Rapid multiple-locus variant-repeat assay (MLVA) for genotyping of Streptococcus agalactiae

Abstract

Several methods have been used for typing of Streptococcus agalactiae (group B streptococci [GBS]). Methods currently in use may provide inadequate resolution (e.g., typing of capsular polysaccharides and surface protein) or are labor-intensive and expensive (e.g., multilocus sequence typing [MLST] or pulsed-field gel electrophoresis). This work describes the construction and use of a multiple-locus variant-repeat assay (MLVA) on 126 well-characterized human GBS strains, consisting mostly of invasive Norwegian strains and international reference strains. Based on in silico whole-genomic analysis of the genomes of strains A909, NEM316, and 2603V/R, 18 candidate loci were selected and investigated by PCR. Eleven loci showed diversity, and the five most diverse loci were used for the construction of an MLVA, consisting of a multiplex PCR followed by fragment analysis with capillary electrophoresis. The assay generated clusters which corresponded well with those observed by other methods. However, it provided a considerably higher degree of diversity, with 70 different MLVA types compared to 36 types generated by MLST. Simpson's index of diversity for the 5-locus MLVA was 0.963, compared to 0.899 for the MLST in this strain collection. MLVA results will generally be available within 2 days, which is usually faster than MLST. In our hands, MLVA of GBS represents a rapid, easy, and comparably inexpensive method for high-resolution genotyping of GBS.

FIG. 1.

Cluster analysis of 126 GBS strains using categorical values and the UPGMA (unweighted-pair group method using average linkages) algorithm, generated with Bionumerics 6.0 software. Numbers in the SATR1 to -5 columns indicate repeat count (−1 repeat, no PCR product). CPS, capsular polysaccharide. The country column gives the origin of the strain: NO, Norway; INT, international reference strain; DK, Denmark; and ZW, Zimbabwe. Strains marked with asterisks were used in the initial analysis of 18 variable loci. There is an overlap of three strains between the two panels.

FIG. 1.

Cluster analysis of 126 GBS strains using categorical values and the UPGMA (unweighted-pair group method using average linkages) algorithm, generated with Bionumerics 6.0 software. Numbers in the SATR1 to -5 columns indicate repeat count (−1 repeat, no PCR product). CPS, capsular polysaccharide. The country column gives the origin of the strain: NO, Norway; INT, international reference strain; DK, Denmark; and ZW, Zimbabwe. Strains marked with asterisks were used in the initial analysis of 18 variable loci. There is an overlap of three strains between the two panels.

FIG. 2.

MLVA cluster analysis of 27 ST17 strains included in the study using categorical values and the UPGMA algorithm, generated with Bionumerics 6.0 software. Numbers in the SATR1 to -5 columns indicate the repeat count.