We report on the development of a scheme for the typing of Pseudomonas aeruginosa, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme SpeI distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of P. aeruginosa based on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P. aeruginosa. A free, online strain identification service based on the genotyping data produced herein has been developed.

BackgroundSome pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies.ResultsThis report presents a database () of tandem repeats from publicly available bacterial genomes which facilitates the identification and selection of tandem repeats. We illustrate the use of this database by the characterization of minisatellites from two important human pathogens, Yersinia pestis and Bacillus anthracis. In order to avoid simple sequence contingency loci which may be of limited value as epidemiological markers, and to provide genotyping tools amenable to ordinary agarose gel electrophoresis, only tandem repeats with repeat units at least 9 bp long were evaluated. Yersinia pestis contains 64 such minisatellites in which the unit is repeated at least 7 times. An additional collection of 12 loci with at least 6 units, and a high internal conservation were also evaluated. Forty-nine are polymorphic among five Yersinia strains (twenty-five among three Y. pestis strains). Bacillus anthracis contains 30 comparable structures in which the unit is repeated at least 10 times. Half of these tandem repeats show polymorphism among the strains tested.ConclusionsAnalysis of the currently available bacterial genome sequences classifies Bacillus anthracis and Yersinia pestis as having an average (approximately 30 per Mb) density of tandem repeat arrays longer than 100 bp when compared to the other bacterial genomes analysed to date. In both cases, testing a fraction of these sequences for polymorphism was sufficient to quickly develop a set of more than fifteen informative markers, some of which show a very high degree of polymorphism. In one instance, the polymorphism information content index reaches 0.82 with allele length covering a wide size range (600-1950 bp), and nine alleles resolved in the small number of independent Bacillus anthracis strains typed here.

We describe an improved multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for genotyping Staphylococcus aureus. We compare its performance to those of multilocus sequence typing (MLST) and spa typing in a survey of 309 strains. This collection includes 87 epidemic methicillin-resistant S. Staphylococcus aureus is a pathogen of worldwide clinical significance. For this reason, it is the subject of intensive investigations in terms of virulence and drug resistance phenotypes and, also, population genetics. Although the latter is not of significant use for short-term patient care, it is essential for understanding the emergence and spread of new phenotypes. For instance, it was initially considered most likely that methicillin (meticillin)-resistant variants were appearing only rarely through the acquisition of a mobile DNA region designated staphylococcal cassette chromosome mec (SCCmec) and that these variants were spreading efficiently worldwide (34). However, the most recent population genetics investigations suggested instead that SCCmec was acquired hundreds of times independently worldwide and that, as a rule, the geographic spread of these resistant strains was limited (28,36). This knowledge could be produced in the past 10 years due to sequence-based approaches, mainly multilocus sequence typing (MLST) analysis, in which approximately 3 kb of coding genome sequence (or 1/1,000 of the whole genome) are scanned for polymorphism. MLST has allowed the creation of shared and high-quality databases which can be easily queried over the Internet, and this has proved to be highly valuable (7). However, as the sequences used in MLST schemes evolve slowly and are highly conserved, the resolution provided by MLST is too low for the investigation of recent evolution and, above all, for short-term epidemiological studies. The sequencing of much larger portions of the genome to increase resolution can only be used in dedicated research projects analyzing a limited number of strains (28). Presently, pulsed-field gel electrophoresis (PFGE) remains the most discriminatory technique for S. aureus typing, but it allows the constitution of shared databases only at the national level and is not appropriate for population studies (1, 35). There is, consequently, still a need for a technology as discriminatory as PFGE and as portable as MLST at a low cost.Tandemly repeated sequences provide a very valuable source of polymorphism. Multilocus variable-number tandemrepeat (VNTR) analysis (MLVA) is now used in genotyping several bacterial species (26,48). MLVA typing relies upon a basic and widespread methodology, the measurement of the length of DNA fragments. It is not a "pattern"-producing method, even when run on agarose gels. The genotype, in the form of a string of numbers corresponding to the number of repeats at each locus, is highly portable and can be readily incorporated in large databases (13).