Evaluation and selection of tandem repeat loci for Streptococcus pneumoniae MLVA strain typing

Abstract

Background: Precise identification of bacterial pathogens at the strain level is essential for epidemiological purposes. In Streptococcus pneumoniae, the existence of 90 different serotypes makes the typing particularly difficult and requires the use of highly informative tools. Available methods are relatively expensive and cannot be used for large-scale or routine typing of any new isolate. We explore here the potential of MLVA (Multiple Loci VNTR Analysis; VNTR, Variable Number of Tandem Repeats), a method of growing importance in the field of molecular epidemiology, for genotyping of Streptococcus pneumoniae.

Results: Available genome sequences were searched for polymorphic tandem repeats. The loci identified were typed across a collection of 56 diverse isolates and including a group of serotype 1 isolates from Africa. Eventually a set of 16 VNTRs was proposed for MLVA-typing of S. pneumoniae. These robust markers were sufficient to discriminate 49 genotypes and to aggregate strains on the basis of the serotype and geographical origin, although some exceptions were found. Such exceptions may reflect serotype switching or horizontal transfer of genetic material.

Conclusion: We describe a simple PCR-based MLVA genotyping scheme for S. pneumoniae which may prove to be a powerful complement to existing tools for epidemiological studies. Using this technique we uncovered a clonal population of strains, responsible for infections in Burkina Faso. We believe that the proposed MLVA typing scheme can become a standard for epidemiological studies of S. pneumoniae.

Figure 1

PCR amplification of 6 VNTRs using DNA of 8 isolates. From left to right R6, RP31, RP28, RP32, RP29, RP33, RP30 and RP34. A 100-bp ladder (100 to 1000 bp) is run on both side of each group of samples. The number of repeat units within each allele is indicated. The rare alleles of intermediate size are coded "#.5" (for instance Strain R6 is coded 1.5 for locus Spneu38).

Figure 2

Dendrogram showing the clustering of 56 isolates by MLVA using 16 markers. The data are expressed as number of repeats for each of the 16 VNTRs. The genotype number is indicated in the first column "Geno". Some strains possess a rare serogroup (RSG) or were not typable (NT). The serotype 1 isolates from Burkina Faso are boxed.