2 Bacteriocins are typically divided into two major, yet diverse, classes: class I, or lantibiotics, contain unusual amino acids and are subject to extensive posttranslational modiﬁcations; and class II, the heat stable nonlantibiotics [21]. Subtilosin has a unique posttranslational structure that is unmatched among bacteriocins, which has led to speculation that it may belong in a distinct and separate class [2, 3]. Bacteriocins have been widely considered for use in medicinal and pharmaceutical applications, particularly for their bactericidal activity against multidrug resistant organisms [22]. They are especially appealing alternatives since their cost of production is so comparatively low to conventional pharmaceutical treatments. For example, commercial grade nisin costs approximately $100/lb through chemical distributors. Much attention has recently been focused on the spermicidal activity of bacteriocins due to their targeted antibacterial activity and lack of eﬀect on human tissues. Nisin A, a well-studied class I bacteriocin produced by Lactococcus lactis subsp. lactis [23], has human spermicidal activity [24, 25], and subtilosin has spermicidal activity against boar, bovine, horse, and rat spermatozoa [26]. The results of these previous studies suggest that subtilosin may also have spermicidal activity against human spermatozoa, prompting our investigation. Since subtilosin has proven antimicrobial activity against the pathogen largely responsible for BV, its toxicity to human tissues was assayed to determine its potential as a safe alternative remedy. For the human in vitro study, the EpiVaginal ectocervical tissue model (MatTek Corpora- tion, Ashland, Mass, USA) was employed to examine the relationship between prolonged exposure to subtilosin and cell viability. Spermicidal evaluations were conducted to investigate the ability of the peptide to restrict or eliminate sperm mobility, leading to its classiﬁcation as a spermicidal agent. 2. MATERIALS AND METHODS 2.1. Production of subtilosin Subtilosin was prepared as previously described [2]. To prepare a cell-free supernatant (CFS), cells were removed by centrifugation (Hermle Z400K; LabNet, Woodbridge, NJ, USA) for 25 minutes at 4500 g and 4◦C. The supernatant was ﬁlter sterilized using 0.45 μm ﬁlters (Fisher, Pittsburgh, Pa, USA). The protein of interest was precipitated from the supernatant by adding 30% ammonium sulfate (w/v) while stirring overnight at 4◦C and was resuspended in 20 mL of double distilled H2O. The column chromatography method described by Sutyak et al. [2] was used to purify subtilosin from the CFS, producing a near-pure isolate in the 90% methanol eluate. The antimicrobial activity of all samples was conﬁrmed by the well-diﬀusion assay according to the protocol of Cintas et al. [27] with additional modiﬁcations [2]. The active fraction was concentrated to dryness using a Savant SC110 Speed Vac and UVS400 Universal Vacuum System (Savant Instruments, Farmingdale, NY, USA), then resuspended in 1.5 mL ddH2O. Infectious Diseases in Obstetrics and Gynecology 2.2. Determination of protein concentration 2.2.1. Column-puriﬁed sample concentration The concentration of subtilosin in the column-puriﬁed frac- tion was determined using the Micro BCA Protein Assay Kit according to the manufacturer’s protocol (Pierce, Rockford, Ill, USA). In brief, the assay measures the reduction of Cu2+ to Cu1+ by colorimetric detection of Cu1+ by bicinchoninic acid. Bovine serum albumin (BSA) was used to develop a standard curve with concentrations ranging from 0.5 to 20 μg/mL; the concentration of subtilosin was calculated using the R value from the trendline of the standard curve graph. 2.2.2. Assay of subtilosin concentration in CFS The concentration of subtilosin in the CFS was not mea- surable with the Micro BCA Protein Assay due to the high level of background proteins in the solvent (MRS medium). As an alternative, the protein concentration was calculated by comparing the antimicrobial activity of known concentrations of column-puriﬁed protein to equal volumes of CFS. Five two-fold dilutions were made from the stock samples of both the CFS and the column-puriﬁed fraction. Well diﬀusion assays were performed using 50 μL of each dilution against Micrococcus luteus ATCC 10420, which is commonly used as a reference microorganism for the determination of a bacteriocin’s biological activity [28]. 2.3. Determination of the presence of weak organic acids As reported previously, the concentration of lactic acid in the CFS was measured to assess its potential eﬀects on antimicrobial activity and cell viability [2]. The quantity of each form of the acid in the sample was measured using a commercially available D-lactic acid/L-lactic acid kit (Roche Boehringer, Mannheim, Germany), according to the manufacturer’s instructions. 2.4. EpiVaginal ectocervical tissue model The EpiVaginal (VEC-100) ectocervical tissue model (Mat- Tek Corporation, Ashland, Mass, USA) was used and maintained as fully described by Dover et al. [29]. The tissues were exposed to 83 μL of subtilosin CFS (∼136 μg/mL) for 4, 24, and 48 hours. For exposure times over 24 hours, the tissues were aerated by placing them on two metal washers (MatTek Corporation, Ashland, Mass, USA) and fed with 5 mL of the assay medium. Double-distilled water (ddH2O) was used as a negative control, and was applied to cells after 6, 24, and 48 hours. A spermicidal product containing 4% Nonoxynol-9 (Ortho Options CONCEPTROL Vaginal Contraception Gel, Advanced Care Products, Skillman, NJ, USA) was used as a positive control based on its documented cytotoxic properties [30, 31]. A cream (Monistat-3, Ortho McNeil Pharmaceutical, Inc., Raritan, NJ, USA) containing 4% of the nontoxic, BV-active compound miconazole nitrate [30, 31], was used as a negative control.