perinatal group b streptococcal infections: virulence factors, … · 2020-03-13 · are...

TrendsGroup B streptococcus (GBS) has toevade genitourinary immune responsesfor successful colonization and infection.

A number of virulence factors promoteGBS vaginal colonization and subse-quent ascension into the pregnantuterus.

The GBS hemolytic pigment and hya-luronidase enable the pathogen toresist immune responses; however,dysregulation of the hemolytic pigmentcan also lead to bacterial clearancefrom the vagina.

GBS invasion of the amniotic fluid isnot absolutely necessary for induction

ReviewPerinatal Group BStreptococcal Infections:Virulence Factors, Immunity,and Prevention StrategiesJay Vornhagen,1,2 Kristina M. Adams Waldorf,3,4 andLakshmi Rajagopal1,2,5,*

Group B streptococcus (GBS) or Streptococcus agalactiae is a b-hemolytic,Gram-positive bacterium that is a leading cause of neonatal infections. GBScommonly colonizes the lower gastrointestinal and genital tracts and, duringpregnancy, neonates are at risk of infection. Although intrapartum antibioticprophylaxis during labor and delivery has decreased the incidence of early-onset neonatal infection, these measures do not prevent ascending infectionthat can occur earlier in pregnancy leading to preterm births, stillbirths, or late-onset neonatal infections. Prevention of GBS infection in pregnancy is complexand is likely influenced by multiple factors, including pathogenicity, host fac-tors, vaginal microbiome, false-negative screening, and/or changes in antibi-otic resistance. A deeper understanding of the mechanisms of GBS infectionsduring pregnancy will facilitate the development of novel therapeutics andvaccines. Here, we summarize and discuss important advancements in ourunderstanding of GBS vaginal colonization, ascending infection, and pretermbirth.

of inflammatory responses and pre-term birth.

Development of a GBS vaccine is cri-tical to reduce or prevent the risk ofinfection during pregnancy, therebyreducing GBS disease burden.

1Department of Global Health,University of Washington, Seattle, WA,USA2Center for Global Infectious DiseaseResearch, Seattle Children’s ResearchInstitute, Seattle, WA, USA3Department of Obstetrics andGynecology, University ofWashington, Seattle, WA, USA4Sahlgrenska Academy, GothenburgUniversity, Gothenburg, Sweden5Department of Pediatrics, Universityof Washington, Seattle, WA, USA

*Correspondence:[email protected](L. Rajagopal).

Infections by GBS during PregnancyGBS is a leading cause of infection during pregnancy, preterm birth, and neonatal infection [1–3]. GBS was first identified in 1887 as a cause of bovine mastitis [4], and later was isolated fromthe human vagina [5] and associated with cases of human disease [6]. Subsequently, GBSvaginal colonization was identified as a risk factor for the development of neonatal GBS disease[7,8] and preterm birth [2,3]. Women who are vaginally colonized during pregnancy are at riskfor ascending infection or transmission of GBS to the newborn during delivery. Ascendinginfection is a widely accepted route by which vaginal bacteria move from the vagina, throughthe cervix, and into the uterus and penetrate gestational tissues (Figure 1). Once GBS hasinvaded the amniotic cavity, or come into contact with the placenta, there is the potential forchorioamnionitis or inflammation of the placental membranes that is frequently associated withpreterm births and stillbirths [9]. Globally, preterm birth is a significant contributor to neonataldeath. Every year, approximately 6 000 000 births are preterm, and more than 500 000neonates die due to prematurity, accounting for 44% of all deaths under the age of 5 years[10,11]. The majority of early preterm births are due to microbial infection [9], and approximately10% are attributable to GBS [12–14]. The bacterial and host determinants that promote GBSvaginal colonization, ascending infection, and adverse perinatal outcomes are poorlyunderstood.

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Choriodecidual space

Uterus

Cervix

Vagina

Group B streptococcus

Endometrium

Decidua

Chorion

Amnion

Amnio�c fluid

Amnio�c epithelium

Figure 1. Ascending Group B Streptococcus (GBS) Infection. GBS vaginal colonization increases the risk ofascending infection during pregnancy. Ascending GBS infection during pregnancy involves bacterial trafficking from thevagina, ultimately leading to bacterial invasion of placental membranes (chorion and amnion), the amniotic cavity, and thefetus. GBS expresses a number of virulence factors that promote vaginal colonization, adhesion and invasion of host cells,and for either activation or suppression of inflammatory responses (Table 1 and Figure 2). These factors increase the risk ofascending infection, fetal injury, or preterm birth.

Recently, significant effort has been dedicated to measuring the global rates of GBS coloniza-tion (Box 1) [15,16], invasive disease [8,17,18], and related risk factors [19,20]. These studiesshow that increased GBS colonization in many low-income countries correlates with increasedneonatal infection and preterm birth [11]. Additionally, women of African descent have a higherincidence of GBS vaginal colonization [15,21,22] and neonatal disease [23–25]. In the USA andmany other countries, women are routinely screened in the late third trimester (between 35 and37 weeks' gestation) for GBS colonization by rectovaginal swab and subsequent culture [26]. Ifthe rectovaginal swab is culture-positive, or if the patient has GBS in the urine, or has a priorhistory of GBS perinatal infection, intrapartum prophylactic antibiotics are administered toprevent vertical transmission of GBS to the neonate during labor and delivery. Unlike the USA,some countries have not adopted the GBS screening program but instead administer anti-biotics upon the development of a risk factor for GBS neonatal disease (e.g., prolonged ruptureof membranes) [26]. However, these approaches have not fully eliminated neonatal GBSinfections. This is because these prevention strategies do not address the risk of ascendinginfection, which can potentially occur anytime during pregnancy, leading to preterm birth orstillbirth. Also, these approaches do not prevent late-onset GBS infections (observed in neo-nates who are older than 1 week of age) where vertical transmission is not the only mode ofacquisition [27]. Overall, prevention of GBS infection in pregnancy is still a complex question,with risk likely imparted by several factors, including: pathogenicity of the GBS strain, hostfactors, influence of the vaginal/rectal microbiome, false-negative screening results, and/orchanges in GBS antibiotic resistance. As current interventions targeting GBS infections arelimited to antibiotic therapy, and given that antibiotic resistance is on the rise [28], a deeperunderstanding of how GBS is able to colonize the vagina and cause neonatal disease is criticalfor the development of new therapeutics. Recently, a number of studies have described hostand bacterial factors important for GBS infections during pregnancy. In this review, we discussrecent advancements in GBS pregnancy-associated infections and therapeutic strategies.

920 Trends in Microbiology, November 2017, Vol. 25, No. 11


Box 1. Limited Knowledge of Colonization Prevalence and Risk

While regional Group B streptococcus (GBS)G vaginal colonization rates have been estimated [1], colonization rates inmany individual countries are unknown. Estimates of GBS vaginal colonization in different countries can be highlyvariable, with interstudy rates differing by as much as 20% [1]. This variability may be due to differences betweensubregions of large countries (e.g., India) or the means of diagnosing colonization (culture-based methods vs. PCR-based methods vs. serology-based methods). More comprehensive country-based and subregional-based studies arenecessary to fully understand the burden of GBS colonization. New studies should focus on areas where preterm birthrates and neonatal mortality rates are especially high, such as sub-Saharan Africa or south Asia. Additionally, fewstudies provide information about GBS serotype prevalence, colonization load, antibiotic-resistance profiles, or valuablegenetic information, such as virulence gene prevalence. While diagnostic technologies exist to evaluate these indicators,they can be time-consuming, expensive, technically challenging, and overall impractical. Diagnostic methodology forGBS colonization has not advanced as rapidly as our understanding of the disease itself, and new technologies need tobe developed to garner more information from future studies to further refine our knowledge of GBS colonization. Finally,more studies need to be designed to conclusively identify risk factors for GBS colonization, ascending infection, andGBS-associated preterm birth. It is clear that previous GBS colonization is a risk factor for colonization during asubsequent pregnancy [2], but few risk factors for initial GBS colonization have been identified. Recent studies haveidentified obesity [3,4] and black ethnicity as possible risk factors for colonization [9]. Future studies should be designedto identify population characteristics beyond ethnic demography and age of GBS-colonized women in an effort toimprove our ability to identify at-risk individuals. Ultimately, universal screening programs are needed in more countriesto measure the burden of GBS colonization and successfully prevent disease.

Mechanisms for GBS Infection during PregnancyIt is long known that GBS vaginal colonization during pregnancy is associated with increasedrates of neonatal infection [29], recurrent maternal colonization [30], early-term birth (weeks37–38, and 6 days of gestation) [31], preterm birth (weeks 14–36, and 6 days of gestation) [3],and stillbirth [32]. GBS is thought to be transmitted from person to person via multiple routes,including fecal–oral, sexual, and vertical transmission [33]. In the same woman, the closeproximity of the vagina and rectum likely enables GBS trafficking from intestinal flora into thevagina. Once GBS enters the vagina, colonization requires the bacteria to overcome a numberof challenges, including: physical barriers created by the mucus and epithelial layers, lowenvironmental pH, antimicrobial peptides, antibodies, microbicidal immune cells, and a vaginalmicrobiome dominated by lactobacilli.

How a nonmotile bacterium, such as GBS, manages to ascend into the uterus while evadinghost responses is not completely understood. The process of ascending infection is challeng-ing to study, as in vitro models are unsuitable, and in vivo models are limited in their ability torecapitulate human pregnancy [34,35]. Despite these limitations, a number of animal models,including pregnant mice and nonhuman primates, have been developed to study the mecha-nisms of ascending GBS infection [36–42], shedding new light on these complicated pro-cesses. While studies using these models have revealed a novel insight into the role of virulencefactors that contribute to ascending infection, more research is needed to fully understand theprocess of ascending GBS infection and adverse neonatal outcomes.

The host immune response evoked in the placenta in response to GBS infection is a keydeterminant of perinatal outcome, microbial invasion of the amniotic cavity (MIAC), and fetalinjury. A variety of fetal and maternal cells within the placental membranes are capable ofpathogen recognition for initiating and sustaining an inflammatory response; these includeamniotic epithelial cells, fetal macrophages, decidual macrophages, decidual NK cells, andneutrophils [9,39,43–46]. While a severe infection leading to early preterm birth is typicallyassociated with MIAC, an inflammatory response confined to the placenta even in the absenceof MIAC is also sufficient to induce preterm labor in some cases [38]. Interestingly, intra-amniotic administration of cytokines, such as tumor necrosis factor-alpha (TNF-a) and inter-leukin (IL)-1b alone (i.e., without any bacteria), can induce preterm labor in pregnant nonhumanprimates [47], and IL-1a, IL-1b, IL-6, and IL-8 drive infection-associated preterm birth inhumans (reviewed in [9,48]). Thus, placental inflammation induced by bacterial infection is

Trends in Microbiology, November 2017, Vol. 25, No. 11 921


likely a critical component of infection-associated preterm birth. Also, bacterial suppression ofplacental immune responses could contribute to MIAC, leading to stillbirths. A better under-standing of the mechanisms by which in utero GBS infections drive preterm births or stillbirthsmay lead to the development of new interventions to reduce the burden of disease. Below, wedescribe key bacterial and host factors that have been identified to influence GBS colonizationand perinatal infection.

Bacterial Factors That Promote GBS Vaginal Colonization, AscendingInfection, and Preterm BirthAdherence and Invasion FactorsGBS encodes a number of virulence factors that allow it to persist in the harsh vaginalenvironment and avoid clearance (Table 1). Many of these factors are involved in adherenceto, and invasion of, host epithelial cells that enable persistent colonization [49]. Adherence andinvasion appear to be mediated by GBS interactions with host extracellular matrix components(ECMs); these interactions may also promote GBS resistance to mechanical clearance,avoidance of immune surveillance, and paracellular transmigration [50]. A few examples ofGBS interaction with host ECMs are discussed below. The GBS extracellular protein BsaB(bacterial surface adhesin of GBS, also known as FbsC) interacts with host laminin [51] andfibrinogen [52], leading to increased adherence to cervicovaginal epithelial cells and biofilmformation [51,52]. The GBS Srr (serine-rich repeat) family of glycoproteins binds to epithelialcells [53] and interacts with host fibrinogen through a unique ‘dock, lock, and latch’ mechanism[54]. Fibrinogen binding leads to an ordered series of conformational changes in Srr1 and Srr2

Table 1. GBS Factors Involved in Vaginal Colonization, Ascending Infection, or Preterm Birth

Virulence factor Host target Function Phenotype/models Refs

HylB Hyaluronic acid Blocks TLR2/4 signaling Mouse vaginal colonization and ascendingInfection

[37,42]

BsaB/FbsC Fibrinogen and laminin Adherence to vaginal epithelial cells Immortalized human cell line [51,52]

Hemolytic pigment Amnion epithelial cellsNeutrophilsMast cellsMacrophages

CytolysisResistance to neutrophilsMast cell degranulationPyroptosis

Mouse vaginal colonization, humanchoriomamnion/placenta, nonhuman primatemodelPrimary human cellsImmortalized human cell line/s

[36,39,46,62,67,69,103]

Srr1/Srr2 Fibrinogen Adherence to vaginal epithelial cells,cervical epithelial cells

Mouse vaginal colonization, immortalized humancell line

[53,55,56,134]

Pili Collagen I Adherence to vaginal epithelial cells Mouse vaginal colonization, immortalized humancell line

[55]

Capsule Siglecsa Adherence to and invasionof cervical epithelial cellsBlunts siglec signaling inamnion epithelial cellsand neutrophils

Immortalized human cell linePrimary human cells

[50][98]

Alpha C protein Host cell surfaceglycosaminoglycan

Invasion of cervical epithelial cells Immortalized human cell line [59]

Two-component system External signal Function Model Refs

CovR/S pH Regulates hemolytic pigmentexpression (apart from other factors),adherence to vaginal epithelial cellsand cervical epithelial cells

Mouse vaginal colonization, immortalized humancell line

[68,69,72]

FspS/R Fructose 6-phosphate Vaginal persistence Mouse vaginal colonization [135]

aSiglecs, Sialic acid-binding immunoglobulin-type lectins.



that result in enhanced adherence [54]. Deletion of the entire Srr1 glycoprotein, or only the latchdomain of Srr1, decreases vaginal colonization [55,56]. The GBS pili also mediate adherenceduring vaginal colonization via the binding of the PilA adhesin to host cell molecules. However,there is a discrepancy in our understanding of the nature of the host cell molecules whereinsome studies indicated that the PilA adhesion binds collagen type 1 [55,57] but others indicatedthat GBS clinical isolates do not bind collagen type 1 but rather bind to fibrinogen [58]. Furtherstudies will provide more insight into these factors during in vivo GBS colonization and infection.Finally, the GBS Alpha C protein, which contains a glycosaminoglycan-binding domain, isthought to mediate GBS invasion of cervical epithelial cells [59,60]; however, the specificglycosaminoglycan that binds Alpha C is not known. GBS invasion in endometrial cells ismediated via lipid rafts and signaling through phosphoinositide 3-kinase [61]. While clinical GBSisolates have been shown to be able to interact with ECM molecules [56,58] (Table 1),interactions between GBS and host ECM components during human vaginal colonizationare unknown. A better understanding of factors that regulate GBS vaginal colonization isnecessary for the development of preventive therapies. Given that there is no known benefit forhumans to be vaginally colonized by GBS, elimination of colonization during pregnancy is idealfor the prevention of GBS disease.

Hemolytic PigmentGBS is a b-hemolytic bacterium, and the hemolytic property of GBS is important for infectionand immune evasion. Hemolytic activity of GBS is due to the ornithine rhamnolipid pigment(hereafter referred to as‘hemolytic pigment’ or ‘pigment’) [62], which is produced by the genesof the cyl operon [63]. Transcription of cyl genes, and therefore production of the hemolyticpigment, is negatively regulated by the CovR/S two-component system (also known as CsrR/S) [62,64,65]. Consequently, deletion of covR/S renders GBS hyperhemolytic and hyperpig-mented [62,64,65]. Conversely, deletion of the cylE gene, which encodes an N-acyltransferasenecessary for pigment production [62], renders GBS nonpigmented and nonhemolytic [62,63].Identification of hemolytic, hyperhemolytic and nonhemolytic GBS strains in human casesallowed for a greater understanding of the role of hemolysin in GBS infection.

Whidbey et al. reported that the hemolytic pigment promotes GBS penetration of humanplacenta (chorioamniotic membranes) and induces loss of barrier function in human amnioticepithelial cells [62]. Furthermore, hyperpigmented GBS strains were isolated from either theamniotic fluid or chorioamniotic membranes of women in preterm labor [62]. Randis et al. alsonoted decreased bacterial dissemination, fetal injury, and preterm birth in mice that werevaginally inoculated with nonhemolytic GBS (i.e., GBS lacking cylE) [36]. Recently, Boldenowet al. showed that the increased hemolytic pigment expression accelerated GBS invasion of theamniotic cavity with significant uterine contractions and inflammatory responses indicative ofpreterm labor in a nonhuman primate model [39]. Although infection with hyperpigmented GBSinduced the formation of neutrophil extracellular traps (NETs) in chorioamniotic membranes ofnonhuman primates [39], these GBS strains were resistant to the antimicrobial activity of NETs,likely through increased pigment-mediated antioxidant activity [66]. Formation of NETs inresponse to GBS infection was also observed in murine models of colonization [67] andascending infection [41]. Collectively, these studies indicate a role for the hemolytic pigmentin promoting GBS dissemination in uterine, placental, and fetal tissues during pregnancy.

The GBS hemolytic pigment also affects vaginal colonization. Absence of the hemolyticpigment reduced the ability of GBS to successfully colonize the vagina, possibly due toincreased susceptibility to neutrophil clearance [36,67]. Surprisingly, hyperpigmented GBSalso exhibited decreased vaginal colonization in mice ([68,69]; see [70] for a visual of the murinevaginal model), likely due to increased pigment-mediated stimulation of neutrophil [68] andmast cell [69] inflammatory pathways. Consistent with these observations, hyperpigmented



strains of GBS were rarely isolated from rectovaginal swabs of asymptomatic pregnant women[69]. These results emphasize the role of vaginal immune responses in pathogen colonization.

Apart from immune cells, pH regulates GBS gene expression and therefore influences vaginalcolonization. For instance, the GBS CovR/S system responds to pH wherein increased CovR/Sregulation was observed under low (acidic) pH [65,71–73]. Changes in pH also influence GBSadhesion [72,74], survival [75], and biofilm formation [76,77]. Of note, high vaginal pH and anon-lactobacilli-dominated vaginal microbiome [78] were associated with a higher incidence ofGBS vaginal colonization [15,21,22] and neonatal disease [23–25] in women of African descent. These studies indicate that GBS responds to environmental cues, such as pH, and evenutilizes regulatory systems such as CovR/S to temporally control virulence factor expression(e.g., hemolytic pigment) during pregnancy-associated infections. As such, this makes the GBSpigment an intriguing target for vaccine development.

HyaluronidaseThe GBS hyaluronidase, known as HylB, promotes vaginal colonization [42]. HylB is secretedby GBS and specifically targets and degrades host hyaluronic acid [79,80]. Hyaluronic acid isan extracellular matrix glycosaminoglycan composed of repeating disaccharide units (N-acetyl-D-glucosamine-D-glucuronic acid) and is important for cell migration, cell signaling, regulation ofinflammation [81], and the prevention of ascending infection [82,83]. GBS HylB degrades hosthyaluronic acid into its disaccharide components, which are immunosuppressive as they bindto TLR2/TLR4 receptors and block signaling [42]. Deletion of hylB led to increased clearance ofGBS from the mouse vagina [42]. Similarly, GBS lacking HylB was less able to ascend from thevagina to the uterus and was diminished for its ability to invade fetal tissues and cause pretermbirth [37]. By contrast, uterine tissue infected with HylB-proficient GBS showed decreasedlevels of inflammatory cytokines such as TNF-a, IL-6, and IL-8, leading to bacterial ascension[37]. Thus, suppression of key inflammatory responses also plays an important role in GBSinfection-associated fetal injury.

Other Virulence FactorsRecent studies have described a role for extracellular membrane vesicles (MVs) in the weak-ening of placental membranes [84]. GBS MVs contained multiple virulence factors, including: (i)HylB; (ii) CAMP factor (Christine, Atkins, Munch-Peterson factor [85]), a secreted pore-formingprotein [86] that may amplify [87], but is not essential for, GBS virulence [88]; (iii) IgA-bindingprotein, with the ability to bind human IgA [89] for host immune evasion [90], and (iv) multipleenzymes that may regulate ECM degradation [84]. Intra-amniotic administration of GBS MVs inpregnant mice caused significant damage to choriodecidual tissues and stimulated leukocyticinfiltration and inflammation, leading to membrane weakening [84]. The specific role played byeach virulence factor in the context of MVs remains unknown. MV weakening of choriodecidualmembranes represents a novel mechanism of GBS fetal injury.

Host Determinants of GBS Vaginal Colonization, Ascending Infection, andPreterm BirthVaginal ColonizationEvasion of the host immune response is essential for successful vaginal colonization. GBSvaginal immunity is mediated by its ability to resist many physical and cellular barriers, includingthe luminal mucus layer, vaginal epithelia, and immune cells in the vagina. Recent work, usinganimal models, has provided new information regarding the host immune response to GBSvaginal colonization [49,67–69]. Vaginal immune responses to GBS are largely mediated byneutrophils [49,67,68], mast cells [69], and macrophages [67] (Figure 2). The role of NK cellsand dendritic cells in GBS colonization is not known. Multiple soluble inflammatory cytokinesand chemokines have been identified as important for reducing GBS vaginal colonization and



Epithelium

Lumen

Neutrophils

Macrophages

Mast cellsGroup Bstreptococcus

Degranula�on IL-5IL-8

TNFαHistamine

IL-1βIL-6IL-8

TNFα

Blocked TLR2/4

NLRP3 ac�va�on

NETforma�on

KEY:

HyIB

Hyaluronic acid

TLR2/4

Figure 2. Interaction of Group B Streptococcus (GBS) with Innate Immune Cells during Genital Infection.Upon primary vaginal colonization, vagina-resident mast cells are activated and degranulated through the hemolyticactivity of the GBS pigment. Degranulation leads to the release of inflammatory mediators, including IL-6, IL-8, TNF-a, andhistamine, which, in turn, recruit other immune cells, such as neutrophils and macrophages, to aid in bacterial clearance[69]. Neutrophils clear GBS through phagocytosis and extrusion of neutrophil extracellular traps (NETs) [67]. Additionally,GBS activates the NLRP3 inflammasome in neutrophils, leading to secretion of IL-1b, as well as other inflammatorycytokines. GBS is able to use the pigment to prevent phagocytic death through sequestration of reactive oxygen species[66], and to avoid being killed by NETs [39]. Macrophages also aid in GBS clearance through phagocytosis. GBS, in turn,activates the NLRP3 inflammasome in macrophages through membrane permeabilization by pigment, leading topyroptosis and fetal damage during pregnancy [46]. Abbreviations: IL, interleukin; TNF-a, tumor necrosis factor-alpha.

include IL-1b, IL-6, IL-8, IL-17, IL-23, and histamine [49,67,68]. Currently, the mucosal T cellresponse to GBS colonization is ill-defined. Studies have shown that IL-17 and IL-17+ cells playan important role in clearance of a hyperadherent and invasive GBS strain from the vagina [49],suggesting that the Th17 differentiation pathway is important for controlling persistent GBScolonization. Similarly, another study found cytokines involved in Th1, Th2, and Th17 differentia-tion pathways as important for decreased colonization [67]; however, T cells were not directlyidentified as being important in either study.



Infection of Placental MembranesMany studies have focused on GBS infection of the placental membranes (chorioamnion) inorder to understand how GBS penetrates these barriers and induces chorioamnionitis. GBS isable to adhere to and invade both chorionic and amniotic epithelial cells [62,91]. Adherence andinvasion to these cells are mediated by several factors: (i) IagA, a glycosyltransferase that helpsanchor lipoteichoic acid to the cell surface [92]; (ii) the hemolytic pigment and its regulatorCovR/S [62]; and (iii) quorum sensing mediated by genes in the rgf operon [93]. GBS has alsobeen shown to induce secretion of multiple cytokines and defensins from placental membranesex vivo, including TNF-a, IL-1a, IL-1b, IL-6, and IL-8 [44,62,94–96]. Inflammation is stimulatedeither through pattern-recognition receptor (PRR) sensing of GBS antigens [44,94] or bypigment-mediated activation of nuclear factor-kB (NF-kB) [62]. An important family of PRRsthat mediate placental membrane immunity are the Siglecs [97,98], a family of cell-surface sialicacid-binding lectins that regulate innate and adaptive immune function [99]. GBS is able to bindSiglecs through the sialic acid capsule or b-protein to suppress immune cell activation [98,100–102] and placental membrane inflammation [98], potentially leading to increased rates ofGBS-associated preterm birth and stillbirth.

Fetal InjuryGBS invasion of the fetus in utero leads to a variety of adverse outcomes, including tissuedamage, inflammation, lung and brain injury, pneumonia, meningitis, sepsis, and fetal death.GBS can invade multiple fetal organs, including the lung, blood, liver, spleen, and gastrovas-cular cavity. Fetal tissue damage has been observed in the presence and absence of bacterialinvasion, which may be due to inflammation in the gestational tissues and amniotic fluid. GBSinvasion of fetal tissues induces inflammation and fetal death [32,36,37,46], and in the case ofthe hemolytic pigment, this involved induction of the NLRP3 inflammasome [46]. GBS stim-ulates the NLRP3 inflammasome in a number of immune cells, such as dendritic cells [103],macrophages [46], and neutrophils [104], which contribute to in vivo inflammation.

Interestingly, fetal injury is not entirely dependent on bacterial invasion of fetal tissues. Fetal lunginjury can also be caused by GBS-induced chorioamnionitis without bacterial invasion [38,105].Also, increases in amniotic fluid cytokines contribute to fetal lung injury [38] and dysregulation offetal lung development [105,106]. Moreover, intra-amniotic administration of MVs leads tosignificantly increased rates of fetal damage and preterm birth [84]. These studies suggest thatfetal injury can occur during transient or limited infection, and further emphasizes the impor-tance of developing therapeutics that prevent vaginal colonization and ascending infection.

Limitations and Next StepsWhile significant research has been committed to understanding the host and bacterial factorsinvolved in GBS vaginal colonization and ascending infection, further research is needed to fullygrasp these complex processes. Additionally, the research that has been completed is notwithout its limitations. These limitations and the next steps for research are discussed below.

Vaginal ColonizationDespite recent advances, there are many limitations in our understanding of GBS vaginalcolonization. Importantly, the complete repertoire of GBS virulence factors, host factors, andenvironmental factors that determine the extent and duration of vaginal colonization remainunknown. This point is key, given that the dynamics of colonization during pregnancy are highlyvariable [107]. In addition, experiments performed in laboratory settings are limited by the use ofanimal models that do not perfectly model the human vaginal environment. To fully understandGBS vaginal colonization, experimental models and outputs that more closely represent humandisease are needed. Finally, it is often assumed that neonates contract GBS from contaminatedvaginal fluids during the birthing process; however, given the kinetics of neonatal disease



(frequently displaying clinical symptoms within 0–24 h of birth [108]), many cases are thought tobe a result of in utero infections caused by ascended GBS. This idea is also supported by thefrequent recovery of GBS from the amniotic fluid and chorioamniotic membranes in cases ofinfection-associated preterm birth [1,109,110]. Given its important implication for understand-ing GBS disease, colonization is an area of active research.

Ascension from the Vagina to the UterusWhile studies have provided some insight into virulence factors that enable GBS to gain accessto the uterine space from the lower genital tract, and host factors involved in this process,significant research is required to fully understand ascending GBS infection. Currently, little isknown about host factors that prevent ascending infection, such as the role of the cervicalbarrier, endocrine signaling, and cellular immunity. Further, host-specific factors and geneticsthat may influence ascending GBS infection are unknown. Studies aimed at filling theseknowledge gaps could lead to the development of therapeutics for preventing ascendinginfection. The choice of animal model is key in answering some of these questions due toimportant differences in pregnancy physiology, mechanism of labor, and placental structurebetween humans and mice [111]. Nonhuman primates are the closest animal model to fullyrecapitulate important aspects of human pregnancy [111] but are limited in their use by ethicalconstraints, availability, and cost. Ideally, a combination of lower animal and nonhuman primatemodels should be used in order to delineate relevant aspects of disease. Finally, clinical studiesshould be designed to identify biomarkers for ascending infection. Viral infection of the cervix[112] and diminished cervical hyaluronic acid levels [83] have been associated with increasedascending infection; however, little is known about the clinical relevance of these or otherfactors in the context of ascending GBS infection.

GBS Prevention and TherapeuticsCurrently, strategies focus on the prevention of GBS transmission during labor and deliverythrough the use of antibiotics. This strategy does not fully capture the biology of GBS infection,nor does it completely address the full burden of GBS disease. Moreover, antibiotic resistanceis increasing [28], and the use of antibiotics during pregnancy has consequential effects forneonatal health that are only now being appreciated [113,114]. To successfully eradicate theburden of disease, interventions need to be specifically targeted, have minimal detrimentaleffects on the microbiome, and target processes upstream of vertical transmission, such ascolonization and ascending infection.

Multiple studies have focused on a probiotic approach to reducing vaginal GBS colonization.Recent studies using probiotic Lactobacillus species have shown that pretreatment of thevagina can block GBS adherence to vaginal epithelial cells [115], and reduce colonization[116,117]. Probiotic administration of the oral colonizer Streptococcus salivarius has also beenshown to have the ability to reduce vaginal GBS burden through a yet unidentified antimicrobialactivity [118]. It is important to note that frequent doses (from three to seven) of probioticbacteria were required for a protective effect in vivo [116,117], raising the question as to thefeasibility of probiotic intervention in humans, and highlighting the need to continue to explorethis field to identify an efficient and feasible probiotic therapy to prevent GBS colonization.Moreover, little is known about the interactions between GBS and the vaginal microbiome, andwhether those interactions have any positive benefits to human health. Further studies that aimto understand these interactions would shed much needed light on this topic.

A vaccine to prevent GBS colonization would be the most effective intervention; however,development of a vaccine has proven to be challenging. Recent work has shown that vaccinationof mice with killed bacteria reduces preterm birth rates [119] and that mucosal vaccine delivery ismore effective than intramuscular delivery [120]. Multiple studies have identified maternal antibody



Outstanding QuestionsWhy are humans intermittently colo-nized with GBS? What determineswhether a GBS strain will colonizethe vagina? How can we eliminateGBS without adverse consequenceson beneficial microbiota?

What factors determine the ability of aGBS strain to ascend into the uterusand invade the placenta and amnioticcavity? Is it environmental factors in thelower genital tract during pregnancy?

How do geographic differences in hostgenetics, environment, and diet con-tribute to differences in the vaginalmicrobiome and GBS colonization?

Can we determine which GBS strainsare likely to invade the amniotic cavityor cause late-onset neonatal sepsis tobetter target antibiotic therapy to ahigher-risk group of pregnant women?

levels to the GBS capsule as being protective against GBS infection [121–124], and it has longbeen known that anticapsular antibodies can confer protection to infection [5]. Unfortunately,vaccines targeting the GBS capsule alone are ineffective due to their poor immunogenicity andthus, conjugate–capsule vaccines, and a vaccine that targets the alpha C/Rib protein family, arenow being tested in clinical trials (reviewed in [125–127]). Despite the promise of these vaccinecandidates,challenges to the eradicationofGBSdisease will existevenafter the implementation ofa vaccine. While ten GBS capsular serotypes have been identified, serotypes Ia, Ib, II, III, and V arepredominantly responsible for GBS disease [127]. Consequently, current vaccine candidatestarget serotypes Ia, Ib, and III, [125], and a pentavalent vaccine targeting serotypes Ia, Ib, II, III, andV is in preclinical development [126]. The development of vaccines targeting various antigens,such as Alpha C/Rib, may prove to be vital, as nontypeable GBS strains can cause disease [128].Additionally, GBS strains can switch capsular serotypes [129], thereby evading host immunityconferred by vaccination.

It is possible that if these vaccines are developed, serotypes or strains that are not typically asignificant cause of GBS disease may emerge in vaccinated populations. Indeed, this phenome-non has been observed with implementation of conjugate vaccines against Streptococcuspneumoniae [130,131]. Thus, GBS surveillance programs would need to remain vigilant evenafter a vaccine becomes widely used. There are also significant challenges in resource-limitedsettingstoconsiderprior to implementation (reviewed in [132]).Regardless, it isclear thata vaccinewould be the most effective means of reducing the global burden of GBS disease [133].

Concluding RemarksGBS has long been recognized as a significant human pathogen [6], yet we are only nowbeginning to fully understand its pathogenesis almost a century later. It is clear that under-standing the interplay between the host and bacteria is vital for understanding how to effectivelyprevent GBS disease while having minimal adverse effects on the human host. Research in thisfield has revealed novel insights into the bacterial virulence factors necessary for successfullyestablishing disease (Table 1) and an appropriate host response to prevent ascending infectionand preterm birth (Figures 1 and 2). Any disturbance in this balance can lead to serious andlasting outcomes for the host, or disadvantageous pressures for the bacteria. Several examplesof how this balance can be perturbed to benefit human health have been described above, butmore research is needed to fully understand what triggers ascending GBS infection, the hostimmune response to colonization and ascending infection, and the physiological drivers of fetaldamage and preterm birth (see Outstanding Questions). Additionally, more epidemiologicaldata are required to describe the global burden of GBS colonization and disease (Box 1). AsGBS colonization during pregnancy is intermittent and variable [107], the current screeningmethodology likely misses a significant portion of colonization during pregnancy. This issueresults in a measurable risk for ascending GBS infection during pregnancy that could lead tostillbirth, preterm birth, and early-term birth. An evaluation of earlier and more frequent GBSscreening during pregnancy may shed light on these issues. With these data, effective andfeasible interventions to prevent GBS disease can be developed. Ultimately, vaccination willprove to be the most effect intervention. A combination of rational vaccine design, intelligentimplementation and monitoring strategies, and strong advocacy [26] may lead to the eradica-tion of GBS as a human pathogen.

AcknowledgmentsWe thank Jan Hamanishi for assistance with graphical design. We apologize to GBS researchers whose work was not

reviewed here due to constraints in space and/or focus of the topic. We acknowledge funding from the National Institutes

of Health, Grant R01AI100989 to L.R and K. A. W., and R21AI125907 and R01AI112619 to L.R. The NIH training grant

T32 AI07509 (PI: Lee Ann Campbell) supported J.V. The content in this manuscript is the responsibility of the authors and

does not necessarily represent the views of the National Institutes of Health.



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