Group B Streptococcus Capsular Serotype Alters Vaginal Colonization Fitness.

BACKGROUND: Group B Streptococcus (GBS) remains a leading cause of infant morbidity and mortality. A candidate vaccine targets 6 GBS serotypes, offering a potential alternative to intrapartum antibiotic prophylaxis to reduce disease burden. However, our understanding of the contributions of specific capsule types to GBS colonization and disease remains limited. METHODS: Using allelic exchange, we generated isogenic GBS strains differing only in the serotype-determining region in 2 genetic backgrounds, including the hypervirulent clonal complex (CC) 17. Using a murine model of vaginal cocolonization, we evaluated the roles of the presence of capsule and of expression of specific capsular types in GBS vaginal colonization fitness independent of other genetic factors. RESULTS: Encapsulated wild-type strains COH1 (CC17, serotype III) and A909 (non-CC17, serotype Ia) outcompeted isogenic acapsular mutants in murine vaginal cocolonization. COH1 wild type outcompeted A909. Notably, expression of type Ia capsule conferred an advantage over type III capsule in both genetic backgrounds. CONCLUSIONS: Specific capsule types may provide an advantage in GBS vaginal colonization in vivo. However, success of certain GBS lineages, including CC17, likely involves both capsule and noncapsule genetic elements. Capsule switching in GBS, a potential outcome of conjugate vaccine programs, may alter colonization fitness or pathogenesis.

Genome-Wide fitness analysis of group B Streptococcus in human amniotic fluid reveals a transcription factor that controls multiple virulence traits.

Streptococcus agalactiae (group B Streptococcus; GBS) remains a dominant cause of serious neonatal infections. One aspect of GBS that renders it particularly virulent during the perinatal period is its ability to invade the chorioamniotic membranes and persist in amniotic fluid, which is nutritionally deplete and rich in fetal immunologic factors such as antimicrobial peptides. We used next-generation sequencing of transposon-genome junctions (Tn-seq) to identify five GBS genes that promote survival in the presence of human amniotic fluid. We confirmed our Tn-seq findings using a novel CRISPR inhibition (CRISPRi) gene expression knockdown system. This analysis showed that one gene, which encodes a GntR-class transcription factor that we named MrvR, conferred a significant fitness benefit to GBS in amniotic fluid. We generated an isogenic targeted deletion of the mrvR gene, which had a growth defect in amniotic fluid relative to the wild type parent strain. The mrvR deletion strain also showed a significant biofilm defect in vitro. Subsequent in vivo studies showed that while the mutant was able to cause persistent murine vaginal colonization, pregnant mice colonized with the mrvR deletion strain did not develop preterm labor despite consistent GBS invasion of the uterus and the fetoplacental units. In contrast, pregnant mice colonized with wild type GBS consistently deliver prematurely. In a sepsis model the mrvR deletion strain showed significantly decreased lethality. In order to better understand the mechanism by which this newly identified transcription factor controls GBS virulence, we performed RNA-seq on wild type and mrvR deletion GBS strains, which revealed that the transcription factor affects expression of a wide range of genes across the GBS chromosome. Nucleotide biosynthesis and salvage pathways were highly represented among the set of differentially expressed genes, suggesting that MrvR may be involved in regulating nucleotide availability.

The Impact of Circulating Antibody on Group B Streptococcus Intestinal Colonization and Invasive Disease.

Gastrointestinal (GI) colonization with group B Streptococcus (GBS) is an important precursor to late-onset (LO) disease in infants. The host-pathogen interactions that mediate progression to invasive disease remain unknown due, in part, to a paucity of robust model systems. Passively acquired maternal GBS-specific antibodies protect newborns from early-onset disease, yet their impact on GI colonization and LO disease is unexplored. Using murine models of both perinatal and postnatal GBS acquisition, we assessed the kinetics of GBS GI colonization, progression to invasive disease, and the role of GBS-specific IgG production in exposed offspring and juvenile mice at age 12 and 14 days, respectively. We defined LO disease as >7 days of life in the perinatal model. We studied the impact of maternal immunization using a whole-cell GBS vaccine on the duration of intestinal colonization and progression to invasive disease after postnatal GBS exposure in offspring. Animals exhibit sustained GI colonization following both perinatal and postnatal exposure to GBS, with 21% and 27%, respectively, developing invasive disease. Intestinal colonization with GBS induces an endogenous IgG response within 20 days of exposure. Maternal vaccination with whole-cell GBS induces production of GBS-specific IgG in dams that is vertically transmitted to their offspring but does not decrease the duration of GBS intestinal colonization or reduce LO mortality following postnatal GBS exposure. Both perinatal and postnatal murine models of GBS acquisition closely recapitulate the human disease state, in which GBS colonizes the intestine and causes LO disease. We demonstrate both endogenous production of anti-GBS IgG in juvenile mice and vertical transfer of antibodies to offspring following maternal vaccination. These models serve as a platform to study critical host-pathogen interactions that mediate LO GBS disease.

High prevalence of Group B Streptococcus colonization among pregnant women in Amman, Jordan.

BACKGROUND: Little is known of the burden of Group B Streptococcus (GBS) colonization among pregnant women in Jordan. We conducted a pilot study to determine the prevalence of GBS among pregnant women in Amman, Jordan, where GBS testing is not routine. We also explored GBS serotypes and the performance of a rapid GBS antigen diagnostic test. METHODS: We collected vaginal-rectal swabs from women who presented for labor and delivery at Al-Bashir Hospital. Three methods were used to identify GBS: Strep B Rapid Test (Creative Diagnostics), blood agar media (Remel) with confirmed with BBL Streptocard acid latex test (Becton Dickinson), and CHROMagar StrepB (Remel). Results were read by a senior microbiologist. We defined our gold standard for GBS-positive as a positive blood agar culture confirmed by latex agglutination and positive CHROMagar. PCR testing determined serotype information. Demographic and clinical data were also collected. RESULTS: In April and May 2015, 200 women were enrolled with a median age of 27 years (IQR: 23-32); 89.0% were Jordanian nationals and 71.9% completed secondary school. Median gestational age was 38 weeks (IQR: 37-40); most women reported prenatal care (median 9 visits; IQR: 8-12). Median parity was 2 births (IQR: 1-3). Pre-pregnancy median BMI was 24.1 (IQR: 21.5-28.0) and 14.5% reported an underlying medical condition. Obstetric complications included gestational hypertension (9.5%), gestational diabetes (6.0%), and UTI (53.5%), of which 84.5% reported treatment. Overall, 39 (19.5%) of women were GBS-positive on blood agar media and CHROMagar, while 67 (33.5%) were positive by rapid test (36% sensitivity, 67% specificity). Serotype information was available for 25 (64%) isolates: III (48%), Ia (24%), II (20%), and V (8%). No demographic or clinical differences were noted between GBS+ and GBS-negative women. CONCLUSIONS: Nearly one in five women presenting for labor in Jordan was colonized with GBS, with serotype group III as the most common. The rapid GBS antigen diagnostic had low sensitivity and specificity. These results support expanded research in the region, including defining GBS resistance patterns, serotyping information, and risk factors. It also emphasizes the need for routine GBS testing and improved rapid GBS diagnostics for developing world settings.

High Rate of Serotype V Streptococcus agalactiae Carriage in Pregnant Women in Botswana.

Maternal rectovaginal colonization is the major risk factor for early-onset neonatal sepsis due to Group B Streptococcus (GBS), a major cause of early life morbidity and mortality. Transmission generally occurs perinatally from colonized mothers to infants. Vaccines targeting a subset of GBS serotypes are under development, but GBS epidemiology remains poorly understood in many African nations. We performed a cross-sectional study of GBS colonization among pregnant women at two sites in Botswana, a country with minimal prior GBS carriage data. We found a rectovaginal colonization rate of 19%, comparable with studies in other regions; however, we also noted a striking predominance of serotype V (> 45% of strains). Although further studies are required to delineate the burden of invasive GBS disease in Botswana and the generalizability of type V epidemiology, these data provide a useful baseline for understanding the potential local impact of GBS prevention strategies, including vaccines.

A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

Improving the Sensitivity of Real-time PCR Detection of Group B Streptococcus Using Consensus Sequence-Derived Oligonucleotides.

Group B Streptococcus (GBS) is a perinatal pathogen and an emerging cause of disease in adults. Culture-independent GBS detection relies on polymerase chain reaction (PCR) of conserved genes, including sip. We demonstrate suboptimal sensitivity of the existing sip PCR strategy and validate an improved method based on consensus sequences from >100 GBS genomes.