Hindawi Publishing Corporation Infectious Diseases in Obstetrics and Gynecology Volume 2007, Article ID 78248, 6 pages doi:10.1155/2007/78248 Research Article Safety Study of an Antimicrobial Peptide Lactocin 160, Produced by the Vaginal Lactobacillus rhamnosus Sara E. Dover,1, 2 Alla A. Aroutcheva,3, 4 S. Faro,5 and Michael L. Chikindas2, 4 1 School of Biotechnology, The Royal Institute of Technology, 100 44 Stockholm, Sweden 2 Rutgers, The State University of New Jersey, New Brunswick, NJ 08901-8520, USA 3 Rush Medical Center, Chicago, IL 60612, USA 4 Health Promoting Naturals, Inc., Highland Park, NJ 08904, USA 5 The Women’s Hospital of Texas, Houston, TX 77054, USA Correspondence should be addressed to Michael L. Chikindas, tchikindas@aesop.rutgers.edu Received 11 October 2007; Accepted 12 November 2007 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. Copyright © 2007 Sara E. Dover et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. INTRODUCTION Lactobacillus rhamnosus 160 was isolated from the vagi- nal microﬂora of a healthy female subject. This bacterium produces the ribosomally synthesized antimicrobial peptide (bacteriocin) lactocin 160, which is active against the most prevalent species associated with bacterial vaginosis (BV). Its bactericidal activity (as shown on model microorganisms) is due to the disturbance of the cellular membrane, probably by pore formation, which leads to eﬄux of ATP [1, 2]. BV is a common condition in women which occurs when the healthy vaginal bacterial ﬂora, consisting mostly of Lac- tobacillus species, is replaced by a ﬂora consisting of several bacterial pathogens [3]. It is associated with pregnancy com- plications such as premature labor as well as with a higher risk of acquisition of HIV infection [4–6]. Since lactocin 160 is active against an array of vaginal bacterial pathogens, it has a potential application as an an- timicrobial treatment of BV. It is, therefore, important to de- termine the safety of the bacteriocin for human application. For in vitro toxicity study, the EpiVaginal tissue model from MatTek Corporation (Ashland, Mass, USA) was employed. This model uses human vaginal ectocervical cells, obtained from healthy adult females, and is free from viral, micro- bial, and yeast infections. The tissue has a three-dimensional structure that mimics that of the human vaginal tissue and the results are highly reproducible, quantiﬁable, and less ex- pensive compared to in vivo studies [7]. The in vitro data was compared with rabbit vaginal irritation (RVI) model. The aim of this study was an in vitro and in vivo testing of antimicrobial peptide lactocin 160 to determine vaginal tis- sue toxicity, hemolytic activity, and inhibition of lactobacilli growth. 2. 2.1. METHODS Production of lactocin 160 Lactobacillus rhamnosus strain 160 was stored in the biofreez- er (−80◦C) until use. Lactocin 160 was puriﬁed as described previously [1]. Brieﬂy, L. rhamnosus 160 was grown anaero- bically overnight in 2000 mL MRS (Difco Lactobacilli MRS broth) at 37◦C. The cells were harvested by centrifugation (5100 xg, 20 minutes, 4◦C), and washed three times in 0.01 M