NATURAL ANTIMICROBIALS AND THEIR ROLE IN VAGINAL HEALTH: A SHORT REVIEW.

Lactobacillus species maintain the vaginal ecosystem in a healthy condition by production of antimicrobial substances. Depletion of lactobacilli in the vagina results in bacterial vaginosis (BV), where the normal flora is replaced by several bacterial pathogens, usually Gardnerella vaginalis and obligate anaerobes. BV may cause complications such as premature labor, low birth weight and increased risk of HIV acquisition. The currently recommended antibiotic treatments for BV are not always effective and often lead to reoccurrence of the infection. In many cases, this is due to the antibiotic-resistant forms of the pathogens. Therefore, there is an interest in the development of treatments using antimicrobials derived primarily from Lactobacillus spp., such as ribosomally produced antimicrobial peptides (bacteriocins) and lactic acid. These substances effectively inhibit pathogenic bacteria, are safe and do not pose any threat to healthy vaginal Lactobacillus spp. It may be possible to find an effective treatment against BV while reducing the infection's reoccurrence and the treatment-related complications through hurdle technology. This would be achieved by combining antimicrobials produced by Lactobacillus spp. with different natural antimicrobials obtained from plants or other non-pathogenic organisms.

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens.

AIMS: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product-derived Bacillus amyloliquefaciens. METHODS AND RESULTS: An unknown bacterial species cultured from the Yogu Farm probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell-free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. CONCLUSION: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of intra-species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin's activity against bacterial vaginosis-associated pathogens.

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. Luke's 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.

Human immunodeficiency virus type 1 stimulatory activity by Gardnerella vaginalis: relationship to biotypes and other pathogenic characteristics.

Stimulation of human immunodeficiency virus (HIV) type 1 expression by Gardnerella vaginalis is one possible cause for an increase in the amount of virus in the genital tract. The ability of G. vaginalis to induce HIV expression in chronically infected U1 cells was investigated, along with its possible relationship to biotype, genotype, and resistance to metronidazole and bacteriocin. Significant HIV stimulatory activity was found in 5 (50%) lysates of G. vaginalis. The ability to induce HIV expression in U1 cells was statistically associated with G. vaginalis biotype (P=.048) but not with genotype or resistance to metronidazole and bacteriocin. Further studies to explore the in vivo relevance of HIV activation by G. vaginalis in the female genital tract are warranted, since prevention strategies of bacterial vaginosis and colonization by certain biotypes of G. vaginalis may be valuable in reducing the risk of sexual transmission of HIV.

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.

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 microg/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 microg/ml (p = 0.014). Concentrations > or = 5000 microg/ml completely inhibited growth of Lactobacillus. Concentrations between 128 and 256 microg/ml stimulated growth of Lactobacillus (p = 0.025 and 0.005, respectively). Concentrations of metronidazole between 64 and 128 microg/ml or > or = 512 microg/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 microg/ml, partially inhibited the growth of Lactobacillus. Concentrations > or = 5000 microg/ml completely suppressed the growth of Lactobacillus. Concentrations between > or = 128 and < or = 256 microg/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.