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.

DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.Bacterial strain typing has several important applications in microbiology. In clinical practice, strain typing is useful for diagnosis and determining treatment strategy and is essential for rapid identification of disease outbreaks and new virulent strains. In the food industry, strain typing is necessary to ensure food safety and for linking cases of food-borne infections to suspected items in the food chain. Classical bacterial identification is based on selective enrichment, followed by plating on selective media. Species identification is mainly by biochemical characterization, and strain identification is primarily based on serology. These methods do not meet the requirement for rapid identification and typing in clinical, epidemiological, and food industry applications. Recent advances in biotechnology have resulted in the development of numerous methods for detection and typing of microorganisms (11-13, 19, 25) which differ in their sensitivity, rapidity, labor intensiveness, complexity, discriminatory power, reproducibility, and cost (5, 32, 43, 49). In principle, by screening a large number of polymorphic sites, genomic methods should be able to provide very accurate discrimination among closely related strains. The total multilocus output of these methods is often termed "DNA fingerprints" or a "DNA bar code."In the pre...