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1.  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.
PMCID: PMC2565553  PMID: 18923673
2.  Safety Study of an Antimicrobial Peptide Lactocin 160, Produced by the Vaginal Lactobacillus rhamnosus  
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.
PMCID: PMC2216118  PMID: 18273406
3.  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.
PMCID: PMC1784636  PMID: 11368258
4.  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.
PMCID: PMC1784632  PMID: 11368257

Results 1-4 (4)