Association of Escherichia coli O157:H7 tir polymorphisms with human infection

Abstract

Background: Emerging molecular, animal model and epidemiologic evidence suggests that Shiga-toxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source.

Results: Five polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T>A and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T>A T allele and lacked RR1-RU3. In contrast, the tir 255 T>A T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p < 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source.

Conclusion: Polymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T>A T allele in human-derived isolates vs the tir 255 T>A A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset.

Figure 1

Physical maps and taxonomical relationships of the tir gene. (A) A physical map of the tir gene from STEC O157 (GenBank accession number BA000007 gene ECs4561). A 1.6-kb region of the tir gene was sequenced from 22 STEC O157 isolates and the polymorphisms mapped on the tir gene along with previously identified functional domains. (B) Consensus tree from seven equally parsimonious trees constructed in PHYLIP (version 3.65) [35] with the program PARS and viewed in TreeView (version 1.6.6) [36]. The tree was generated from seven variable sites (255 T>A, RR1-RU2, RR1-RU3, RR1-RU4, RR2, RR3 and RR4) and describes the taxonomical relationship of the tir nucleotide sequence with associated phenotypic and genotypic information. Ten genotypes resulting from one nucleotide polymorphism and four repeat region variations were identified from the 185 isolates. Colored boxes identify the different nucleotide variations and repeat units, while white boxes indicate missing repeat units. The sequences of the repeat regions are located below the colored boxes with the top line as the consensus sequence. A variant repeat unit (*) in repeat region 1 was identified that had the following sequence "AAAGGTGCTGGGGAGTTG".