Kim Ziebell

Association of Genomic O Island 122 of Escherichia coli EDL 933 with Verocytotoxin-Producing Escherichia coli Seropathotypes That Are Linked to Epidemic and/or Serious Disease

ABSTRACT The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five “seropathotypes” (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an “incomplete” OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001).

Genome evolution in major Escherichia coli O157:H7 lineages

BackgroundGenetic analysis of Escherichia coli O157:H7 strains has shown divergence into two distinct lineages, lineages I and II, that appear to have distinct ecological characteristics, with lineage I strains more commonly associated with human disease. In this study, microarray-based comparative genomic hybridization (CGH) was used to identify genomic differences among 31 E. coli O157:H7 strains that belong to various phage types (PTs) and different lineage-specific polymorphism assay (LSPA) types.ResultsA total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7 strains and 1,751 were variably present or absent. Based on this data, E. coli O157:H7 strains were divided into three distinct clusters, which consisted of 15 lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221), respectively. Eleven different genomic regions that were dominant in lineage I strains (present in ≥80% of lineage I and absent from ≥ 92% of lineage II strains) spanned segments containing as few as two and up to 25 ORFs each. These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78, 83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic backbone region. All four lineage I/II strains were of PT 2 and possessed eight of these 11 lineage I-dominant loci. Several differences in virulence-associated loci were noted between lineage I and lineage II strains, including divergence within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II strains.ConclusionCGH data suggest the existence of two dominant lineages as well as LSPA type and PT-related subgroups within E. coli O157:H7. The genomic composition of these subgroups supports the phylogeny that has been inferred from other methods and further suggests that genomic divergence from an ancestral form and lateral gene transfer have contributed to their evolution. The genomic features identified in this study may contribute to apparent differences in the epidemiology and ecology of strains of different E. coli O157:H7 lineages.

Distribution of Core Oligosaccharide Types in Lipopolysaccharides from Escherichia coli

ABSTRACT In the lipopolysaccharides of Escherichia coli there are five distinct core oligosaccharide (core OS) structures, designated K-12 and R1 to R4. The objective of this work was to determine the prevalences of these core OS types within the species. Unique sequences in the waa (core OS biosynthesis) gene operon were used to develop a PCR-based system that facilitated unequivocal determination of the core OS types in isolates of E. coli. This system was applied to the 72 isolates in the E. coli ECOR collection, a compilation of isolates that is considered to be broadly representative of the genetic diversity of the species. Fifty (69.4%) of the ECOR isolates contained the R1 core OS, 8 (11.1%) were representatives of R2, 8 (11.1%) were R3, 2 (2.8%) were R4, and only 4 (5.6%) were K-12. R1 is the only core OS type found in all four major phylogenetic groups (A, B1, B2, and D) in the ECOR collection. Virulent extraintestinal pathogenic E. coli isolates tend to be closely related to group B2 and, to a lesser extent, group D isolates. All of the ECOR representatives from the B2 and D groups had the R1 core OS. In contrast, commensal E. coli isolates are more closely related to group A, which contains isolates representing each of the five core OS structures. R3 was the only core OS type found in 38 verotoxigenic E. coli (VTEC) isolates from humans and cattle belonging to the common enterohemorrhagic E. coliserogroups O157, O111, and O26. Although isolates from other VTEC serogroups showed more core OS diversity, the R3 type (83.1% of all VTEC isolates) was still predominant. When non-VTEC commensal isolates from cattle were analyzed, it was found that most possessed the R1 core OS type.

Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes

BackgroundAdherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohns Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype.ResultsWe sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohns Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype.ConclusionsUp to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

Molecular subtypes of Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman River watershed, Alberta, Canada

Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from 898 faecal, 43 sewage, and 342 surface water samples from the Oldman River were characterized using bacterial subtyping methods in order to investigate potential sources of contamination of the watershed. Among these pathogens, Campylobacter spp. were the most frequently isolated from faecal, sewage, and surface water samples (266/895, 11/43, and 91/342, respectively), followed by Salmonella (67/898, 8/43, and 29/342, respectively), and E. coli O157:H7 (16/898, 2/43, and 8/342, respectively). Salmonella Rubislaw was the most common serovar isolated from water. This serovar was also isolated from two wild bird species. Most other serovars isolated from water were either not isolated from animals or were isolated from multiple species. E. coli O157:H7 was predominantly isolated from cattle. The most common phage-types of this pathogen from cattle were also the most common among water isolates, and there were exact pulsed field gel electrophoresis and comparative genomic fingerprint matches between cattle, sewage, and water isolates. Campylobacters were commonly isolated from surface waters and faeces from most animal species. Restriction fragment length polymorphism of the Campylobacter flaA gene identified several location and host species-specific (cattle, goose, pig) fingerprints. Molecular subtyping of these bacterial pathogens shows considerable promise as a tool for determining the sources of faecal pollution of water.

Escherichia coli O157:H7 infection in cows and calves in a beef cattle herd in Alberta, Canada

Escherichia coli O157:H7 infection of cows and calves in a naturally-infected beef cattle herd in Alberta, Canada, was investigated over 2 years, encompassing two calf production cycles. In both years of the study, E. coli O157:H7 was isolated from the faeces of cows shortly after but not before parturition in late winter: 6/38 (16%) in 1996 and 13/50 (26%) in 1997. At < 1 week post-partum, 13/52 (25%) calves born in 1997 were shedding the organism. Faecal shedding of E. coli O157:H7 by cows and calves continued over the 7 weeks that they were in the calving pens, with the organism being isolated from the faeces of 2-18% of cows and 23-26% of calves during this period. Five weeks after they were moved onto a native grass pasture, all the calves and all but one cow in 1997 had ceased shedding the organism. When the calves were weaned in the fall, E. coli O157:H7 was isolated from the faeces of 0-1.5% of the calves 1 week prior to weaning and from 6-14% of the calves within 2 weeks after weaning. Parturition, calving pens and weaning appear to be important factors in maintaining E. coli O157:H7 infections in this beef cattle herd. Isolates from cows and calves during the immediate post-partum period were mostly of the same pulsed-field gel electrophoresis (PFGE) type of E. coli O157:H7. Similarly, at weaning a common PFGE type of E. coli O157:H7, which differed slightly from the post-partum PFGE type, was isolated from the calves. These typing data suggest a common source of infection for the animals as well as demonstrate clonal turnover of resident populations of this pathogen.

Genotypic Characterization and Prevalence of Virulence Factors among Canadian Escherichia coli O157:H7 Strains

ABSTRACT In this study, the association between genotypic and selected phenotypic characteristics was examined in a collection of Canadian Escherichia coli O157:H7 strains isolated from humans and cattle in the provinces of Alberta, Ontario, Saskatchewan, and Quebec. In a subset of 69 strains selected on the basis of specific phage types (PTs), a strong correlation between the lineage-specific polymorphism assay (LSPA6) genotype and PT was observed with all strains of PTs 4, 14, 21, 31, 33, and 87 belonging to the LSPA6 lineage I (LSPA6-LI) genotype, while those of PTs 23, 45, 67, and 74 belonged to LSPA6 lineage II (LSPA6-LII) genotypes. This correlation was maintained when additional strains of each PT were tested. E. coli O157:H7 strains with the LSPA6-LI genotype were much more common in the collection than were the LSPA6-LII or lineage I/II (LSPA6-LI/II)-related genotypes (82.6, 11.2, and 5.8%, respectively). Of the strains tested, proportionately more LSPA6-LI than LSPA6-LII genotype strains were isolated from humans (52.7% versus 19.7%) than from cattle (47.8% versus 80.2%). In addition, 96.7% of the LSPA6-LII strains carried the stx2c variant gene, while only 50.0% of LSPA6-LI/II and 2.7% of LSPA6-LI strains carried this gene. LSPA6-LII strains were also significantly more likely to possess the colicin D gene, cda (50.8% versus 23.2%), and have combined resistance to streptomycin, sulfisoxazole, and tetracycline (72.1% versus 0.9%) than were LSPA6-LI strains. The LSPA6 genotype- and PT-related characteristics identified may be important markers of specific ecotypes of E. coli O157:H7 that have unique epidemiological and virulence characteristics.

BACTERIAL PATHOGENS IN RURAL WATER SUPPLIES IN SOUTHERN ALBERTA, CANADA

Raw river and irrigation water in the Oldman River Basin in southern Alberta was tested for the presence of two bacterial pathogens, Escherichia coli O157:H7 and Salmonella spp., over the last 2 yr (2000–2001). The number of E. coli O157:H7 and Salmonella spp. isolated from raw water peaked during the summer months. While E. coli O157:H7 was only isolated from 11/802 (1.3%) of raw water samples over the entire sampling season in 2000 and from 16/806 (2.0%) of the samples in 2001, the pathogen was isolated one or more times from 10/35 (28.5%) sampling sites in 2000 and from 13/40 (32.5%) sampling sites in 2001. Salmonella was isolated from 44/802 (5.5%) of raw water samples in 2000 and from 122/822 (14.9%) of the samples in 2001; the pathogen was isolated one or more times from 25/35 (71.4%) sampling sites in 2000 and from 29/40 (72.5%) sampling sites in 2001. Certain sites had multiple pathogen isolations in the same year and from year to year. Salmonella Rublislaw was the most common Salmonella serovar isolated in both years, accounting for 52.4% of isolates.

Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

ABSTRACT Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated “mobile genetic elements” such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx2 flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx2) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx2, whereas all LII strains carried variant stx2c and 4 of 14 LI/II strains had copies of both stx2 and variant stx2c. Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx2 mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

Characterization of Escherichia coli isolated from gut biopsies of newly diagnosed patients with inflammatory bowel disease

Background: Mucosal‐associated Escherichia coli may play a role in the pathogenesis of inflammatory bowel diseases (IBDs). In this study we assessed mucosal‐associated E. coli in adults at the time of first diagnosis. Materials and Methods: E. coli were isolated from 59 right colon biopsies of 34 newly diagnosed adult IBD patients (Crohns disease [CD] = 23, ulcerative colitis [UC] = 11) and 25 healthy controls (HC). Strains were serotyped, phylotyped into A, B1, B2, or D, and tested for their ability to survive in macrophages. The presence of various virulence factors was also assessed. The fimH subunit of type 1 fimbriae was sequenced and phylogenetically analyzed. Results: A total of 65 E. coli were isolated from CD (29 isolates from 23 patients), UC (11 isolates from 11 patients), and HC (25 isolates from 25 subjects). All E. coli were positive for fimH, crl, and cgsA and negative for vt1, vt2, hlyA, cnf, and eae. Significant positive associations were between CD and in between CD and afae (P = 0.002), and between UC and ompA (P = 0.02), afae (P = 0.03), and USP (P = 0.04). The B2+D phylotype was significantly associated with inflammation (P = 0.04) as it was with serine protease autotransporters (SPATE), malX, ompA, and kpsMTII (P < 0.05). Macrophage survival was the highest in UC‐isolated E. coli (P = 0.04). FimH amino acid substitutions N91S, S99N, and A223V were associated with IBD (P < 0.05). Conclusions: Adherent invasive E. coli are present at first diagnosis, suggesting that they may have a role in the early stages of disease onset. (Inflamm Bowel Dis 2010;)