Phylogenetic classification of Escherichia coliO157:H7 strains of human and bovine origin using a novel set of nucleotide polymorphisms
- Michael L Clawson†1,
- James E Keen†1, 4,
- Timothy PL Smith1,
- Lisa M Durso1,
- Tara G McDaneld1,
- Robert E Mandrell2,
- Margaret A Davis3 and
- James L Bono1Email author
© Clawson et al.; licensee BioMed Central Ltd. 2009
Received: 21 January 2009
Accepted: 22 May 2009
Published: 22 May 2009
Cattle are a reservoir of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157), and are known to harbor subtypes not typically found in clinically ill humans. Consequently, nucleotide polymorphisms previously discovered via strains originating from human outbreaks may be restricted in their ability to distinguish STEC O157 genetic subtypes present in cattle. The objectives of this study were firstly to identify nucleotide polymorphisms in a diverse sampling of human and bovine STEC O157 strains, secondly to classify strains of either bovine or human origin by polymorphism-derived genotypes, and finally to compare the genotype diversity with pulsed-field gel electrophoresis (PFGE), a method currently used for assessing STEC O157 diversity.
High-throughput 454 sequencing of pooled STEC O157 strain DNAs from human clinical cases (n = 91) and cattle (n = 102) identified 16,218 putative polymorphisms. From those, 178 were selected primarily within genomic regions conserved across E. coli serotypes and genotyped in 261 STEC O157 strains. Forty-two unique genotypes were observed that are tagged by a minimal set of 32 polymorphisms. Phylogenetic trees of the genotypes are divided into clades that represent strains of cattle origin, or cattle and human origin. Although PFGE diversity surpassed genotype diversity overall, ten PFGE patterns each occurred with multiple strains having different genotypes.
Deep sequencing of pooled STEC O157 DNAs proved highly effective in polymorphism discovery. A polymorphism set has been identified that characterizes genetic diversity within STEC O157 strains of bovine origin, and a subset observed in human strains. The set may complement current techniques used to classify strains implicated in disease outbreaks.
Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) recently emerged as a cause of diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) . STEC O157 cause an estimated 73,480 illnesses each year in the United States  and probably evolved from a progenitor of E. coli O55:H7, a source of infantile diarrhea . The STEC O157 5.5-Mb genome contains a 4.1-Mb backbone that is shared with E. coli K-12 and thought to be conserved across most E. coli serotypes [4, 5]. Much of the remaining genome originates from horizontal transfer, with a significant contribution from bacteriophages [4, 5]. The loss and gain of genes through horizontal transfer, coupled with nucleotide variation distributed throughout the STEC O157 genome, serve in both recording the evolution and defining the diversity of this pathogenic serotype [6–8].
Detection of genetic diversity between STEC O157 strains is an important component of outbreak investigations. Heterogeneity between STEC O157 strains has been detected through multilocus sequence tagging , octamer and PCR-based genome scanning [10, 11], phage typing [12, 13], multiple-locus variable-number tandem repeat analysis , microarrays [15, 16], nucleotide polymorphism assays , phage integration patterns coupled with genome polymorphisms [17, 18], and pulsed-field gel electrophoresis (PFGE) [19, 20]. Of these, PFGE is currently the method of choice for distinguishing between STEC O157 strains implicated in outbreaks , and entails standardized chromosome digestions with XbaI, and separation of DNA segments through gel electrophoresis. Differing banding patterns are used to identify genetic diversity between STEC O157 strains. However, PFGE does not effectively show evolutionary descent between epidemiologically related strains [8, 23]. Additionally, the standardized PFGE method is unreliable for determining genetic relatedness between STEC O157 strains that are epidemiologically unrelated .
Nucleotide polymorphisms, if sufficiently present within microbial populations, such as STEC O157, are highly amenable for determining genetic relatedness and descent between either epidemiologically related or unrelated strains . Single nucleotide polymorphisms (SNPs) have been recently identified throughout the STEC O157 genome [15, 16, 26] and some have been used to identify variation between STEC O157 strains originating from clinically ill humans . Thirty-nine SNP-based STEC O157 genotypes were identified that defined nine phylogenetic clades, of which one associated with increased hemolytic uremic syndrome, a serious complication of STEC O157 infection . Thus, SNPs have been employed in the classification of STEC O157 by phylotype, and for distinguishing a subpopulation with increased human virulence.
Cattle are a reservoir of STEC O157 and harbor subtypes that are not typically observed in humans [10, 26]. Consequently, SNPs ascertained exclusively with strains associated with human outbreaks  may be ineffective at distinguishing a greater proportion of STEC O157 genetic diversity present in cattle. Given that strains of any genetic subtype may be drawn into a human outbreak investigation, and/or food recall, an ability to detect the fullest spectrum of STEC O157 genetic diversity with nucleotide polymorphisms would be useful in any STEC O157 investigation. Additionally, a greater understanding of STEC O157 genetic diversity, and how it relates to human pathogenesis, may lead to the identification of alleles that are directly involved with increased human virulence.
The main goal of this study was to sequence the genomes (1×) of 193 diverse STEC O157 strains and identify a set of nucleotide polymorphisms that classify STEC O157 of either bovine or human origin by genotype. Reported here are 42 unique polymorphism-derived STEC O157 genotypes that are tagged by a minimal set of 32 polymorphisms. Phylogenetic trees produced by the genotypes are split into clades that represent strains of cattle origin, or cattle and human origin. These results indicate that heterologous members of the STEC O157 serotype are distinguishable through nucleotide polymorphisms, and support the notion that a subset of STEC O157 harbored in cattle causes the majority of human disease.
Sequencing coverage of 193 STEC O157 strains
Approximately 1× genome coverage of 193 STEC O157 strains was obtained through 454 GS FLX shotgun sequencing of three STEC O157 DNA pools (see Additional data file 1 for supplementary strain, PFGE, and genotype information). The DNA pools were designed to account for: host origin; and the alleles of a polymorphism in the translocated intimin receptor gene (tir 255T>A), as STEC O157 with the tir 255T>A A allele are rarely isolated from clinically ill humans . A total of 1.306 Gb of genomic sequence was obtained from DNA pools of: 51 strains of bovine origin, tir 255 T>A A allele (346.2 Mb); 51 strains of bovine origin tir 255T>A T allele (402.6 Mb); and 91 strains of human origin, of which all had the tir 255T>A T allele (557.5 Mb). Given that the STEC O157 genome is approximately 5.5 Mb, the depth of sequence obtained for each of the three pools averages to slightly more than 1× whole genome coverage for each of the 193 strains sequenced in this study.
Polymorphism identification and validation
Matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) genotype validation assays were developed for 227 putative polymorphisms based on their minor allele frequencies in one or more of the STEC O157 DNA pools. The minor alleles of 169 polymorphisms were observed exclusively in one of the three DNA pools at a frequency of 15% or higher (human strain DNA pool (n = 18), bovine strain DNA pool, tir 255T>A T allele (n = 81), bovine strain DNA pool, tir 255T>A A allele (n = 70)). Additionally, 58 polymorphisms were included where the minor allele was observed in both bovine DNA pools with a minor allele frequency of 10% or higher in one or both pools. MALDI-TOF genotyping of the 227 polymorphisms across 261 STEC O157 strains (Additional data file 1) indicated that 21 putative polymorphisms resided in duplicated genomic regions as a subset of STEC O157 strains yielded heterozygous genotypes. Another 28 putative polymorphisms proved either intractable for genotyping or yielded monomorphic genotypes. Of 178 polymorphisms validated by MALDI-TOF genotyping, 139 reside in open reading frames with 86 predicted non-synonymous or premature stop codon allele variants. Additionally, 154 reside on the conserved genomic backbone of E. coli (see Additional data file 2 for supplementary polymorphism information).
Identification of polymorphism-derived genotypes in STEC O157 strains of human and cattle origin
Phylogenetic analyses of STEC O157 polymorphism-derived genotypes
Comparison of PFGE and polymorphism-derived genotype diversity
GS FLX sequences are clonal in origin because they are ultimately derived from a single strand of DNA. Consequently, high- and low-frequency polymorphism alleles can be detected through GS FLX sequencing of pooled DNA libraries. We took advantage of this attribute by designing STEC O157 DNA pools that were sorted by host origin phenotype (cattle or human), and genotype for the tir 255 T>A polymorphism. Because the tir 255 T>A A allele is rarely observed in STEC O157 isolated from humans, STEC O157 DNAs of cattle origin could be separated into two pools, one representing a portion of STEC O157 diversity that appears primarily in cattle, and one representing a portion of STEC O157 diversity that may or may not appear in clinically ill humans. These two pools were complemented with the DNA pool of STEC O157 strains isolated from clinically ill humans. By selecting polymorphisms where the minor allele was observed at a relatively high frequency in either the STEC O157 DNA pool of human strains or at least one of the two cattle strain DNA pools, 42 polymorphism genotypes were identified that cover a large spectrum of STEC O157 diversity present in cattle, and a subset of genotypes that manifests in clinically ill humans.
While this study defined a sub-lineage of STEC O157 that is poorly represented in humans, a genetic mechanism for causing this host restriction is unknown. The tir 255T>A polymorphism is a likely candidate as the translocated intimin receptor protein is part of the STEC O157 type-three secretion system and facilitates bacterium attachment to enterocyte cells within the colon and subsequent effacement . Additionally, the T>A substitution encodes a non-synonymous replacement of aspartate for glutamate in the translocated intimin receptor protein. However, the tir 255T>A polymorphism is not known to directly affect STEC O157 virulence in humans , and this study identified 1,735 putative polymorphisms (outside of phage integration sites) with minor alleles exclusively observed in the human STEC O157 DNA pool. This finding complicates interpretations regarding a tir 255T>A polymorphism effect on human virulence. Regardless of knowing which alleles directly impact the ability of STEC O157 to cause human disease, an ability to track and identify variation linked with the tir 255T>A A allele is important, as one human strain in this study had the tir 255T>A A allele and a polymorphism-derived genotype that fell within the monophyletic clade typically found in cattle.
A majority of STEC O157-induced human disease sampled in this study was caused by strains that have followed one of two overarching lines of descent, as 92% of all human strain polymorphism-derived genotypes were placed within two large subclades (Figures 3 and 4; Additional data file 5). These two clades are separated from one another by both orthologous descent and probable recombination (Figure 4) and may be split out further into smaller clade sets . It is likely that variation between the subclades, or variation among genotypes within a subclade, may associate with human virulence, as this has been previously demonstrated with the US spinach outbreak strain of 2006 , which had a higher rate of hospitalization and hemolytic uremic syndrome than other outbreak strains . The US spinach outbreak strain was included in this study and has a polymorphism-derived genotype (genotype 21) that differs from all others, in that only it contains the minor alleles of two non-synonymous polymorphisms (Additional data files 2 and 3, N-acetylglutamate synthase: alanine to serine (position 3,672,410), and cytochrome c nitrite reductase: arginine to histidine (position 5,141,169)). These polymorphisms were not characterized in a previous study of STEC O157 polymorphism-derived genotypes and human virulence  as only four polymorphisms used in that study coincide with the 178 described here.
The polymorphisms validated in this study primarily reside in the conserved backbone of E. coli and some may be informative across Escherichia species. PFGE, the current gold standard for assessing STEC O157 genetic diversity , primarily detects insertions and/or deletions within genomic regions specific to STEC O157 . Consequently, PFGE and the polymorphism-derived genotypes described in this study target different regions of the STEC O157 genome that do not share a common phylogeny. It is not surprising that PFGE diversity surpassed polymorphism-derived genotype diversity overall, given that PFGE patterns are known to change between subcultures of the same strain of STEC O157:H7  and that plasmid migration within PFGE can be unpredictable . Future studies should be conducted that compare STEC O157 diversity assessed with the polymorphism-derived genotypes and PFGE using outbreak samples. However, given that ten different PFGE patterns were each observed in two or more strains with different polymorphism genotypes, the 42 polymorphism-derived genotypes identified in this study have immediate potential to resolve genetically distinct STEC O157 strains comprising an outbreak investigation that may be indistinguishable by PFGE.
The method of pooling large numbers of phenotyped STEC O157 strain DNAs and subsequent high throughput 454 sequencing proved extremely efficient for the identification of variation within and between pooled populations, and resulted in the identification of 178 polymorphisms that collectively define 42 unique STEC O157 genotypes. The genotypes characterize genetic diversity and relatedness within STEC O157 strains of bovine origin, and a subset observed in human strains. We identified a minimal set of 32 polymorphisms that tag all 42 genotypes, and show that this set can detect genetically diverse STEC O157 strains that are indistinguishable by PFGE.
Materials and methods
STEC O157 strains of bovine origin (n = 102) that varied by source, and epidemiologically unrelated human clinical STEC O157 strains (n = 91) were used for polymorphism discovery (Additional data file 1) [4, 31–37]. Each strain was characterized as STEC O157 by an enzyme-linked immunosorbent assay using an O157 monoclonal antibody and multiplex PCR for stx1, stx2, eae, hlyA, rfbO157 and fliCH7 [38–41]. Additionally, each strain was genotyped for a polymorphism residing within the translocated intimin receptor gene (tir 255 T>A) . A total of 261 STEC O157 strains, 164 isolated from cattle and 97 isolated from human were targeted for genotyping of: tir 255T>A; 178 polymorphisms identified in this study; and PFGE (Additional data file 1).
Genomic DNA was extracted from STEC O157 strains using Qiagen Genomic-tip 100/G columns (Valencia, CA, USA) and a modified manufacturer's protocol. Following overnight growth in 5 ml of Luria broth, bacteria were pelleted by centrifugation at 5,000 × g for 15 minutes, re-suspended in Qiagen buffer B1 containing RNase A (0.2 mg/ml), and vortexed per the manufacturer's instructions. Importantly, the samples were then incubated at 70°C for 10 minutes, vortexed, and equilibrated at 37°C (failure to include the 70°C step frequently resulted in the columns becoming plugged and/or a significant decrease in DNA yield). Following the addition of 80 μl lysozyme (100 mg/ml), 100 μl proteinase K (Qiagen), and a 37°C incubation for 30 minutes, the DNAs were extracted and air dried per the manufacturer's protocol. Purified DNAs were suspended in 500 μl TE (10 mM Tris pH 8.0, 0.1 mM EDTA) and incubated for 2 hours at 50°C, followed by an overnight incubation at room temperature with gentle mixing. Strain DNA preparations were assessed by 260 nm/280 nm absorptions, which were determined with a NanoDrop Technologies ND-1000 spectrophotometer (Wilmington, DE, USA), and by gel electrophoresis.
STEC O157 DNA pools, GS FLX sequencing, and polymorphism identification
Three STEC O157 DNA pools were created for GS FLX sequencing and polymorphism discovery. One consisted of DNAs from 51 STEC O157 strains (3 μg/strain), all of cattle origin and all with the tir 255 T>A A allele. Another consisted of DNAs from 51 STEC O157 strains (3 μg/strain), all of cattle origin and all with the tir 255 T>A T allele. Another consisted of DNAs from 91 STEC O157 strains (3 μg/strain) originating from clinically ill humans, all with the tir 255 T>A T allele. Genomic libraries were prepared from each of the three DNA pools for Roche 454 GS FLX shot-gun sequencing according to the manufacturer's protocol (Nutley, NJ, USA). A total of 11 emulsion-based PCRs and sequencing runs were performed, three for the DNA pool of cattle origin, tir 255T>A A allele, three for the DNA pool of cattle origin, tir 255T>A T allele, and five for the DNA pool of human origin. SNPs were mapped to a reference sequence of STEC O157 (Sakai strain) and identified with Roche GS Reference Mapper Software (version 1.1.03).
A file containing all targeted polymorphisms was prepared for assay design and multiplexing by MassARRAY® assay design software as recommended by the manufacturer (Sequenom, Inc., San Diego, CA, USA). A target of maximum 36 and minimum 21 polymorphisms per multiplex was set for design, with default settings for all other parameters. Seven multiplexes containing 225 polymorphisms were designed (average 32 polymorphisms per multiplex, range 21 to 36). Assays were performed using iPLEX Gold® chemistry on a MassARRAY® genotyping system as recommended by the manufacturer (Sequenom Inc.). Genotypes designated as high confidence by the Genotyper® software were accepted as correct; those with lower confidence (marked 'aggressive' in the software) were manually inspected. Replicate iPLEX assays and/or Sanger sequencing were used to verify genotypes.
Polymorphism-derived genotype analyses
The alleles of 178 polymorphisms were concatenated by physical order along the STEC O157 genome for 261 STEC O157 strains and aligned using Clustal X (version 1.83) . Redundant polymorphism-derived genotypes were identified using TreePuzzle (version 5.2) [43, 44], and removed from Clustal X alignments. Neighbor-joining and parsimony phylogenetic trees were generated using a collection of software programs in PHYLIP (version 3.65, Consense, DnaDist, DnaPars, Neighbor, Retree, Seqboot) . To construct a neighbor-joining tree, a distance matrix was first produced in DnaDist using an F84 distance model of substitution and a transition/transversion ratio of 2. The output of DnaDist was used to construct a neighbor-joining tree in Neighbor, which was mid-point rooted using Retree. Neighbor-joining bootstraps (1,000) were determined with Seqboot, DnaDist, Neighbor, and Consense. A parsimony tree with 1,000 bootstraps was generated with Seqboot, DnaPars (best tree thorough search) and Consense. Maximum-likelihood trees were generated in Tree-Puzzle (version 5.2) with 10,000 puzzling steps and an HKY model of substitution. Neighbor-joining, parsimony, and maximum-likelihood trees were all viewed in TreeView (version 1.6.6) .
Haploview v 4.1  was used to identify a minimal set of polymorphisms (tagging polymorphisms) that distinguish each of the unique polymorphism-derived genotypes observed in this study. All 178 polymorphism genotypes were used to infer STEC O157 haplotypes in Haploview at a haplotype frequency threshold of 0% or higher. Neighbor-joining, parsimony, and maximum-likelihood trees were generated from concatenated tagging polymorphism genotypes using model assumptions identical to those used for the full genotype data sets. Additionally, a median-joining network was constructed in Network (version 22.214.171.124)  for the concatenated tagging polymorphism genotypes.
Pulsed field gel electrophoresis
The standardized PFGE method  was performed on 261 STEC O157 strains that were also targeted for SNP genotyping (Additional data file 1). Gel images were analyzed using Bionumerics (Applied Maths, Sint-Martens-Latem, Belgium), and banding patterns were clustered using an unweighted pair-group method with arithmetic mean algorithm and a band-based Dice coefficient. Default tolerance settings were used. No restriction enzymes additional to XbaI were used. Strains were assigned to the same PFGE group only if XbaI banding patterns were indistinguishable.
Additional data files
The following additional data are available with the online version of this paper: a table of STEC O157 strains used in this study with their corresponding PFGE patterns and polymorphism-derived genotypes (Additional data file 1); a table of nucleotide polymorphism allele frequencies in STEC O157 strains of bovine and human origin (Additional data file 2); a table of STEC O157 genotypes defined by 178 nucleotide polymorphisms (Additional data file 3); a table of STEC O157 genotypes defined by a minimal set of 32 nucleotide polymorphisms (Additional data file 4); a figure showing neighbor-joining tree of polymorphism-derived genotypes tagged with a minimal set of 32 polymorphisms (Additional data file 5).
matrix-assisted laser desorption-ionization time-of-flight
pulsed-field gel electrophoresis
single nucleotide polymorphism
- STEC O157:
Shiga toxin-containing Escherichia coli O157:H7.
We thank Renee Godtel, Bob Lee, Sandy Fryda-Bradley, Gennie Schuller-Chavez, Kevin Tennill, Linda Flathman, Ron Mlejnek, Scott Schroetlin, and Casey Trambly for outstanding technical support for this project; Dr Thomas E Besser for his generous donations of STEC O157 strains; Dr Michael P Heaton for helpful discussions on experimental design; Jim Wray, Phil Anderson, and Randy Bradley for computer support; Joan Rosch for secretarial support, and Drs Jeffrey Gawronski and David Benson for reviewing the manuscript. This work was supported in part by The Beef Checkoff (MLC JEK, JLB) and the Agricultural Research Service (MLC, JEK, TPLS, LMD, TGM, REM, JLB). The use of product and company names is necessary to accurately report the methods and results; however, the USDA neither guarantees nor warrants the standard of the products, and the use of names by the USDA implies no approval of the product to the exclusion of others that may also be suitable.
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