Characterization of a Putative Hemolysin Expressed by Sneathia amnii, a Preterm Birth-associated Pathogen

The gram-negative bacteria Sneathia amnii is a poorly-characterized commensal of the female urogenital tract frequently associated with adverse clinical outcomes such as bacterial vaginosis (BV), amnionitis, and preterm labor. To investigate its potential role in virulence, we sought to identify and characterize virulence determinants produced by S. amnii in an effort to better understand the pathogenesis of infectious preterm birth. Through sequencing of the Sn35 genome (type strain of S. amnii), we identified two genes with amino acid sequence similarity and structural similarity to the filamentous hemagglutinin (FHA) protein of Bordetella pertussis and its Type Vb transporter. Because S. amnii requires human blood components for growth and lyses human red blood cells, we hypothesized that this two-partner system was involved in hemolysis. To characterize the function of the FHA-like protein, a purified, recombinant peptide was used to induce an antibody response. The polyclonal rabbit serum against the antigenic peptide was incubated with S. amnii to block the FHA-like protein prior to the addition of red blood cells. Pre-treatment with the antiserum inhibited hemolytic activity against human erythrocytes suggesting that the FHA-like protein is somehow involved in hemolysis. Additionally, we found that the hemolytic activity of S. amnii was highly specific against human red blood cells; it did not lyse horse or rabbit red blood cells and only minimally lysed sheep red blood cells. Further research efforts will focus on purifying functional FHA-like protein for further characterization and to determine whether it is sufficient to induce hemolysis.https://scholarscompass.vcu.edu/uresposters/1249/thumbnail.jp

Characterization of Protein Involved in Hemolysis Expressed by Sneathia amnii, a Pathogen of the Female Urogenital Tract

Sneathia amnii is a poorly characterized gram-negative anaerobe that commonly colonizes the vagina. It has been linked to many obstetric disorders, including preterm labor, preeclampsia, and chorioamnionitis. S. amnii lyses human red blood cells, and we aimed to identify the hemolysin. We identified two genes that appear to encode transporter and effector components of a two-partner secretion system. The putative effector, which we refer to as SaFHA, contains a domain with amino acid similarity to the filamentous hemagglutinin (FHA) of Bordetella pertussis and its predicted structure suggests it may form a transmembrane channel or pore. Thus, we hypothesized that SaFHA would be secreted by S. amnii and that it would play a role in hemoglobbin release. To test this, a portion of the gene encoding the SaFHA protein in S. amnii was expressed in E. coli and used as an immunogen in rabbits. Western analysis using anti-SaFHA revealed that the protein is secreted and localizes to the bacterial surface. Pre-treatment of S. amnii with anti-SaFHA blocked the hemolytic activity whereas antiserum against an irrelevant protein had no effect. We partially purified SaFHA from S. amnii using cation exchange chromatography and the partially purified protein mediated hemoglobin release from human RBC, supporting our hypothesis. Further characterization of SaFHA will help provide more insight on the virulence of S. amnii, and perhaps shed light on the etiology of Sneathia-associated vaginal conditions, as well as future treatment options.https://scholarscompass.vcu.edu/gradposters/1046/thumbnail.jp

How Proactive Measures Reduce Patient Handling Injuries Among Healthcare Workers In A Hospital Setting: A Systematic Review To Determine Best Practice

The purpose of this study is to determine the best proactive measures in a hospital setting to reduce patient handling injuries among healthcare workers. Improper patient handling techniques have been found to be a major cause of workplace injuries among health care workers in the hospital setting. A systematic review was performed to assess programs that have implemented various methods of improving patient handling techniques. There are numerous programs or components that were found to be successful in reducing patient handling injuries. Among these program components are lift teams, lifting equipment, ceiling lifts, education, training and an active multidisciplinary team. The costs to the organization resulting from lost work days, workers compensation claims, turnover and modified duty days was found to be decreased in those organizations that have implemented a safe patient handling program

Phase Variation of Poly-N-Acetylglucosamine Expression in Staphylococcus aureus

Polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-β-(1–6)-glucosamine (PIA/PNAG) is an important component of Staphylococcus aureus biofilms and also contributes to resistance to phagocytosis. The proteins IcaA, IcaD, IcaB, and IcaC are encoded within the intercellular adhesin (ica) operon and synthesize PIA/PNAG. We discovered a mechanism of phase variation in PIA/PNAG expression that appears to involve slipped-strand mispairing. The process is reversible and RecA-independent, and involves the expansion and contraction of a simple tetranucleotide tandem repeat within icaC. Inactivation of IcaC results in a PIA/PNAG-negative phenotype. A PIA/PNAG-hyperproducing strain gained a fitness advantage in vitro following the icaC mutation and loss of PIA/PNAG production. The mutation was also detected in two clinical isolates, suggesting that under certain conditions, loss of PIA/PNAG production may be advantageous during infection. There was also a survival advantage for an icaC-negative strain harboring intact icaADB genes relative to an isogenicicaADBC deletion mutant. Together, these results suggest that inactivation of icaC is a mode of phase variation for PIA/PNAG expression, that high-level production of PIA/PNAG carries a fitness cost, and that icaADB may contribute to bacterial fitness, by an unknown mechanism, in the absence of an intact icaC gene and PIA/PNAG production

Resident perspectives on green infrastructure in an experimental suburban stormwater management program

Municipalities are increasingly promoting green infrastructure in residential neighborhoods as a strategy to manage stormwater runoff, but the extent to which residents are willing to adopt these alternatives to conventional landscaping remains unclear. This study examines the West Creek Ecosystem Restoration Project in Parma, Ohio, a suburban green infrastructure demonstration site for a new regional stormwater management program in the Cleveland metropolitan region. Residents were offered free installation of green infrastructure (e.g., rain gardens, right-of-way bioretentions, and rain barrels) on their property. Through a mixed method case-study, we analyze the socio-cultural factors that influence participation including resident’s self-reported landscaping behaviors, environmental knowledge and values, and attitudes and perceptions toward green infrastructure and stormwater management. Results suggest that attitudes and perceptions most strongly influenced participation, residents generally disconnect local stormwater management from regional water resource issues, and that trusted peers may be more likely to encourage participation than official promotional strategies. These findings point to the importance of context-dependent framing and neighborhood partners in outreach activities

Untargeted Lipidomic Analysis to Broadly Characterize the Effects of Pathogenic and Non-Pathogenic Staphylococci on Mammalian Lipids

Modification of the host lipidome via secreted enzymes is an integral, but often overlooked aspect of bacterial pathogenesis. In the current era of prevalent antibiotic resistance, knowledge regarding critical host pathogen lipid interactions has the potential for use in developing novel antibacterial agents. While most studies to date on this matter have focused on specific lipids, or select lipid classes, this provides an incomplete picture. Modern methods of untargeted lipidomics have the capacity to overcome these gaps in knowledge and provide a comprehensive understanding of the role of lipid metabolism in the pathogenesis of infections. In an attempt to determine the role of lipid modifying enzymes produced by staphylococci, we exposed bovine heart lipids, a standardized model for the mammalian lipidome, to spent medium from staphylococcal cultures, and analyzed lipid molecular changes by MS/MSALLshotgun lipidomics. We elucidate distinct effects of different staphylococcal isolates, including 4 clinical isolates of the pathogenic species Staphylococcus aureus, a clinical isolate of the normally commensal species S. epidermidis, and the non-pathogenic species S. carnosus. Two highly virulent strains of S. aureus had a more profound effect on mammalian lipids and modified more lipid classes than the other staphylococcal strains. Our studies demonstrate the utility of the applied untargeted lipidomics methodology to profile lipid changes induced by different bacterial secretomes. Finally, we demonstrate the promise of this lipidomics approach in assessing the specificity of bacterial enzymes for mammalian lipid classes. Our data suggests that there may be a correlation between the bacterial expression of lipid-modifying enzymes and virulence, and could facilitate the guided discovery of lipid pathways required for bacterial infections caused by S. aureus and thereby provide insights into the generation of novel antibacterial agents

Genetic heterogeneity and taxonomic diversity among Gardnerella species

Gardnerella spp. are the most frequent organisms found in bacterial vaginosis (BV). However, colonization by Gardnerella spp. does not always lead to BV. Developments in molecular genetics have shed new light on the diversity of the genus Gardnerella, leading to an emended description of Gardnerella vaginalis and, for the first time, the inclusion of more species of Gardnerella, namely, Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii. While no association had been made between the novel Gardnerella species and virulence potential, there is genomic evidence of a higher prevalence of some genotypes in BV cases, as compared with colonization in women in whom BV has been clinically ruled out. Gardnerella vaginalis has been considered a pivotal player in the progression of bacterial vaginosis (BV), a condition associated with serious health complications. However, G. vaginalis is also commonly found in asymptomatic or BV-negative women. This has generated interest in the question of whether genetic differences among isolates might distinguish pathogenic from commensal isolates. G. vaginalis was the only recognized species in its genus for four decades, but recently an emended description of G. vaginalis and descriptions of three new species Gardnerella leopoldii, Gardnerella piotii, and Gardnerella swidsinskii have been proposed. This review provides background on the heterogeneity and diversity within the genus Gardnerella, highlighting the main features that distinguish species and clades, and how these features may impact BV development.J.C. and N.C. acknowledge the projects PTDC/BIA-MIC/28271/2017 (POCI-01-0145-FEDER-028271), and the strategic funding of unit UID/BIO/04469/2019, supported by the Portuguese Foundation for Science and Technology.info:eu-repo/semantics/publishedVersio

Introduction of plasmid DNA into Sneathia vaginalis; the first step to genetic manipulation

Many bacterial pathogens are capable of secreting one or more toxins, all of which can be categorized based on their function, target, or mechanism of action. Pore-forming toxins, for instance, are characterized based on their ability to perforate the host membrane. This may result in the delivery of bacterial substances into the host cell, release of compounds from the host cell, and/or death of the host cell. Research from our lab has demonstrated that Sneathia vaginalis, a gram-negative anaerobic bacterial species that is associated with bacterial vaginosis and preterm birth, produces a pore-forming toxin referred to as cytopathogenic toxin A. CptA is capable of lysing human red blood cells and permeabilizing chorionic trophoblasts and other epithelial cells. CptA appears to contribute to the traversal of Sneathia vaginalis across fetal membranes and may therefore contribute towards preterm birth and bacterial vaginosis. In order to confirm its role in fetal membrane traversal, an isogenic mutant of Sneathia vaginalis, in which the cptA gene has been functionally deleted, is needed. To accomplish this, we tested both plasmids and linear PCR products for the ability to insertionally inactivate the cptA gene. If the mutant is no longer able to traverse fetal membranes, then this will confirm that CptA is necessary for the virulence of Sneathia vaginalis.https://scholarscompass.vcu.edu/uresposters/1405/thumbnail.jp

Quantitative analysis of initial adhesion of bacterial vaginosis-associated anaerobes to ME-180 cells

Bacterial vaginosis is the leading vaginal disorder but the transition from health to this dysbiotic condition remains poorly characterized. Our goal was to quantify the ability of BV-associated anaerobes to adhere to epithelial cells in the presence of lactobacilli. Gardnerella vaginalis outcompeted Lactobacillus crispatus and Lactobacillus iners actually enhanced its adherence.We want to generously thank to Melissa Jamerson and Guy A. Cabral, from the Department of Microbiology and Immunology (Virginia Commonwealth University), for the AMG EVOSfl fluorescence microscope usage and procedure advices. This work was supported by European Union funds (FEDER/COMPETE) and by national funds (FCT) under the project with reference FCOMP-01-0124-FEDER-008991 (PTDC/BIA-MIC/098228/2008)

Environmental regulation of the pgaABCD locus in Escherichia coli

Background : The staphylococci are a major cause of catheter-related infections, due, in part, to their ability to form a biofilm. The major constituent of the biofilm matrix is the polysaccharide PNAG, synthesized by the proteins encoded in the icaADBC locus. Recently, it has been demonstrated that the Escherichia coli locus ycdSRQP (re-named pgaABCD) has homology to the staphylococcal icaADBC locus, and also encodes proteins that direct the synthesis of a polysaccharide that is biochemically identical to PNAG. Furthermore, PNAG promotes biofilm formation in E. coli. The aim of this work was to determine environmental factors that regulate transcription of the pgaABCD locus and the production of PNAG. Methods: We cloned the promoter of the locus pgaABCD into a green fluorescent protein (GFP) reporter plasmid, and transformed the plasmid into a competent E. coli strain. The transformed cells were grown in TSB supplemented with a variety of factors, including glucose, sucrose, mannose, magnesium, manganese and ethanol. The production of GFP was detected with a fluorimeter. Factors that augmented the production of GFP were further tested for their influence on PNAG production and biofilm formation in 18 E. coli urinary tract clinical isolates by immunoblot using IgG raised against staphylococcal PNAG and by microtiter well biofilm assay, respectively. Results: Transcription of pgaABCD was augmented by glucose, manganese, potassium, and ethanol. PNAG production, as detected by immunoblot, was also increased by the same factors. However, glucose was the only factor to significantly increase biofilm formation. Conclusions: Environmental regulation of transcription of the E. coli pgaABCD locus, as determined by reporter gene activation was correlated with the production of PNAG in E. coli clinical isolates. However, biofilm formation by E.coli may not be entirely dependent on PNAG production