Sustained Simultaneous Delivery of Metronidazole and Doxycycline From Polycaprolactone Matrices Designed for Intravaginal Treatment of Pelvic Inflammatory Disease.

Poly(ɛ-caprolactone) (PCL) intravaginal matrices were produced for local delivery of a combination of antibacterials, by rapidly cooling a mixture of drug powders dispersed in PCL solution. Matrices loaded with different combinations of metronidazole (10%, 15%, and 20% w/w) and doxycycline (10% w/w) were evaluated in vitro for release behavior and antibacterial activity. Rapid "burst release" of 8%-15% of the doxycycline content and 31%-37% of the metronidazole content occurred within 24 h when matrices were immersed in simulated vaginal fluid at 37°C. The remaining drug was extracted gradually over 14 days to a maximum of 65%-73% for doxycycline and 62%-71% for metronidazole. High levels of antibacterial activity up to 89%-91% against Gardnerella vaginalis and 84%-92% against Neisseria gonorrhoeae were recorded in vitro for release media collected on day 14, compared to "nonformulated" metronidazole and doxycycline solutions. Based on the in vitro data, the minimum levels of doxycycline and metronidazole released from PCL matrices in the form of intravaginal rings into vaginal fluid in vivo were predicted to exceed the minimum inhibitory concentrations for N. gonorrhea (reported range 0.5-4.0 µg/mL) and G. vaginalis (reported range 2-12.8 µg/mL) respectively, which are 2 of the major causative agents for pelvic inflammatory disease.

Thymbra capitata essential oil as potential therapeutic agent against Gardnerella vaginalis biofilm-related infections.

AIM: To evaluate the antibacterial activity of Thymbra capitata essential oil and its main compound, carvacrol, against Gardnerella vaginalis grown planktonically and as biofilms, and its effect of vaginal lactobacilli. MATERIALS & METHODS: Minimal inhibitory concentration, minimal lethal concentration determination and flow cytometry analysis were used to assess the antibacterial effect against planktonic cells. Antibiofilm activity was measured through quantification of biomass and visualization of biofilm structure by confocal laser scanning microscopy. RESULTS: T. capitata essential oil and carvacrol exhibited a potent antibacterial activity against G. vaginalis cells. Antibiofilm activity was more evident with the essential oil than carvacrol. Furthermore, vaginal lactobacilli were significantly more tolerant to the essential oil. CONCLUSION: T. capitata essential oil stands up as a promising therapeutic agent against G. vaginalis biofilm-related infections.

Inhibitory effects of seaweed extracts on the growth of the vaginal bacterium Gardnerella vaginalis.

Of 44 species of seaweed screened for potential anti-Gardnerella vaginalis activity, 27 (61.4%) showed antimicrobial activity by the agar disk-diffusion method. Among them, the strongest activities against the pathogen were exhibited by Chlorophyta, with Ulva pertusa producing an 11.3-mm zone of inhibition at 5 mg disk⁻¹. The MIC values of U. pertusa extracts against both G. vaginalis KCTC 5096 and KCTC 5097, the main cause of vaginosis, were 312 µg ml⁻¹, while the MIC values against both Candida albicans KCTC 7270 and KCTC 7965, the main cause of candidiasis, were 2.5 mg ml⁻¹. Against Lactobacillus gasseri KCTC 3173 and Lactobacillus jensenii KCTC 5194, members of the normal vaginal microflora, no inhibitory effect was seen even at 10 mg ml⁻¹. To identify the primary active compounds, a U. pertusa powder was successively fractionated according to polarity, and the main active agents against G. vaginalis KCTC 5096 were determined to be nitrogenous compounds (156 µg ml⁻¹ of the MIC value). According to these results, it was suggested that extracts of the seaweed U. pertusa are valuable for the development of natural therapeutic agents for treating women with bacterial vaginosis.

Seasonal variation of antibacterial activities in the green alga Ulva pertusa Kjellman.

The present study was performed to screen out the extracts of algae and assess the seasonal variation in antimicrobial activity of Ulva pertusa against Gardnerella vaginalis. Seasonal variation in antibacterial activity was observed, with the extracts showing no activity during summer and autumn, and showing antibacterial activity from early winter (December) to middle spring (April). The maximum value of antimicrobial activity (6.5 mm inhibition zone at 5 mg disk(-1)) of U. pertusa against G. vaginalis was observed in April. Otherwise, for both chlorophyll a and b, the highest content (2.87 mg g(-1) and 1.37 mg g(-1)) was observed in March 2009. These results may reflect variation in cellular chemical compositions such as secondary metabolite(s) rather than chlorophyll and biological activities according to season.

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.

[Role and place of antibacterial therapy in prophylaxis of disturbances in reproductive function of women].

One-stage retrospective analysis of 350 primary medical documents of the female patients treated under hospital conditions for salpingo-oophoritis in 2010-2011 was performed. The results were compared with those of the investigation of the present etiological pattern of pelvic inflammatory diseases (PID) by the data of the microbiological examination of 117 patients with PID and susceptibility of the isolates to the antibacterials. The frequency and efficiency of the use of antibacterials alone or in combinations were analysed in the treatment of various clinical forms of PID. The ovarian reserve was estimated in 87 patients after recovery from salpingo-oophoritis. 52 of them had an episode of the chronic process exacerbation and 35 had the first episode of acute PID. The ovarian reserve was estimated by determination of the anti-Mullerian hormone (AMH), basal FSH level, ovarian volume and antral follicle count. A statistically significant decrease of the ovarian reserve in the patients with chronic salpingo-oophoritis confirmed the necessity of rational treatment of the acute inflammatory process.

Bacterial vaginosis, Atopobium vaginae and nifuratel.

As bacterial vaginosis (BV) is a potential cause of obstetric complications and gynecological disorders, there is substantial interest in establishing the most effective treatment. Standard treatment - metronidazole or clindamycin, by either vaginal or oral route � is followed by relapses in about 30% of cases, within a month from treatment completion. This inability to prevent recurrences reflects our lack of knowledge on the origins of BV. Atopobium vaginae has been recently reported to be associated with BV in around 80% of the cases and might be involved in the therapeutic failures. This review looks at the potential benefits of nifuratel against A. vaginae compared to the standard treatments with metronidazole and clindamycin. In vitro, nifuratel is able to inhibit the growth of A. vaginae, with a MIC range of 0.125-1 µg/mL; it is active against G. vaginalis and does not affect lactobacilli. Metronidazole is active against A. vaginae only at very high concentrations (8-256 µg/mL); it is partially active against G. vaginalis and also has no effect on lactobacilli. Clindamycin acts against A. vaginae with an MIC lower than 0.125 µg/mL and is active on G. vaginalis but it also affects lactobacilli, altering the vaginal environment. These observations suggest that nifuratel is probably the most valid therapeutic agent for BV treatment.

Artemisia princeps Pamp. Essential oil and its constituents eucalyptol and α-terpineol ameliorate bacterial vaginosis and vulvovaginal candidiasis in mice by inhibiting bacterial growth and NF-κB activation.

To investigate the inhibitory effects of Artemisia princeps Pamp. (family Asteraceae) essential oil (APEO) and its main constituents against bacterial vaginosis and vulvovaginal candidiasis, their antimicrobial activities against Gardnerella vaginalis and Candida albicans in vitro and their anti-inflammatory effects against G. vaginalis-induced vaginosis and vulvovaginal candidiasis were examined in mice. APEO and its constituents eucalyptol and α-terpineol were found to inhibit microbe growths. α-Terpineol most potently inhibited the growths of G. vaginalis and C. albicans with MIC values of 0.06 and 0.125 % (v/v), respectively. The antimicrobial activity of α-terpineol was found to be comparable to that of clotrimazole. Intravaginal treatment with APEO, eucalyptol, or α-terpineol significantly decreased viable G. vaginalis and C. albicans numbers in the vaginal cavity and myeloperoxidase activity in mouse vaginal tissues compared with controls. These agents also inhibited the expressions of proinflammatory cytokines (IL-1 ß, IL-6, TNF- α), COX-2, iNOS, and the activation of NF- κB and increased expression of the anti-inflammatory cytokine IL-10. In addition, they inhibited the expressions of proinflammatory cytokines and the activation of NF- κB in lipopolysaccharide-stimulated peritoneal macrophages, and α-terpineol most potently inhibited the expressions of proinflammatory cytokines and NF- κB activation. Based on these findings, APEO and its constituents, particularly α-terpineol, ameliorate bacterial vaginosis and vulvovaginal candidiasis by inhibiting the growths of vaginal pathogens and the activation of NF- κB.

Response of Gardnerella vaginalis biofilm to 5 days of moxifloxacin treatment.

Polymicrobial communities are often recalcitrant to antibiotics. We tested whether the polymicrobial Gardnerella vaginalis biofilm can be eradicated with moxifloxacin. Twenty women with bacterial vaginosis were treated with 400 mg moxifloxacin for 5 days. The changes in the occurrence and proportions of Gardnerella, Atopobium and Lactobacillus spp. were assessed using FISH. The bacterial biofilm was investigated using desquamated epithelial cells of spontaneously voided urine and sections of vaginal biopsies. Fifteen of 20 women showed a significant and sustained clinical response to moxifloxacin according to Amsel and Nugent criteria. The concentrations of adherent bacteria decreased significantly. The incidence and proportion of Atopobium declined sustainably. The proportions of Lactobacillus in the biofilm mass increased following therapy. Initially, Gardnerella was the main component of the polymicrobial biofilm. Following treatment, Gardnerella was not accessible to FISH in the urine and vaginal samples of 75% of all women. Ten to 12 weeks after the end of therapy, Gardnerella biofilm was cumulatively present in 40%. This was not due to newly acquired disease, but due to reactivation of the persisting, but biochemically inactive biofilm. Despite clear clinical efficacy, and initially definite suppression of the biofilm, moxifloxacin was, similar to metronidazole, not able to eradicate the Gardnerella vaginalis biofilm in all patients.

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.

Activity of Novispirin G-10, a novel antimicrobial peptide against Chlamydia trachomatis and vaginosis-associated bacteria.

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptide's activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.

Effects of Cetyltrimethylammonium naproxenate on the adherence of Gardnerella vaginalis, Mobiluncus curtisii, and Lactobacillus acidophilus to vaginal epithelial cells.

BACKGROUND AND OBJECTIVES: A decreased concentration or total disappearance of Lactobacillus acidophilus in the vagina constitutes a frequent observation in bacterial vaginosis. GOAL OF THE STUDY: Cetyltrimethylammonium naproxenate has been evaluated in vitro to detect antiadhesive properties at subinhibitory concentrations for Gardnerella vaginalis and Mobiluncus curtisii to vaginal epithelial cells (VEC). STUDY DESIGN: Bacterial strains 14C- and or 3H-labeled were tested for adherence and competition to binding sites in VECs before and after treatment at sub-MIC with cetyltrimethylammonium naproxenate. RESULTS: In control tests of adherence, G. vaginalis and M. curtisii had their maximal adhesion at pH 5.4, L. acidophilus at pH 4.4. Preincubation of G. vaginalis and M. curtisii with cetyltrimethylammonium naproxenate 2.5 mg/mL (subinhibitory concentration) at pH 5.4 reduced adherence to VECs respectively by 48.3% and 34.1%. The same treatment of L. acidophilus showed no statistically significant difference. Treatment of VECs alone did not modify adherence. Competition tests between L. acidophilus and G. vaginalis and between L. acidophilus and M. curtisii showed that, in small quantities, L. acidophilus could compete with G. vaginalis and M. curtisii for binding sites in VECs at pH 4.4, when pretreated with cetyltrimethylammonium naproxenate. At a higher pH (4.8 and 5.4), L. acidophilus in higher quantities did not compete for binding sites occupied by G. vaginalis and M. curtisii. CONCLUSIONS: Cetyltrimethylammonium naproxenate at subinhibitory concentrations modifies the adhesiveness of G. vaginalis and M. curtisii to VECs, reducing it by 48.3% and 34.1%, respectively. Adhesion of L. acidophilus to VECs is not impaired by pretreatment with cetyltrimethylammonium naproxenate at pH 4.4, even if they are in low number and compete for binding sites against pathogens. At higher pH levels, L. acidophilus did not compete for binding sites occupied by G. vaginalis and M. curtisii.

The role of benzydamine in the topical treatment of the so-called non-specific vaginitis.

The authors report the preliminary results of their investigation of the efficacy of benzydamine in the treatment of the so-called non-specific vaginitis. An initial in vitro study to test its bacteriostatic and/or bactericidal activity on Gardnerella vaginalis showed that the drug has high activity even at the lowest concentrations, and completely inhibits this micro-organism at 1000 micrograms/ml which is the usual concentration employed in therapy. The first pilot study performed in vivo on 14 selected vaginitis cases which were found positive for G. vaginalis by culture test appears to confirm the therapeutic efficacy of benzydamine; however, this confirmation must await the results of a double-blind clinical trial which is still in progress.

Comparison of two different metronidazole regimens in the treatment of Gardnerella vaginalis infection with or without trichomoniasis.

Gardnerella vaginalis infection confirmed by culture was treated either by a 2 g dose or divided doses of metronidazole in 100 and 200 female patients respectively. Both dosages were equally effective. We recommend a single dose of 2 g metronidazole in the treatment of Gard. vaginalis infection, particularly when association with trichomoniasis is confirmed or suspected.

Susceptibility of Gardnerella vaginalis to thiamphenicol: clinical experience with nonspecific vaginitis.

We compared the in-vitro activity of thiamphenicol against 100 strains of Gardnerella vaginalis with the activity of 11 other antimicrobial agents. The MICs for thiamphenicol ranged from 0.39 micrograms/ml to 6.25 micrograms/ml. The concentration at which 50% of strains were inhibited (MIC50) was 1.96 micrograms/ml, and the concentration at which 90% of strains were inhibited (MIC90) was 3.93 micrograms/ml. All strains were very susceptible to erythromycin, chloramphenicol, beta-lactam antibiotics, and clindamycin. Tetracycline and metronidazole were only moderately active. In an attempt to cure G. vaginalis-associated vaginitis with a single-dose treatment, we administered 2.25 g of thiamphenicol to 20 volunteers; 17 were clinically and bacteriologically cured. In two cases we observed that G. vaginalis was not eliminated immediately (i.e., at the first follow-up visit), but we saw a progressive disappearance of the strain without further treatment. In one case the treatment seemed to have failed but reinfection could not be ruled out. The results show that a single dose of thiamphenicol can cure G. vaginalis-associated vaginitis.

Bacterial vaginosis. An evaluation of treatment.

Various treatment regimens for bacterial vaginosis are reviewed. Orally administered metronidazole and tinidazole have been consistently successful, whereas widely variable results have been published from use of peroral ampicillin or tetracycline and from locally applied sulphonamide cream. The rationale for different treatments is discussed, with particular regard to the pathogenesis of the disease.

Influence of metronidazole treatment on the vaginal microbiological flora.

In order to study the effects of metronidazole on the microbiology of the vagina in general and on Gardnerella vaginalis infection in particular, quantitative aerobic and anaerobic bacterial cultures were performed before and 4 weeks after initiation of metronidazole treatment, 400 mg three times daily for five days. Bacteriological results were compared with microscopic findings and with the results of "amine testing" with potassium hydroxide and with other clinical variables. We found a reasonably good correlation between the finding of clue cells, positive amine test and symptomatic Gardnerella infection. Treatment with metronidazole resulted in most cases in recolonization with lactobacilli and disappearance of clinical symptoms and findings, including clue cells, although G. vaginalis could still be detected.

Metronidazole in treatment against Haemophilus vaginalis (Corynebacterium vaginale).

The rate of bactericidal activity and inactivation of metronidazole was studied in time-kill curves with Haemophilus vaginalis (Corynebacterium vaginale). The minimum inhibitory concentrations of metronidazole for the eight strains tested ranged from 4 to 16 micrograms/ml. At a concentration of 20 micrograms/ml, metronidazole demonstrated a slow cidal effect against exponential-phase organisms, requiring 24 to 48 h for completion. Inactivation of metronidazole during the time-kill curve was quite variable and averaged 28% of the starting concentration after 48 h. Against stationary-phase organisms (inoculum, 10(10) to 10(11) colony-forming units per ml), a slow cidal effect was also seen, with an average inactivation of metronidazole of 38% after 48 h. At a subinhibitory concentration of 5 micrograms/ml, metronidazole was inactivated to the greatest degree (57% after 48 h). Therefore, in contrast to earlier studies in which metronidazole was rapidly and consistently cidal within 4 h against obligate anaerobes and was almost completely inactivated by 8 h, the bactericidal effect of metronidazole againsts H. vaginalis in this study was much slower and was associated with a variable and slower rate of inactivation.