Streptococcus agalactiae (Group B Streptococcus or GBS) is an opportunistic bacterial pathogen that asymptomatically colonizes the recto-vaginal tract of up to 35% of pregnant people. GBS colonization during pregnancy is a risk factor for adverse pregnancy outcomes including chorioamnionitis, preterm and stillbirths, as well as severe neonatal disease. GBS disease in neonates has two presentations: early (EOD) and late onset disease (LOD), which occur within the first week or first three months after birth, respectively. Prevention protocols for GBS disease include screening for recto-vaginal colonization during late stages of pregnancy (35-37 weeks) and, if positive, administering intrapartum antibiotic prophylaxis (IAP) treatment. IAP has only been successful in reducing EOD and is not effective in preventing LOD, preterm births, or stillbirths. Importantly, there are increasing observations of persistent colonization after IAP intervention, indicating that the pathogen can survive and rebound following antibiotic exposure. Intriguingly, strains classified as a hypervirulent genotype are better able to withstand these stressors and persistently colonize the vaginal tract, while others are easily cleared by host immune cells or a course of antibiotics. The mechanisms by which these GBS strains avoid antibiotic-mediated killing to persistently colonize the vaginal tract, however, are poorly understood. I sought to investigate these mechanisms by examining the impact of IAP on 1) GBS genomic evolution and 2) the biogenesis of membrane vesicles (MVs). I employed whole genome sequencing analyses on 97 clinical GBS isolates previously obtained from the vaginal tracts of pregnant individuals before (35-37 weeks’ gestation) and after (6 weeks postpartum) IAP and childbirth. One goal was to identify key genomic signatures associated with persistent colonization. Using both reads-based and assembly-based methods, I observed substantial evidence of genomic variation between pairs (prenatal-postpartum) of isolates, leading to the discovery of mutators among the postpartum isolates. These mutators have exceptionally high mutation rates due to disruptions in DNA mismatch repair systems and provide a reservoir of beneficial mutations that enhance fitness. Indeed, I observed evidence of genes under positive selection in mutator isolates after IAP exposure, including those that encode attachment and regulatory proteins. Moreover, we observed stronger biofilms in most of the postpartum isolates compared to their respective prenatal isolates. These findings demonstrate that antibiotic treatment impacts GBS evolution in vivo by selecting for mutations that promote persistent colonization and survival. The presence of mutators may lead to the emergence of more resilient strains in the vaginal tract, increasing the risk of invasive GBS infection. To further elucidate mechanisms of survival in the presence of antibiotics, I examined the production and composition of MVs produced by a hypervirulent GBS strain that persistently colonized the vaginal tract after IAP. MVs are biologically active, lipid-enclosed entities that have been shown to play a role in bacterial persistence and survival of antibiotic stress in other species. No prior studies of GBS MVs in the context of antibiotics have been conducted and thus, I isolated MVs produced by GBS after exposure to β-lactam and macrolide antibiotics. Quantification of MVs revealed that antibiotic treatment significantly increases the abundance of MVs produced regardless of the antibiotic class. Using proteomics to characterize the proteins packaged within MVs revealed protein compositions that were distinct for each antibiotic treatment when compared to the untreated (control) group. Furthermore, increased abundances of antibiotic targets that were specific to each respective antibiotic treatment were detected, suggesting that GBS MVs protect the bacteria from antibiotic killing by disseminating decoy targets outside of the cell. Both the excess quantities and distinct compositions of MVs produced in the presence of antibiotics may enhance GBS survival and contribute to persistent colonization despite IAP intervention. Improving our understanding of GBS MVs and their role in persistent infections will aid in the development of more targeted and effective treatments for GBS disease during pregnancy. Together, this work considerably improves our understanding of persistent colonization despite antibiotic exposure and is a fundamental step towards improving GBS treatment and prevention strategies to effectively reduce the incidence of GBS disease.
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