Characterization and Partial Purification of a Bacteriocin-like Inhibitor Produced by Enterococcus faecium strain 62-6 Antagonistic to the Growth of Vaginal Lactobacilli: Potential Significance for the Pathogenesis of Bacterial Vaginosis
Mequio, Michael J.
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Bacterial vaginosis is a ploymicrobial syndrome characterized by an alteration in the normal vaginal microflora. This includes a decline in the concentration of Lactobacillus populations which are generally dominant in the healthy micro flora. Currently, mechanisms that could account for the disappearance of lactobacilli are not well understood. Previous studies conducted in this laboratory tested vaginal isolates of genera of bacteria associated with the normal microflora for the ability to inhibit the growth of vaginal lactobacilli and found that a strain of Enterococcus faecium, 62-6, with strong inhibitory activity. Using inhibitor containing culture supernatants we determined that the inhibitor was heat (100°C), cold (4°C), and pH (4-7) stable, was retained on a cellulose filter of 0.22 µm pore size, and was sensitive to pepsin and 𝛼-chymotrypsin, but resistant to trypsin and proteinase K. Sensitivity to proteolytic enzymes and precipitation of the inhibitor with ammonium sulfate indicated that the inhibitor had an essential proteinaecous component. These properties, in addition to its sensitivity to lipase suggested classification as a complex bacteriocin-like inhibitor. Concentration and partial purification of the inhibitor was achieved using Sepharose cation-exchange chromatography. SDS-PAGE analysis of fractions with bacteriocin-like activity showed the presence of a protein band with an estimated molecular mass of between 14 and 20 kDa. Kinetic assays showed that inhibitor production coincided with a minimum concentration of strain 62-6 of 10 7 cfu/ml in broth culture. This result may have substantial clinical significance for both the establishment and recurrence of bacterial vaginosis. Additionally, this is the first in-depth characterization of a bacteriocin-like inhibitor produced by an enterococcal strain of vaginal origin.With honors.