Phylogeny and Strain Typing ofEscherichiacoli, Inferred from Variation at Mononucleotide RepeatLoci

Abstract

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

“…Variations of these loci were due to repeat variability in the MNR tract and due to the SNPs in the flanking sequences. Similar results were observed for MNR loci in E. coli (20,30,54) or in Mycobacterium avium subsp. paratuberculosis (1).…”

“…Each of these strains presented a unique L-SSR type and a unique PFGE type, thus indicating that the L-SSR have higher mutation rates compared to MNR-MLST in V. cholerae. Similar results were found in E. coli, where MNR-MLST could not differentiate among O157 strains (20) opposed to the LSSRs (VNTRs) that have higher mutational rate and therefore have higher resolution power of closely related O157 E. coli strains (41). Interestingly, in Bacillus anthracis the mutation rate for L-SSR and MNR was found to be the same (40).…”

“…The original MLST method is based on sequence variation at housekeeping genes that are conserved and therefore usually present in all strains but that have limited variation (21,59,69). On the other hand, the MNR loci that present high levels of polymorphism have the disadvantage of missing data from a portion of the strains due to nonamplified ("null") alleles (20,75) as a result of sequence mismatch (SNPs) at the priming sites, as well as insertions or deletions and genome rearrangements (32). We have solved the problem of "no data" from part of the tested strains by using a nonparametric sequence type NST analysis.…”

“…The variation in SSR tracts, generally having several alleles, enables their use for strain typing in several bacterial species (30,38,41,59,80). SSR composed of mononucleotide repeats (MNR) in Escherichia coli were found to be abundant and highly polymorphic but stable at the strain level, making them a valuable tool for bacterial typing and phylogenetics studies (20,30). Larger SSRs (L-SSR) consisting of two to 9-bp motifs were successfully used to analyze variation among strains by size determination of the PCR amplicon (39,81).…”

“…Hence, it has been suggested to apply MLST analysis to additional highly polymorphic genomic regions, such as virulence genes, stress response genes, and intergenic regions (11,16,44,61). Recently, MLST of loci harboring MNR at noncoding regions were shown in E. coli to contain high sequence variation, including single-nucleotide polymorphisms (SNPs) in the flanking area of the MNR (20). This approach was termed MNR-MLST.…”