Faculty Bibliography

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 demonstrate 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 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.

Group B Streptococcus (GBS) is a neonatal pathogen frequently transmitted from maternal asymptomatic vagino-rectal colonization. Co-colonization with multiple GBS serotypes, which has implications for type-specific vaccination strategies, is difficult to detect with standard microbiologic techniques. We designed a nested real-time PCR assay to detect vaginal co-colonization in samples from a cohort of non-pregnant women (N = 433). 6/91 (6.6%) GBS-positive samples harbored ≥2 GBS serotypes, with over-representation of serotype V among co-colonized samples. Serotype IV GBS was more prevalent (>10%) in this cohort than in previously reported United States studies. Ongoing surveillance of GBS serotype epidemiology and co-colonization is indicated.

BACKGROUND/AIMS/OBJECTIVE:Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS:Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS:Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION/CONCLUSIONS:Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.

Background. Controlling methicillin-resistant Staphylococcus aureus (MRSA) colonization is a common strategy to prevent transmission and recurrent infection. Standard decolonization regimens include nasal application of mupirocin ointment; however, increasing rates of mupirocin-resistance (Mup-R) have been noted globally. At our institution there has been an increase in community-acquired MRSA (CA-MRSA) infections among children living in Brooklyn, New York. A genotypic geographic cluster of an outbreak clone of the CA-MRSA strain USA 300 with a high rate (>85%) of mupirocin resistance, mediated by the plasmid borne mupA gene, was identified prompting investigation into an alternative decolonizing agent. We sought to investigate retapamulin, a topical pleuromutilin antibiotic, which has been shown to be effective against S. aureus with in vitro and in vivo activity against MRSA and a low propensity to develop resistance. Methods. Broth microdilution was used to determine the minimum inhibitory concentrations (MIC) of retapamulin against 53 Mup-R MRSA isolates collected from pediatric patients (aged 9 months-17 years) presenting to our institution over an 18 month period with clinical MRSA infection. Susceptibility defined as <=0.5 mg/L susceptible (EUCAST). Whole genome sequence data were analyzed for the presence of rplC and cfr gene mutations known to confer resistance to retapamulin. Results. All 53 isolates were susceptible to retapamulin. 49/53 (92%) strains were inhibited at MIC 0.25 mg/L, 2/53 (4%) at MIC 0.125 mg/L, and 2/53 (4%) at MIC 0.5 mg/L. DNA sequence analysis showed that one isolate had a first-step mutation in the rplC gene, but it was not associated with reduced phenotypic susceptibility to retapamulin, as the MIC of that isolate was 0.25 mg/L. Conclusion. Retapamulin demonstrated excellent in vitro activity against a genotypic cluster of Mup-R isolates from pediatric patients presenting to our institution with MRSA infection. These data suggest that retapamulin may be a promising alternative decolonization therapy for MRSA and a viable option to prevent the spread of mupirocin-resistant MRSA clones. Further research includes an ongoing randomized, placebo-controlled trial testing the in vivo efficacy of retapamulin as a nasal and perirectal decolonizing agent in children

PROBLEM/OBJECTIVE:Bacterial chorioamnionitis causes adverse pregnancy outcomes, yet host-microbial interactions are not well characterized within gestational membranes. The decidua, the outermost region of the membranes, is a potential point of entry for bacteria ascending from the vagina to cause chorioamnionitis. We sought to determine whether paracrine communication between decidual stromal cells and macrophages shaped immune responses to microbial sensing. METHOD OF STUDY/METHODS:production. RESULTS:in amniotic fluid, suggesting such paracrine effects might hold relevance in vivo. CONCLUSION/CONCLUSIONS:.

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.

Vaginolysin (VLY), a cytotoxic protein produced by Gardnerella vaginalis, may contribute to bacterial vaginosis (BV). Women with G. vaginalis, low levels of lactobacilli, history of vaginal douching, higher Nugent scores, and higher vaginal pH had increased VLY. Inflammatory markers were not highly expressed with increasing VLY. VLY's role in BV warrants further evaluation.

The cholesterol dependent cytolysins (CDCs) are a family of pore-forming toxins produced by a wide range of bacteria. Some CDCs are important virulence factors for their cognate organisms, but their activity must be tightly regulated to ensure they operate at appropriate times and within the appropriate subcellular compartments. pH-dependent activity has been described for several CDCs, but the mechanism of such regulation has been studied in depth only for listeriolysin O (LLO), which senses environmental pH through a triad of acidic residues that mediate protein unfolding. Here we present data supporting a distinct mechanism for pH-dependence for inerolysin (INY), the CDC produced by Lactobacillus iners. Inerolysin (INY) has an acidic pH optimum with loss of activity at neutral pH. INY pH-dependence is characterized by reversible loss of pore formation with preservation of membrane binding. Fluorescent membrane probe assays indicated that INY insertion into host cell membranes, but not oligomerization, was defective at neutral pH. These data support the existence of a newly appreciated form of CDC pH-dependence functioning at a late stage of pore formation.

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.

We report a case of cutaneous cryptococcosis due to Cryptococcus neoformans in a pediatric patient with hyper IgM syndrome with scalp lesions that resembled tinea capitis on gross examination and mimicked juvenile xanthogranuloma on histologic examination. This case highlights the importance of considering cutaneous cryptococcosis in patients with hyper IgM syndrome.