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.

Bacteriocin susceptibility of Gardnerella vaginalis and its relationship to biotype, genotype, and metronidazole susceptibility.

OBJECTIVE: Our purpose was to investigate the bacteriocin susceptibility of Gardnerella vaginalis and its relationship to biotype, genotype, and resistance to metronidazole. STUDY DESIGN: Bacteriocin susceptibility of 36 G vaginalis clinical isolates was tested against a vaginal strain of Lactobacillus acidophilus by a growth-inhibition method. The relationship to biotype, genotype, and resistance to metronidazole were analyzed by the chi2 test and Fisher exact test. RESULTS: Eight G vaginalis strains (22%) were bacteriocin-resistant. Biotypes 5 and 7 were found to be the most frequent among these resistant strains. Eight (42%) of the 19 isolates classified as biotype 5, 6, or 7 were bacteriocin-resistant compared with none of the isolates that were classified as other biotypes (P <.01). Biotype 5 was found in higher prevalence among the isolates resistant to bacteriocin (62%) than among the susceptible isolates (14%) (P =.01). Genotype B was found more frequently among the bacteriocin-resistant strains, but this finding was not statistically significant (P =.71). Seven (88%) bacteriocin-resistant strains were also resistant to metronidazole. CONCLUSION: An association between biotype and an increased resistance to bacteriocin was found. The ability of G vaginalis to resist the antibacterial activity of Lactobacillus bacteriocin may be a pivotal factor in understanding bacterial vaginosis.

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.

Defense factors of vaginal lactobacilli.

OBJECTIVE: To determine the antagonistic relationship between vaginal lactobacilli and endogenous vaginal microflora. STUDY DESIGN: Twenty-two Lactobacillus strains were studied for the production of lactic acid, hydrogen peroxide, and bacteriocin. RESULTS: Under standardized growth conditions, most strains increased their biomass by more than 4 times. Lactobacillus species grew best at a pH > or = 4.5, and growth was retarded at a pH < 4.5. Lactic acid levels were 0.68 to 2.518 mg/mL and were not related to the number of cells or the pH of media. The pH of the media was caused by the secretion of lactic and other organic acids. Approximately 80% of the strains produced H(2)O(2) and were graded as 2+ in one third of the strains and 1+ in others. No statistical correlation was found between H(2)O(2) lactic acid and bacteriocin production. Bacteriocin activity was tested on 4 strains of Gardnerella vaginalis. Approximately 80% of the lactobacilli tested produced bacteriocin that inhibited growth of G vaginalis. Six of the strains did not produce bacteriocin. Thirteen strains produced all 3 defense factors, whereas the others lacked 1 or 2 properties. CONCLUSIONS: Lactobacillus species grow best at a pH > 4.5. The pH of the media is dependent on the cell mass and on all organic acids produced by Lactobacillus species. Although all species produce organic acids, not all produce H(2)O(2) and bacteriocin. Not all strains of G vaginalis can be inhibited by lactobacilli-producing bacteriocin.

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.

The inhibitory effect of clindamycin on Lactobacillus in vitro.

OBJECTIVE: To evaluate the in vitro effect of varying concentrations of clindamycin on Lactobocillus spp. METHODS: Concentrations of clindamycin ranging from 1.95-20,000 microg/ml were studied for their effect on the growth of six strains of Lactobacillus. RESULTS: Clindamycin concentrations between 1.95-31.25 microg/ml had no statistically significant effect on growth of lactobacilli (p > 0.05). Concentrations 125 and 250 microg/ml had a bacteriostatic effect. The mean minimum inhibitory concentration (MIC) for studied Lactobacillus strains was determined as 1,000 microg/ml. CONCLUSION: High concentrations of clindamycin achieved in the vagina by intravaginal application might be inhibitory for Lactobacillus.

Induction of human immunodeficiency virus type 1 expression by anaerobes associated with bacterial vaginosis.

Bacterial vaginosis (BV) is a common disorder characterized by increased levels of anaerobic bacteria in the genital tract. BV has been associated with an increased rate of sexual transmission of human immunodeficiency virus (HIV). The effects of BV-associated anaerobic bacteria on HIV expression in monocytoid cells and T cells were examined. Peptostreptococcus asaccharolyticus and Prevotella bivia stimulated HIV expression in monocytoid cells, whereas Bacteroides ureolyticus, Peptostreptococcus anaerobius, and Lactobacillus acidophilus did not enhance HIV expression. P. asaccharolyticus also enhanced HIV expression in T cells and activated HIV long-terminal-repeat transcription in U38 cells. This report suggests a mechanism by which disturbances in vaginal flora could lead to a higher rate of sexual transmission of HIV. Furthermore, this study supports the idea that treatment of BV might serve as a preventive measure to reduce the risk of HIV transmission.

Evaluation of the Strep B OIA test compared to standard culture methods for detection of group B streptococci.

OBJECTIVE: This study evaluated the accuracy of the commercial product Strep B OIA (optical immunoassay) compared to the standard agar and broth culture methods for detecting vaginal colonization with group B streptococcus (GBS). METHODS: Preoperative vaginal cultures were obtained from 141 nonpregnant gynecological patients undergoing major gynecologic surgery. Major gynecologic surgery was defined as benign gynecologic, gyne-oncology, and urogynecologic procedures. The results of the Strep B OIA test were compared to the results obtained from SXT agar (selective for GBS), colistin-nalidixic acid ((CNA) agar, and Todd-Hewitt broth cultures. RESULTS: The prevalence of vaginal GBS colonization in this population was 20.6%. The sample sensitivity and specificity of the OIA method were 58.6% and 85.7%, respectively. These values are lower than the sensitivity and specificity of 85.4% and 91.5%, respectively, given in the OIA package insert. Although the sample negative predictive value was fairly high (88.9%), the positive predictive value was low (51.5%). CONCLUSION: Although a previous study stated that the product Strep B OIA reduces the time required to obtain results (30 minutes versus days) and can, therefore, function as a useful diagnostic tool in the management of early-onset GBS disease, the present study's finding of low sensitivity and low positive predictive value indicates that this test may have very limited clinical value.

The role of bacterial vaginosis in infection after major gynecologic surgery.

PURPOSE: Previous studies have reported an association between bacterial vaginosis (BV) and postoperative fever and infection. This prospective study investigated whether the intermediate or definite stages of BV are risk factors for postoperative infection after major gynecologic surgery. METHODS: Vaginal cultures were obtained preoperatively from 175 women undergoing gynecologic surgery. The diagnostic criteria for BV were based on Nugent's standardized method of Gram stain interpretation. Postoperative fever was defined as at least one temperature equal to 101.0 degrees F or greater, or two or more temperatures more than 6 hours apart equal to 100.4 degrees F or greater. RESULTS: Thirty-six percent of the positive-BV group developed a postoperative fever, compared with 20% of the Lactobacillus-predominant group and 12% of the intermediate-BV group (P = 0.017). The differences between the positive-BV group and the Lactobacillus-predominant group, and between the positive-BV group and the intermediate-BV group, with respect to postoperative fever, were statistically significant (P = 0.045 and P = 0.007, respectively). The difference between the intermediate-BV group and the Lactobacillus-predominant group was not statistically significant (P = 0.28). CONCLUSIONS: Although the association between BV and postoperative febrile morbidity could be a spurious result of confounding with other variables, it may be prudent for the surgeon to identify patients with BV and treat them preoperatively.