Lineage-specific Polymorphism Assay Research Articles

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle.

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

Genetic features of human and bovine Escherichia coli O157:H7 strains isolated in Argentina

Shiga toxin-producing Escherichia coli (STEC) are important food-borne pathogens associated with human diseases. In Argentina, O157:H7 is the dominant serotype in hemolytic uremic syndrome (HUS) cases. Previously, we have described the almost exclusive circulation of human E. coli O157 strains belonging to the hypervirulent clade 8 in Neuquén Province. The aim of the present study was to investigate, by a broad molecular characterization, if this particular distribution of E. coli O157 clades in Neuquén is similar to the situation in other regions of the country and if it may be originated in a similar profile in cattle, its main reservoir. Two-hundred and eighty O157 strains (54 bovine and 226 human) isolated between 2006 and 2008 in different regions of Argentina were studied. All strains harbored rfbO157, fliCH7, eae, and ehxA genes. The predominant genotype was stx2a/stx2c in human (76.1%) and bovine (55.5%) strains. All human isolates tested by Lineage-Specific Polymorphism Assay (LSPA-6), were lineage I/II; among bovine strains, 94.1% belonged to lineage I/II and 5.9% to lineage I. No LSPA-6 lineage II isolates were detected. Single nucleotide polymorphism (SNP) analysis has revealed the existence of nine clade phylogenetic groups. In our clinical strains collection, 87.6% belonged to the hypervirulent clade 8, and 12.4% were classified as clade 4/5. In bovine isolates, 59.3% strains were clade 8, 33.3% clade 4/5 and 7.4% clade 3. More than 80% of human strains showed the presence of 6 of the 7 virulence determinants described in the TW14359 O157 strain associated with the raw spinach outbreak in the U.S. in 2006. More than 80% of bovine strains showed the presence of 3 of these factors. The q933 allele, which has been related to high toxin production, was present in 98.2% of clinical strains and 75.9% of the bovine isolates. The molecular characterization of human STEC O157 strains allows us to conclude that the particular situation previously described for Neuquén Province, may actually be a characteristic of the whole country. These genetic features are quite similar to those observed in the bovine reservoir and may be derived from it. This data confirms that, unlike the rest of the world, in Argentina most of the STEC O157 strains present in cattle may cause human infections of varying severity and the marked virulence described for these strains may be related to the high incidence of HUS in our country.

Model-based clustering of Escherichia coli O157:H7 genotypes and their potential association with clinical outcome in human infections

This study addresses the potential association of Escherichia coli O157:H7 genetic clusters with severe clinical manifestations in humans. The genotypes used in this model-based clustering had been delineated on the basis of lineage-specific polymorphism assay, Shiga toxin–encoding bacteriophage insertion site assay, clade typing, tir (A255T) polymorphism, variant analysis of Shiga toxin 2 gene, and antiterminator Q genes. Based on this model, the distribution of genotypes among tested strains suggested the presence of 6 main genetic clusters of E. coli O157:H7 strains. Clusters 1 and 3 were observed to be more frequent among E. coli O157:H7 strains isolated from bloody diarrhea and hemolytic uremic syndrome, respectively. Consequently, our findings supported the growing evidence of the existence of distinct genotypes of E. coli O157:H7 that differ in their virulence levels to human.

Multilocus genotypic characterization of Escherichia coli O157:H7 recovered from food sources.

Escherichia coli O157:H7 strains (n = 33) recovered from different food sources in Egypt were characterized using molecular assays to identify strain genotypes associated with various levels of pathogenic potential. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), Shiga-toxin-encoding bacteriophage insertion site assay (SBI), clade 8 typing, Tir (A255 T) polymorphism, and variant analysis of Shiga toxin 2 gene (Stx 2a and Stx 2c), and anti-terminator Q genes (Q 933 and Q 21). Genotypes LI/II (76%), SBI 1 (60·6%), clade 8 (69·7%), Tir (255 T) (72·7%) and Stx 2c (45·5%) were found to be significantly more frequent compared to other genetic markers in the strains analysed. Multivariable analysis revealed a significant association between LPSA-6 and clade types as well as Tir (A255 T). To the best of our knowledge, this is the first study to report the characterization of these genetic markers in E. coli O157:H7 strains in the Middle East and Africa.

Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources.

The aim of this study was to identify an epidemiological association between Shiga toxin-producing Escherichia coli O157 : H7 strains associated with human infection and with food sources. Frequency distributions of different genetic markers of E. coli O157 : H7 strains recovered from human and food sources were compared using molecular assays to identify E. coli O157 : H7 genotypes associated with variation in pathogenic potential and host specificity. Genotypic characterization included: lineage-specific polymorphism assay (LSPA-6), clade typing, tir (A255T) polymorphism, Shiga toxin-encoding bacteriophage insertion site analysis and variant analysis of Shiga toxin 2 gene (stx2a and stx2c) and antiterminator Q genes (Q933 and Q21). The intermediate lineage (LI/II) dominated among both food and human strains. Compared to other clades, clades 7 and 8 were more frequent among food and human strains, respectively. The tir (255T) polymorphism occurred more frequently among human strains than food strains. Q21 and Q933 + Q21 were found at significantly higher frequencies among food and human strains, respectively. Moreover, stx2a and stx2a+c were detected at significantly higher frequencies among human strains compared to food strains. Bivariate analysis revealed significant concordance (P<0.05) between the LSPA-6 assay and the other typing methods. Multivariable regression analysis suggested that tir (255T) was the most distinctive genotype that can be used to detect bacterial clones with potential risk for human illness from food sources. This study supported previous reports of the existence of diversity in genetic markers among different isolation sources by including E. coli O157 : H7 strains from both food and human sources. This might enable tracking genotypes with potential risk for human illness from food sources.

Molecular typing of Escherichia coli O157:H7 isolates from Swedish cattle and human cases: population dynamics and virulence.

While all verotoxin-producing Escherichia coli O157:H7 bacteria are considered potential pathogens, their genetic subtypes appear to differ in their levels of virulence. The aim of this study was to compare the distribution of subtypes of E. coli O157:H7 in the cattle reservoir and in human cases with and without severe complications in order to gain clues about the relationship between subtype and relative virulence. A lineage-specific polymorphism assay (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a novel real-time PCR assay to identify clade 8 were applied to a large and representative set of isolates from cattle from 1996 to 2009 (n = 381) and human cases from 2008 to 2011 (n = 197) in Sweden. Draft genome sequences were produced for four selected isolates. The E. coli O157:H7 isolates in Swedish cattle generally belonged to four groups with the LSPA-6 profiles 211111 (clade 8/non-clade 8), 213111, and 223323. The subtype composition of the cattle isolates changed dramatically during the study period with the introduction and rapid spread of the low-virulence 223323 subtype. The human cases presumed to have been infected within the country predominantly carried isolates with the profiles 211111 (clade 8) and 213111. Cases progressing to hemolytic-uremic syndrome (HUS) were mostly caused by clade 8, with MLVA profiles consistent with Swedish cattle as the source. In contrast, infections contracted abroad were caused by diverse subtypes, some of which were associated with a particular region. The work presented here confirms the high risk posed by the clade 8 variant of E. coli O157:H7. It also highlights the dynamic nature of the E. coli O157:H7 subtype composition in animal reservoirs and the importance of this composition for the human burden of disease.

Phylogenetic characterization of Escherichia coli O157 : H7 based on IS629 distribution and Shiga toxin genotype

Shiga toxin (stx)-producing Escherichia coli O157 : H7 is a prominent food-borne pathogen. Symptoms in human infections range from asymptomatic to haemorrhagic colitis and haemolytic uraemic syndrome, and there is a need for methods that yield information that can be used to better predict clinical and epidemiological outcomes. IS629 is an insertion sequence notable for its prevalence and variable distribution in the chromosome of E. coli O157 : H7, which has been exploited for subtyping and strain characterization. In particular, IS629 distribution is closely aligned with the major phylogenetic lineages that are known to be distinctive in their genome structure and virulence potential. In the present study, a comprehensive subtyping method in which IS629-typing was combined with stx genotyping was developed using a conventional PCR approach. This method consisted of a set of 32 markers based on the unique distribution of IS629 in the three major phylogenetic lineages of E. coli O157 : H7 and six additional markers to determine the stx genotype, a key virulence signature associated with each lineage. The analysis of IS629 loci variation with the 32 markers allowed us to determine the IS629 distribution profile (IDP), phylogenetic lineage and genetic relatedness of the 31 E. coli O157 : H7 strains examined. An association between IDP typing and stx genotype was observed. The use of both IDP and the stx genotype for strain characterization provided confirmative and complementary data in support of lineage placement of closely related strains. In addition, IS629/stx profiles were in agreement with strain segregation based on LSPA-6 (lineage-specific polymorphism assay) and PFGE subtyping, demonstrating its potential as a subtyping and strain tracking method.

Are Super-Shedder Feedlot Cattle Really Super?

The objective of this study was to determine the frequency and duration of super-shedding in cattle by enumerating Escherichia coli O157:H7 in feces and to compare lineage and pulsed-field gel electrophoresis (PFGE) subtypes from super- and low-shedders. E. coli O157:H7 was enumerated from fecal samples obtained from the rectums of 400 feedlot cattle. Super-shedding steers (N=11) were identified, transported, and penned individually. Freshly voided fecal pats were sampled 2 h before and 6 h after feeding for 7 d, then once daily for an additional 19 d. Isolates (N=126) were subtyped using PFGE, and lineage was typed using a lineage-specific polymorphism assay. Of the 11 super-shedders identified at the commercial feedlot, only five were confirmed as super-shedders at the research feedlot, with no super-shedders identified 6 d after sampling at the commercial feedlot. Super-shedding was not consistent in fecal pats collected from the same individual at different times of the day. Isolates exhibited three distinct PFGE subtypes, with most isolates (97.6%) displaying the same subtype, including those obtained from steers that transitioned from super- to low-shedding. The short duration of super-shedding and its lack of continuance suggest that these individuals may not play as great a role in the dissemination of E. coli O157:H7 within the feedlot as previously proposed.

Characterization of Escherichia coli O157:H7 Strains Isolated from Supershedding Cattle

Previous reports have indicated that a small proportion of cattle shedding high levels of Escherichia coli O157:H7 is the main source for transmission of this organism between animals. Cattle achieving a fecal shedding status of 10(4) CFU of E. coli O157:H7/gram or greater are now referred to as supershedders. The aim of this study was to investigate the contribution of E. coli O157:H7 strain type to supershedding and to determine if supershedding was restricted to a specific set of E. coli O157:H7 strains. Fecal swabs (n = 5,086) were collected from cattle at feedlots or during harvest. Supershedders constituted 2.0% of the bovine population tested. Supershedder isolates were characterized by pulsed-field gel electrophoresis (PFGE), phage typing, lineage-specific polymorphism assay (LSPA), Stx-associated bacteriophage insertion (SBI) site determination, and variant analysis of Shiga toxin, tir, and antiterminator Q genes. Isolates representing 52 unique PFGE patterns, 19 phage types, and 12 SBI clusters were obtained from supershedding cattle, indicating that there is no clustering to E. coli O157:H7 genotypes responsible for supershedding. While being isolated directly from cattle, this strain set tended to have higher frequencies of traits associated with human clinical isolates than previously collected bovine isolates with respect to lineage and tir allele, but not for SBI cluster and Q type. We conclude that no exclusive genotype was identified that was common to all supershedder isolates.

Molecular and Antimicrobial Susceptibility Analyses Distinguish Clinical from Bovine Escherichia coli O157 Strains

A population-based study combining (i) antimicrobial, (ii) genetic, and (iii) virulence analyses with molecular evolutionary analyses revealed segregative characteristics distinguishing human clinical and bovine Escherichia coli O157 strains from western Canada. Human (n = 50) and bovine (n = 50) strains of E. coli O157 were collected from Saskatchewan and Manitoba in 2006 and were analyzed by using the six-marker lineage-specific polymorphism assay (LSPA6), antimicrobial susceptibility analysis, the colicin assay, plasmid and virulence profiling including the eae, ehxA, espA, iha, stx1, stx2, stx2c, stx2d, stx2d-activatable, stx2e, and stx2f virulence-associated genes, and structure analyses. Multivariate logistic regression and Fisher's exact test strongly suggested that antimicrobial susceptibility was the most distinctive characteristic (P = 0.00487) associated with human strains. Among all genetic, virulence, and antimicrobial determinants, resistance to tetracycline (P < 0.000) and to sulfisoxazole (P < 0.009) were the most strongly associated segregative characteristics of bovine E. coli O157 strains. Among 11 virulence-associated genes, stx2c showed the strongest association with E. coli O157 strains of bovine origin. LSPA6 genotyping showed the dominance of the lineage I genotype among clinical (90%) and bovine (70%) strains, indicating the importance of lineage I in O157 epidemiology and ecology. Population structure analysis revealed that the more-diverse bovine strains came from a unique group of strains characterized by a high degree of antimicrobial resistance and high frequencies of lineage II genotypes and stx2c variants. These findings imply that antimicrobial resistance generated among bovine strains of E. coli O157 has a large impact on the population of this human pathogen.

Variation in Stress Resistance Patterns among stx Genotypes and Genetic Lineages of Shiga Toxin-Producing Escherichia coli O157

To evaluate the relationship between bacterial genotypes and stress resistance patterns, we exposed 57 strains of Shiga toxin-producing Escherichia coli (STEC) O157 to acid, freeze-thaw, heat, osmotic, oxidative, and starvation stresses. Inactivation rates were calculated in each assay and subjected to univariate and multivariate analyses, including principal component analysis (PCA) and cluster analysis. The stx genotype was determined for each strain as was the lineage-specific polymorphism assay (LSPA6) genotype. In univariate analyses, strains of the stx(1) stx(2) genotype showed greater resistance to heat than strains of the stx(1) stx(2c) genotype; moreover, strains of the stx(1) stx(2) genotype showed greater resistance to starvation than strains of the stx(2) or stx(2c) genotypes. LSPA6 lineage I (LI) strains showed greater resistance to heat and starvation than LSPA6 lineage II (LII) strains. PCA revealed a general trend that a strain with greater resistance to one type of stress tended to have greater resistance to other types of stresses. In cluster analysis, STEC O157 strains were grouped into stress-resistant, stress-sensitive, and intermediate clusters. In stx genotypes, all strains of the stx(1) stx(2) genotype were grouped with the stress-resistant cluster, whereas 72.7% (8/11) of strains of the stx(1) stx(2c) genotype grouped with the stress-sensitive cluster. In LI strains, 77.8% (14/18) of the strains were grouped with the stress-resistant cluster, whereas 64.7% (11/17) of LII strains were grouped with the stress-sensitive cluster. These results indicate that the genotypes of STEC O157 that are frequently associated with human illness, i.e., LI or the stx(1) stx(2) genotype, have greater multiple stress resistance than do strains of other genotypes.

Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtyping of virulence factors and markers [stx1, stx(2a)/stx(2c), q21/Q933, tir(A255T), and rhsA(C3468G)]. LSPA6 lineage II dominated among bovine isolates (63%), followed by lineage I/II (35.6%) and lineage I (1.4%). In contrast, the majority of the human isolates were typed as lineage I/II (77.6%), followed by lineage I (14.1%) and lineage II (8.2%). Multivariate analysis revealed that the tir(A255T) SNP and the stx(2a)/stx(2c) gene variants were the genetic features most differentiating human from bovine isolates. Bovine and food isolates were dominated by stx(2c) (86.4% and 65.5%, respectively). Among human isolates, the frequency of stx(2c) was 36.5%, while the frequencies of stx(2a) and stx(2a) plus stx(2c) were 41.2% and 22.4%, respectively. Bovine isolates showed equal distribution of tir(255A) (54.8%) and tir(255T) (45.2%), while human isolates were dominated by the tir(255T) genotype (92.9%). LSPA6 lineage I isolates were all genotype stx(2c) and tir(255T), while LSPA6 lineage II was dominated by tir(255A) (86.4%) and stx(2c) (90.9%). LSPA6 lineage I/II isolates were all genotype tir(255T) but showed more variation in stx(2) types. The results support the hypothesis that in The Netherlands, the genotypes primarily associated with human disease form a minor subpopulation in the bovine reservoir. Comparison with published data revealed that the distribution of LSPA6 lineages among bovine and human clinical isolates differs considerably between The Netherlands and North America.

Escherichia coliO157:H7 of Genotype Lineage-Specific Polymorphism Assay 211111 and Clade 8 Are Common Clinical Isolates Within Pennsylvania

Molecular subtyping methods have previously shown that there is a nonrandom distribution of Escherichia coli O157:H7 strains among clinical and nonclinical isolates. Two examples include the lineage-specific polymorphism assay (LSPA) and clade typing assay. The clade typing method was previously used to identify a phylogenetic group of E. coli O157:H7 designated clade 8, which is believed to be more virulent than non-clade 8 isolates, and the LSPA previously indicated that clade 8 isolates are LSPA genotype 211111. Published screens have suggested that LSPA 211111 comprise anywhere from 3.9% to greater than 46% of clinical isolates. To determine the prevalence of such isolates within Pennsylvania, we applied LSPA and screened 52 clinical isolates. We found that 31% of isolates were LSPA 211111 and that 13/16 of these could be classified as clade 8. A rapid polymerase chain reaction screen for clade 8 isolates was developed and shown to have a specificity and sensitivity of 0.92 and 1.0, respectively. Polymerase chain reaction screens indicated that all isolates carried hlyA and eaeA and that all but one of the isolates carried katP. The most common LSPA genotype seen within our collection was 111111, and 29 of 30 of these carried both stx1 and stx2. Clade 8 isolates were more diverse, with four different Shiga toxin profiles observed. We conclude that E. coli O157:H7 of LSPA 211111 and clade 8 are common to clinical isolates in Pennsylvania and suggest that further studies are needed to determine whether their prevalence is increasing as observed elsewhere.

Multivariate Analyses Revealed Distinctive Features Differentiating Human and Cattle Isolates of Shiga Toxin-Producing Escherichia coli O157 in Japan

Genotypes of Shiga toxin-producing Escherichia coli (STEC) O157 isolated from humans and cattle were analyzed by uni- and multivariable logistic regression, and population structure methods, to gain insight into transmission and the nature of human infection. Eleven genotyping assays, including PCR typing of five virulence factors (stx(1), stx(2), stx(2c), eae, and ehxA) and a lineage-specific polymorphism assay using six markers (LSPA6), were considered in the analyses. The prevalence of the stx(1), stx(2), and stx(2c) virulence factors was significantly different between human and cattle isolates. However, multivariable regression revealed that the presence of only the stx(2) gene was significantly associated with human isolates after controlling for confounding effects. LSPA6 typing demonstrated an apparent difference in the distribution of LSPA6 lineages between human and cattle isolates and a strong association between stx genotypes and LSPA6 genotypes. Population genetics tools identified three genetically distinct clusters of STEC O157. Each cluster was characterized by stx genotypes and LSPA6 genotypes. The human isolates typically comprised LSPA6 lineage I with stx(1) stx(2) strains and LSPA6 lineage I/II with stx(2c) or stx(2) stx(2c) strains [corrected]. In contrast, the cattle isolates comprised LSPA6 lineage II strains withstx(2c) or stx(1) stx(2c) strains [corrected] in addition to the clusters identified for the human isolates. Our analyses provide new evidence that the stx(2) gene is the most distinctive feature in human isolates compared to cattle isolates in Japan, and only a subset of the genetically diverse population isolated from cattle is involved in human illnesses. Our results may contribute to international comparisons and risk assessments of STEC O157.

Escherichia coli O157:H7 lineages in healthy beef and dairy cattle and clinical human cases in Alberta, Canada.

The aim of this study was to examine the prevalence and distribution of Escherichia coli O157:H7 lineage-specific polymorphism assay (LSPA) 6 genotypes from cattle (n = 313) and clinical human (n = 203) isolates from northern and southern Alberta, Canada, to understand possible associations of genotypes with host and geographic location. The majority of cattle isolates (feedlot and dairy) typed as LSPA-6 111111 (72.2%), with proportionately higher LSPA-6 222222 (19.4%) than other LSPA-6 genotypes (10.7%). Clinical human isolates also typed primarily as LSPA-6 111111 (90.1%), but a higher percentage of genotypes (6.8%) other than LSPA-6 222222 (3.1%) was observed. A significantly higher frequency of LSPA-6 111111 in southern Alberta cattle (P < 0.0001) and a significant difference in LSPA-6 genotypes between human versus feedlot cattle from northern Alberta (P < 0.0001) were detected. LSPA-6 211111 genotype was third and second most common in cattle and humans, respectively, and several new LSPA-6 genotypes (n = 19) were also discovered. Despite avoiding over-representation of isolates from specific farms or outbreaks, higher strain diversity among cattle by pulsed-field gel electrophoresis (PFGE; 50 genotypes) in contrast to human (9 PFGE genotypes) isolates was observed. The majority of cattle (74.4%) and human (90.6%) isolates were susceptible to the antimicrobials tested. Within resistant cattle isolates, sulfisoxazole-tetracycline resistance was common (62.5%) and was accounted for by the presence of sul1 and sul2, and tet(A) and tet(B) determinants. An association between LSPA-6 and PFGE genotypes but not between geographic location and PFGE genotype for both hosts was evident.

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

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 stx(2c) 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.

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.

Identification of Common Subpopulations of Non-Sorbitol-Fermenting, β-Glucuronidase-Negative Escherichia coli O157:H7 from Bovine Production Environments and Human Clinical Samples

Non-sorbitol-fermenting, beta-glucuronidase-negative Escherichia coli O157:H7 strains are regarded as a clone complex, and populations from different geographical locations are believed to share a recent common ancestor. Despite their relatedness, high-resolution genotyping methods can detect significant genome variation among different populations. Phylogenetic analysis of high-resolution genotyping data from these strains has shown that subpopulations from geographically unlinked continents can be divided into two primary phylogenetic lineages, termed lineage I and lineage II, and limited studies of the distribution of these lineages suggest there could be differences in their propensity to cause disease in humans or to be transmitted to humans. Because the genotyping methods necessary to discriminate the two lineages are tedious and subjective, these methods are not particularly suited for studying the large sets of strains that are required to systematically evaluate the ecology and transmission characteristics of these lineages. To overcome this limitation, we have developed a lineage-specific polymorphism assay (LSPA) that can readily distinguish between the lineage I and lineage II subpopulations. In the studies reported here, we describe the development of a six-marker test (LSPA-6) and its validation in a side-by-side comparison with octamer-based genome scanning. Analysis of over 1,400 O157:H7 strains with the LSPA-6 demonstrated that five genotypes comprise over 91% of the strains, suggesting that these subpopulations may be widespread.