The effect of vaginal probiotic therapy on the outcome of papillomavirus infection

Aim. To study the effect of vaginal probiotic therapy on the outcome of human papillomavirus (HPV) infection.Materials and methods. The study included HPV-infected patients: 29 patients with normal vaginal flora and 146 patients with a deficiency of vaginal lactobacilli, of which 117 patients received vaginal probiotic therapy. In samples obtained before and after the therapy, the effect of the probiotic on the change in the ratio of living, apoptotic, and necrotic vaginal epithelial cells after preliminary exposure to oxidative stress was studied.Results. It was found that probiotics reduce the number of infected epithelial cells that survived the oxidative damage and shift the balance of cell death forms towards apoptosis. Vaginal probiotic therapy in patients with a deficiency of lactobacilli increased the frequency of HPV elimination by 2.5 times and reduced the likelihood of treatment failure from 1.5 to 4 times, depending on the viral load. The probiotic therapy made the structure of HPV outcomes in Lactobacillus-deficient patients similar to that in patients with normal vaginal flora.Conclusion. Vaginal probiotic therapy improves outcomes of HPV infection in patients with a deficiency of lactobacilli by reducing the number of survived infected cells and shifting the cell death pattern towards apoptosis.

Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.

Lactobacillus iners Cell-Free Supernatant Enhances Biofilm Formation and Hyphal/Pseudohyphal Growth by Candida albicans Vaginal Isolates

Candida albicans is a commensal fungus of the vaginal mucosa and the principal etiological agent of vaginal candidiasis. Vaginal dysbiosis has been reported during vulvovaginal candidiasis (VVC), with a progressive decrease in Lactobacillus crispatus population and an increase in L. iners population. To date, the role of L. iners in VVC pathogenesis remains scarcely explored. Herein we investigated the in vitro effect of L. iners cell-free supernatant (CFS) on the ability of C. albicans to form biofilms. Biomass and metabolic activity were measured by crystal violet and XTT assays. Further, light microscopy was performed to determine the effect of L. iners CFS on biofilm cellular morphology. We found that L. iners CFS induced a significant increase in biofilm formation by C. albicans clinical isolates which were categorized as moderate or weak biofilm producers. This effect was associated with an enhancement of hyphal/pseudohyphal growth, and the expression levels of HWP1 and ECE1, which are typical hyphae-associated genes, were upregulated. Overall, these results suggest that L. iners contributes to the pathogenesis of VVC and highlight the complexity of the interaction between C. albicans and vaginal lactobacilli. Understanding these interactions could prove essential for the development of new strategies for treating VVC.

Anti-Candida Activity of Hyaluronic Acid Combined with Lactobacillus crispatus Lyophilised Supernatant: A New Antifungal Strategy

Vulvovaginal candidiasis (VVC) and recurrencies are common in reproductive-aged women. The emergence of Candida strains resistant to conventional antimycotic drugs prompted the search for alternative therapies. Hyaluronic acid (HA), a uniform and linear glycosaminoglycan, has been proposed as an anti-Candida agent. Vaginal lactobacilli and their derivatives, including cell free culture supernatants (CFS), represent potential strategies for the treatment of Candida infections. In the present paper, the anti-Candida potential of HA and lyophilised CFS (LCFS), obtained from the vaginal strain Lactobacillus crispatus BC5, was investigated. HA and LCFS proved to be active towards a panel of clinical Candida isolates belonging to different species in a dose dependent manner and their association maintained the antifungal activity. Notably, also Candida species generally resistant to conventional antifungals resulted sensitive. A vaginal matrix based on microcrystalline cellulose and containing effective doses of both agents was developed and characterised. This vaginal formulation showed mucoadhesive ability and almost abrogated Candida albicans growth. In conclusion, HA and LCFS from L. crispatus BC5 are thus good candidates to design a new therapeutic strategy to counteract VVC, and the proposed vaginal matrix represents a promising prototype.

Probiotic and Metabolic Characterization of Vaginal Lactobacilli for a Potential Use in Functional Foods

The main aim of this work was to verify the metabolic and functional aptitude of 15 vaginal strains belonging to Lactobacillus crispatus, Lactobacillus gasseri, and Limosilactobacillus vaginalis (previously Lactobacillus vaginalis), already characterized for their technological and antimicrobial properties. In order to evaluate the metabolic profile of these vaginal strains, a phenotype microarray analysis was performed on them. Functional parameters such as hydrophobicity, auto-aggregation, deconjugation of bile salts, adhesion to an intestinal cell line (Caco-2), and a simulated digestion process were evaluated for these strains. A good number of these strains showed hydrophobicity values higher than 70%. Regarding the auto-aggregation assay, the most promising strains were L. crispatus BC9 and BC1, L. gasseri BC10 and BC14, and L. vaginalis BC17. Moreover, L. crispatus BC4, BC6, BC7, and BC8 were characterized by strong bile salts hydrolase activity (BHS). In addition, L. crispatus BC8 and L. vaginalis BC17 were characterized by a medium ability to adhere to Caco-2 cells. Data related to digestion process showed a strong ability of vaginal lactobacilli to withstand this stress. In conclusion, the data collected show the metabolic versatility and several exploitable functional properties of the investigated vaginal lactobacilli.

Distinct Functional Traits of Lactobacilli from Women with Asymptomatic Bacterial Vaginosis and Normal Microbiota

Asymptomatic bacterial vaginosis (BV) in reproductive-age women has serious obstetric and gynecological consequences. Despite its high incidence, the behavior of vaginal lactobacilli in asymptomatic BV is unknown. We analyzed the functional properties of previously isolated vaginal lactobacilli from asymptomatic women with normal, intermediate, and BV microbiota. Lactic acid and antimicrobial activity against seven urogenital pathogens were evaluated from lactobacilli cell-free culture supernatants (CFCs) (n = 207) after 48 h incubation in MRS. Lactobacilli isolates were used to evaluate H2O2, autoaggregation and coaggregation with C. albicans. Lactobacilli from normal microbiota produced more d-lactate than lactobacilli from intermediate and asymptomatic BV (p = 0.007). L. plantarum, L. fermentum and L. reuteri produced greater d-lactate whereas L. rhamnosus, L. crispatus, L. johnsonii were greater producers of l-lactate. Interspecies positive correlation was observed in the lactic acid contents of CFCs. Distribution of H2O2-producing lactobacilli did not vary significantly among the groups. When lactic acid isomers were considered, species from intermediate and BV microbiota clustered together with each other and distinctly from species of normal microbiota. Broad-spectrum antagonism (≥90% inhibition) against E. coli, C. albicans, S. aureus, P. aeruginosa, G. vaginalis, N. gonorrhoeae, S. agalactiae were displayed by 46.86% (97) of isolates. Our study highlights the differential functional properties of vaginal lactobacilli from women with normal microbiota and asymptomatic BV.

Caerin 1 Antimicrobial Peptides that Inhibit HIV and Neisseria May Spare Protective Lactobacilli

Although acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is a manageable disease for many, it is still a source of significant morbidity and economic hardship for many others. The predominant mode of transmission of HIV/AIDS is sexual intercourse, and measures to reduce transmission are needed. Previously, we showed that caerin 1 antimicrobial peptides (AMPs) originally derived from Australian amphibians inhibited in vitro transmission of HIV at relatively low concentrations and had low toxicity for T cells and an endocervical cell line. The use of AMPs as part of microbicidal formulations would expose the vaginal microbiome to these agents and cause potential harm to protective lactobacilli. Here, we tested the effects of caerin 1 peptides and their analogs on the viability of two species of common vaginal lactobacilli (Lactobacillus rhamnosus and Lactobacillus crispatus). Several candidate peptides had limited toxicity for the lactobacilli at a range of concentrations that would inhibit HIV. Three AMPs were also tested for their ability to inhibit growth of Neisseria lactamica, a close relative of the sexually transmissible Neisseria gonorrhoeae. Neisseria lactamica was significantly more sensitive to the AMPs than the lactobacilli. Thus, several candidate AMPs have the capacity to inhibit HIV and possible N. gonorrhoeae transmission at concentrations that are significantly less harmful to the resident lactobacilli.

Survival of Beneficial Vaginal Lactobacilli (BVL) to Different Gastrointestinal Tract Conditions

Background: Lactobacilli are the dominant bacteria in the healthy vaginal tract, preventing the income of pathogenic microorganisms, either sexually or not transmitted. Probiotics are used to restore the vaginal microbiome by local administration. However, the ascendant colonization is proposed as a way to restore the vaginal balance, and to exert some complementary effects on the host, situation that requires that probiotic strains resist the gastrointestinal tract passage. Objective: To determine which probiotic vaginal strains were able to resist different gastrointestinal factors (pH, bile salts, and enzymes) to advance in the design of oral formulas. Methods: Different protocols were applied to evaluate the growth of 24 beneficial vaginal lactic bacteria (BVL) strains at low pH and high bile salts (individually evaluated) and in combined protocols. The viability of the strains in simulated gastrointestinal tract conditions was studied to select the most resistant strains. Results: A low number of BVL was able to grow at low pH. Most of the strains did not survive at high bile salts concentration. The passage through pH first and bile salts later showed that only three strains were able to survive. In the simulated intestinal conditions, only Lactobacillus gasseri CRL1290, L. jensenii CRL1313, and L. jensenii CRL1349 decrease one or two logarithmic growth units (UFC/ml) at the end of the assay, maintaining their beneficial properties. Conclusion: The behavior of BVL in the conditions assayed is not related to specific strain or metabolic group, because the resistance is strain-specific. The results highlight the importance of the screening performed in a way to select the most adequate strains to be included in the oral designed formula for the restoration of the vaginal tract microbiome.

Novel Selective Inhibition of Lactobacillus iners by Lactobacillus-Derived Bacteriocins

ABSTRACT Lactobacillus iners is often associated with vaginal dysbiosis and bacterial vaginosis (BV), which are risk factors for adverse gynecological and obstetric outcomes. To discover natural inhibitors of L. iners, cell-free culture supernatants (CFSs) from 77 vaginal human Lactobacillus strains and 1 human intestinal strain were screened for inhibitory activity. Three active strains were identified, and Lactobacillus paragasseri K7 (K7), a human intestinal strain, produced the most potent L. iners-inhibitory activity. The active material was purified from the K7 CFS and yielded three active peptides, identified as components of two different class IIb, two-peptide bacteriocins, gassericin K7A (GasK7A) and gassericin K7B (GasK7B). The peptides corresponded to the GasK7A α peptide and the GasK7B α and β peptides. While all three peptides exhibited individual activity against L. iners, GasK7B α was the most potent, with an MIC of 23 ng/ml (4 nM). When combined in equal amounts, the GasK7B α and β peptides showed synergistic inhibition, with an MIC of 2 ng/ml (each peptide at 0.4 nM). Among the four major vaginal Lactobacillus species, the K7 bacteriocins selectively inhibited L. iners. All 21 strains of L. iners tested (100%) were inhibited by the K7 bacteriocins, whereas <20% of the vaginal Lactobacillus crispatus, L. jensenii, and L. gasseri strains were inhibited. The combination of the BV treatment metronidazole and K7 bacteriocins completely killed both L. iners and Gardnerella vaginalis in a coculture experiment to mimic BV conditions. In contrast, this treatment did not inhibit L. crispatus cultures. IMPORTANCE Lactobacillus iners is a prevalent species of the vaginal microbiome, but unlike other major vaginal Lactobacillus species, it is not considered protective against BV and can coexist with BV-associated bacteria. L. iners is generally the first Lactobacillus species to emerge following the treatment of BV with metronidazole, and mounting evidence suggests that it may contribute to the onset and maintenance of vaginal dysbiosis. The discovery of highly potent bacteriocins that selectively kill L. iners while sparing protective vaginal lactobacilli may provide novel pharmacological tools to better understand the roles of this enigmatic bacterium in vaginal ecology and potentially lead to new and improved therapies for dysbiosis-related conditions such as BV.