CRISPR Typing Increases the Discriminatory Power of Streptococcus agalactiae Typing Methods

Abstract

We explored the relevance of a Clustered regularly interspaced short palindromic repeats (CRISPR)-based genotyping tool for Streptococcus agalactiae typing and we compared this method to current molecular methods [multi locus sequence typing (MLST) and capsular typing]. To this effect, we developed two CRISPR marker schemes (using 94 or 25 markers, respectively). Among the 255 S. agalactiae isolates tested, 229 CRISPR profiles were obtained. The 94 and 25 markers made it possible to efficiently separate isolates with a high diversity index (0.9947 and 0.9267, respectively), highlighting a high discriminatory power, superior to that of both capsular typing and MLST (diversity index of 0.9017 for MLST). This method has the advantage of being correlated with MLST [through analysis of the terminal direct repeat (TDR) and ancestral spacers] and to possess a high discriminatory power (through analysis of the leader-end spacers recently acquired, which are the witnesses of genetic mobile elements encountered by the bacteria). Furthermore, this "one-shot" approach presents the benefit of much-reduced time and cost in comparison with MLST. On the basis of these data, we propose that this method could become a reference method for group B Streptococcus (GBS) typing.

Keywords: CRISPR-Cas; Streptococcus agalactiae; group B Streptococcus; molecular subtyping; typing.

FIGURE 1

CRISPR clustering according to TDR and ancestral spacers. The CRISPR1 arrays are represented using a macro-enabled Excel tool, whereby spacers are converted into two-color symbols based on spacer sequence. Repeats are not shown except terminal direct repeats (TDRs), which are represented by different colored borders according to their sequence. Spacers were identified by a number attributed following the spacer dictionary (http://crispr.i2bc.paris-saclay.fr/CRISPRcompar/Dict/Dict.php). Arrays are oriented with respect to the leader sequence located on the left. A total of 14 clusters were obtained. The six groups were defined by TDR and ancestral spacers. These groups were then divided into subgroups, based on more recent spacers.

FIGURE 2

Minimum spanning tree (MST) representation of the 25 CRISPR1 markers scheme clustering. Each circle represents a CRISPR1 genotype and its size is proportional to the number of strains. Each color represents CC or singleton defined by MLST (e.g., yellow for CC17). A high level of correlation between this marker selection and MLST type was observed; circles (representing CRISPR1 genotype) are characterized mostly by a same color, especially for CC17 and CC23, whereas isolates belonging to CC1 and CC19 are more dispersed.