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IN VITRO SELECTION OF VAGINAL LACTOBACILLI FOR UROGENITAL APPLICATION

IN VITRO SELECTION OF VAGINAL LACTOBACILLI FOR UROGENITAL APPLICATION. S vetla Danova 1 , Silvia Dimitonova 1 , Maria Petrova 1 , Jordanka Dermendjieva 1 , Rositsa Tropcheva 1 and Nina Ivanovska 2 1- Department of Microbial Genetics, 2 - Department of Immunology.

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IN VITRO SELECTION OF VAGINAL LACTOBACILLI FOR UROGENITAL APPLICATION

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  1. IN VITRO SELECTION OF VAGINAL LACTOBACILLI FOR UROGENITAL APPLICATION SvetlaDanova1, Silvia Dimitonova1, Maria Petrova1, JordankaDermendjieva1, RositsaTropcheva1 and Nina Ivanovska2 1-Department of Microbial Genetics, 2- Department of Immunology std@microbio.bas.bg or stdanova@yahoo.com

  2. . Human desire for well-being and a long healthy life is creating needs for new approaches in PROPHYLAXIS AND THERAPY OF DIFFERENT DISORDERS PROBIOTICS Lactobacilli – a major part of protective microflora of human body VAGINAL LACTOBACILLI – a native barrier against invading pathogens and the major protective component of defensive biofilm of the vagina Vaginal microflora of healthy women Vaginal microflora of women with bacterial vaginosis (BV) If ever a bacteria has loved us, the Lactobacillus is that bacteria.

  3. TO CHARACTERIZE NEWLY ISOLATED LACTOBACILLI FROM BULGARIAN WOMEN, AS POTENTIAL VAGINAL PROBIOTICS FOR UROGENITAL HEALTH AIM Age, race social and culture traditions Nutrition, smoking, personal style of life and hygiene vaginal MICROBIOME HEALTH DESEASSES STRESS factors; Antibiotics, spermicides Others

  4. HOW TO PRE-SELECT PUTATIVE VAGINAL PROBIOTICS? IDENTITY SAFETY IN VITRO SELECTION CRITERIA FUNCTUANILTY

  5. Biodiversity of vaginal microbiota L. acidophilus L. gasseri L. crispatus L. iners L. jensenii L. plantarum L. caseri L. paracasei L. fermentum L. reuteri L. vaginalis L. brevis L. salivarius L. delbrueckii L. fornicalis L. coleohominis Normal flora varies in female genital tract

  6. Biodiversity of Lactobacillus microbiota of healthy Bulgarian women at childbearing age • Non culturable- DNA based approaches: Collected 50 vaginal swabs of healthy volunteers in reproductive ages 25 samples of total DNA DNA Dot-blot hybridization assay with new DIG- labelled probes from total DNA Lactobacillus species presented in mixed vaginal simples (1) DIG labeled – genomic DNA probes Danova S. et al. (2005)World Journal of Microbiology & Biotechnology, 2005, 21: 835-842 Dot hybridization assay with the sp.-specific DIG labeled DNA probes; PCR analysis with species–specific primers for L. crispatus . L. fermentum L. johnsoniii, L. crispatus, L. salivarius, L. casei subsp. ramnosus in healthy Bulgarian women (2) Direct PCR analyses of collected samples of total DNA • Monitoring the possible disturbance in Lactobacillus microflora in women with HPV

  7. Lactobacillus diversity in vaginal microbiota of healthy Bulgarian women • Results from 16S rDNA sequence analyses L. johnsonii Lactobacillus spp. L. crispatus L. brevis L. fermentum L. jensenii L. rhamnosus L. gasseri L. plantarum

  8. A polyphasic taxonomy approach combining classical phenotypic and molecular methods (ARDRA, Ribotyping, PCR and 16S rDNA sequences) Lactobacillus sp.

  9. Urogenital tract health reproductive health well- being >300 millions cases of urinary tract infections (UTI), bacterial vaginosis (BV) and yeast vaginitis worldwide per annum

  10. Antimicrobial activity of vaginal LAB strains • Pre-selection of active strains against E. coli HB101 Ps.a = Pseudomonas aeruginosa ATCC 27853, Ec.BLN = E. coli ATCC 25922 SaMSSA = Staphylococcus aureus ATCC 25923; SaMRSA = Staphylococcus aureus ATCC 39592 Anti- E. coli • Pre-selection of vaginal strains -effective antagonists of human pathogens. • In vitro antimicrobial tests with cells-free cultures (acid and neutralized) and whey fractions of 46 LAB against clinical reference strains • In vitro assays for activity against BV -associated and other microbes related to uro-genital disorders (Gardnerella vaginalis, Candida albicans, Streptococcus spp: Selected active vaginal LAB with a broad spectrum of activity against Gram (+), Gram (-) and Candida sp. : L. salivarius S16 strain, active against HSV -2; L. fermentum strains - antagonists of Gardnerellavaginalis and L. gasseri, L. jensenii strains with anti-Candida activity Petrova M. et al. 2009; Dimitonova S. et al. 2007; Serkedjieva J. et al, 2004

  11. In vitro assay for activity against multidrug resistant out-patient clinical isolates Tests with acid supernatants of vaginal LAB • Acinetobacter baumanii C№ 2762 • R-AmC, AmP, CXM, FOX, CTX, CRO, CAZ, FEP, ATM and S- IPM, MEM, G, AN, BIS, CIP, Sul) • Acinetobacter baumanii C№4383 • R-AmC, AmP, CXM, FOX, CTX, CRO, CAZ, FEP, ATM , IPM, MEM, G, AN, BIS, CIP, Sul) • E.coli ESBL C№ 2747 • R-AmC, AmP, FOX, CTX, CRO, CAZ, AN, ATM, FEP, BIS, CIP, G and S- IMP, MEM Tests with catalase treated and neutralized supernatants Petrova M. 2007, MSc thesis

  12. In vitro estimation of anti - E. coli activity in model systems • Activity of pre-selected L. fermentum in simulated vaginal fluids medium (pH 5.5) • H202 production (according to Eschenbasch et al., 1989) ~80% of tested vaginal lactobacili are H202producers Mode of action of non-purified catalase-treated and neutralized filtered cells-free supernatants of vaginal LAB • E. coli HB101 growth inhibition in Luria Bertran broth (A) and in simulated vaginal fluids medium (B), supplemented with 5% v/v of active – non purified FSC of vaginal strains

  13. In Luria-Bertran broth Time (h) Characterization and mode of action of bacteriocin-like substances (BLIS) • The effects estimated in vitro were strain- and concentration-dependent. • The antimicrobials produced during the cultivation are:lactic acid and hydrogen peroxidein combination with proteinase-sensitive to different agentsbacteriocin-like metabolites (BLIS). • Pre-selected L. fermentum and L. salivariusactive strains inhibited the growth of E. coli in different model systems and the produced BLISs express a synergic effect against E. coli E. Coli + FCS – L. sal FSC E. Coli + FCS L. ferm. E. Coli + FCS L. ferm. + FSC L. saliv.

  14. Production study of pre-selected bacteriocinogenic vaginal strains and characterisation of produced BLIS In MRS broth • Spectrum of activity of non-purified FCS • L. fermentum * Legend: As= acid FCS; Ns= neutralised and catalase-treated • BLIS produced by L. salivarius with bactericidal effect on E. coli cells A, B andC) AFMof E.coliHB101 cells treated with active cells-free culture of vaginal L. salivarius D) AFMof non-treatedE.coliHB101 (control) C A B D (Acknowledgments to Dr. S. Todorov and prof. LMT Dicks, Stellenboch University, South Africa *Legend: ( + ) detected activity after the treatment –the inhibition zone ≥ 11 mm; (-) no activity after the treatment ; test-culture used – E. coli HB 101

  15. Bioprotective role of vaginal lactobacilli • Effects of Lactobacillus filtrates (FSC) on biofilm formation of E. coli strains * The results in the table represent the mean percent from the three separate colonies. Effects of Lactobacillus FSCs (supplemented to M63 broth) on biofilmproduction of uropathogenic E. coli by the CV test, presented as % of control values. The biofilms were coloured with 0.1% crystal violet, washed, solubilised and absorbance (at 550 nm) was measured on ELISA reader. For each of the variants, 6 wells were inoculated and the means were re-calculated as per cent of the mean value of the control for the respective single colony. The results in the table represent the mean percent from the three separate colonies.

  16. In vitroestimation the biofilm formation capacity of vaginal lactobacilli • In vitro assay for biofilm-formation of vaginal LAB, in the laboratory model of healthy vagina (vaginal fluids mediumVfM pH 4.4) and in the model of BV (VfM pH 5.9) LAB strain LAB strain LAB strain + E. coli LAB strain + E. coli • Biofilm formation (in VfM) of selected vaginal lactobacilli with a broad spectrum of activity and probiotic potential L. fermentum

  17. Auto-aggregation inPBS ( pH 6.0) in VfM ( pH 6.0) Co-aggregation (E.coli- vag LAB) Aggregation phenotype of vaginal lactobacilli = protection against invading pathogens 56% 81% 75% Important factors of the stable colonization of the UTI *R= rapid (15 min); N =Normal (15-120 min) vs a control

  18. Adhesion ability of vaginal lactobacilli Important factors of the stable colonization of the UTI (Human cervix epithelial like cells line)

  19. Mechanisms and determinants of the stable colonization of the mucous Mub M-100 bp; 1-L. fermentum, 2-4L. gasseri, 5- L. plantarum, 6-7L. jensenii, 8- L. salivarius, 9- L. jensenii,11-neg. PCR control MapA • Presence of Mucous binding proteins (Mub), mucus adhesion promoting protein (MapA) genes in vaginal Lactobacillus sp. • Characterization of cell-surface properties of vaginal lactobacilli Surface layer protein (SLP ) SLP ~ 44 kDafrom a vaginal strainL. salivarius

  20. L.salivarius S16 S16 Adhesion study of a bacteriocinogenic strain L. salivarius (S16) to HeLa cell line • Role of S-layer protein of Lactobacillus S16 for the adhesion to HeLa cells Light microscopy of L. salivarius S16 adhesion to HeLa cell line (Olympus 400x) a) Exclusion test- HeLa cell monolayer was cultured with L. salivarius S16 cells for 1h. Non-adhering lactobacilli were removed by washing with PBS three times; E. coli HB101was added and incubated for another 1h. b) Competition test- L. salivarius S16 and E. coli HB101 (1:1)were incubated simultaneously for 2 h. c)Depletion test- E. coli was incubated with monolayer for 1h and, after removing non-adhering cells by washing with PBS three times, L. salivarius S16 cells were added and incubation was continued for another 1h. Ability of lactobacilli to block the adherence of E. coli to HeLa cells

  21. Immunomodulation activity Influence of Lactobacillus live cells and filtered cultures supernatants (Sn) on the in vitro release of TNF-a and IL-10 by murine peritoneal macrophages Cytokine assay: Effect of Sn and cells on TNF- production (A) and IL-10 production (B). Values are means  SD from three determinations in two experiments. *** P<0.001 vs LPS stimulated group, # P<0.001 vs nonstimulated group. All 4 samples inhibited LPS-induced TNF- secretion most powerfully by Sn of vaginal strains L. fermentum, as the effect of cells was weaker. The release of IL-10 was also diminished less exerted by both supernatants compared to the cells. The both supernatants were able to stimulate IL-10 secretion in the absence of LPS. The observed inhibition of the both cytokines, points on nonspecific suppression of macrophage activity

  22. IF EVER A BACTERIA HAS LOVED WOMEN, THE VAGINAL LACTOBACILLUSIS THAT BACTERIA. Acknowledgment • Kendy Pharma • Dr Nasja Hadjieva - Clinical laboratory of University hospital ISUL, Sofia • Dr. S. Todorov, Stellenbosh University, South Africa • My colleagues: Prof. Nina Ivanovska, Prof. Stoyanka Stoitcova, Prof. Julia Serkedjieva, The Stephan Angeloff Institute of Microbiology, BAS, Sofia • My group: • PhD students: Silvia Dimitonova (2007) and RositsaTropcheva (2013); • MSc students – Maria Petrova (2008) and JordankaDermendjieva (2011).

  23. “WHEN MOVING FORWARD TOWARD THE DISCOVERY OF THE UNKNOWN, THE SCIENTIST IS LIKE A TRAVELER WHO REACHES HIGER AND HIGER SUMMITS FROM WHICH HE SEES IN THE DISTANCE NEW COUNTRIES TO EXPLORE” Thank you for your attention Luis Pasteur

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