To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in-vitro.
Methods and Results
Bacillus coagulans ATCC 7050 was grown at 37 °C for 18 hours. The cell free supernatant was concentrated 10-fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Δψ) and transmembrane pH (ΔpH) of the sensitive cells. Lactosporin caused efflux of potassium ions from M. luteus cells and dissipation of ΔpH in G. vaginalis while it had no effect on the Δψ. The safety of lactosporin was evaluated by using EpiVaginal™ ectocervical (VEC-100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 hours.
Lactosporin potentially exerts its antimicrobial activity by selective dissipation of ΔpH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a non-cytotoxix antimicrobial for vaginal application.
Significance and Impact of the Study
This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for control of bacterial vaginosis.
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.
Bacterial vaginosis is a common vaginal infection associated with numerous gynecological and obstetric complications. This condition is characterized by the presence of thick adherent vaginal biofilms, composed mainly of Gardnerella vaginalis. This organism is thought to be the primary aetiological cause of the infection paving the way for various opportunists to colonize the niche. Previously, we reported that the natural antimicrobials subtilosin, ε-poly-L-lysine, and lauramide arginine ethyl ester selectively inhibit the growth of this pathogen. In this study, we used plate counts to evaluate the efficacy of these antimicrobials against established biofilms of G. vaginalis. Additionally, we validated and compared two rapid methods (ATP viability and resazurin assays) for the assessment of cell viability in the antimicrobial-treated G. vaginalis biofilms. Out of the tested antimicrobials, lauramide arginine ethyl ester had the strongest bactericidal effect, followed by subtilosin, with clindamycin and polylysine showing the weakest effect. In comparison to plate counts, ATP viability and resazurin assays considerably underestimated the bactericidal effect of some antimicrobials. Our results indicate that these assays should be validated for every new application.
Lactocin 160 is a vaginal probiotic-derived bacteriocin shown to selectively inhibit the growth of Gardenerella vaginalis and some other pathogens commonly associated with bacterial vaginosis. The natural origin of this peptide, its safety, and selective antimicrobial properties make it a promising candidate for successful treatment and prophylaxis of bacterial vaginosis (BV). This study evaluated interactions between lactocin 160 and four other natural antimicrobials in the ability to inhibit G. vaginalis. We report that zinc lactate and soapnut extract act synergistically with lactocin 160 against this pathogen and therefore have a potential to be successfully used as the components of the multiple-hurdle antimicrobial formulation for the treatment of BV.
Bacteriocin; Natural antimicrobial; Antimicrobial synergy
Bacterial vaginosis (BV) is the most common vaginal infection among women of childbearing age. This condition is notorious for causing severe complications related to the reproductive health of women. Five decades of intense research established many risk factors for acquisition of BV, however due to the complexity of BV and due to lack of a reliable animal model for this condition, its exact etiology remains elusive. In this manuscript we use a historical perspective to critically review the development of major theories on the etiology of BV, ultimately implicating BV-related pathogens, healthy vaginal microbiota, bacteriophages and the immune response of the host. None of these theories on their own can reliably explain the epidemiological data. Instead, BV is caused by a complex interaction of multiple factors, which include the numerous components of the vaginal microbial ecosystem and their human host. Many of these factors are yet to be characterized because a clear understanding of their relative contribution to the etiology of BV is pivotal to formulation of an effective treatment for and prophylaxis of this condition.
bacterial vaginosis; etiology; Gardnerella vaginalis; causes; immune response; lactobacilli; Lactobacillus
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.
Bacteriocin; Subtilosin; Mode of action; Vaginal pathogen
Objective. To evaluate the safety of the antimicrobial peptide, lactocin 160. Methods. Lactocin 160, a product of vaginal probiotic Lactobacillus rhamnosus 160 was evaluated for toxicity and irritation. An in vitro human organotypic vaginal-ectocervical tissue model (EpiVaginal) was employed for the safety testing by determining the exposure time to reduce tissue viability to 50% (ET-50). Hemolytic activity of lactocin160 was tested using 8% of human erythrocyte suspension. Susceptibility of lactobacilli to lactocin160 was also studied. Rabbit vaginal irritation (RVI) model was used for an in vivo safety evaluation. Results. The ET-50 value was 17.5 hours for lactocin 160 (4.9 hours for nonoxynol 9, N9). Hemolytic activity of lactocin 160 was 8.2% (N9 caused total hemolysis). Lactobacilli resisted to high concentrations of peptide preparation. The RVI model revealed slight vaginal irritation. An average irritation index grade was evaluated as “none.” Conclusions. Lactocin 160 showed minimal irritation and has a good potential for intravaginal application.
Bacterial vaginosis (BV) is a commonly occurring vaginal infection that is associated with a variety of serious risks related to the reproductive health of women. Conventional antibiotic treatment for this condition is frequently ineffective because the antibiotics tend to inhibit healthy vaginal microflora along with the pathogens. Lactocin 160, a bacteriocin produced by healthy vaginal lactobacilli, is a promising alternative to antibiotics; this compound specifically inhibits the BV-associated vaginal pathogens such as Gardnerella vaginalis and Prevotella bivia without affecting the healthy microflora. This study investigates the molecular mechanism of action for lactocin 160 and reveals that this compound targets the cytoplasmic membrane of G. vaginalis, causing the efflux of ATP molecules and dissipation of the proton motive force.
Probiotics; Lactocin 160; Bacterial vaginosis; Bacteriocin; Mode of action
BioRad's Rotofor® system has been frequently used for the purification of proteins and smaller peptides such as bacteriocins. In this study, we report that some commercially available ampholytes used with the Rotofor® isoelectric focusing system possess antimicrobial activity, which may interfere with the purification of bacteriocins and bacteriocin-like substances.
Ampholytes; Rotofor; isoelectric focusing; bacteriocins
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.
This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR+) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR+ cells had lower transmembrane pH (ΔpH) and electric potential (Δψ) than the control (ATR−) cells. The decreased proton motive force (PMF) of ATR+ cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR+ cells were ∼7-fold higher than those in ATR− cells. This suggested a role for the FoF1 ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the FoF1 ATPase enzyme complex by N′-N′-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR− but not in ATR+ cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR+ listeriae, the downregulation of the proton-translocating c subunit of the FoF1 ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of FoF1 ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.
OBJECTIVES: To determine the mechanism of antimicrobial action of lactocin 160, a bacteriocin produced by the healthy vaginal strain of Lactobacillus rhamnosus, using an established model, with Micrococcus luteus ATCC 10420 as a test organism. METHODS: Sensitivity of M. luteus to lactocin 160 was determined by the diffusion assay. Loss of cellular ATP in the lactocin-treated cells was elucidated using a commercially available ATP determination kit (luciferin-luciferase bioluminescence assay). Luminescence intensity as a reflection of ATP quantity was determined using a luminometer. Dissipation of membrane potential (Deltapsi) was studied using fluorophore DiSC3(5) with the fluorescence spectrum sensitive to changes in Deltapsi. RESULTS: Lactocin 160 inhibited growth of M. luteus ATCC 10420 at a concentration of 5 microg/ml. There were no significant changes in the intracellular ATP level of M. luteus upon the addition of 20 microg/ml of lactocin 160. However, the extracellular ATP level increased significantly. This means that the treatment of cells with lactocin 160 resulted in an efflux of ATP from inside the cells. Therefore, a partially purified lactocin 160 preparation (16 microg /ml of the bacteriocin in the sample) killed sensitive cells and dissipated 3.12 +/- 0.36% of Deltapsi. CONCLUSION: Lactocin 160 has a mode of action typical for bacteriocins. It disturbs the cellular membrane (Deltapsi dissipation) and induces ATP efflux, most likely because of the pore formation, which is a common mechanism of action for many bacteriocins.
Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10°C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30°C, those of L. monocytogenes grown at 10°C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30°C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.
This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nisr) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nisr cells. Wild-type cells grown in 100% CO2 were two to five times longer than cells grown in air. Nisin (2.5 μg/ml) did not decrease the viability of Nisr cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO2. There was a quantifiable synergistic action between nisin and CO2 in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 μg of nisin per ml increased from 3.1 to 12.5 μg/ml over 35 days, but this increase was markedly delayed for cultures in CO2. This synergism between nisin and CO2 was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO2 and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO2 atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO2 and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO2 is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.