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1.  The Natural Antimicrobial Peptide Subtilosin Acts Synergistically with Glycerol Monolaurate, Lauric Arginate, and ε-Poly-l-Lysine against Bacterial Vaginosis-Associated Pathogens but Not Human Lactobacilli 
Subtilosin is a cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens that has antimicrobial activity against the bacterial vaginosis-associated human pathogen Gardnerella vaginalis. The ability of subtilosin to inhibit G. vaginalis alone and in combination with the natural antimicrobial agents glycerol monolaurate (Lauricidin), lauric arginate, and ε-poly-l-lysine was tested using a checkerboard approach. Subtilosin was found to act synergistically with all of the chosen antimicrobials. These promising results indicate that lower concentrations of subtilosin in combination with other compounds could effectively be used to inhibit growth of the pathogen, thereby decreasing the risk of developed antimicrobial resistance. This is the first report on the effects of subtilosin combined with other natural antimicrobials against G. vaginalis.
doi:10.1128/AAC.05861-11
PMCID: PMC3318360  PMID: 22252803
2.  Spermicidal Activity of the Safe Natural Antimicrobial Peptide Subtilosin 
Bacterial vaginosis (BV), a condition affecting millions of women each year, is primarily caused by the gram-variable organism Gardnerella vaginalis. A number of organisms associated with BV cases have been reported to develop multidrug resistance, leading to the need for alternative therapies. Previously, we reported the antimicrobial peptide subtilosin has proven antimicrobial activity against G. vaginalis, but not against the tested healthy vaginal microbiota of lactobacilli. After conducting tissue sensitivity assays using an ectocervical tissue model, we determined that human cells remained viable after prolonged exposures to partially-purified subtilosin, indicating the compound is safe for human use. Subtilosin was shown to eliminate the motility and forward progression of human spermatozoa in a dose-dependent manner, and can therefore be considered a general spermicidal agent. These results suggest subtilosin would be a valuable component in topical personal care products aimed at contraception and BV prophylaxis and treatment.
doi:10.1155/2008/540758
PMCID: PMC2565553  PMID: 18923673
3.  Elucidation of the Molecular Mechanisms of Action of the Natural Antimicrobial Peptide Subtilosin Against the Bacterial Vaginosis-associated Pathogen Gardnerella vaginalis 
Subtilosin A is a 35-amino acid long cyclical peptide produced by Bacillus amyloliquefaciens that has potent antimicrobial activity against a variety of human pathogens, including the bacterial vaginosis-related Gardnerella vaginalis. The specific mode of action of subtilosin against G. vaginalis was elucidated by studying its effects on the proton motive force’s (PMF) components: transmembrane electric potential (ΔΨ), transmembrane pH gradient (ΔpH), and intracellular ATP levels. The addition of subtilosin to G. vaginalis cells caused an immediate and total depletion of the ΔpH, but had no effect on the ΔΨ. Subtilosin also triggered an instant but partial efflux of intracellular ATP that was twofold higher than that of the positive control bacteriocin, nisin. Taken together, these data suggest that subtilosin inhibits G. vaginalis growth by creating transient pores in the cells’ cytoplasmic membrane, leading to an efflux of intracellular ions and ATP and eventually cell death.
doi:10.1007/s12602-010-9061-4
PMCID: PMC3176456  PMID: 21949544
Bacteriocin; Subtilosin; Mode of action; Vaginal pathogen
4.  Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens 
Journal of applied microbiology  2007;104(4):1067-1074.
Aims
To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product-derived Bacillus amyloliquefaciens.
Methods and Results
An unknown bacterial species cultured from the Yogu Farm™ probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell-free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp.
Conclusion
The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis.
Significance and Impact of the Study
This is the first report of intra-species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis-associated pathogens.
doi:10.1111/j.1365-2672.2007.03626.x
PMCID: PMC2322860  PMID: 17976171
antimicrobial; Bacillus amyloliquefaciens; Bacillus subtilis; bacteriocin; subtilosin
5.  Polyethylene Glycol-Based Hydrogels for Controlled Release of the Antimicrobial Subtilosin for Prophylaxis of Bacterial Vaginosis 
Current treatment options for bacterial vaginosis (BV) have been shown to be inadequate at preventing recurrence and do not provide protection against associated infections, such as that with HIV. This study examines the feasibility of incorporating the antimicrobial peptide subtilosin within covalently cross-linked polyethylene glycol (PEG)-based hydrogels for vaginal administration. The PEG-based hydrogels (4% and 6% [wt/vol]) provided a two-phase release of subtilosin, with an initial rapid release rate of 4.0 μg/h (0 to 12 h) followed by a slow, sustained release rate of 0.26 μg/h (12 to 120 h). The subtilosin-containing hydrogels inhibited the growth of the major BV-associated pathogen Gardnerella vaginalis with a reduction of 8 log10 CFU/ml with hydrogels containing ≥15 μg entrapped subtilosin. In addition, the growth of four common species of vaginal lactobacilli was not significantly inhibited in the presence of the subtilosin-containing hydrogels. The above findings demonstrate the potential application of vaginal subtilosin-containing hydrogels for prophylaxis of BV.
doi:10.1128/AAC.02446-14
PMCID: PMC3993243  PMID: 24566190
6.  DNase Inhibits Gardnerella vaginalis Biofilms In Vitro and In Vivo 
The Journal of Infectious Diseases  2013;207(10):1491-1497.
Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.
doi:10.1093/infdis/jit047
PMCID: PMC3627197  PMID: 23431033
Bacterial vaginosis; biofilm; extracellular DNA; Gardnerella vaginalis
7.  Glycerol Monolaurate Inhibits Candida and Gardnerella vaginalis In Vitro and In Vivo but Not Lactobacillus▿  
We investigated the effects of glycerol monolaurate (GML) on Lactobacillus, Candida, and Gardnerella vaginalis human vaginal microflora. Our previous work demonstrated that 6 months of GML treatment vaginally does not alter lactobacillus counts in monkeys. Candida and G. vaginalis are commonly associated with vaginal infections in women, many becoming chronic or recurrent. In vitro growth inhibition studies determined the effects of GML (0 to 500 μg/ml) against multiple Candida species and G. vaginalis. A randomized, double-blind study investigated the effects of GML on vaginal microflora Lactobacillus, Candida, and G. vaginalis in colonized or infected women (n = 36). Women self-administered intravaginal gels containing 0% (n = 14), 0.5% (n = 13), or 5% (n = 9) GML every 12 h for 2 days. Vaginal swabs were collected before and immediately after the first gel administration and 12 h after the final gel administration. Swabs were tested for Lactobacillus, Candida, G. vaginalis, and GML. In vitro GML concentrations of 500 μg/ml were candicidal for all species tested, while a concentration of 10 μg/ml was bactericidal for G. vaginalis. Control and GML gels applied vaginally in women did not alter vaginal pH or Lactobacillus counts. Control gels reduced G. vaginalis counts but not Candida counts, whereas GML gels reduced both Candida and G. vaginalis. No adverse events were reported by participating women. GML is antimicrobial for Candida and G. vaginalis in vitro. Vaginal GML gels in women do not affect Lactobacillus negatively but significantly reduce Candida and G. vaginalis.
doi:10.1128/AAC.01151-09
PMCID: PMC2812150  PMID: 20008774
8.  Safety, formulation, and in vitro antiviral activity of the antimicrobial peptide subtilosin against herpes simplex virus type 1 
In the present study the antiviral properties of the bacteriocin subtilosin against Herpes simplex virus type 1 (HSV-1) and the safety and efficacy of a subtilosin-based nanofiber formulation were determined. High concentrations of subtilosin, the cyclical antimicrobial peptide produced by Bacillus amyloliquefaciens, were virucidal against HSV-1. Interestingly, at non-virucidal concentrations, subtilosin inhibited wild type HSV-1 and aciclovir-resistant mutants in a dose-dependent manner. Although the exact antiviral mechanism is not fully understood, time of addition experiments and western blot analysis suggest that subtilosin does not affect viral multiplication steps prior to protein synthesis.
Poly(vinyl alcohol) (PVOH)-based subtilosin nanofibers with a width of 278 nm were produced by the electrospinning process. The retained antimicrobial activity of the subtilosin-based fibers was determined via an agar well diffusion assay. The loading capacity of the fibers was 2.4 mg subtilosin/g fiber, and loading efficiency was 31.6%. Furthermore, the nanofibers with and without incorporated subtilosin were shown to be nontoxic to human epidermal tissues using an in vitro human tissue model.
Taking together these results subtilosin-based nanofibers should be further studied as a novel alternative method for treatment and/or control of HSV-1 infection.
doi:10.1007/s12602-012-9123-x
PMCID: PMC3637976  PMID: 23637711
subtilosin; bacteriocin; antiviral; nanofiber
9.  Analysis of adherence, biofilm formation and cytotoxicity suggests a greater virulence potential of Gardnerella vaginalis relative to other bacterial-vaginosis-associated anaerobes 
Microbiology  2010;156(Pt 2):392-399.
Worldwide, bacterial vaginosis (BV) is the most common vaginal disorder in women of childbearing age. BV is characterized by a dramatic shift in the vaginal microflora, involving a relative decrease in lactobacilli, and a proliferation of anaerobes. In most cases of BV, the predominant bacterial species found is Gardnerella vaginalis. However, pure cultures of G. vaginalis do not always result in BV, and asymptomatic women are sometimes colonized with low numbers of G. vaginalis. Thus, there is controversy about whether G. vaginalis is an opportunistic pathogen and the causative agent of many cases of BV, or whether BV is a polymicrobial condition caused by the collective effects of an altered microbial flora. Recent studies of the biofilm-forming potential and cytotoxic activity of G. vaginalis have renewed interest in the virulence potential of this organism. In an effort to tease apart the aetiology of this disorder, we utilized in vitro assays to compare three virulence properties of G. vaginalis relative to other BV-associated anaerobes. We designed a viable assay to analyse bacterial adherence to vaginal epithelial cells, we compared biofilm-producing capacities, and we assessed cytotoxic activity. Of the BV-associated anaerobes tested, only G. vaginalis demonstrated all three virulence properties combined. This study suggests that G. vaginalis is more virulent than other BV-associated anaerobes, and that many of the bacterial species frequently isolated from BV may be relatively avirulent opportunists that colonize the vagina after G. vaginalis has initiated an infection.
doi:10.1099/mic.0.034280-0
PMCID: PMC2890091  PMID: 19910411
10.  Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity 
Journal of Antimicrobial Chemotherapy  2012;67(12):2870-2872.
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.
doi:10.1093/jac/dks305
PMCID: PMC3494843  PMID: 22855857
defensin; vaginolysin; bacterial vaginosis; biofilm
11.  Comparative Genomics of Gardnerella vaginalis Strains Reveals Substantial Differences in Metabolic and Virulence Potential 
PLoS ONE  2010;5(8):e12411.
Background
Gardnerella vaginalis is described as a common vaginal bacterial species whose presence correlates strongly with bacterial vaginosis (BV). Here we report the genome sequencing and comparative analyses of three strains of G. vaginalis. Strains 317 (ATCC 14019) and 594 (ATCC 14018) were isolated from the vaginal tracts of women with symptomatic BV, while Strain 409-05 was isolated from a healthy, asymptomatic individual with a Nugent score of 9.
Principal Findings
Substantial genomic rearrangement and heterogeneity were observed that appeared to have resulted from both mobile elements and substantial lateral gene transfer. These genomic differences translated to differences in metabolic potential. All strains are equipped with significant virulence potential, including genes encoding the previously described vaginolysin, pili for cytoadhesion, EPS biosynthetic genes for biofilm formation, and antimicrobial resistance systems, We also observed systems promoting multi-drug and lantibiotic extrusion. All G. vaginalis strains possess a large number of genes that may enhance their ability to compete with and exclude other vaginal colonists. These include up to six toxin-antitoxin systems and up to nine additional antitoxins lacking cognate toxins, several of which are clustered within each genome. All strains encode bacteriocidal toxins, including two lysozyme-like toxins produced uniquely by strain 409-05. Interestingly, the BV isolates encode numerous proteins not found in strain 409-05 that likely increase their pathogenic potential. These include enzymes enabling mucin degradation, a trait previously described to strongly correlate with BV, although commonly attributed to non-G. vaginalis species.
Conclusions
Collectively, our results indicate that all three strains are able to thrive in vaginal environments, and therein the BV isolates are capable of occupying a niche that is unique from 409-05. Each strain has significant virulence potential, although genomic and metabolic differences, such as the ability to degrade mucin, indicate that the detection of G. vaginalis in the vaginal tract provides only partial information on the physiological potential of the organism.
doi:10.1371/journal.pone.0012411
PMCID: PMC2928729  PMID: 20865041
12.  Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung 
There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a >128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.
doi:10.3791/3857
PMCID: PMC3471314  PMID: 22711026
Immunology;  Issue 64;  Microbiology;  Pseudomonas aeruginosa;  antimicrobial susceptibility;  artificial sputum media;  lung infection;  cystic fibrosis;  diagnostics;  plankton
13.  Resolution and Characterization of Distinct cpn60-Based Subgroups of Gardnerella vaginalis in the Vaginal Microbiota 
PLoS ONE  2012;7(8):e43009.
Bacterial vaginosis (BV), characterized by a shift of the vaginal microbiota from a Lactobacillus-dominated community to a dense biofilm containing a complex mixture of organisms, is an important risk factor in poor reproductive health outcomes. The Nugent score, based on Gram stain, is used to diagnose BV and Gardnerella vaginalis abundance in the sample is one factor determining Nugent score. A high Nugent score is indicative of BV but does not always correspond to the presence of clinical symptoms. G. vaginalis is recognized as a heterogeneous group of organisms, which can also be part of the normal, healthy vaginal microbiome. In addition, asymptomatic BV and non-Gardnerella types of BV are being recognized. In an attempt to resolve the heterogeneous group of G. vaginalis, a phylogenetic tree of cpn60 universal target sequences from G. vaginalis isolates was constructed that indicates the existence of four subgroups of G. vaginalis. This subdivision, supported by whole genome similarity calculation of representative strains using JSpecies, demonstrates that these subgroups may represent different species. The cpn60 subgroupings did not correspond with the Piot biotyping scheme, but did show consistency with ARDRA genotyping and sialidase gene presence. Isolates from all four subgroups produced biofilm in vitro. We also investigated the distribution of G. vaginalis subgroups in vaginal samples from Kenyan women with Nugent scores consistent with BV, Intermediate and Normal microbiota (n = 44). All subgroups of G. vaginalis were detected in these women, with a significant difference (z = −3.372, n = 39, p = 0.001) in frequency of G. vaginalis subgroup B between BV and Normal groups. Establishment of a quantifiable relationship between G. vaginalis subgroup distribution and clinical status could have significant diagnostic implications.
doi:10.1371/journal.pone.0043009
PMCID: PMC3416817  PMID: 22900080
14.  Isolation of a Variant of Subtilosin A with Hemolytic Activity▿  
Journal of Bacteriology  2009;191(18):5690-5696.
Bacillus subtilis produces an anionic bacteriocin called subtilosin A that possesses antibacterial activity against certain gram-positive bacteria. In this study, we uncovered a hemolytic mutant of B. subtilis that produces an altered form of subtilosin A. The mutant bacteriocin, named subtilosin A1, has a replacement of threonine at position 6 with isoleucine. In addition to the hemolytic activity, subtilosin A1 was found to exhibit enhanced antimicrobial activity against specific bacterial strains. The B. subtilis albB mutant that does not produce a putative immunity peptide was more sensitive to both subtilosin A and subtilosin A1. A spontaneous suppressor mutation of albB that restored resistance to subtilosin A and subtilosin A1 was obtained. The sbr (subtilosin resistance) mutation conferring the resistance is not linked to the sboA-alb locus. The sbr mutation does not increase the resistance of B. subtilis to other cell envelope-targeted antimicrobial agents, indicating that the mutation specifically confers the resistance to subtilosins. The findings suggest possible bioengineering approaches for obtaining anionic bacteriocins with enhanced and/or altered bactericidal activity. Furthermore, future identification of the subtilosin-resistant mutation could provide insights into the mechanism of subtilosin A activity.
doi:10.1128/JB.00541-09
PMCID: PMC2737964  PMID: 19633086
15.  Phase I Randomized Safety Study of Twice Daily Dosing of Acidform Vaginal Gel: Candidate Antimicrobial Contraceptive 
PLoS ONE  2012;7(10):e46901.
Background
Acidform gel, an acid-buffering product that inactivates spermatozoa, may be an effective topical non-hormonal contraceptive. This study was designed to evaluate the safety of vaginal dosing and effects of Acidform on mucosal immune mediators, antimicrobial properties of genital secretions, and vaginal microbiota.
Methods
Thirty-six sexually abstinent U.S. women were randomized to apply Acidform or hydroxyethylcellulose (HEC) placebo gel twice daily for 14 consecutive days. Safety was assessed by symptoms and pelvic examination. The impact of gel on mucosal immunity was assessed by quantifying cytokines, chemokines, antimicrobial proteins and antimicrobial activity of genital secretions collected by cervicovaginal lavage (CVL) at screening, 2 hours after gel application, and on days 7, 14 and 21. Vaginal microbiota was characterized at enrollment and day 14 using species-specific quantitative PCR assays.
Results
The median vaginal and cervical pH was significantly lower 2 hours after application of Acidform and was associated with an increase in the bactericidal activity of CVL against E. coli. However, 65% of women who received Acidform had at least one local adverse event compared with 11% who received placebo (p = 0.002). While there was no increase in inflammatory cytokines or chemokines, CVL concentrations of lactoferrin and interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory protein, were significantly lower following Acidform compared to HEC placebo gel application. There were no significant changes in Lactobacillus crispatus or Lactobacillus jensenii in either group but there was a decrease in Gardnerella vaginalis in the Acidform group (p = 0.08).
Conclusions
Acidform gel may augment mucosal defense as evidenced by an increase in bactericidal activity of genital secretions against E. coli and a decrease in Gardnerella vaginalis colonization. However, Acidform was associated with more irritation than placebo and lower levels of antimicrobial (lactoferrin) and anti-inflammatory (IL-1ra) proteins. These findings indicate the need for additional safety studies of this candidate non-hormonal contraceptive.
Trial Registration
ClinicalTrials.gov NCT00850837
doi:10.1371/journal.pone.0046901
PMCID: PMC3466198  PMID: 23056520
16.  Interactions between Lactobacillus crispatus and Bacterial Vaginosis (BV)-Associated Bacterial Species in Initial Attachment and Biofilm Formation 
Certain anaerobic bacterial species tend to predominate the vaginal flora during bacterial vaginosis (BV), with Gardnerella vaginalis being the most common. However, the exact role of G. vaginalis in BV has not yet been determined. The main goal of this study was to test the hypothesis that G. vaginalis is an early colonizer, paving the way for intermediate (e.g., Fusobacterium nucleatum) and late colonizers (e.g., Prevotella bivia). Theoretically, in order to function as an early colonizer, species would need to be able to adhere to vaginal epithelium, even in the presence of vaginal lactobacilli. Therefore, we quantified adherence of G. vaginalis and other BV-associated bacteria to an inert surface pre-coated with Lactobacillus crispatus using a new Peptide Nucleic Acid (PNA) Fluorescence In Situ Hybridization (FISH) methodology. We found that G. vaginalis had the greatest capacity to adhere in the presence of L. crispatus. Theoretically, an early colonizer would contribute to the adherence and/or growth of additional species, so we next quantified the effect of G. vaginalis biofilms on the adherence and growth of other BV-associated species by quantitative Polymerase Chain Reaction (qPCR) technique. Interestingly, G. vaginalis derived a growth benefit from the addition of a second species, regardless of the species. Conversely, G. vaginalis biofilms enhanced the growth of P. bivia, and to a minor extent of F. nucleatum. These results contribute to our understanding of BV biofilm formation and the progression of the disorder.
doi:10.3390/ijms140612004
PMCID: PMC3709769  PMID: 23739678
Lactobacillus spp.; Gardnerella vaginalis; BV anaerobes; initial adhesion; epithelial cell line; fluorescence in situ hybridization; peptide nucleic acid; quantitative-PCR
17.  Adhesion of Human Probiotic Lactobacillus rhamnosus to Cervical and Vaginal Cells and Interaction with Vaginosis-Associated Pathogens 
Objectives. The ability of a probiotic Lactobacillus rhamnosus strain (Lcr35) to adhere to cervical and vaginal cells and to affect the viability of two main vaginosis-associated pathogens, Prevotella bivia, Gardnerella vaginalis, as well as Candida albicans was investigated. Methods. Adhesion ability was determined in vitro with immortalized epithelial cells from the endocervix, ectocervix, and vagina. Coculture experiments were performed to count viable pathogens cells in the presence of Lcr35. Results. Lcr35 was able to specifically and rapidly adhere to the three cell lines. In coculture assays, a decrease in pathogen cell division rate was observed as from 4 hours of incubation and bactericidal activity after a longer period of incubation, mostly with P. bivia. Conclusion. The ability of Lcr35 to adhere to cervicovaginal cells and its antagonist activities against vaginosis-associated pathogens suggest that this probiotic strain is a promising candidate for use in therapy.
doi:10.1155/2008/549640
PMCID: PMC2631649  PMID: 19190778
18.  Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp. 
BMC Microbiology  2013;13:82.
Background
Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples.
Results
Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH.
Conclusions
This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.
doi:10.1186/1471-2180-13-82
PMCID: PMC3637831  PMID: 23586331
Fluorescence in situ Hybridization (FISH); Peptide Nucleic Acid Probe (PNA probe); Lactobacillus spp.; Gardnerella vaginalis; Bacterial vaginosis
19.  Oleate lipase activity in Gardnerella vaginalis and reconsideration of existing biotype schemes 
BMC Microbiology  2009;9:78.
Background
Gardnerella vaginalis is a facultative gram positive organism that requires subculture every 1–2 days to maintain viability. It has been linked with bacterial vaginosis (BV), a syndrome that has been associated with increased risk for preterm delivery, pelvic inflammatory disease and HIV acquisition. About 10% of the G. vaginalis isolates have been reported to produce sialidase, but there have not been any studies relating sialidase production and biotype. Sialidase activity is dramatically increased in the vaginal fluid of women with BV and bacterial sialidases have been shown to increase the infectivity of HIV in vitro. There are 8 different biotypes of G. vaginalis. Biotypes 1–4 produce lipase and were reported to be associated with BV and the association of these biotypes with BV is under dispute. Other studies have demonstrated that G. vaginalis biotype 1 can stimulate HIV-1 production. Because of the discrepancies in the literature we compared the methods used to biotype G. vaginalis and investigated the relationship of biotype and sialidase production.
Results
A new medium for maintenance of Gardnerella vaginalis which allows survival for longer than one week is described. Some isolates only grew well under anaerobic conditions. Sialidase producing isolates were observed in 5 of the 6 biotypes tested. Using 4-methylumbelliferyl-oleate to determine lipase activity, instead of egg yolk agar, resulted in erroneous biotypes and does not provide reliable results.
Conclusion
Previous studies associating G. vaginalis biotype with bacterial vaginosis were methodologically flawed, suggesting there is not an association of G. vaginalis biotypes and bacterial vaginosis. Sialidase activity was observed in 5 of the 8 biotypes.
doi:10.1186/1471-2180-9-78
PMCID: PMC2680412  PMID: 19386125
20.  Effect of biofilm phenotype on resistance of Gardnerella vaginalis to hydrogen peroxide and lactic acid 
Objectives
Bacterial vaginosis (BV) is the most common vaginal disorder worldwide. Certain lactobacilli produce H202 and lactic acid, which normally suppress growth of anaerobes, but in BV, G. vaginalis and other anaerobes proliferate, and the number of lactobacilli decreases. Gardnerella vaginalis colonizes the vaginal epithelium as a biofilm, which likely plays a role in colonization and relapsing infection.
Study Design
We developed an in vitro model for G. vaginalis biofilm formation, and compared susceptibilities of biofilms vs. planktonic cultures to H2O2 and lactic acid. The structure and composition of the biofilm matrix were studied in order to design a method for biofilm dissolution.
Results
Biofilms tolerated 5-fold and 4–8 fold higher concentrations of H2O2 and lactic acid (respectively) than planktonic cultures. Proteolytic dissolution of biofilms reduced sensitivity to H202 and lactic acid.
Conclusions
Increased tolerance to H2O2 and lactic acid suggests that biofilm formation contributes to survival of G. vaginalis in the presence of lactobacilli.
doi:10.1016/j.ajog.2007.02.027
PMCID: PMC2020809  PMID: 17689638
Vaginosis; Gardnerella; Biofilm
21.  Microbiologic Response to Treatment of Bacterial Vaginosis with Topical Clindamycin or Metronidazole 
Journal of Clinical Microbiology  2005;43(9):4492-4497.
To compare the frequencies, concentrations, and antimicrobial susceptibilities of vaginal microbes isolated from women with bacterial vaginosis (BV) before and after therapy, 119 nonpregnant women aged 18 to 45 with clinical and Gram stain evidence of BV were randomized to receive intravaginal clindamycin or metronidazole. Vaginal swabs were collected at baseline and 7 to 12 days, 35 to 45 days, and 70 to 90 days following therapy for quantitative vaginal culture. For the 99 women completing all four visits, statistical analyses were performed comparing differences in vaginal microflora between the two treatment arms and between visits in the same treatment group. Antimicrobial susceptibility testing using the agar dilution method was performed for anaerobic gram-negative rods. Although both therapies resulted in decreased colonization by Gardnerella vaginalis and Mycoplasma hominis, only metronidazole treatment resulted in a significant decrease in the frequency and concentration of Prevotella bivia and black-pigmented Prevotella species. Of the 865 anaerobic gram-negative rods evaluated for susceptibility, only 3 (0.3%) were resistant to metronidazole, whereas clindamycin resistance increased significantly for P. bivia and black-pigmented anaerobic gram-negative rods persisting following clindamycin therapy. Clindamycin-resistant subpopulations of P. bivia and black-pigmented Prevotella species emerged 7 to 12 days after therapy even among women colonized initially by clindamycin-susceptible strains. These resistant subpopulations persisted at high frequencies (42 to 50%) 70 to 90 days following therapy. The two topical agents for treatment of BV have differing microbiologic effects on the vaginal microflora. The emergence of clindamycin-resistant anaerobic gram-negative rods following therapy is of concern.
doi:10.1128/JCM.43.9.4492-4497.2005
PMCID: PMC1234077  PMID: 16145097
22.  Reversible Deficiency of Antimicrobial Polypeptides in Bacterial Vaginosis  
Infection and Immunity  2006;74(10):5693-5702.
Bacterial vaginosis is a common condition associated with increased risk of sexually transmitted diseases, including human immunodeficiency virus infections. In contrast, vulvovaginal candidiasis has a much weaker association with sexually transmitted diseases. We found that vaginal lavage fluid from women with bacterial vaginosis is deficient in antimicrobial polypeptides and antimicrobial activity compared to fluid from healthy women or women with vulvovaginal candidiasis. Effective treatment normalized the concentrations of antimicrobial polypeptides in both bacterial vaginosis and in vulvovaginal candidiasis, suggesting that the abnormalities were a result of the diseases. Unlike in vulvovaginal candidiasis, the neutrophil attractant chemokine interleukin-8 (IL-8) was not increased in bacterial vaginosis, accounting for low concentrations of neutrophil-derived defensins in vaginal fluid. In organotypic cultures of human vaginal epithelium containing dendritic cells, treatment with Lactobacillus jensenii, a typical vaginal resident, induced the synthesis of IL-8 mRNA and the epithelial human β-defensin-2 mRNA, but a typical bacterial vaginosis pathogen, Gardnerella vaginalis, had no effect. When the two bacteria were combined, Gardnerella vaginalis did not interfere with the immunostimulatory effect of Lactobacillus jensenii. The loss of normal immunostimulatory flora in bacterial vaginosis is thus associated with a local deficiency of multiple innate immune factors, and this deficiency could predispose individuals to sexually transmitted diseases.
doi:10.1128/IAI.00524-06
PMCID: PMC1594936  PMID: 16988245
23.  Complexity of vaginal microflora as analyzed by PCR denaturing gradient gel electrophoresis in a patient with recurrent bacterial vaginosis. 
OBJECTIVE: Gardnerella vaginalis has long been the most common pathogen associated with bacterial vaginosis (BV). We aimed to test our hypothesis that symptoms and signs of BV do not necessarily indicate colonization by this organism, and often will not respond to standard metronidazole or clindamycin treatment. METHODS: Using a relatively new molecular tool, PCR denaturing gradient gel electrophoresis (DGGE), the vaginal microflora of a woman with recalcitrant signs and symptoms of BV was investigated over a 6-week timeframe. RESULTS: The vagina was colonized by pathogenic enterobacteriaceae, staphylococci and Candida albicans. The detection of the yeast by PCR-DGGE is particularly novel and enhances the ability of this tool to examine the true nature of the vaginal microflora. The patient had not responded to antifungal treatment, antibiotic therapy targeted at anaerobic Gram-negative pathogens such as Gardnerella, nor daily oral probiotic intake of Lactobacillus rhamnosus GG. The failure to find the GG strain in the vagina indicated it did not reach the site, and the low counts of lactobacilli demonstrated that therapy with this probiotic did not appear to influence the vaginal flora. CONCLUSIONS: BV is not well understood in terms of its causative organisms, and further studies appear warranted using non-culture, molecular methods. Only when the identities of infecting organisms are confirmed can effective therapy be devized. Such therapy may include the use of probiotic lactobacilli, but only using strains which confer a benefit on the vagina of pre- and postmenopausal women.
doi:10.1080/10647440400025504
PMCID: PMC1784553  PMID: 16040324
24.  Use of a Continuous-Culture Biofilm System To Study the Antimicrobial Susceptibilities of Gardnerella vaginalis and Lactobacillus acidophilus 
Gardnerella vaginalis and Lactobacillus acidophilus have been shown to grow to high titers in a simple biofilm system. This system was used in the present investigation to compare the biofilm-eradicating concentrations (BECs) of amoxicillin, clindamycin, erythromycin, and metronidazole to standard tube MIC and minimum bactericidal concentration (MBC) results. With the lactobacillus, the BEC/tube MBC ratio was at least 16:1, while for G. vaginalis the ratio varied from 2:1 to 512:1. The simple continuous-culture system used in the present investigation is ideal for investigating the BEC for bacteria involved in complex ecological situations such as bacterial vaginosis and may be useful for the identification of the most effective and selective antibiotic therapy.
PMCID: PMC105617  PMID: 9624489
25.  Benzoyl Peroxide Formulated Polycarbophil/Carbopol 934P Hydrogel with Selective Antimicrobial Activity, Potentially Beneficial for Treatment and Prevention of Bacterial Vaginosis 
The human vagina is colonized by a variety of indigenous microflora; in healthy individuals the predominant bacterial genus is Lactobacillus while those with bacterial vaginosis (BV) carry a variety of anaerobic representatives of the phylum Actinobacteria. In this study, we evaluated the antimicrobial activity of benzoyl peroxide (BPO) encapsulated in a hydrogel against Gardnerella vaginalis, one of the causative agents of BV, as well as indicating its safety for healthy human lactobacilli. Herein, it is shown that in well diffusion assays G. vaginalis is inhibited at 0.01% hydrogel-encapsulated BPO and that the tested Lactobacillus spp. can tolerate concentrations of BPO up to 2.5%. In direct contact assays (cells grown in a liquid culture containing hydrogel with 1% BPO or BPO particles), we demonstrated that hydrogels loaded with 1% BPO caused 6-log reduction of G. vaginalis. Conversely, three of the tested Lactobacillus spp. were not inhibited while L. acidophilus growth was slightly delayed. The rheological properties of the hydrogel formulation were probed using oscillation frequency sweep, oscillation shear stress sweep, and shear rate sweep. This shows the gel to be suitable for vaginal application and that the encapsulation of BPO did not alter rheological properties.
doi:10.1155/2013/909354
PMCID: PMC3870611  PMID: 24382940

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