Design and safety of phytobiotics with beneficial vaginal lactobacilli for the prevention of urogenital tract infections

Abstract

The vegetal extracts are applied to decrease or counteract the Urogenital Tract Infections (UGTI) symptom. Most of them are produced as a consequence of the desequilibrium of the vaginal microbioma, and by the genetic polymorphism of the host. Lactobacilli are the dominant bacteria in human vagina, exerting different type of effects both in healthy, sexually active and pregnant women. The complementation of the two biological compounds, phytoderivatives and BVL, can be used to restore the ecological equilibrium of vaginal microbioma and prevent the UGTI, which represent very high costs in public health systems for their effect mainly in pregnant and newborn. The objective of this work is: evaluate the compatibility between phytoderivatives and BVL and their safety and inocuity to advance in the design of phytobiotic formulas the urogenital tract. Materials and MethodsCompatibility between phytocompounds and BVLB: Different BVL (24 different strains identified as Lactobacillus reuteri, rhamnosus, delbruekii, jhonsoiii, salivarius, gasseri, paracasei) with pure phytocompounds and drops (Hydrocotile asiática L., Caléndula officinalis L., Peumus boldu., Hamamelis virginiana L., Atropa belladona L., Cyclolepis genistoides., Equisetum arvence L., Aesculus hippocastanum L., Amaranthus muricatus., Acrtostaphylos uva-ursi L., Matricaria recutita L., Urtica dioica L., Smilax aspera L., Echinacea purpúrea L., Chelidonium majus L., Plantago lanceolata L., Thymus vulgaris L., Minthostachys mollis., Aloysia polystachya) were incubated in polystyrene microplates at 37°C during 24 h. Optical density (OD560nm) at 3, 6, 9, 12 and 24 h were used to determine the growth parameters, behavior and degree of compatibility. Inhibition of pathogens by phytocompounds: The inhibitory effect of phytocompounds was determined by the plate diffusion technique against urogenital pathogens: Cándida albicans, Cándida tropicalis, Streptococcus aureus, Streptococcus agalactiae, Staphylococcus saprophyticus, Staphylococcus aureus, Escherichia coli and Enterococcus faecalis. Safety assays: Antimicrobial resistance of BVL to Ampicillin, Tetracycline, Chloramphenicol, Gentamicin, Streptomycin, Kanamycin, Clyndamicin, Vancomycin and Erythromycin was determined by microdilution technique, and the MIC (minimum inhibitory concentration) in polystirene microplates with BVL in LSM broth, incubated in microaerophyllic conditions at 37ºC for 48 h, and results for each antibiotic defined by EFSA directions. Virulence factors were evaluated in BVL in agarized media containing specific substrates for the expression of Lecithinase, Gelatinase and Hemolysin.Animal assays: Strogenized mice were intravaginally inoculated once/day with different phytocompounds- BVL during 7 days. The estral cycle was defined through vaginal washing. The absence of inflammatory response was determined by Giemsa stains and histological studies. Result and Discussion. Most of the phytocompounds exerted different type of effect on the BVL growth. Some of them (Smilax aspera L.) stimulated the growth of VBL: L.ga. 1263, L.ga. 1509, L.ga. 1320, L.rh. 1261, L.rh. 1508 while some other showed no effect on: L.rh. 1511, L.re. 1327, L.re. 1324, L.ga. 1264. The growth parameters were calculated and compared with standar growth conditions. Some phytocompounds had a stimulating effect agains BVL as: drops of Betula, Urtica dioica L., Hydrocotile asiática L. and Carica papaya; while in presence of others phytocompounds develop inhibitory effect as: Echinacea purpúrea L., drop of Lippia, Chelidonium majus L., Hamamelis virginiana L., Acrtostaphylos uva-ursi L. and drop of Acrtostaphylos uva-ursi L.. These extract were able to inhibit growth most of the pathogens assayed. Most of the BVL were sensitive to CHLOR/STREP/GEN/CLYN, frequently applied for UGTI therapy, being most of the BVL resistant to ERY. No virulence factors were detected or expressed in all the strains. The in vivo mice assays did not evidence inflammatory response or adverse effect after the vaginal administration of 7 daily doses of phytobiotics formulated with selected phytoderivatives and BVL. The results obtained support the design of safe phytobiotics containing different BVL and phytoderivatives for the prevention of women urogenital infections.