1. std@microbio.bas.bgstd@microbio.bas.bg or stdanova@yahoo.comstdanova@yahoo.com

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

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

4. In vitro In vitro selection of vaginal lactobacilli Isolation of lactobacilli from vaginal swabs of healthy women Polyphasic taxonomic study of biodiversity of vaginal microbiota Antagonistic activity against pathogens Adhesion ability in model systems simulated UGT Aggregation phenotype Biofilm formation Immuno- modulation activity Antibiotic resistance Technological relevance and compatibility HOW TO PRE-SELECT PUTATIVE VAGINAL PROBIOTICS? IN VITRO SELECTION CRITERIA

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: A)Dot hybridization assay with the sp.-specific DIG labeled DNA probes; B)PCR analysis with species–specific primers for L. crispatus. (1) DIG labeled – genomic DNA probes L. fermentum L. johnsoniii, L. crispatus, L. salivarius, L. casei subsp. ramnosus in healthy Bulgarian women 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 Danova S. et al. (2005)World Journal of Microbiology & Biotechnology, 2005, 21: 835-842 Direct PCR analyses of collected samples of total DNA (2) Direct PCR analyses of collected samples of total DNA  Monitoring the possible disturbance in Lactobacillus microflora in women with HPV

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

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

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

10.  In vitro antimicrobial tests with cells-free cultures (acid and neutralized) and whey fractions of 46 LAB against clinical reference strains Anti- E. coli  Pre-selection of vaginal strains -effective antagonists of human pathogens. Antimicrobial activity of vaginal LAB strains  In vitro assays for activity against BV -associated and other microbes related to uro-genital disorders (Gardnerella vaginalis, Candida albicans, Streptococcus spp:  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 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 Gardnerella vaginalis 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.  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 acid supernatants of vaginal LAB Tests with catalase treated and neutralized supernatants In vitro assay for activity against multidrug resistant out-patient clinical isolates Petrova M. 2007, MSc thesis

12.  Activity of pre-selected L. fermentum in simulated vaginal fluids medium (pH 5.5) In vitro estimation of anti - E. coli activity in model systems  H 2 0 2 production ( according to Eschenbasch et al., 1989) ~80% of tested vaginal lactobacili are H 2 0 2 producers  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 Mode of action of non-purified catalase-treated and neutralized filtered cells-free supernatants of vaginal LAB Mode of action of non-purified catalase-treated and neutralized filtered cells-free supernatants of vaginal LAB

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

14. *Legend: ( + ) detected activity after the treatment –the inhibition zone ≥ 11 mm; (-) no activity after the treatment ; test-culture used – E. coli HB 101 Test - microorganisms L. ferm. As L. ferm Ns L. saliv. As L. saliv. Ns E.coli HB101 25142515 E.coli WF+ 2950nd109 L.innocuaF CIP T82 12015 Pr. vulgaris 139300160 B. megatherium 9885 12030nd S. aureus 209 001817 G. vaginalis 14018 171500 Kl. pneumoniae 4669 171400 Kl. pneumoniae 4603 0000 S.pyogenes 15436 002824 * Legend: As= acid FCS; Ns= neutralised and catalase-treated In MRS broth  L. fermentum Production study of pre-selected bacteriocinogenic vaginal strains and characterisation of produced BLIS  Spectrum of activity of non-purified FCS A, B and C) AFM of E.coli HB101 cells treated with active cells-free culture of vaginal L. salivarius D) AFM of non-treated E.coli HB101 (control) A B C D  BLIS produced by L. salivarius with bactericidal effect on E. coli cells ( Acknowledgments to Dr. S. Todorov and prof. LMT Dicks, Stellenboch University, South Africa

15.  Effects of Lactobacillus filtrates (FSC) on biofilm formation of E. coli strains Effects of Lactobacillus FSCs (supplemented to M63 broth) on biofilm production 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. Bioprotective role of vaginal lactobacilli * The results in the table represent the mean percent from the three separate colonies.

16. In vitro estimation 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)  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)  Biofilm formation (in VfM) of selected vaginal lactobacilli with a broad spectrum of activity and probiotic potential L. fermentum LAB strain LAB strain + E. coli

17. Vainal strainsAuto-aggregation In PBS pH 6.0 In VfM (pH 4.5) L. crispatus VD526 +R*- L.plantarum VD339/1 +N- L. jensenii VD5132/1 -- L. gasseri VD2996 -- L. gasseri Vel -- L. fermentum VD505 -- L. fermentum VD2421 -- L. johnsonii VD026 +N- L. fermentum VD4553/B1 ++R- L. fermentum VL2 -+/-N L. fermentum VD265/5 -+++R L. gasseri VAG1 -+N L. plantarum VD265/4 +/-N+R Lactobacillus spp. VD1369 ++N++R Co-aggregation (E.coli- vag LAB) in PBS ( pH 6.0) in VfM ( pH 6.0) Auto-aggregation E. coli aggregation Co-aggregation In PBS In VfM In PBSIn VfM +R+R--- +R+R--- ++R--- +R+R--- -+R- ++R--- --- +R+R- +N +R+R-+R- ---- ---- --- ---++R --- *R= rapid (15 min); N =Normal (15-120 min) vs a control Aggregation phenotype of vaginal lactobacilli = protection against invading pathogens Important factors of the stable colonization of the UTI 81% 56% 75%

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

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

20. Adhesion study of a bacteriocinogenic strain L. salivarius (S16) to HeLa cell line L.salivarius S16 S16  Role of S-layer protein of Lactobacillus S16 for the adhesion to HeLa cells Ability of lactobacilli to block the adherence of E. coli to HeLa cells 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 HB101 was 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. Light microscopy of L. salivarius S16 adhesion to HeLa cell line (Olympus 400x)

21. 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 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. Cytokine assay: 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. Immunomodulation activity The observed inhibition of the both cytokines, points on nonspecific suppression of macrophage activity

22.  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:  My group: PhD students PhD students: Silvia Dimitonova (2007) and Rositsa Tropcheva (2013); MSc students MSc students – Maria Petrova (2008) and Jordanka Dermendjieva (2011). IF EVER A BACTERIA HAS LOVED WOMEN, THE VAGINAL LACTOBACILLUS IS THAT BACTERIA.

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” Luis Pasteur