Vaginal biofilm forming lactobacilli as probiotic pharmaceutic candidates. Characterizacion of the biofilm matrix

MARIA CECILIA LECCESE TERRAF; MARIA SILVINA JUAREZ TOMAS; ELENA BRU; MARIA CLAUDIA OTERO; MARIA ELENA FÁTIMA NADER; CLARA SILVA DE RUIZ

Rosario

Congreso; 2da Reunón Internacional de Ciencias Farmacéuticas RICIFA; 2012

UNR-UNC

Introduction Lactobacilli are the predominant microorganisms of the healthy women vagina and participate in the maintenance of the ecological balance of the urogenital tract. They decrease in number by different causes, and the intravaginal administration of beneficial strains in pharmaceutical products has been proposed to restore the vaginal microbiome. 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 beneficial characteristic to favor their mucosal colonization. Biofilms are defined as communities of bacteria, included 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 to inhibit the growth of urogenital pathogens(3).   Materials and methods The characterization of the biofilm matrix was performed by using a factorial experimental design 2x9x2: two Lactobacillus strains (L.rhamnosusCRL1332 and L.reuteriCRL1324), nine different treatments with enzymes or chemicals (proteinase K, protease, trypsin, α-amylase, bovine DNaseI, recombinant human DNasaI, α-chymotrypsin, cellulose and sodium metaperiodate) at two levels (presence or absence (solvent control) of each agent). Biofilms were evaluated in MRSbroth without Tween80 inoculated with 200µl of Lactobacillus in polystyrene microplates (at 37ºC for 72h). Biofilms were treated later with different agents. Control wells were treated with the corresponding solvent. After 1h at 37°C, treated biofilms were rinsed with buffer PBS. The remaining bacteria attached were stained with crystal violet and the dye extracted with acid acetic. The OD was determined at 570nm. For data analysis, a linear mixed model was applied (SoftwareInfostatvs2012).   Results All the factors assayed (strain, treatment and agent level) 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 effects on the attachment of the biofilm formed by L. reuteri CRL 1324. Among the nine treatments applied, none of them produced a significant detachment of L. rhamnosus CRL1332 biofilm from the microplate surface.   Conclusions The results obtained indicate the chemical nature of the matrix of the biofilm formed by vaginal L. reuteri CRL 1324, being proteins one of the main components of the biofilm. However, the biofilm matrix of L. rhamnosus CRL1332 was resistant to the different treatments applied in this study, and further assays will be performed to characterize this matrix. The knowledge of the chemical composition of the biofilm matrix will help to promote the lactobacilli biofilm and to study its stability in presence of the different components of the vaginal environment as a way to predict the functionality of the beneficial microorganism   References. 1) FAO/WHO Report. Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria.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. EurJObstetGynecolReprodBiol.2011;159(2):399-406