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Department of Bacterial Diseases, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB,1 Molecular Microbiology Group, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom2
Received 27 January 2003/ Returned for modification 29 March 2003/ Accepted 15 May 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Strains of this serogroup are also of veterinary importance, as they have been isolated from a variety of animals, including healthy cattle and pigs (5, 20) as well as diarrheic calves (17), diarrheic lamb and goats (8), and mastitic cattle (9). Therefore, animals not only act as an important reservoir for O26 strains but may also be infected by these pathogens. In contrast, E. coli O157 does not cause disease in animals.
The clonal diversity of isolates from different food products as well as from human and animal subjects has been studied to determine the reservoirs and routes of transmission of O26 strains through the food chain. However, most research has concentrated only on VT-positive O26 strains, reflecting the seriousness of hemolytic-uremic syndrome infection and the rising numbers of these isolates. These studies imply considerable genetic heterogeneity within the O26 serogroup, but the data from different typing methods (multilocus enzyme electrophoresis, random amplification of polymorphic DNA, and pulsed-field gel electrophoresis) do not allow direct comparison (29, 33, 42, 44, 45). Our aim was to assess the clonal diversity of the O26 serogroup, including both VT-positive and -negative strains, using an approach we call comparative genomic indexing (CGI). In this study we used an E. coli K-12 microarray as the baseline for determining the genomic variation between O26 isolates.
Comparisons of the genome sequences of the pathogenic O157 strain EDL933 with the laboratory E. coli K-12 strain MG1655 revealed that they have a common K-12 chromosomal backbone punctuated by unique genomic regions reflecting deletion and insertion events (30). It was expected that CGI would allow the definition of the core genes common to pathogenic strains and the commensal E. coli K-12 and also identify regions of differences between these strains.
Strains chosen in this study were mainly of veterinary origin and were isolated in the United Kingdom, where these zoonotic pathogens are found in the food chain and associated with human and animal disease. It was known at the outset that half of the O26 strains were verotoxin positive, while the other half were not. Additional strains whose toxin status was known were randomly chosen from serotypes associated with EPEC and enterohemorrhagic E. coli (EHEC) infection (O157, O86, O55, O111, O126, and untypeable); a control commensal strain (O29) was also examined.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA isolation and PCR amplification of protein coding sequences.
For preparation of genomic DNA, cells were grown overnight in Luria-Bertani (LB) broth at 37°C, and DNA was isolated with the Qiagen DNeasy Tissue kit (no. 69504; Qiagen). Oligonucleotide primers and PCR conditions used for amplification of the eae 
, eae ß, eae 
, and eae 
genes were essentially as described by McGraw et al. (26) and Adu-Bobie et al. (1). PCR amplification of the bfp gene was performed with primers (with restriction enzyme sites underlined) bfpAF (CGGCGGATTCTGGTTTCTAAAATCATGAATAAG) and bfpAR (CGGCAAGCTTCTTCATAAAATATGTAACTTTAT). PCR amplification of the hlyA gene was performed with primers hlyCF (GCTATGGGCCTGTTCTCCTCTG) and hlyAR (TGTCTTGCGTCATATCCATTCTCA).
E. coli microarray construction. The microarrays used in this study featured 4,262 of the 4,279 protein-coding sequence (CDS) identified in E. coli K-12 strain MG1655 (http://www. ncbi.nlm.nih.gov). Entire CDS were amplified with specific primer pairs (Sigma-Genosys) with some minor modifications. PCRs were performed in a total volume of 100 µl with 40 ng of E. coli MG1655 chromosomal DNA, 60 pmol of each primer, 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphate mix, and HotStart Taq DNA polymerase (Qiagen). PCR amplifications were performed with an MWG RoboAmp 4200 liquid handling robot for 30 cycles for 1 min at 94°C, 0.5 min at 60°C, and 3 min at 72°C, following an initial enzyme activation step at 95°C for 15 min.
Agarose gel electrophoresis was used to perform quality control on all PCR products Oligonucleotides were removed from the PCR mix by isopropanol precipitation. DNA was resuspended in 40 µl of spotting solution containing 50% dimethyl sulfoxide and 0.3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). PCR products were spotted onto to gamma amino propylsilane-coated (GAP) slides (Corning) with a Stanford arrayer (40). The DNA was UV cross-linked to slides with a Stratalinker at 300 mJ (Stratagene). Subsequently, the slides were washed in a 95°C water bath for 2 min and in 95% ethanol for 1 min and dried by centrifugation at 185 x g prior to storage at room temperature.
Probe preparation and hybridization. For each microarray hybridization reaction, genomic DNAs from a reference strain (E. coli K-12 MG1655) and a test strain were fluorescently labeled with indodicarbocyanine and indocarbocyanine, respectively, with the protocol of DeRisi (http://www.microarrays.org/pdfs/GenomicDNALabel_A.pdf). The genomic DNA was not sheared or digested with restriction enzymes prior to labeling. Labeled reference and test DNAs were combined in a 15-µl hybridization solution (3x SSC, 25 mM HEPES [pH 7.0], 1.87 µg of E. coli tRNA per µl, 0.2% sodium dodecyl sulfate, and 5x Denhardt's solution) and added to a microarray slide. Hybridizations were performed for 14 to 18 h at 63°C. The slides were then washed in 2x SSC-0.1% sodium dodecyl sulfate at 65°C for 5 min, followed by 1x SSC at room temperature for 5 min, and finally in 0.2x SSC at room temperature for 5 min. They were dried by centrifugation at 185 x g for 5 min. At least two hybridization reactions were performed for each test strain.
Microarray data analysis. The processed slides were scanned with a GenePix 4000A scanner (Axon Instruments, Inc.). Fluorescent spots and the local background intensities were quantified with Genepix Pro software (Axon Instruments, Inc.). The data were filtered so that spots with a reference signal lower than the background plus 2 standard deviations of the background were discarded. Signal intensities were corrected by subtracting the local background, then the red/green (indodicarbocyanine/indocarbocyanine) ratios were calculated. To compensate for unequal dye incorporation, data centering was performed by bringing the median Ln(red/green) for each block to 0 (one block being defined as the group of spots printed by the same pin) with the following equation: ln(Ti) = ln(Ri/Gi) - c, where T is the centered ratio, i is the gene index, R and G are the red and green intensities, respectively, and c is the 50th percentile of all red/green ratios. Centered data (i.e., normalized) from all strains were used for all subsequent analyses and subjected to average-linkage hierarchical clustering with the Pearson correlation coefficient (13), in the GeneSpring microarray analysis software version 5.0 (Silicon Genetics). Only those CDS or genes with a good reference signal (or reading) in at least 22 of the 26 test strains upon hybridization to the microarray were considered for clustering. Approximately 70% of the CDS passed this test, leaving a total of 3,039 CDS in the data set. The CDS data set is available as supplemental data at http://www.defra.gov.uk/corporate/vla/aboutus/publicat.htm.
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RESULTS |
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Categorization of strains with conventional virulence markers.
Our collection of E. coli strains whose toxin status was already known were further characterized with respect to selected virulence determinants: bfpA, hlyA, eae , eae ß, eae , and eae (Table 1). Toxin testing had already shown that half of the O26 strains and all of the O157s were VT positive, a diagnostic feature of EHEC strains. However, typical EHECs also possess the plasmid-encoded hlyA gene (27) and the gamma intimin ( eae) gene (1). All four O157 strains fitted this criterion, but the VT-positive O26 strains possessed the ß eae and not the eae gene and were designated atypical EHECs. The VT-negative O26 strains also possessed the ß eae gene, a typical EPEC feature, but did not possess the plasmid-encoded bfp gene typical of EPEC strains (18).
Two VT-negative strains also possessed the EHEC-associated hlyA gene. Therefore, these strains were also designated atypical EPECs. In fact, only one strain in this study showed typical EPEC features and belonged to the O111 serogroup. All other strains were characterized as either atypical EPECs (O55, O126, and O86) or neither EPEC or EHEC (O55 and O29) based on their virulence characteristics. Two O86 strains were designated atypical EPECs because they were positive for the cytolethal distending toxin, a feature common to many EPEC strains, but showed the presence of the eae gene associated with EHECs.
In a separate study, this variant intimin was further characterized (23). An untypeable VT-positive strain (EC720/98) of bovine origin also included in the study, showed virulence characteristics similar to those of other VT-positive O26 strains and was designated an atypical EHEC. In summary, our pathogenic E. coli strains were a heterogeneous group of organisms with respect to their virulence characteristics. This heterogeneity has been observed previously in clinical isolates (27, 28), although virulence characteristics are still routinely used for pathotype determination.
Comparative genomic indexing of E. coli strains. Microarrays were used to compare the relatedness of the 26 E. coli strains by CGI (Table 1). Following microarray hybridization, the scanned data was centered (i.e., normalized; see Materials and Methods), and the presence or absence of genes was determined (see supplementary data). In the first dimension or vertical axis of the hierarchical clustering, the relationship among genomes of each strain was assessed with the Pearson coeffecient correlation pairwise similarity function, where the linkage distance between strains is represented by branch lengths or distance score in the resulting hierarchical cluster (13). The higher the correlation between strains, the smaller the distance score (Fig. 2). The second dimension of the hierarchical clustering was used to group genes with a similar profile for each strain along the horizontal axis, also with the Pearson correlation coefficient. This enabled the clear identification of groups of genes absent in the majority of E. coli field strains as a central cluster (shown in red in Fig. 2; branches for this clustering have not been shown).
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In general, VT-negative and VT-positive O26 E. coli strains (with the exception of EC622/99) were found to be 92.7% similar, and on average 7.3% of the total number of CDS present in MG1655 were absent from this group (Table 2). More than 94% of the absent CDS were conserved within each VT group and between 87% and 94% were conserved between the VT-negative and VT-positive strains. In fact, most large regions of genes absent from the K-12 chromosomal backbone were commonly missing from the majority of E. coli strains included in this study, and not only strains of the O26 serogroup (Table 3). One region was found to differentiate the VT-positive from the VT-negative strains; all VT-positive strains lacked a 5.2-kb region (yagP-yagT) which was present in all VT-negative strains (see supplemental data at http://www.defra.gov.uk/corporate/vla/aboutus/publicat.htm). This could prove to be a useful diagnostic feature.
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All four O157 strains formed a discrete cluster reflecting their close relationship (distance score = 0.046), in agreement with PCR and toxin data (Fig. 2). It was noted that the O157 strains had several unique regions missing from the K-12 chromosomal backbone. These included a 9-kb fragment from yiaJ to sgbE and a much larger 24-kb fragment from yghD to yghT (Table 3). An untypeable strain, EC720/98, which showed virulence characteristics similar to those of the VT-positive O26 strains (Table 1), was positioned close to the O157 cluster; the number of absent CDS was slightly higher in the EC720/98 strain (11%) than the O157 strains included in this study (9.8%; Table 2).
The CGI approach allowed detection of all K-12 genes missing from the chromosomal backbone, for each strain included in this study (see supplemental data). The absent genes are represented in red in Fig. 2. Further analysis of the missing genes showed that many could be grouped into regions that were absent from several strains. Table 3 shows regions of the K-12 chromosome where 10 or more consecutive genes were absent in at least two strains. Five regions of the K-12 chromosome had genes missing in 22 or more strains. These included b0245 to perR; intA to yfjY/yfjP; rplW/rplC/rpsJ/pinO to yheB; waaL (rfaL) to waaQ (rfaQ); and insA7/yjhU to yjhR. These regions were mainly composed of genes expressing hypothetical proteins, with the exception of the waa locus, which is involved in lipopolysaccharide biosynthesis. Three of the regions (bO245, intA, and yjhU) were also flanked by (or contained) transposases, insertion sequence elements, and/or a tRNA-like genes at the 5' end. Such genes are commonly found at the sites of integration of foreign DNA, such as pathogenicity islands. The rfb locus, which is involved in synthesis of the structurally diverse O antigen polymer, had at least eight consecutive genes of the locus missing in all strains (see supplemental data), and 10 or more genes missing in the four O157 strains as well as SO55, EC38/99, and EC622/00.
Therefore, our data clearly demonstrate that comparative genomic indexing is a valuable tool for studying the phylogenomics of E. coli pathogens and for defining the core genes present in all strains included in this study.
Functional analysis of core genes. To gain more information concerning the absent genes and also to define the core genes present in all strains, the E. coli CDS were grouped into functional groups defined by the clusters of orthologous groups of proteins (COGs; http://www.ncbi.nlm.nih.gov/COG). Each COG includes orthologous proteins, which are proteins connected through vertical evolutionary descent, and serves as a platform for functional annotation. The COG information is based on 30 genomes and breaks down into 17 broad functional categories, which include function unknown (39). Genes in each COG category for the E. coli genome were analyzed against our data set with GeneSpring software. The results show that every gene in our data set assigned to the following COG functional categories were present for all strains (Table 4): cell division and chromosome partitioning; coenzyme metabolism; energy production and conversion; nucleotide transport and metabolism; posttranslational modification; protein turnover and chaperones; and translation, ribosome structure, and biogenesis. For groups involved with information storage and processing, only the DNA replication, recombination, and repair functional category showed a high number of genes to be absent. Many genes in the general function prediction, function unknown, and not in COGs categories were missing from our data set, as they did not pass our filtering criteria (see Materials and Methods), so their status was not determined (data not shown).
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The data summarized in Table 4 show that a core pool of genes involved with metabolism, various cellular processes (excluding cell motility and secretion), and information storage and processing (excluding DNA replication, recombination, and repair) have been conserved and maintained in all strains. Conversely, many genes of unknown function, external origin, or facultative function were absent from the chromosomal backbone of a majority of strains and could be assigned as expendable. Such patterns of gene stability within E. coli populations are consistent with suggestions by Dobrindt et al. (10), Lan and Reeves (22), and Woodward and Charles (43). Preliminary analysis of the expendable regions suggest that gene insertions with no similarity to the K-12 genome are harbored in many of these regions (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results (Fig. 2) clearly divided the strains into EHECs (O157), EPECs (O111, O126, O26, and O55), and others (O86 and O29). Furthermore, all O26 strains (excluding EC622/99) clustered within the EPEC cluster irrespective of their pathotype designation (most strains in this study had been shown to possess a mixture of EHEC and EPEC virulence features; Table 1). The CGI results showed a similar percentage of the K-12 chromosomal backbone to be absent in the O26 serogroup and the majority of absent genes were missing from both VT-positive and VT-negative O26 strains (Table 2); only one region on the K-12 chromosome was found to differentiate them (yagP-yagT). These results indicate greater genetic homogeneity within this serogroup than previously proposed and suggest a common clonal lineage of both EPEC and EHEC O26 strains.
Also included in the study were two VT-negative O26 strains (EC335/98 and EC225/00) which possessed the EHEC-associated hlyA gene. Interestingly, both of these strains clustered with the VT-positive O26 strains, showing higher correlation to these strains than to the remaining VT-negative strains. Several studies (29, 45) have shown that O26 strains classed as atypical EPECs (stx eae+) belonged to the classical EHEC O26 serotypes (O26:H11 and O26:H-). This suggests that classification of O26:H11 and O26:H- strains as EHECs may be misleading. Based both on our findings and those of others (29, 45), the proposition that stx eae+ O26 strains could be EHECs which have lost their stx genes or the progenitors of EHEC O26 strains (45) is more likely. This suggestion is further substantiated by a demonstration by Schmidt et al. (35) that an Stx2-converting phage isolated from E. coli O157 was able to infect and lysogenize various E. coli strains, including both EPEC and EHEC O26 strains. Therefore it is likely that stx hlyA O26 strains are O26 strains which have yet to acquire (or have lost) the verotoxin-producing prophage and hlyA plasmid, while stx hlyA+ strains are at an intermediate stage, i.e., have already acquired the virulence plasmid but not the VT-expressing prophage. Therefore, division of this serotype into the pathotypes EPEC and EHEC may be misleading, as VT-positive O26 strains are likely to arise from VT-negative strains and vice versa. Such an environment of genomic exchange would also result in coevolution of VT-positive and VT-negative O26 strains.
Conversely, it could be postulated that in the absence of VTEC genes in the environment, the O26 subpopulation would accumulate different niche-adaptive genes. Studies involving VTEC and O157 isolation from animals (3, 7) have shown the prevalence of these bacteria to be much lower in pigs (7.5% VTEC and 4% O157) than in sheep (66.6% VTEC and 22% O157) and cattle (21.1% VTEC and 15.7% O157). As a result, the O26 subpopulation in pigs (which are less likely to encounter VTECs) have probably accumulated different gene transfer and recombination events than found in the O26 bacteria from ruminants. The resulting difference in genotype could explain our CGI results, which showed that EC622/99, the only strain of porcine origin included in this study, clustered outside the O26 cluster; the O26 cluster included strains of mostly bovine and ovine origin (Fig. 2).
With CGI and the COG database, we identified the core gene pool involved with essential cellular functions, which was maintained in all 26 E. coli field strains used in this study (Table 4 and supplemental data). This collection of genes, which were common to all the pathogenic and nonpathogenic field strains examined as well as the laboratory-adapted MG1655, is the minimal requirement for these bacteria to be classed as the same species. The expendable genes, which were regions of the K-12-chromosomal backbone missing in the field strains, were regions representing adaptation and evolution of these organisms to a different host environment and/or an ecological niche. Six regions of expendable genes missing from the K-12 chromosomal backbone for the majority of strains were identified (Table 3 and supplemental data); this included genes in the O-antigen locus (rfb) and the core oligosaccharide domain (waa locus). Other variable regions of the E. coli chromosome were also identified, e.g., intA, pinO, and yjhU (Table 3).
In our study, most of the E. coli strains were clustered by their serotype, showing serotyping to be an useful indicator of genetic diversity in a clonally structured population such as E. coli, where associations between loci are nonrandom (41). The localized horizontal gene transfer in E. coli populations would not destroy linkage disequilibrium due to its low frequency but would maintain useful variation within a subpopulation (25), as evidenced by our CGI data. Therefore, these variable regions help make the bacterial genome a dynamic structure and contribute to intraspecies variation. Future analyses of these regions of variability will provide further insight into the clonal diversity within the O26 serogroup and will give a better understanding of the genomic transition between VT-positive and VT-negative O26 strains. It will also identify the genomic differences between serogroups which enable them to adapt to distinct host environments.
Therefore, the CGI approach has provided a valuable tool for understanding the clonality of pathogenic E. coli, by defining the core genome and identifying regions of variation. CGI overcomes the limitations inherent in focusing on a particular set of related proteins or group of genes, which may reflect a partial phylogeny. However, because this method relies on CDS microarrays, it cannot detect the single nucleotide changes which cause protein polymorphism and allelic variation.
In summary, we have clearly demonstrated that strains of serogroup O26, both VT positive and negative, have a common clonal lineage and that VT-negative strains are likely to have lost their stx genes or to be progenitors of EHEC O26 strains. Further insight into clonality may be gained by focusing on the hypervariable regions that we have identified on the E. coli chromosome. Future epidemiological studies of outbreaks of this important food-borne pathogen should consider genetic analysis of both VT-positive and VT-negative O26 strains, as the latter may well be an important environmental reservoir that can give rise to EHEC O26 infections.
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ACKNOWLEDGMENTS |
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We thank Mary Berlyn for E. coli K-12 strains and VLA, Weybridge Diagnostics Unit, for veterinary strains. We are also very grateful to S. Gordon at VLA for many helpful discussions and suggestions.
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FOOTNOTES |
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2) are shown in red, and CDS with ln(red/green) between 1.5 and 2.0 are shown in yellow. Strains of the O26 serotype are labeled in red; other strains in the EPEC cluster are labeled in green, O157 strains are labeled in blue, and O86 strains are labeled in orange. The scale for distance score (ds) is also shown.