Genomic Analysis of Group B Streptococcus Carriage Isolates From Botswana Reveals Distinct Local Epidemiology and Identifies Novel Strains

Abstract In pregnant people colonized with group B Streptococcus (GBS) in Botswana, we report the presence/expansion of sequence types 223 and 109, a low rate of erythromycin resistance, and 3 novel sequence types. These data highlight the importance of local epidemiologic studies of GBS, a significant source of neonatal disease.

In pregnant people colonized with group B Streptococcus (GBS) in Botswana, we report the presence/expansion of sequence types 223 and 109, a low rate of erythromycin resistance, and 3 novel sequence types.These data highlight the importance of local epidemiologic studies of GBS, a significant source of neonatal disease.
Group B Streptococcus (GBS) is a major cause of neonatal sepsis and meningitis and a leading cause of death in infants globally [1,2].Rectovaginal colonization in late pregnancy can lead to transmission to newborns and is the major risk factor for early-onset GBS disease.Sub-Saharan African countries carry much of the burden of invasive GBS disease [1].In the United States and some other high-income countries, pregnant people are routinely screened for rectovaginal colonization in late gestation, and targeted intrapartum antibiotic prophylaxis (IAP) is highly effective for prevention of early-onset GBS disease [3].
However, IAP has no significant effect in preventing late-onset disease and has not been implemented widely in low-and middle-income countries.Furthermore, rates of GBS resistance to second-line drugs such as erythromycin and clindamycin have increased, and strains with reduced β-lactam susceptibility (RBLS), though rare, have become more common [4,5].
Vaccines targeting GBS capsular polysaccharide are in development and offer a promising alternative to IAP.However, there are 10 known GBS serotypes (Ia, Ib, and II-IX), and current protein-capsular polysaccharide conjugate vaccine candidates cover only a subset of these [6].In addition, immunogenicity of multivalent GBS vaccines may vary by serotype [7].There is significant geographic variability in GBS serotype distribution, strain backgrounds as determined by multilocus sequence typing (MLST), clonal complex (CC) assignment, and antibiotic resistance [1,8].Therefore, local epidemiologic studies are important for understanding the potential utility of candidate vaccines.Our group previously reported GBS rectovaginal colonization rate and serotype distribution in a cohort of pregnant people in Botswana, demonstrating local predominance of serotype V (>45% of isolates compared with approximately 20% worldwide) [9,10].Here, we further characterize GBS isolates from that cohort by whole-genome sequencing (WGS) to determine MLST/CC, presence of specific bacterial virulence determinants, and antimicrobial susceptibility.

METHODS
Streptococcus agalactiae isolates were grown on selective agar (CHROMagar StrepB) at 37°C for 18-24 hours.Purple colonies, indicating GBS growth, were used to inoculate overnight cultures in 5 ml of tryptic soy broth.Bacteria were pelleted, resuspended in 300 mL of Tissue & Cell Lysis Solution (LGC Biosearch), and underwent bead beating with 0.1-mm zirconia/silica beads for 6 minutes.Genomic DNA was extracted using the MagMAX Viral/Pathogen Ultra Nucleic Acid Isolation Kit (Applied Biosystems) on a KingFisher Flex platform.Library preparation and WGS were performed on the Illumina NovaSeq 6000 platform (paired end; 150-base pair reads).Trimmomatic 0.36 software was used for adapter sequence removal and quality trimming, with a minimum read length of 120 base pairs.We used SRST2 0.2.0 software for read mapping to assign genomic serotype using the GBS-SBG database [11], MLST/CC using S agalactiae allele sequences and MLST definition files from PubMLST [12], and antimicrobial resistance and protein profiles using databases from Metcalf et al [13].We detected the presence of AlpST-1, a more distantly related member of the Alp protein family, by read-mapping to the AlpST-1 open reading frame from GBS SS1 [14].Phenotypic determination of antimicrobial susceptibility was not performed.We used Mashtree 1.2.0 software to calculate a kmer distance tree and visualized it on the Microreact web server [15,16].Raw sequencing reads are available in the National Center for Biotechnology Information Sequence Read Archive (under BioProject identifier PRJNA986888).Isolate data are in the PubMLST database (PubMLST.org;accession nos.25868-25908).This study of deidentified samples did not include factors necessitating patient consent.
Among the serotype III GBS isolates, 10 of 10 (100%) were members of the hypervirulent CC17 group.Most of these (7 of 10 [70%]) were ST17, but 2 were ST109, a GBS type previously described as a cause of invasive disease among children in Mozambique and Angola [17,18].ST109 has been reported to have an RBLS phenotype owing to the presence of a G398A mutation in penicillin-binding protein 2x [18].Like the ST109 strains from the Mozambique study, both ST109 strains described here were found to have the G398A mutation.No other strains from the current study had G398A or other penicillin-binding protein 2x mutations known to be associated with a RBLS phenotype.
Nearly all Botswana GBS isolates (39/41 [95.1%]) harbored a tetM gene tetracycline resistance allele.Four isolates (4 of 41[9.8%]),all of which were serotype V/ST1, contained an ermTR gene that confers resistance to macrolides, lincosamides, and streptogramin B (MLS).No other MLS resistance determinants were identified, and no mutations in the 23S ribosomal subunit or the rpo genes were found.No changes in predicted quinolone resistance determinants were predicted by the analysis pipeline.

DISCUSSION
Africa has the highest estimated burden of invasive neonatal GBS cases, but epidemiologic data on circulating strains are still limited [1,20].GBS capsular serotype and MLST distributions differ among countries, with implications for potential efficacy of candidate vaccines.Here, we used bacterial WGS to determine serotypes, MLST, and the presence of genes encoding potential virulence factors and antimicrobial resistance genes in GBS strains from a previously described cohort of pregnant people in Botswana [9].We noted an abundance (>20%) of serotype V, ST223 GBS strains.These findings differ from those of a prior study in coastal Kenya, in which <1% of GBS isolates from pregnant people were ST223 [21].Similarly, in a GBS pangenome-wide association study, 6% of Malawian isolates were identified as ST223 [22].In a sample of >6000 invasive GBS isolates from the United States, only 3 (<0.1%)were ST223 [23].Notably, all of those US-based ST223 isolates were serotype Ia [23].Local expansion of specific GBS types has been noted, including emergence of ST283 as a cause of invasive infections in humans and fish in southeast Asia and of  ST459 in North America [24,[25][26].Of the 6 major GBS CCs, CC17 is highly associated with sepsis and bacterial meningitis in infants in the first 90 days of life.Within CC17, ST109 strains are not commonly found but are of potential concern, given their association with both invasive disease and RBLS [17,18].Two ST109 isolates were identified in the current study, representing approximately 5% of GBS strains and raising the possibility that this GBS type may cause neonatal disease in Botswana and could be more widespread in the region than previously appreciated.We found that a low percentage of colonizing isolates (<10%) were predicted to be resistant to erythromycin, a rate lower than that reported in a recent meta-analysis from African nations (25.1%) [27].We speculate that this finding may reflect a low rate of exposure to this class of antibiotics among the sampled population.In the United States, more than half of GBS strains are resistant to erythromycin [13,23].Fluoroquinolone resistance, which was not detected in this cohort, is rare in the United States (<2% of strains) [23].The high rate of tetracycline resistance noted is consistent with findings that most human colonizing and invasive GBS isolates derive from tetracycline-resistant clones [28].Taken together, these data carry messages of both hope and caution.All of the rectovaginal colonization isolates from this cohort of pregnant people in Botswana have capsular serotypes that are contained in the hexavalent vaccine candidate currently in clinical trials [7,29].In addition, rates of GBS resistance to erythromycin appear to be low in Botswana.However, local proliferation of the CC17/ST223 strain and detection of 2 ST109 isolates in this cohort underscore the need for expanded molecular epidemiologic studies of GBS in this region and for assessment of the role of emerging local strains in neonatal disease.

Figure 1 .
Figure 1.A, Serotype, sequence type (ST), and clonal complex (CC) of group B Streptococcus (GBS) isolates from pregnant people in Botswana.B, GBS whole-genome similarity was assessed using Mashtree 1.2.0 and visualized using the Microreact web server.Nodes are shaded by CC.Branch lengths and scale bar represent Mash distances.Specific ST groupings discussed in the text (ST109 and ST223) are indicated.