Genomic profiling of Streptococcus agalactiae (Group B Streptococcus) isolates from pregnant women in northeastern Mexico: clonal complexes, virulence factors, and antibiotic resistance

Introduction

Streptococcus agalactiae (S. agalactiae), commonly known as Group B Streptococcus (GBS), is an encapsulated Gram-positive bacterium that is part of the normal microbiota of the human gastrointestinal and genitourinary tracts (Furfaro, Chang & Payne, 2018). Despite its commensal nature, S. agalactiae is a significant pathogen, particularly in newborns, where it can cause invasive infections when colonization occurs in pregnant women during the later stages of pregnancy (Alsheim et al., 2024). In Latin America, S. agalactiae colonization rates range from 2% to 20.4%, with an estimated neonatal infection incidence of 0.3% to 1% (Palacios-Saucedo et al., 2017).

The pathogenicity of S. agalactiae is mediated by several virulence factors that enhance colonization and contribute to antimicrobial resistance (Burcham et al., 2019). Among these, the sialic acid capsular polysaccharide (Cps), encoded by the cps loci, is one of the most extensively studied. This polysaccharide is used to classify the bacterium into serotypes Ia, Ib, and II to IX and is known to facilitate immune evasion (Teatero et al., 2014). Other important virulence factors include laminin binding protein (Lmb), fibrinogen-binding proteins (Fbs), hypervirulent adhesin (HvgA), and alpha C protein (αC protein), all of which are associated with adherence and cell invasion (Bobadilla et al., 2021; Lacasse et al., 2022). The pili virulence factor, encoded by PI-1, PI-2a and PI-2b genes, confers resistance to antimicrobial peptides (Lu et al., 2015). Additionally, β-hemolysin/cytolysin, encoded by the CylE gene, acts as a pore-forming toxin (Shimizu et al., 2020). Several other virulence factors also contribute to immune evasion, cell adhesion and invasion, antimicrobial resistance, and toxin production, making them relevant to S. agalactiae pathogenesis (Rajagopal, 2009). Antibiotic resistance in S. agalactiae is an increasing concern. Macrolide resistance is conferred by the mreA, mefA, mefE and ermB genes, while tetracycline resistance is primarily associated with the tetM gene, which are commonly found in GBS (Mudzana, Mavenyengwa & Gudza-Mugabe, 2021; Liang et al., 2023).

Although numerous studies have examined the epidemiology and prevalence of S. agalactiae in pregnant women, further research is needed to characterize its molecular features to improve therapeutic and monitoring strategies (Delgado-Arévalo et al., 2020; Cabrera-Reyes et al., 2021; van Kassel et al., 2021). This study first aimed to determine the prevalence of S. agalactiae colonization in pregnant women attending a secondary referral hospital in Northeastern Mexico. Additionally, we sought to characterize the genetic diversity of S. agalactiae isolates by analyzing their sequence type (ST), clonal complex (CC), capsular polysaccharide (Cps) genotype, virulence factors, and antibiotic resistance genes.

Materials and Methods

Wet lab procedures

Dry lab (in silico) procedures

Results

Assemblages generation and bioinformatics data analysis

Draft genome assemblies were generated for all 51 isolates following quality control (Table S1). Genomic data are available under NCBI BioProjects PRJNA892112 and PRJNA551699. MLST analysis identified 13 distinct ST, grouped into six CC’s: CC12 (23.5%), CC452 (19.6%), CC23 (17.6%), CC19 (9.8%), CC1 (7.8%), and CC17 (7.8%). Seven isolates (13.7%) were unassigned to any CC (Fig. 1).

Discussion

S. agalactiae, or GBS, is a leading cause of neonatal infections due to vertical transmission from mother to child during birth (Alsheim et al., 2024). In this study, we first determined the prevalence of S. agalactiae colonization among pregnant women attending a secondary referral hospital in Northeastern Mexico, identifying a colonization rate of 2.7% (51/1,924). Compared to other studies in Mexico, Cabrera-Reyes et al. (2021) reported a slightly higher prevalence of 4.3% (145/3,347) in pregnant women. Additionally, a meta-analysis by Reyna-Figueroa et al. (2007) documented a wide range of S. agalactiae prevalence rates (0.46% to 38%) across different regions of Mexico, with an average of 9.5%. Our findings fall within the lower range of this spectrum, suggesting potential regional differences in colonization rates or variations in detection methodologies. The observed discrepancies may be attributed to differences in population characteristics, clinical and environmental factors, sampling techniques, and microbiological detection methods. Furthermore, to gain deeper insight into the genetic diversity of S. agalactiae in this population, we characterized the isolates by analyzing their ST, CC, Cps genotype, virulence factors, and antibiotic resistance genes.

The most common serotypes in developed countries are Ia (31%), III (27%), V (19%), Ib (14%), and II (5%) (Hall et al., 2017). In our study, serotypes II (19.6%), III (17.6%), IV (17.6%), and Ia (15.7%) were the most frequent. The variation in serotype distribution may be influenced by geographical factors, as well as intra-country differences (Bobadilla et al., 2021). Additionally, we identified one pregnant woman colonized with serotype VIII, which is primarily reported in Asia (Lachenauer et al., 1999; Genovese et al., 2020). GBS capsular serotypes (Ia, Ib and II to IX) can be identified using latex agglutination and molecular methods such as PCR or sequencing. In this study, serotypes were identified in 47 (92.16%) of 51 isolates using serological tests, whereas capsular genotypes were identified in 50 (98.04%) of the 51 strains. A high agreement rate (82.98%, 39 of 47) was observed between the two methods, consistent with reports indicating >80% concordance between serological and molecular approaches (Yao et al., 2013; Brigtsen et al., 2015; Suhaimi et al., 2017). Some studies suggest the possibility of capsular polysaccharide switching, although clear evidence is lacking (Martins, Melo-Cristino & Ramirez, 2010; Bellais et al., 2012). These findings highlight the potential limitations of serological methods and underscore the importance of molecular approaches for accurate genotyping. The most frequent CCs associated with serotypes Ia, Ib, II, III, IV, and V are 23, 8, 22, 17, 459, and 1, respectively (McGee et al., 2021). Our results were consistent for serotypes Ia and III, but for serotype II, CC12 was predominant, while for serotypes IV and V, CC452 and CC19 were most common, respectively.

The GBS genome harbors various virulence factors. Consistent with previous studies, genes involved in immune evasion, such as scpB, sodA and cspA, were present in 100% of the isolates, with sodA commonly used as a housekeeping gene (Rosenau et al., 2007; Slotved et al., 2021; Koide et al., 2022). Genes associated with adhesion and invasion, such as lmb, were present in all isolates. Fibrinogen-binding protein genes were detected at rates of 54.9% for fbsA+/fbsB+, 35.3% for fbsA+/fbsB−, and 9.8% for fbsA−/fbsB−, aligning with previously reported data (Rosenau et al., 2007). The bca gene, which exhibits variable prevalence (21% to 88.6%), was detected in 72.5% of our isolates (Bobadilla et al., 2021; Lacasse et al., 2022). The ssr-1 and ssr-2 genes were found in 79.5% and 15.4% of isolates, respectively, compared to reported rates of 70.6% and 37.3%. The bibA gene, rarely detected in GBS, was present in 35.3% of our isolates, similar to the previously reported 34% (Lacasse et al., 2022). The hvgA gene, associated with hypervirulent strains, was identified in 39.2% of our isolates, exceeding the reported 12.8% (Burcham et al., 2019).

Pili are surface structures that contribute to S. agalactiae adhesion, host colonization, and biofilm formation, facilitating bacterial persistence in the host. In S. agalactiae, three pilus islands (PI-1, PI-2a, and PI-2b) have been identified, which can be present individually or in combination. Previous reports indicate the presence of PI-1 in 43.1%, PI-2a in 85.6%, and PI-2b in 14.4% of S. agalactiae isolates. In our study, the prevalence of PI-1 (76.5%) and PI-2b (35.3%) was higher, while PI-2a (68.8%) was lower compared to previous reports (Lu et al., 2015). While pili are primarily associated with bacterial adherence, previous studies suggest they may also play a role in antimicrobial peptide resistance by influencing bacterial interactions with the host immune system and antimicrobial compounds, although the precise mechanisms remain unclear (Rajagopal, 2009).

The dltA-D operon, which enhances bacterial resistance to host defenses, was present in 100% of our isolates, in agreement with studies indicating increased susceptibility in its absence (Armistead et al., 2019). The cfb gene, encoding pore-forming proteins, is universally present in GBS strains, with rare exceptions due to chromosomal deletions (Bobadilla et al., 2021; Creti et al., 2023). The cylE gene, which confers hemolytic properties and enhances virulence, was present in all hemolytic strains. Non-hemolytic strains, which are less virulent, constitute only 3% to 4% of reported isolates (Shimizu et al., 2020).

GBS resistance to clindamycin and erythromycin is primarily mediated by ribosomal methylation genes such as ermB or ermA. In this study, only one isolate (2.0%) carried ermB, while the isaE gene, associated with clindamycin resistance, was detected in isolates lacking erm genes. The mreA gene, encoding an antibiotic efflux pump that confers erythromycin resistance, was present in all isolates (Liu et al., 2023). Tetracycline resistance is commonly mediated by tetM, tetL, or tetO, with tetM being the most prevalent. In our study, 31 (60.1%) of isolates carried tetM, while tet(W/N/W) and tet(45) were detected in only 2 (2.0%) of cases (Gizachew et al., 2020).

Overall, our findings contribute important prevalence and genomic epidemiology data on S. agalactiae in northeastern Mexico. However, some limitations should be considered. First, the study was conducted at a single referral hospital, which may limit the generalizability of the findings to the broader population. Second, while whole-genome sequencing allowed for precise genetic characterization, phenotypic antimicrobial susceptibility testing was not performed to correlate resistance genes with actual resistance profiles. Lastly, the relatively low prevalence observed suggests that a larger sample size across multiple healthcare centers would be beneficial for a more comprehensive epidemiological assessment.

Conclusion

This study determined the prevalence of S. agalactiae colonization among pregnant women attending a secondary referral hospital in northeastern Mexico, identifying a colonization rate of 2.7% (51/1,924), which falls within the lower range reported in previous studies across Mexico. To further understand the genetic diversity of S. agalactiae in this population, we characterized the isolates based on their ST, CC, Cps genotype, virulence factors, and antibiotic resistance genes. The predominant CC identified were CC452, CC23, and CC19. While Cps genotyping showed overall concordance between serological and molecular methods, some discrepancies were observed, including the detection of two Cps genotypes in the same isolate. All isolates harbored key virulence genes (scpB, sodA, lmb, and fbsA) at varying frequencies, and pili-encoding genes were present in distinct combinations. Additionally, all isolates carried mreA and tetM, highlighting the persistence of antimicrobial resistance determinants in S. agalactiae.

Supplemental Information