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ABSTRACT The Gardnerella genus, comprising at least 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species, including Gardnerella spp., are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all tested Gardnerella strains, representing at least 10 different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of Neisseria gonorrhoeae and commensal Lactobacillus species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127–173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in Gardnerella phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. Gardnerella species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that Gardnerella species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for Gardnerella infection and immune avoidance.

The association between Sneathia vaginalis and preterm birth is emerging. The Gram-negative anaerobe produces a large exotoxin, the cytopathogenic toxin A (CptA), that forms pores in human epithelial cells and red blood cells. The structure of the toxin has not been determined, but in silico analysis predicts that a large amino-terminal region of the protein is globular and separated from the carboxy-terminal tandem repeats by a disordered region. We found that a recombinant protein consisting of the predicted structured amino-terminal portion of CptA and devoid of the repeat region was sufficient to permeabilize epithelial cells and red blood cells. The repeat region was capable of binding to epithelial cells but did not permeabilize them or lyse red blood cells. CptA is the only S. vaginalis virulence factor that has been examined mechanistically to date, and this analysis sets the foundation for an understanding of how this novel pore-forming toxin exerts its activity.

Wright et al. apply biometric genetic approach to identify the extent to which host genetic factors influence species-level variation in the vaginal microbiota. Their study suggests that Lactobacillus crispatus, a major determinant of vaginal health, is heritable among European American women, consistent with the reduced prevalence of adverse reproductive disorders in them.

Abstract. The vaginal microbiome likely influences host signaling compounds within the reproductive tract, including pro-inflammatory signals, which may play an important role during pregnancy. Vaginal lactobacilli are associated with positive pregnancy outcome, whereas bacterial vaginosis, a dysbiosis of the vaginal microbiome, is associated with an increased risk of adverse pregnancy outcomes including preterm birth. If the host response could be predicted based on the taxonomic composition of the vaginal microbiome, particularly early in pregnancy, then those predictions could potentially be used to personalize intervention methods to reduce preterm birth and other adverse events. In this proof of principle study, we apply multivariate strategies to analyze 16S rRNA-based taxonomic surveys in conjunction with targeted immuno-proteomic and lipidomic data from vaginal samples from 58 women enrolled in the Multi-Omic Microbiome Study-Pregnancy Initiative during early pregnancy. Relationships between the vaginal microbiome and the vaginal lipidome have not been previously reported. Results from this study reveal significant multiple pairwise associations between microbial taxa, specific eicosanoids and sphingomyelins, and cytokines. While the biologic significance of these associations is not yet known, these results support the utility of such multi-omic approaches as a means to predict the impact of the microbiome on the host.

Bacterial lipases play important roles during infection. The Staphylococcus aureus genome contains several genes that encode well-characterized lipases and several genes predicted to encode lipases or esterases for which the function has not yet been established. In this study, we sought to define the function of an uncharacterized S. aureus protein, and we propose the annotation S. aureus lipase 3 (SAL3) (SAUSA300_0641). We confirmed that SAL3 is a lipase and that it is surface associated and secreted through an unknown mechanism. We determined that SAL3 specifically hydrolyzes short chain (4-carbon and fewer) fatty acids and specifically binds negatively charged lipids including phosphatidic acid, phosphatidylinositol phosphate, and phosphatidylglycerol, which is the most abundant lipid in the staphylococcal cell membrane. Mutating the catalytic triad S66-A, D167-A, S168-A, and H301-A in the recombinant protein abolished lipase activity without altering binding to host lipid substrates. Taken together we report the discovery of a novel lipase from S. aureus specific to short chain fatty acids with yet to be determined roles in host pathogen interactions.

Vaginal infection: Gene activity protects biofilms Altered gene activity may promote chronic and recurrent infections in the most common gynecological disorder in women of reproductive age. Bacterial vaginosis (BV) frequently involves the formation of biofilms by Gardnerella vaginalis bacteria. Nuno Cerca and colleagues at the University of Minho in Portugal, with co-workers elsewhere in Portugal and in the USA, studied changes in gene activity in BV biofilms. Their results linked differences in the activity of 815 genes to an ability of the bacteria to enter a protected form of growth when producing biofilms. The differences included increased activity of specific genes associated with antibiotic resistance. This insight into the bacterial ability to undergo drastic changes in gene activity could guide work to understand chronic and recurrent BV. It may also lead to new ways to treat this common and troublesome condition.

Gardnerella vaginalis has recently been split into 13 distinct species. In this study, we tested the hypotheses that species-specific variations in the vaginolysin (VLY) amino acid sequence could influence the interaction between the toxin and vaginal epithelial cells and that VLY variation may be one factor that distinguishes less virulent or commensal strains from more virulent strains. This was assessed by bioinformatic analyses of publicly available Gardnerella spp. sequences and quantification of cytotoxicity and cytokine production from purified, recombinantly produced versions of VLY. After identifying conserved differences that could distinguish distinct VLY types, we analyzed metagenomic data from a cohort of female subjects from the Vaginal Human Microbiome Project to investigate whether these different VLY types exhibited any significant associations with symptoms or Gardnerella spp.-relative abundance in vaginal swab samples. While Type 1 VLY was most prevalent among the subjects and may be associated with increased reports of symptoms, subjects with Type 2 VLY dominant profiles exhibited increased relative Gardnerella spp. abundance. Our findings suggest that amino acid differences alter the interaction of VLY with vaginal keratinocytes, which may potentiate differences in bacterial vaginosis (BV) immunopathology in vivo.

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/MSALL shotgun 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.

BACKGROUND:Bacterial vaginosis (BV) is the leading dysbiosis of the vaginal microbiome. The pathways leading towards the development of BV are not well understood. Gardnerella vaginalis is frequently associated with BV. G. vaginalis produces the cholesterol-dependent cytolysin (CDC), vaginolysin, which can lyse a variety of human cells and is thought to play a role in pathogenesis. Because membrane cholesterol is required for vaginolysin to function, and because HMG-CoA reductase inhibitors (statins) affect not only serum levels of cholesterol but membrane levels as well, we hypothesized that statins might affect the vaginal microbiome. METHODS:To investigate the relationship between use of the statins and the vaginal microbiome, we analyzed 16S rRNA gene taxonomic surveys performed on vaginal samples from 133 women who participated in the Vaginal Human Microbiome Project and who were taking statins at the time of sampling, 152 women who reported high cholesterol levels but were not taking statins, and 316 women who did not report high cholesterol. To examine the effect of statins on the cytolytic effect of vaginolysin, the cholesterol-dependent cytolysin (CDC) produced by Gardnerella vaginalis, we assessed the effect of simvastatin pretreatment of VK2E6/E7 vaginal epithelial cells on vaginolysin-mediated cytotoxicity. RESULTS:The mean proportion of G. vaginalis among women taking statins was significantly lower relative to women not using statins. Women using statins had higher mean proportions of Lactobacillus crispatus relative to women with normal cholesterol levels, and higher levels of Lactobacillus jensenii relative to women with high cholesterol but not taking statins. In vitro, vaginal epithelial cells pretreated with simvastatin were relatively resistant to vaginolysin and this effect was inhibited by cholesterol. CONCLUSIONS:In this cross-sectional study, statin use was associated with reduced proportions of G. vaginalis and greater proportions of beneficial lactobacilli within the vaginal microbiome. The negative association between statin use and G. vaginalis may be related to inhibition of vaginolysin function.

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 isogenic icaADBC 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.

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as "Mnola." In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name "Candidatus Mycoplasma girerdii" for this potential new pathogen.

Objectives: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures.Methods: Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic.Results: In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays.Conclusions: This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis. [ABSTRACT FROM AUTHOR]