Lactobacillus crispatus as the etiological agent in cytolytic vaginosis

Introduction: Lactobacillus spp. dominate the vaginal niche but can also be involved in other vaginal dysbiosis, such as cytolytic vaginosis (CV), which remains poorly studied. It is characterized by a cryptic symptomatology, that often confounds the clinic. Goals: The aim of this work was to search for the etiological agent of CV, by studying the vaginal microbiome and metabolomics of women afflicted with this disease and compare it with women with other clinical diagnostic. Methods: Twenty-one vaginal washes have been collected from women attending a gynaecology consultation of a private clinic. The samples were categorized according with clinical diagnosis at the time of sampling (CV, 11; vulvovaginal candidosis, 8; Healthy, 2). The distribution of bacterial species, and their prevalence was assessed by next-generation sequencing of the 16S V4 region. In addition, total lactate D-lactic acid and L-lactic acid was quantified in all washes by a commercial kit, as well as lactate dehydrogenase (LDH) activity. Results: L. crispatus was dominant (>70%) in all CV samples. Lactate was increased in CV in comparison with other cases. The presence of D-lactic acid isomer was associated with presence of L. crispatus. LDH activity was increased in vaginal washes that tested positive for the presence of L. crispatus, however no direct association was found with CV cases. Discussion/Conclusions: The microbiome of women afflicted with CV was dominated in all cases by L. crispatus, contrarily with the results obtained for women diagnosed with other clinical symptomatology. In addition, the finding that an increase in D-lactic acid is associated with CV patients can be related to the role of L. crispatus in CV. The determination of LDH activity did not correlate exclusively with CV cases. On the other hand, D-lactic acid and total lactate quantification could be used as a valuable biomarker to diagnose this cryptic vaginal infection.info:eu-repo/semantics/publishedVersio

Assessment of Lactobacillus crispatus´ role in vaginal infections: friend or foe?

Lactobacillus spp. are the most prevalent microorganisms in the vaginal milleu. Several probiotic mechanisms have been associated with Lactobacillus, but the most relevant one is lactate production – resulting in a low pH value, typical of the healthy vagina. However, its pathogenic state is still unknown. We aim to elucidate the role of a commensal vaginal microorganism, Lactobacillus crispatus, in vaginal infections. Twenty-four vaginal washes have been collected from women attending a gynaecology consultation of a private clinic. The samples were categorized according with clinical diagnosis at the time of sampling. The distribution of bacterial species, and their prevalence was assessed by Next-Generation Sequencing of the 16S rRNA V4 region. In addition, L- and D-lactate concentration, and LDH enzymatic activity was quantified in all washes by a commercial kit. Detection of Candida spp. was performed by PCR. L. crispatus was dominant (>70%) in 11 out of 24 samples – diagnosed for vaginal atrophy (VA, 3), cytolytic vaginosis (CV, 2) and lactobacillosis (LB, 2). Lactate was increased in CV, LB and VA cases only. The remaining samples, diagnosed for vulvovaginal candidosis in its majority, had lower prevalence of L. crispatus; and lower to moderate lactate metabolite. There was not a direct relationship between LDH enzymatic activity and clinical diagnosis. L. crispatus dominance, associated with increased lactate production, was observed in CV, LB and VA cases. These results indicate that this microorganism might have a role in dysbiosis of the vagina associated with these specific pathologies.info:eu-repo/semantics/publishedVersio

Role of Lactobacillus crispatus in vaginal infections: insights from metagenomics and metabolomics studies

Objective: Lactobacillus spp. constitute the most prevalent bacterial microorganism in the vaginal milleu. Several probiotic mechanisms have been associated with Lactobacillus, but the most relevant one is lactate production – resulting in a low pH value, typical of the healthy vagina. We aim to elucidate the role of a native vaginal microorganism, Lactobacillus crispatus, in vaginal infections. Methods: Twenty-four vaginal washes have been collected from women (mean 38±14 years of age) attending a gynecology consultation of a private clinic. The samples were categorized according with clinical diagnosis at the time of sampling. The distribution of bacterial species, and their prevalence was assessed by next-generation sequencing of the 16S V4 region. In addition, lactate was quantified in all washes by a commercial kit. Results: L. crispatus was dominant (>70%) in 11 out of 24 samples – diagnosed for vaginal atrophy (VA, 3), cytolytic vaginosis (CV, 2), lactobacillosis (LB, 2) and vulvovaginal candidosis (VVC, 1). For three samples, diagnostic was inconclusive. Lactate was increased in CV, LB and VA cases only. One sample, diagnosed with VVC had 23% dominance of L. crispatus; and lower lactate metabolite. L. crispatus was not detected in the remaining 12 cases (6 VVC, 4 VA, 1 healthy and 1 inconclusive). Of these, only 5 samples had moderate lactate metabolite. Conclusions: L. crispatus dominance, associated with increased lactate production, was observed in CV, LB and VA cases. These results indicate that this microorganism might have a role in dysbiosis of the vagina associated with these specific pathologies.info:eu-repo/semantics/publishedVersio

Bacteriocin production of the probiotic Lactobacillus acidophilus KS400

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.Project POCI-01-0145-FEDER-007491; Medinovainfo:eu-repo/semantics/publishedVersio

Evaluating the Concentration of a Candida albicans Suspension

Objective: The objective of this study was to develop a reproducible method of establishing the concentration of yeast cells per milliliter of solution

Preliminary studies for the aplication of Thymbra capitata essential oil as potential antimicrobial agent in Bacterial Vaginosis

Bacterial vaginosis (BV) is the most common gynecological clinical condition in women in reproductive age, and has been associated with an increased risk of development of preterm labor, spontaneous abortion, and several sexually transmitted diseases such as HIV [1]. BV is not only a condition that involves excessive growth of anaerobic microorganisms, but also involves the presence of an adherent biofilm [2]. Despite its high prevalence, the aetiology of BV remains unknown, but frequently Gardnerella vaginalis is the main microorganism associated with this disorder [3], that is often the main species in biofilms presented in BV [4]. These results led to the theory that the microorganisms that form biofilms may be relevant for the aetiology of BV [5]. Traditionally, the treatment of BV involves the antibiotics metronidazole, clindamicin or tinidazole [6]. However, the recurrence rate remains high and this can be related to the biofilm-forming ability of G. vaginalis [5]. In this context, recently it was reported that natural compounds, such as Thymbra capitata essential oil, can reduce the activity of biofilms [7]. To test if T. capitata could be used in the treatment of BV, we determined the biofilm-forming ability and the effect of essential oil in seven strains of G. vaginalis isolated from women with BV. Thus, the biofilm-forming ability was assessed under anaerobic conditions for 48 hours, allowing each strain to form biofilm. These assays revealed that in fact, G. vaginalis strains produced moderate to tenacious biofilm. We then determined the sensibility of the tested bacteria to the common antibiotics used to treat BV. Not surprisingly, most of strains were resistant of metronidazole and tinidazole which are more commonly administered. However half of strains were sensitive to clindamicin. We then evaluated the antibacterial activity of essential oil upon G. vaginalis strains, determining minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC). The T. capitata essential oil exhibited a potent anti-G. vaginalis effect, which was confirmed by the low values of MIC (0.16 µL.mL-1) and MLC (0.16 - 0.31 µL.mL-1). To conclude, these results showed that T. capitata essential oil presents a strong antibacterial activity upon antibiotic resistant and biofilm forming of G. vaginalis strains, reinforcing the potential interest of this essential oil as antimicrobial agent in the treatment of BV

In Vitro Effect of Local Anesthetics on Candida albicans Germ Tube Formation

Objective: This study was planned to clarify the in vitro effect of lidocaine and bupivacaine on germ tube formation by Candida albicans isolates from cases of clinical vaginal candidiasis

Antifungal activity of local anesthetics against Candida species.

OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity. METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains). The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry. The fungicidal activity of the three drugs was assayed by viability counts. Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts. RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively. The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes. A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively. CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane