Thomas A. Hooven

Group B Streptococcus β-hemolysin/Cytolysin Breaches Maternal-Fetal Barriers to Cause Preterm Birth and Intrauterine Fetal Demise in Vivo

BACKGROUND Maternal vaginal colonization with Streptococcus agalactiae (Group B Streptococcus [GBS]) is a precursor to chorioamnionitis, fetal infection, and neonatal sepsis, but the understanding of specific factors in the pathogenesis of ascending infection remains limited. METHODS We used a new murine model to evaluate the contribution of the pore-forming GBS β-hemolysin/cytolysin (βH/C) to vaginal colonization, ascension, and fetal infection. RESULTS Competition assays demonstrated a marked advantage to βH/C-expressing GBS during colonization. Intrauterine fetal demise and/or preterm birth were observed in 54% of pregnant mice colonized with wild-type (WT) GBS and 0% of those colonized with the toxin-deficient cylE knockout strain, despite efficient colonization and ascension by both strains. Robust placental inflammation, disruption of maternal-fetal barriers, and fetal infection were more frequent in animals colonized with WT bacteria. Histopathologic examination revealed bacterial tropism for fetal lung and liver. CONCLUSIONS Preterm birth and fetal demise are likely the direct result of toxin-induced damage and inflammation rather than differences in efficiency of ascension into the upper genital tract. These data demonstrate a distinct contribution of βH/C to GBS chorioamnionitis and subsequent fetal infection in vivo and showcase a model for this most proximal step in GBS pathogenesis.

Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity

BACKGROUND Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown. METHODS We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro. RESULTS RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation. CONCLUSIONS These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention.

CD59 signaling and membrane pores drive Syk-dependent erythrocyte necroptosis.

Mature erythrocytes (red blood cells (RBCs)) undergo the programmed cell death (PCD) pathway of necroptosis in response to bacterial pore-forming toxins (PFTs) that target human CD59 (hCD59) but not hCD59-independent PFTs. Here, we investigate the biochemical mechanism of RBC necroptosis with a focus on the mechanism of induction and the minimal requirements for such RBC death. Binding or crosslinking of the hCD59 receptor led to Syk-dependent induction of vesiculated morphology (echinocytes) that was associated with phosphorylation of Band 3 and was required for Fas ligand (FasL) release. FasL-dependent phosphorylation of receptor-interacting protein kinase 1 (RIP1) in combination with plasma membrane pore formation was required for execution of RBC necroptosis. RIP1 phosphorylation led to the phosphorylation of RIP3, which was also critical for RBC necroptosis. Notably, RBC necroptosis was mediated by FasL and not by other candidate inducers, including tumor necrosis factor alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL). Other types of RBC damage, such as eryptotic damage, failed to induce necroptosis when combined with hCD59 crosslinking. This work sheds light on the requirements for this recently discovered PCD in RBCs and provides a clear picture of the biochemical mechanism of induction of RBC necroptosis.

Complete Genome Sequence of Streptococcus agalactiae CNCTC 10/84, a Hypervirulent Sequence Type 26 Strain

ABSTRACT Streptococcus agalactiae (group B Streptococcus [GBS]) is a human pathogen with a propensity to cause neonatal infections. We report the complete genome sequence of GBS strain CNCTC 10/84, a hypervirulent clinical isolate frequently used to study GBS pathogenesis. Comparative analysis of this sequence may shed light on novel pathogenic mechanisms.

What’s the harm? Risks and benefits of evolving rule-out sepsis practices

Asymptomatic term and late-preterm newborns with risk factors for early onset sepsis commonly undergo laboratory evaluation and receive empiric antibiotic therapy. Some have challenged the rationale for current “rule-out sepsis” practices, arguing that they lead to unnecessary overtreatment and healthcare costs. A series of recent clinical studies has explored scheduled serial observations as an alternative to laboratory testing and empiric antibiotics for asymptomatic newborns with historical risk factors for sepsis. These studies have shared the conclusion that serial observation is safe and cost-effective for well-appearing term and late-preterm babies, but they are also somewhat speculative because culture-proven early onset sepsis is an extremely low prevalence diagnosis. Here, we review the evolving consensus of optimal rule-out sepsis practices. We examine chorioamnionitis as an example of a problematic risk factor that has contributed to the controversy surrounding this topic. We also discuss how introduction of online sepsis risk calculators has allowed more precise delineation of a patient’s chances of developing culture-proven infection. Finally, we analyze existing data from published studies to estimate the number needed to harm (NNH) when an observation-based strategy is used instead of a risk-based approach. We conclude that, if harm is defined as death or serious sepsis complications such as hemodynamic instability or neurologic injury, the NNH is 1610, compared to an NNH of 7 and 2.9 for IV infiltrates and delayed breastfeeding, respectively—two common and potentially consequential complications of NICU admission for a rule-out sepsis. We believe that the differential between risk of serious harm from observing a well-appearing term or late-preterm newborn with risk factors for sepsis and the risk of less significant but common NICU complications argues in favor of the ongoing trend toward less aggressive management of newborns with sepsis risks.

Time To Overhaul the “Rule Out Sepsis” Workup

For neonatologists, decisions about how to manage well-appearing newborns with risk factors for sepsis remain fraught. Maternal chorioamnionitis (inflammation of the chorion and amnion) caused by intrauterine bacterial infection affects 0.1% to 2% of pregnancies1,2 and increases the newborn’s risk of early-onset sepsis.1,3 For this reason, clinical guidelines published by the Centers for Disease Control and Prevention in 20104 and the American Academy of Pediatrics in 2012 (with a subsequent clarifying supplement in 2013)5,6 recommended that all well-appearing term newborns whose mothers were diagnosed with chorioamnionitis undergo laboratory screening for sepsis, including blood culture, and receive at least 48 hours of broad-spectrum antibiotic therapy. However, consensus has shifted in the years since these recommendations were published. Multiple studies and commentaries have suggested that performing sepsis evaluations on all well-appearing term newborns with a maternal history of chorioamnionitis is unnecessary and may have significant downsides, including: interference with breastfeeding and bonding; alteration of the neonatal microbiome (with uncertain long-term consequences); and risk of … Address correspondence to Richard A. Polin, MD, NewYork-Presbyterian Morgan Stanley Children’s Hospital, 3959 Broadway, CHC102, New York, NY 10032. E-mail: rap32{at}cumc.columbia.edu

Neonatal Bacterial Infections

The neonate is uniquely susceptible to bacterial infections, which can range from a subtle to a flagrant presentation. Early identification and treatment of bacterial infection in the newborn can prevent major complications and often depend on consideration of risk factors—such as chorioamnionitis and prolonged rupture of the membranes—in combination with careful, repeated physical examinations.

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

Abstract 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.

The Streptococcus agalactiae Stringent Response Enhances Virulence and Persistence in Human Blood

ABSTRACT Streptococcus agalactiae (group B Streptococcus [GBS]) causes serious infections in neonates. We previously reported a transposon sequencing (Tn-seq) system for performing genomewide assessment of gene fitness in GBS. In order to identify molecular mechanisms required for GBS to transition from a mucosal commensal lifestyle to bloodstream invasion, we performed Tn-seq on GBS strain A909 with human whole blood. Our analysis identified 16 genes conditionally essential for GBS survival in blood, of which 75% were members of the capsular polysaccharide (cps) operon. Among the non-cps genes identified as conditionally essential was relA, which encodes an enzyme whose activity is central to the bacterial stringent response—a conserved adaptation to environmental stress. We used blood coincubation studies of targeted knockout strains to confirm the expected growth defects of GBS deficient in capsule or stringent response activation. Unexpectedly, we found that the relA knockout strains demonstrated decreased expression of β-hemolysin/cytolysin, an important cytotoxin implicated in facilitating GBS invasion. Furthermore, chemical activation of the stringent response with serine hydroxamate increased β-hemolysin/cytolysin expression. To establish a mechanism by which the stringent response leads to increased cytotoxicity, we performed transcriptome sequencing (RNA-seq) on two GBS strains grown under stringent response or control conditions. This revealed a conserved decrease in the expression of genes in the arginine deiminase pathway during stringent response activation. Through coincubation with supplemental arginine and the arginine antagonist canavanine, we show that arginine availability is a determinant of GBS cytotoxicity and that the pathway between stringent response activation and increased virulence is arginine dependent.