In this study, we applied the multi locus VNTR analysis (MLVA) typing method to S. agalactiae. VNTR analysis, a method based on tandem repeat polymorphisms at multiple loci, has been successfully applied to many other bacterial species [30, 41]. We investigated the relevance of this tool for the genotyping of S. agalactiae, by testing this method on six VNTR loci in 189 strains previously characterized by MLST and serotyping. The MLVA-6 scheme is inexpensive and can be carried out with the equipment routinely used for PCR amplification and agarose gel electrophoresis. For the six VNTR loci, amplification was achieved with all the strains tested. For SAG7, a second PCR targeting a larger flanking region was required for 14% of the strains, which did not have a 16 kb genomic island encompassing the VNTR. The repeat sizes of the six VNTRs were sufficiently large for evaluation of the number of repeats on agarose gels. Moreover, the conversion of results into allelic profiles should make it possible to construct databases for exchange between laboratories. The MLVA-6 scheme includes a set of markers with different diversity indices, making it suitable for epidemiological studies. Markers with a moderate diversity and small number of alleles (presumably reflecting their slow rate of evolution) define clusters, whereas markers displaying more rapid evolution reflect variability within clusters. The MLVA-6 method described here is a rapid, reproducible and epidemiologically meaningful typing tool.
Three loci studied in the present MLVA scheme are in common with the MLVA scheme proposed by Radtke et al. . The 3 additional loci studied here provide more weight to clusters while maintaining a high discrimination power. Moreover, in the MLVA scheme proposed here, only one locus (SAG7) was missing in some strains (14%), and another primer pair targeting larger consensual flanking region confirmed the absence of this locus with a specific amplification. Unlike Radtke et al., we sought to develop a MLVA scheme in which a PCR product was amplified in all strains whether the VNTR was present or absent. In fact, negative amplification may result from the lack of a VNTR locus or modification of the flanking regions, especially as some VNTRs are close to transposases or insertion sequences such as SAG4 (alias SATR1) which is close to IS1381. Thus, the possibility of negative amplification for 3 out of 5 VNTR loci in the Radtke et al. MLVA analysis could be a real problem in terms of resolution and reproducibility of the genotyping method. Nevertheless, cumulative works allow to define the best set of VNTR loci, as has already been done for other bacterial species such as Mycobacterium tuberculosis [22, 42–46] and Staphylococcus aureus [30, 47–49]. Finally, the study of 34 isolates of bovine origin provided information about their distribution, especially those belonging to MLST CC17.
Population analysis by MLVA revealed a clonal distribution of the strains similar to that obtained by MLST. The greater discriminatory index of MLVA (0.96) made it possible to distinguish between strains within the clonal complexes defined by MLST. Thus, MLVA divided CC23 into two groups: one associated with serotype III and the other associated with serotype Ia. Moreover, MLVA also separated CC17 into two groups: one corresponding to strains of human origin and the other, containing several related STs (ST-61, ST-64, ST-301 etc.), corresponding to strains of animal origin only. A previous study analyzing 75 strains of S. agalactiae of human and animal origin by whole-genome DNA-array hybridization also separated ST-23 strains into two clusters, one associated with serotype III and the other with serotype Ia . Each of these two clusters was associated with a particular pattern of surface protein expression. This previous study also separated the bovine and human CC17 strains . These results are consistent with an ancient divergence of these clusters, whereas other observations based on MLST analysis suggest that ST-17 strains may have arisen from a bovine ancestor . The lack of a strict correlation between the results of MLST and MLVA may be accounted for by differences in the markers used for MLST (targeting housekeeping genes) and MLVA (targeting a set of diverse regions that may or may not be conserved). Unlike MLST, MLVA targets several types of markers: genes involved in metabolism, genes associated with virulence and a genomic island. Indeed, SAG2 is located upstream from the gene encoding the ribosomal protein S10 which is involved in transcription and translation, and SAG3 is located within dnaJ, which encodes a member of the Hsp70 family, a co-chaperone protein (Hsp40). The SAG21 locus encodes a surface protein involved in virulence, FbsA. The SAG7 locus is located on a genomic island and belongs to a gene encoding a hypothetical protein whose function has not yet been identified, like most of the genes of genomic islands . Clustering based on MLVA data was almost identical with the UPGMA and MST algorithms except for cluster 1. The differences in mathematical calculation between the two methods may account for the observed differences in strain clustering. This phenomenom has been previously observed in MLVA studies .
Some VNTRs for the alpha C protein have already been described in S. agalactiae [41, 53, 54]. One of these VNTRs is involved in regulating gene expression: a pentanucleotide repeat located upstream from the promoter regulates expression in vitro by phase variation. Another is an intragenic VNTR that modifies the size of the alpha C protein, thereby altering its antigenicity and strain virulence . These two VNTR loci were not included in the MLVA method proposed here, in one case because the small size of the repeat unit (5 bp) complicates the mode of PCR fragment size assessment . The amplicons of the second VNTR locus not included were more than 2000 bp in size, again making it difficult to evaluate repeat number. Tandem repeats were also found in the gene encoding another surface protein, FbsA, which interacts with epithelial cells and is involved in invasion of the central nervous system of colonized neonates. Its ability to bind to fibrinogen depends on the number of repeats of a unit of 16 amino acids present at its N-terminus . A particular number of repeats is associated with the greater potential of the ST-17 strains implicated in neonatal meningitis to adhere to fibrinogen . This major marker was included in our MLVA method and corresponds to SAG21.