Sebastian Faro

Gardnerella vaginalis Isolated from Patients with Bacterial Vaginosis and from Patients with Healthy Vaginal Ecosystems

The differences in the phenotype and genotype of Gardnerella vaginalis isolates from patients with bacterial vaginosis (BV) and from patients without BV are unknown. In our study, 43 isolates of G. vaginalis were examined for biotype (hippurate hydrolysis, lipase, and beta-galactosidase activity), sensitivity to metronidazole, and genotype. Of the 117 women visiting the gynecology clinic at Rush-Presbyterian-St. Lukes Medical Center who were included in the study, 27.4% were found to have BV. G. vaginalis was found in samples from 87.5% of women with BV, from 34.0% of women with intermediate BV, and from 26.4% of women with healthy vaginal ecosystems. Among patients with G. vaginalis, biotypes 7 and 8 were isolated from 32% and 20% of patients, respectively. Biotype 5 was predominantly associated with a healthy vaginal ecosystem (P=.0004). Biotypes 5 and 7 were the most resistant to metronidazole. No specific phenotype or genotype of G. vaginalis causes BV.

Antimicrobial protein produced by vaginal Lactobacillus acidophilus that inhibits Gardnerella vaginalis

Objective: To isolate bacteriocin from a vaginal strain of Lactobacillus acidophilus. Methods: L. acidophilus 160 was grown on two media. The first was MRS broth for 18 hours; the cells were harvested, washed, and placed into a chemically defined medium. The second medium resembled vaginal fluid minus protein. Bacteriocin was precipitated from both media using ammonium sulfate. The growth-inhibiting activity of bacteriocin was determined by a bioassay using nine different isolates of Gardnerella vaginalis. Results: MRS broth is not a suitable medium for extracting bacteriocin, because it binds with Tween 80. Bacteriocin was isolated, without contaminating constituents, from chemically defined medium and identified as a single band by electrophoresis. Bacteriocin has a molecular weight of 3.8 kDa. All nine isolates of Gardnerella were inhibited by the bacteriocin isolated from L. acidophilus 160. Conclusions: Bacteriocin produced by L. acidophilus 160 was isolated from the chemically defined medium (starvation medium) in a partially pure form. L. acidophilus 160 bacteriocin inhibited growth of all nine isolates of Gardnerella vaginalis.

Two Novel Vaginal Microbicides (Polystyrene Sulfonate and Cellulose Sulfate) Inhibit Gardnerella vaginalis and Anaerobes Commonly Associated with Bacterial Vaginosis

ABSTRACT This is the first report demonstrating the in vitro inhibitory activity of two novel microbicides (cellulose sulfate and polystyrene sulfonate) against bacterial vaginosis (BV)-associated bacteria. Vaginal application of these microbicides not only may reduce the risk of acquisition of human immunodeficiency virus and other sexually transmitted infection-causing organisms but may also decrease the incidence of BV.

Antibiotic prophylaxis: Is there a difference?

Seven antibiotics, administered in 10 different regimens for prophylaxis, were randomly assigned to 1580 patients who were delivered by cesarean section. Cefazolin 1 gm, administered for three doses, served as the control group. Cefazolin 1 gm, cefazolin 2 gm, cefoxitin 1 gm, cefoxitin 2 gm, cefonicid 1 gm, cefotetan 1 gm, ceftizoxime 1 gm, ampicillin 2 gm, and piperacillin 4 gm were all administered in a single dose. Four antibiotics proved to be superior in preventing postpartum endometritis: ampicillin 2 gm (p = 0.03), cefazolin 2 gm (p = 0.005), piperacillin 4 gm (p = 0.0007), and cefotetan 1 gm (p = 0.0001). Single-dose cephalosporin antibiotic prophylaxis was found to result in approximately a twofold increase in Enterococcus faecalis colonization of the vagina (p less than 0.01). This may be significant in patients in whom postpartum endometritis develops and who have failure of initial treatment with a broad-spectrum cephalosporin, e.g., cefoxitin or cefotetan, or a combination such as clindamycin or metronidazole plus an aminoglycoside. Rupture of amniotic membranes for a half hour or more was associated with an increased risk for postpartum endometritis. The use of internal fetal monitoring was associated with an increased risk of soft tissue pelvic infection.

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.

Comparison of the activities of penicillin G and new beta-lactam antibiotics against clinical isolates of Bacteroides species.

MICs were determined for 218 clinical isolates of Bacteroides by a broth microdilution method. Imipenem was the most active antibiotic tested. Azlocillin, mezlocillin, and cefoxitin had comparable activities, with resistance among members of the B. fragilis group and B. capillosus. Ceftizoxime was the most active cephalosporin tested. Members of the B. fragilis group showed high levels of resistance to cefotetan and ceftazidime. Resistance to penicillin G varied from 0 to 14%.

Vaginal flora and pelvic inflammatory disease

Forty-one patients with acute pelvic inflammatory disease were evaluated for the coexistence of bacterial vaginosis. Because all patients had a copious purulent vaginal discharge, microscopic criteria could not be used and microbiologic criteria were employed. The vaginal bacterial flora were not consistent with that of bacterial vaginosis, because Lactobacillus and other gram-positive bacteria dominated with colony counts of 10(3) to 10(5) cfu/ml (colony-forming units per milliliter). Endocervical specimens yielded Neisseria gonorrhoeae from 20 patients and Chlamydia trachomatis from 11 patients. Anaerobes were not dominant in any site sampled. A total of 147 bacteria were isolated from the endometrium, 16 (11%) of which were anaerobes. Thus the endogenous bacterial flora were not consistent with that of the microbiologic definition of bacterial vaginosis. N. gonorrhoeae was the most common isolate from the endocervix and endometrium; it was isolated three times more frequently from the endocervix and two times more frequently from the endometrium than was C. trachomatis.

Effect of Metronidazole on the Growth of Vaginal Lactobacilli in vitro

Objective: To determine whether metronidazole has an adverse effect on the growth of Lactobacillus. Methods: Hydrogen peroxide- and bacteriocin-producing strains of Lactobacillus were used as test strains. Concentrations of metronidazole used ranged from 128 to 7000 μg/ml. Susceptibility to metronidazole was conducted by the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards. Results: Growth of Lactobacillus was partially inhibited at concentrations between 1000 and 4000 μg/ml (p = 0.014). Concentrations ≥ 5000 μg/ml completely inhibited growth of Lactobacillus. Concentrations between 128 and 256 μg/ml stimulated growth of Lactobacillus (p = 0.025 and 0.005, respectively). Concentrations of metronidazole between 64 and 128 μg/ml or ≥ 512 μg/ml did not have an inhibitory or a stimulatory effect on the growth of Lactobacillus compared to the control. Conclusions: High concentration of metronidazole, i.e. between 1000 and 4000 μg/ml, partially inhibited the growth of Lactobacillus. Concentrations ≥ 5000 μg/ml completely suppressed the growth of Lactobacillus. Concentrations between ≥ 128 and ≤ 256 μg/ml stimulated the growth of Lactobacillus. Further investigation to determine the ideal concentration of metronidazole is needed in order to use the antimicrobial agent effectively in the treatment of bacterial vaginosis.

Lactocin 160, a Bacteriocin Produced by Vaginal Lactobacillus rhamnosus, Targets Cytoplasmic Membranes of the Vaginal Pathogen, Gardnerella vaginalis

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.

Mode of action of lactocin 160, a bacteriocin from vaginal Lactobacillus rhamnosus.

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.