Shiga toxin-producing E. coli (STEC) isolates, and Shigella spp. The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx(1) and stx(2) for STEC isolates, and ipaH for EIEC isolates and Shigella spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.     

Molecular characterization of strains of enteroinvasive Escherichia coli O143, including isolates from a large outbreak in Houston, Texas.

A large diarrhea outbreak due to enteroinvasive Escherichia coli (EIEC) serogroup O143 occurring in Houston, Tex., provided the opportunity to investigate aspects of the molecular epidemiology of this and related organisms. This was done by comparing the plasmid patterns and the chromosomal restriction endonuclease digestion patterns by pulsed-field gel electrophoresis (PFGE) of EIEC from the outbreak, other E. coli from the same serogoup (O143), and EIEC isolated from other patients with diarrhea. Among the isolates studied, there was marked restriction fragment length polymorphism. All 3 non-O143 EIEC isolates had very different restriction endonuclease digestion patterns, as did 5 of 5 O143 non-EIEC isolates and 6 of 15 O143 EIEC isolates. Four Houston outbreak O143 EIEC isolates had the same restriction pattern as an O143 EIEC strain isolated 2 months before in Mexico and was nearly identical to another two O143 EIEC Mexican isolates. These related strains also had the same plasmid pattern; however, the presence of only a few plasmid bands, versus the 21 to 30 chromosomal bands seen with PFGE, suggests that plasmid patterns could be a less specific way to distinguish different strains. These results demonstrate that PFGE can distinguish between different E. coli strains of the same serogroup and phenotype. This technique can also identify relatedness within O143 EIEC, and our data suggest the spread of a strain of EIEC from Mexico to Houston, where it caused a large outbreak. PFGE may be useful to study the epidemiology of EIEC.     

Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli.

The products of the ipaB, ipaC, and ipaD genes are involved in the expression of the invasive phenotype in all species of Shigella and enteroinvasive Escherichia coli (EIEC). DNA probes derived from these genes are accurate indicators of the invasive phenotype (M. Venkatesan, J. M. Buysse, E. V. Vandendries, and D. J. Kopecko, J. Clin. Microbiol. 26:261-266, 1988); however, spontaneous loss of the invasion plasmid or selective deletion of invasion-associated genes may restrict the usefulness of such probes as general diagnostic tools. In this study, we report that laboratory-passaged strains of Shigella spp. and EIEC that were invasion and Sereny test negative were unable to hybridize to the ipaC DNA probe. However, a second DNA probe, derived from the Shigella flexneri ipaH gene, a multiple-copy element found on the chromosome and invasion plasmid that encodes a 60-kilodalton antigen, was more sensitive in its ability to detect virulent as well as avirulent shigellae and EIEC. Analysis of colony blots and stool blots from pediatric patients with diarrhea indicated that the ipaH probe was more effective in detecting shigellae and EIEC than was either the ipaC or 17-kilobase EcoRI fragment probe.     

Curli loci of Shigella spp

An unstable chromosomal element encoding multiple antibiotic resistance in Shigella flexneri serotype 2a was found to include sequences homologous to the csg genes encoding curli in Escherichia coli and Salmonella enterica serovar Typhimurium. As curli have been implicated in the virulence of serovar Typhimurium, we investigated the csg loci in all four species of Shigella. DNA sequencing and PCR analysis showed that the csg loci of a wide range of Shigella strains, of diverse serotypes and different geographical distributions, were almost universally disrupted by deletions or insertions, indicating the existence of a strong selective pressure against the expression of curli. Strains of enteroinvasive E. coli (EIEC), which share virulence traits with Shigella spp. and cause similar diseases in humans, also possessed insertions or deletions in the csg locus or were otherwise unable to produce curli. Since the production of curli is a widespread trait in environmental isolates of E. coli, our results suggest that genetic lesions that abolish curli production in the closely related genus Shigella and in EIEC are pathoadaptive mutations.     

Sensitivity and performance characteristics of a direct PCR with stool samples in comparison to conventional techniques for diagnosis of Shigella and enteroinvasive Escherichia coli infection in children with acute diarrhoea in Calcutta, India

As the sensitivity of the conventional techniques for identifying Shigella spp. and enteroinvasive Escherichia coli (EIEC) causing dysentery cases is low, a PCR assay was evaluated in this study. Analytical sensitivity (2 x 10(2) cfu) of the PCR technique was obtained by artificially spiking negative stool samples with a standard strain of S. flexneri type 2, then determining the detection limit. Specificity (100%) of the method was determined by testing a number of known Shigella and EIEC strains and organisms other than Shigella spp. A total of 300 stool samples collected from children with acute diarrhoea was plated on to two selective agar media after enrichment in Luria broth. Shigella spp. were isolated from 7.7% (23 of 300) and EIEC from 1% (3 of 300) patients. All enriched stool samples were subjected to PCR to amplify the target sequence of invasive plasmid antigen (ipa)H locus, a multicopy element found on the chromosome and invasion plasmid. The stool PCR was positive in 24 of the 26 culture-positive and in 22 culture-negative stools, thus detecting the presence of Shigella spp. or EIEC in 15.3% (46 of 300) of diarrhoea cases. When an ial probe was used for colony hybridistion with enriched stool cultures blotted on to membranes, 9.6% (29 of 300) of dysentery cases were identified as being caused by Shigella spp. or EIEC. Thus the sensitivity of enriched stool culture, colony hybridisation and enriched stool PCR was found to be 54%, 60% and 96%, respectively, when each of the methods was compared to the total microbiologically confirmed cases of dysentery. It was also observed that only 38% (48 of 126) of acute bloody dysentery cases actually had shigella or EIEC infection, as confirmed by laboratory methods. Moreover, this PCR assay could identify a number of untypable Shigella strains (Sh OUT), which would have remained undiagnosed had this assay not been used.     

A novel Shigella dysenteriae serovar isolated in Canada

The etiological agent most commonly associated with bacillary dysentery is Shigella. As part of its mandate, the Bacteriology and Enteric Disease Program of Health Canada identifies and serotypes unusual isolates of Shigella received from provincial laboratories of public health. In this report, six unusual isolates from three provinces were analyzed biochemically and serologically using slide and tube agglutinations and molecularly using standard pulsed-filed gel electrophoresis (PFGE), PCR, and PCR-restriction fragment length polymorphism (RFLP) techniques. All six isolates were identical. PFGE analysis grouped these strains; biochemically, they were mannitol negative and consistent with the profile of Shigella. Serologically, these strains produced weak reactions in Shigella dysenteriae serovars 4 and 16 and Escherichia coli O159 and O173 antisera. Molecular serotyping by PCR-RFLP of the rfb gene produced an S. dysenteriae serovar 2/E. coli O112ac pattern. They were positive by PCR for ipaH and ial enteroinvasive genes but negative for all other genes tested. Antiserum was prepared from one of the isolates and tested against Shigella and E. coli reference strains as well as the other isolates. The antiserum reacted with the five remaining isolates and showed cross-reactivity with S. dysenteriae serovars 1, 4, and 16; Shigella flexneri type 3; and E. coli O118, O159, O168, O172, and O173 antigens. Absorbing the sera with E. coli O159 and S. dysenteriae serovar 4 antigen removed all cross-reactions and only slightly reduced the homologous titer. Based on biochemical, molecular, and complete serological analysis, we propose that these six isolates represent a new provisional serovar of S. dysenteriae, type strain BEDP 02-5104.     

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory

A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.     

A new genetic test for the rapid identification of shiga-toxines producing (STEC), enteropathogenic (EPEC) E. coli isolates from children

Routine bacteriological techniques do not allow detection of the most frequent enteric pathogens in young children: enteropathogenic Escherichia coli (EPEC) and shigatoxinogenic E. coli (STEC/EHEC). Since there is no correlation between serotype and pathotype, a genotypic determination is therefore necessary for the identification of these pathogenic strains. We evaluated the Genotype EHEC test (Hain Life Science, Germany), a new rapid system based on DNA multiplex amplification and further hybridization for the detection of shigatoxin stx1, stx2 genes, intimin eae gene and invasin ipaH gene harbored by Shigella and enteroinvasive E. coli (EIEC). E. coli strains of various serogroups isolated from children with acute gastroenteritis, hemorrhagic colitis or hemolytic-uremic syndrome were tested. Their genotypes were first determined by standard in-house PCR. The strains collection included 11 STEC/EHEC (serogroups O157, O111, O26, O91, O-untypable) and nine EPEC (serogroups O26, O157, O55, O126, O127, O-untypable). The same strains were tested with Genotype EHEC. For all the strains, the hybridization banding pattern obtained by Genotype EHEC correlated with their expected genotypic characteristics. No specific equipment is required, except a thermocycler. Absence of electrophoresis system, of ethidium bromide staining and imaging system is a clear-cut advantage of Genotype EHEC. In addition, the short testing time (less than 2 h) optimizes treatment orientation. The Genotype EHEC test allows an easy and reliable identification of EHEC, STEC, EPEC and also EIEC. As such, it is a useful tool for the rapid diagnosis of diarrheal diseases.