Isolation and identification techniques for GBS v1
Authors: not provided lydiariver 
Published: February 2022

Abstract Introduction Group B Streptococcus (GBS) causes infections in women during pregnancy and puerperium and invasive infections in newborns. The genes lmb, cylE, scpB, and hvgA are involved with an increased virulence of GBS and hypervirulent clones have been identified in different regions. In addition, increasing resistance of GBS to macrolides, lincosamides, and tetracyclines has been reported, so knowing the patterns of resistance to antibiotics may be necessary to prevent and treat GBS infections adequately. This study aimed to identify virulence genes and antibiotic resistance associated with GBS colonization in pregnant women from northeastern Mexico. Methods Pregnant women with 35-37 weeks of gestation underwent recto-vaginal swabbing. One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin and nalidixic acid, a second swab was inoculated into LIM enrichment broth, and a third swab was submerged into a transport medium. All samples were subcultured onto blood agar. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification by Gram staining, catalase test, hippurate hydrolysis, CAMP test, and incubation in a chromogenic medium. We used latex agglutination to confirm and serotype GBS isolates. Antibiotic resistance patterns were assessed by Vitek 2 and disk diffusion. Periumbilical, rectal and nasopharyngeal swabs were collected from some newborns of colonized mothers. All colonized women and their newborns were followed-up for three months to assess the development of disease attributable to GBS. Draft genomes of all GBS isolates were obtained by whole-genome sequencing. In addition, bioinformatic analysis to identify genes encoding capsular polysaccharides and virulence factors was performed using BRIG, while antibiotic resistance genes were identified using the CARD database. Results We found 17 GBS colonized women out of 1154 pregnant women (prevalence: 1.47%). None of the six newborns sampled were colonized, and no complications due to GBS were detected in pregnant women or newborns. Serotyping showed that 3 isolates were serotype I, 5 serotype II, 3 serotype III, 4 serotype IV, and 2 serotype V. Ten distinct virulence gene profiles were identified, being scpB, lmb, fbsA, acp, PI-1, PI-2a, cylE the most common (1/14, 21%). The virulence genes identified were namely scpB 100% (14/14), lmb 100% (14/14), cylE 100% (14/14), PI-1 86% (12/14), fbsA 77% (11/14), PI-2a 77% (11/14), acp 71% (10/14), fbsB 46%(6/14), PI-2b 36% (5/14), and hvgA 36% (5/14). We identified resistance to tetracycline in 65% (11/17) of the isolates, intermediate susceptibility to clindamycin 41% (7/17), and reduced susceptibility to ampicillin 23.5% (4/17). The tetM gene associated to tetracyclines resistance was found in 79% (11/14) and the mel and mefA genes associated to macrolides resistance in 7% (1/14). Conclusions The low prevalence of colonization and the non-occurrence of mother-to-child transmission suggest that the intentional search for GBS colonization in this population is not justified. Our results also suggest that risk factors should guide the use of intrapartum antibiotic prophylaxis. The detection of strains with genes coding virulence factors means that clones with pathogenic potential circulates in this region. On the other hand, the identification of decreased susceptibility to antibiotics from different antimicrobial categories shows the importance of adequately knowing the resistance patterns to prevent and to treat GBS perinatal infection.