Faculty Bibliography

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.

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.

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.

Bordetella holmesii causes respiratory and invasive diseases in humans, but its pathogenesis remains poorly understood. We report here the genome sequences of seven bacteremia isolates of B. holmesii, including the type strain. Comparative analysis of these sequences may aid studies of B. holmesii biology and assist in the development of species-specific diagnostic strategies.

Background: Streptococcus agalactiae (group B Streptococcus [GBS]) is an important neonatal pathogen and emerging cause of disease in adults. The major risk factor for neonatal disease is maternal vaginal colonization. However, little is known about the relationship between GBS and vaginal microbiota. Methods: Vaginal lavage samples from nonpregnant women were tested for GBS, and amplicon-based sequencing targeting the 16S ribosomal RNA V3-V4 region was performed. Results: Four hundred twenty-eight of 432 samples met the high-quality read threshold. There was no relationship between GBS carriage and demographic characteristics, alpha-diversity, or overall vaginal microbiota community state type (CST). Within the non-Lactobacillus-dominant CST IV, GBS positive status was significantly more prevalent in CST IV-A than CST IV-B. Significant clustering by GBS status was noted on principal coordinates analysis, and 18 individual taxa were found to be significantly associated with GBS carriage by linear discriminant analysis. After adjusting for race/ethnicity, 4 taxa were positively associated with GBS, and 6 were negatively associated. Conclusions: Vaginal microbiota CST and alpha-diversity are not related to GBS status. However, specific microbial taxa are associated with colonization of this important human pathogen, highlighting a potential role for the microbiota in promotion or inhibition of GBS colonization.

Cardiomyopathy frequently complicates sepsis and is associated with increased mortality. Increased cardiac oxidative stress and mitochondrial dysfunction have been observed during sepsis, but the mechanisms responsible for these abnormalities have not been determined. We hypothesized that NADPH oxidase 2 (NOX2) activation could be responsible for sepsis-induced oxidative stress and cardiomyopathy. Treatment of isolated adult mouse cardiomyocytes with low concentrations of the endotoxin lipopolysaccharide (LPS) increased total cellular reactive oxygen species (ROS) and mitochondrial superoxide. Elevated mitochondrial superoxide was accompanied by depolarization of the mitochondrial inner membrane potential, an indication of mitochondrial dysfunction, and mitochondrial calcium overload. NOX2 inhibition decreased LPS-induced superoxide and prevented mitochondrial dysfunction. Further, cardiomyocytes from mice with genetic ablation of NOX2 did not have LPS-induced superoxide or mitochondrial dysfunction. LPS decreased contractility and calcium transient amplitude in isolated cardiomyocytes, and these abnormalities were prevented by inhibition of NOX2. LPS decreased systolic function in mice, measured by echocardiography. NOX2 inhibition was cardioprotective in 2 mouse models of sepsis, preserving systolic function after LPS injection or cecal ligation and puncture (CLP). These data show that inhibition of NOX2 decreases oxidative stress, preserves intracellular calcium handling and mitochondrial function, and alleviates sepsis-induced systolic dysfunction in vivo. Thus, NOX2 is a potential target for pharmacotherapy of sepsis-induced cardiomyopathy.