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Phylogeny and Strain Typing of Escherichia coli, Inferred from Variation at Mononucleotide Repeat Loci

Eran Diamant, Yniv Palti,^, Riva Gur-Arie, Helit Cohen, Eric M. Hallerman, and Yechezkel Kashi^*

Department of Food Engineering and Biotechnology, Technion—Israel Institute of Technology, Haifa 32000, Israel

Received 10 September 2003/ Accepted 13 January 2004

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.

E.D. and Y.P. contributed equally to this article.

Present address: National Center for Cool and Cold Water Aquaculture, USDA-ARS, Kearneysville, WV 25430.

Permanent address: Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.

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