Group B Streptococcus Vaginal Carriage in Pregnant Women as Deciphered by Clustered Regularly Interspaced Short Palindromic Repeat Analysis

Abstract

We evaluated the diversity of group B Streptococcus (GBS) vaginal carriage populations in pregnant women. For this purpose, we studied each isolate present in a primary culture of a vaginal swab using a new approach based on clustered regularly interspaced short palindromic repeats (CRISPR) locus analysis. To evaluate the CRISPR array composition rapidly, a restriction fragment length polymorphism (RFLP) analysis was performed. For each different pattern observed, the CRISPR array was sequenced and capsular typing and multilocus sequence typing (MLST) were performed. A total of 970 isolates from 10 women were analyzed by CRISPR-RFLP. Each woman carrying GBS isolates presented one to five specific "personal" patterns. Five women showed similar isolates with specific and unique restriction patterns, suggesting the carriage of a single GBS clone. Different patterns were observed among isolates from the other five women. For three of these, CRISPR locus sequencing highlighted low levels of internal modifications in the locus backbone, whereas there were high levels of modifications for the last two women, suggesting the carriage of two different clones. These two clones were closely related, having the same ancestral spacer(s), the same capsular type and, in one case, the same ST, but showed different antibiotic resistance patterns in pairs. Eight of 10 women were colonized by a single GBS clone, while two of them were colonized by two strains, leading to a risk of selection of more-virulent and/or more-resistant clones during antibiotic prophylaxis. This CRISPR analysis made it possible to separate isolates belonging to a single capsular type and sequence type, highlighting the greater discriminating power of this approach.

Keywords: CRISPR. Streptococcus agalactiae; carriage; diversity; genotyping.

FIG 1

Spacer composition of CRISPR1 arrays for GBS isolates from 10 women. Only one array is represented by different patterns observed after RFLP. The CRISPR1 arrays are represented using a macro-enabled Excel tool whereby spacers are converted into two-color symbols based on the spacer sequence. Gaps (indicating missing spacers) are shown with a boxed “×” symbol after alignment of identical spacers between strains of the same group. Terminal direct repeats (TDRs) are represented by different color borders according to their sequence. The leader sequence (L) is represented by its last two nucleotides. For each woman, the main CRISPR array was represented first. Each woman presented a specific CRISPR array. Three of the 10 women carried isolates belonging to a single CRISPR type with internal modification of the CRISPR1 array (women 1, 3, and 9). Two of the 10 women carried isolates with different CRISPR1 arrays, indicating the presence of two clones (women 8 and 10).