Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the Whole Genome PCR scanning.

Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains.     

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.     

Chromosome of the enterohemorrhagic Escherichia coli O157:H7; comparative analysis with K-12 MG1655 revealed the acquisition of a large amount of foreign DNAs.

A complete Xba I and Bln I cleavage map was constructed for the chromosome of an enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain isolated from an outbreak in Sakai City, Japan, in 1996. A comparative chromosome analysis with E. coli K-12 strain MG1655 was made. The EHEC chromosome was approximately 5600 kb in length, 1 Mb larger than that of MG1655. Despite the marked difference in chromosome length, the location and direction of seven rRNA operons of the EHEC strain were similar to those for MG1655. Overall organization of genes common in both strains is also highly conserved. Chromosome expansion was observed throughout the EHEC chromosome, albeit in an uneven manner. A large portion of the chromosome enlargement was observed in the region surrounding the replication terminus, particularly in a segment containing the terA locus. Sample sequencing of 3627 random shotgun clones suggested the presence of approximately 1550 kb strain-specific DNAs on the EHEC chromosome, most of which are likely to be of foreign origin.     

Low level of cross-resistance between triclosan and antibiotics in Escherichia coli K-12 and E. coli O55 compared to E. coli O157

Misuse of biocides has encouraged the emergence of resistance and cross-resistance in certain strains. This study investigated resistance of triclosan-adapted Escherichia coli K-12 and E. coli O55 to antimicrobial agents and compared these to E. coli O157:H7. Cross-resistance in E. coli K-12 and E. coli O55 was observed however to a lesser extent than in E. coli O157:H7. Triclosan-adapted E. coli K-12 demonstrated cross-resistance to chloramphenicol, whereas triclosan-adapted E. coli O55 exhibited resistance to trimethoprim. In comparison, E. coli O157:H7 was resistant to chloramphenicol, tetracycline, amoxicillin, amoxicillin/clavulanic acid, trimethoprim, benzalkonium chloride and chlorohexidine suggesting strain specific rather than general resistance mechanisms.     

Characterization of the distal tail fiber locus and determination of the receptor for phage AR1, which specifically infects Escherichia coli O157:H7

Phage AR1 is similar to phage T4 in several essential genes but differs in host range. AR1 infects various isolates of Escherichia coli O157:H7 but does not infect K-12 strains that are commonly infected by T4. We report here the determinants that confer this infection specificity. In T-even phages, gp37 and gp38 are components of the tail fiber that are critical for phage-host interaction. The counterparts in AR1 may be similarly important and, therefore, were characterized. The AR1 gp37 has a sequence that differs totally from those of T2 and T4, except for a short stretch at the N terminus. The gp38 sequence, however, has some conservation between AR1 and T2 but not between AR1 and T4. The sequences that are most closely related to the AR1 gp37 and gp38 are those of phage Ac3 in the T2 family. To identify the AR1-specific receptor, E. coli O157:H7 was mutated by Tn10 insertion and selected for an AR1-resistant phenotype. A mutant so obtained has an insertion occurring at ompC that encodes an outer membrane porin. To confirm the role of OmpC in the AR1 infection, homologous replacement was used to create an ompC disruption mutant (RM). When RM was complemented with OmpC originated from an O157:H7 strain, but not from K-12, its AR1 susceptibility was fully restored. Our results suggest that the host specificity of AR1 is mediated at least in part through the OmpC molecule.     

EhaA is a novel autotransporter protein of enterohemorrhagic Escherichia coli O157:H7 that contributes to adhesion and biofilm formation

Autotransporter (AT) proteins have been identified in many Gram-negative pathogens and are unique in that their primary sequence is sufficient to direct their transport across the bacterial membrane system. Where characterized they are uniformly associated with virulence. Using conserved AT motifs as a search tool, four putative AT proteins were identified in the Enterohemorrhagic Escherichia coli O157:H7 EDL933 genome. The genes encoding these proteins (z0402/ ehaA , z0469/ ehaB , z3487/ ehaC and z3948/ ehaD ) were PCR amplified, cloned and expressed in an E. coli K-12 MG1655 flu background. Preliminary characterization revealed that ehaA , ehaB and ehaD encode proteins associated with increased biofilm formation. One of these genes ( ehaA ) resides on a genomic island in E. coli O157:H7 strains EDL933 and Sakai. Over-expression of EhaA in E. coli K-12 demonstrated it is located at the cell surface and resulted in the formation of large cell aggregates, promoted significant biofilm formation and mediated adhesion to primary epithelial cells of the bovine terminal rectum. The expression of ehaA was demonstrated in E. coli EDL933 by RT-PCR. An EhaA-specific antibody revealed the EhaA protein was expressed in 24/50 generic Shiga toxin-producing E. coli (STEC) strains of various serotypes including O157:H7. However, the deletion of ehaA from E. coli EDL933 and a STEC strain from serotype O111:H^– did not affect biofilm growth. Our results suggest that EhaA may contribute to adhesion, colonization and biofilm formation by E. coli O157:H7 and possibly other STEC serotypes.     

Collagen-like proteins in pathogenic E. coli strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Molecular characterization of Irish E. coli O157:H7 isolates of human, bovine, ovine and porcine origin

Aims:  To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin.
Methods and Results:  Escherichia coli O157:H7 isolates were compared using (i) PFGE Xba I patterns, (ii) PCR profiles of virulence genes and (iii) the DNA sequences of genes reported to play a role in pathogenicity. The 77 E. coli O157:H7 isolates demonstrated 49 different PFGE patterns of which, eight were common to multiple isolates, and the remaining 41 were distinct. Isolates of different origin did not correlate, except for one cluster consisting of two human and two beef isolates. The majority of animal isolates had the same PCR profiles of virulence genes as those isolated from clinical patients. Single nucleotide polymorphisms (SNPs) were identified in the sequence of a 255-bp region of the vtx2 subunit A gene.
Conclusions:  Six SNPs were detected in the vtx2 A gene, defining four different haplotypes. One nonsynonymous substitution encoded for an amino acid change from glutamic to aspartic acid.
Significance and Impact of the Study:  Results indicate that although E. coli O157:H7 isolates of differing origin were distinct by PFGE, the DNA sequences of the main virulence genes associated with human clinical illness were conserved.     

Genomic divergence of Escherichia coli strains: evidence for horizontal transfer and variation in mutation rates.

This report describes the sequencing in the Escherichia coli B genome of 36 randomly chosen regions that are present in most or all of the fully sequenced E. coli genomes. The phylogenetic relationships among E. coli strains were examined, and evidence for the horizontal gene transfer and variation in mutation rates was determined. The overall phylogenetic tree indicated that E. coli B and K-12 are the most closely related strains, with E. coli O157:H7 being more distantly related, Shigella flexneri 2a even more, and E. coli CFT073 the most distant strain. Within the B, K-12, and O157:H7 clusters, several regions supported alternative topologies. While horizontal transfer may explain these phylogenetic incongruities, faster evolution at synonymous sites along the O157:H7 lineage was also identified. Further interpretation of these results is confounded by an association among genes showing more rapid evolution and results supporting horizontal transfer. Using genes supporting the B and K-12 clusters, an estimate of the genomic mutation rate from a long-term experiment with E. coli B, and an estimate of 200 generations per year, it was estimated that B and K-12 diverged several hundred thousand years ago, while O157:H7 split off from their common ancestor about 1.5-2 million years ago.     

Gene conservation and loss in the mutS-rpoS genomic region of pathogenic Escherichia coli

The extent and nature of DNA polymorphism in the mutS-rpoS region of the Escherichia coli genome were assessed in 21 strains of enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) and in 6 strains originally isolated from natural populations. The intervening region between mutS and rpoS was amplified by long-range PCR, and the resulting amplicons varied substantially in length (7.8 to 14.2 kb) among pathogenic groups. Restriction maps based on five enzymes and sequence analysis showed that strains of the EPEC 1, EPEC 2, and EHEC 2 groups have a long mutS-rpoS region composed of a approximately 6.0-kb DNA segment found in strain K-12 and a novel DNA segment ( approximately 2.9 kb) located at the 3' end of rpoS. The novel segment contains three genes (yclC, pad1, and slyA) that occur in E. coli O157:H7 and related strains but are not found in K-12 or members of the ECOR group A. Phylogenetic analysis of the common sequences indicates that the long intergenic region is ancestral and at least two separate deletion events gave rise to the shorter regions characteristic of the E. coli O157:H7 and K-12 lineages.     

Identification of a type III secretion system in uropathogenic Escherichia coli

To determine virulence-related genes in uropathogenic Escherichia coli (UPEC) showing invasiveness to T-24 bladder cancer cells, genomic subtractive hybridization was performed between a highly invasive and a less invasive strain. Forty-nine DNA fragments were isolated from the invasive strain. One of them showed homology with Salmonella invA gene. By chromosomal walking of the strain, a type III secretion system that has been described in E. coli O157:H7 was identified on the genome of the invasive strains. Three strains out of 100 UPEC isolates had a type III secretion system inserted at 64 min of the chromosome, corresponding to E. coli K-12 MG1655. This finding suggested that the type III secretion system could play a part in uropathogenicity of UPEC.     

Design and evaluation of PCR primers which differentiate Escherichia coli O157:H7 and related serotypes

Aims:  To develop methods to differentiate Escherichia coli O157:H7 and related serotypes by the use of amplicon length polymorphism (ALP) analysis based on identifying DNA sequence deletions within highly homologous regions of three sequenced E. coli strains.
Methods and Results:  Potential primer locations along the ancestral genomic backbone were identified and evaluated against three sequenced genomes and then applied to a reference set of pathogenic E. coli strains. All 16 primer combinations generated the expected diagnostic fragments as predicted for the E. coli K12 MG1655, O157:H7 EDL933, and O157:H7B Sakai genomes.
Conclusions:  This study defines a collection of primers distributed along the length of the E. coli genome that were applied to ALP analysis methods to successfully differentiate between serotypes of E. coli O157:H7 and other E. coli serotypes.
Significance and Impact of the Study:  ALP-PCR analysis method was validated as an independent method of classification when compared with that of rep-PCR. The principles underlying ALP analysis can be readily applied for the detection and differentiation of other closely related microbial species because of the abundance of complete DNA sequence data for a large number of microbial genomes.     

Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells

When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12 strains of E. coli do not bind to sprouts under similar conditions. The roles of E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and exopolysaccharides in binding to sprouts were examined. An LPS mutant had no effect on the binding of the pathogenic strain. Cellulose synthase mutants showed a significant reduction in binding; colanic acid mutants were more severely reduced, and binding by poly-beta-1,6-N-acetylglucosamine (PGA) mutants was barely detectable. The addition of a plasmid carrying a cellulose synthase gene to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps biosynthesis genes had only a marginal effect on the binding of K-12 bacteria. However, the introduction of the same plasmid allowed Sinorhizobium meliloti and a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments. These results suggest that although multiple redundant protein adhesins are involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides required for binding are not redundant and each polysaccharide may play a distinct role. PGA, colanic acid, and cellulose were also required for biofilm formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7 to mammalian cells.     

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.     

In vitro selection of Escherichia coli O157:H7-specific RNA aptamer

Escherichia coli (E. coli) O157:H7 is a major foodborne pathogen that causes life-threatening symptoms in humans worldwide. To rapidly and properly identify the pathogen and avoid its toxic effects, ligands which can directly and specifically bind to the virulent E. coli O157:H7 serotype should be identified. In this study, a RNA aptamer-based ligand which can specifically distinguish the pathogen E. coli O157:H7 from others was developed by a subtractive cell-SELEX method. To this end, an RNA library was first incubated with the E. coli K12 strain, and the RNAs binding to the strain were discarded. The precluded RNAs were then used for the selection of O157:H7-specific aptamers. After 6 rounds of the subtractive cell-SELEX process, the selected aptamer was found to specifically bind to the O157:H7 serotype, but not to the K12 strain. This was evidenced by aptamer-immobilized ELISA, real-time PCR analysis, or an aptamer-linked precipitation experiment. Importantly, the isolated RNA aptamer that distinguishes between the virulent serotype and the nonpathogenic strain specifically bound to an O157:H7-specific lipopolysaccharide which includes the O antigen. This novel O157:H7-specific aptamer could be of potential application as a diagnostic ligand against the pathogen-related food borne illness.     

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.     

The IS1 elements in Shigella boydii: horizontal transfer,vertical inactivation and target duplication

IS1(SB) and its two variants were identified as the major and minor IS1 elements in Shigella boydii. The nucleotide sequences of IS1(SB), IS1(O157:H7) from Escherichia coli O157:H7 and IS1F from E. coli K12 suggest that these IS1 elements had been horizontally transferred among S. boydii and E. coli O157:H7 and K12. The two IS1(SB) variants and IS1(O157:H7) have transposition activities 7- to 86-fold less than that of IS1(SB), whereas IS1F has little transposition activity. Analysis of the flanking sequences of IS1(SB) and its two variants in S. boydii revealed the nature of regional specificity of the target sites and the sequence dependence of 8 and 9 bp target duplications, for which a model is presented.