Vaginal Biofilm Forming Lactobacilli as Probiotic Pharmaceutical Candidates. Characterization of the Biofilm Matrix

LECCESE TERRAF, MC; JUÁREZ TOMÁS, MS; BRU, E; OTERO, MC; NADER, MEF

Rosario de Santa Fe

Congreso; 2da Reunión Internacional de Ciencias Farmacéuticas; 2012

Univ. Nac. de Rosario-Univ. Nac de Córdoba

CHARACTERIZATION OF VAGINAL LACTOBACILLI? BIOFILM MATRIX AS CANDIDATES FOR POTENTIAL PHARMACEUTICAL PROBIOTIC PRODUCTS   Leccese Terraf MC1, Juárez Tomás MS1, Bru E1, Otero MC1, Nader MEF1, Silva C2.   1CERELA-CONICET, Chacabuco 145, San Miguel de Tucumán. 2Facultad de Bqca., Qca. yFcia., Universidad Nacional de Tucumán, Ayacucho 471, San Miguel de Tucumán, Tucumán, CP: T4000ILC, Argentina #   Introduction Lactobacilli are the predominant microorganisms of healthy women vagina and participate in maintaining the ecological balance of the urogenital tract. They decrease in number by different causes; therefore pharmaceutical probiotic administration is suggested to restore the vaginal microbiota. Probiotics are defined as ?live microorganisms which, when administered in adequate amounts, exert a beneficial physiological effect in the host health? (1). Biofilm formation of lactobacilli is a desirable characteristic to ensure mucosal colonization of probiotic strains. Biofilms are defined as communities of bacteria, encased in a self-synthesized extracellular polymeric matrix (2). The aim of this study was to characterize the biofilm matrix formed by Lactobacillus rhamnosus CRL1332 and Lactobacillus reuteri CRL1324, isolated from human vagina. These strains were selected previously for their ability to form biofilm and for to inhibit the growth of urogenital pathogens (3).   Materials and methods The characterization of biofilm matrix was performed using a factorial experimental design 2x9x2: two Lactobacillus strains (L. rhamnosus CRL1332 and L. reuteri CRL1324), nine treatments with different chemicals (proteinase K, protease, trypsin, α-amylase, bovine DNase I, recombinant human DNasa I, α-chymotrypsin, cellulose and sodium metaperiodate) and two chemical concentrations (presence or absence (solvent control) of each chemical). Biofilms were evaluated in MRS broth without Tween 80 inoculated with 200 µl of Lactobacillus in polystyrene microplates (at 37ºC for 72 hours). Biofilms were treated with different agents. Control wells were treated with the corresponding solvent for each chemical. After 1 h at 37°C, treated biofilms were rinsed with buffer PBS. The remaining bacteria attached were stained with crystal violet and the dye was extracted with acid acetic. The OD was determined at 570 nm. For data analysis, a linear mixed model was applied (Software Infostat vs 2012).   Results All factors assayed (strain, treatment and chemical concentration) significantly affected the biofilm matrix of lactobacilli. Proteinase K, protease and trypsin efficiently detached L. reuteri CRL1324 biofilm. None of the other enzymes had significant effect on the attachment of L. reuteri CRL 1324 biofilm. Among nine treatments used in this study, none caused significant detachment of L. rhamnosus CRL1332 biofilm from the microplate surface.   Conclusions Biochemical studies have revealed the chemical nature of the matrix of the biofilm formed by one vaginal L. reuteri strain. These findings suggest that proteins are a major component matrix in L. reuteri CRL1324 biofilm. However, biofilm matrix of L. rhamnosus CRL1332 was resistant to the different treatments used in this study. Further studies will be performed to characterize the biofilm matrix of L. rhamnosus CRL1332.   References. 1) FAO/WHO. Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria. Food and Agriculture Organization of the United Nations and World Health Organization Report. 2001; http://www.fao.org/es/ESN/Probio/probio.htm 2) Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annu Rev Microbiol. 1995;49:711-45. 3) Juárez Tomás MS, Saralegui Duhart CI, De Gregorio PR, Vera Pingitore E, Nader-Macías ME. Urogenital pathogen inhibition and compatibility between vaginal Lactobacillus strains to be considered as probiotic candidates. Eur J Obstet Gynecol Reprod Biol. 2011;159(2):399-406. # Corresponding author. Tel +54 381 4310465, fax +54 381 4005600; e-mail: fnader@cerela.org.ar