The Complexity of Bacterial Vaginosis

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Do Gardnerella vaginalis biofilms contribute to colonization by other anaerobes in bacterial August 5, 2008 vaginosis?

Annica Stull-Lane BBSI Summer 2008

VCU Mentor: Kimberly Jefferson Home Mentor: Taylor Allen

Bacterial Vaginosis
Most common vaginal disorder in reproductive-age women, world-wide Symptoms: abnormal homogeneous discharge, sometimes “fishy” odor Many cases are asymptomatic, >50% Imbalance of vaginal flora, characterized by:
  

Raised pH Overgrowth of unhealthy anaerobes Reduction of lactobacilli

http://www.yamagiku.co.jp/pathology/photo/photo138-1.htm

chemblogs.org/archives/2006/11/23/drug-of-the-week-metronidazole .Complications with BV Preterm delivery Increased risk of PID Increased acquisition & transmission of HIV Recurring infections!  Treatment http://the-half-decent-pharmaceutical-chemistry-blog.

. Managing recurrent bacterial vaginosis.80:8-11. is the presence of a certain species necessary for BV?? Wilson. Sex Transm Infect 2004. J.Etiology of BV: Causes? Etiology unknown Sexually transmitted? Reduction of lactobacilli Reduction in H2O2 production Important connection between the characterization factors Overgrowth of BV associated bacteria Raised pH Generally considered a polymicrobial disorder with a variety of anaerobes. however.

106.1012-23. Obstet Gynecol 2005.5. Adherent biofilms in bacterial vaginosis. but not sole factor. Loening-Baucke V. as does not fulfill Koch’s Postulates Adheres to vaginal epithelium as a biofilm---perhaps allows other anaerobes to colonize Many other anaerobes  Strict anaerobes Prevotella bivia Mobiluncus mulieris Atopobium vaginae Gardnerella vaginalis biofilm Swidsinski A. . et al.most prevalent    Gram-positive or variable rod-shaped anaerobe Present in up to 95% of BV cases.Bacteria Associated with BV Gardnerella vaginalis --. Mendling W.

asp . and antibiotics  Related to recurrence of BV? S. host immune defenses. proteins and/or nucleic acids Resistance to chemicals.cdc. aureus biofilm on a catheter http://phil.gov/phil/details. pH extremes.Bacterial Biofilms Complex matrix of polysaccharides.

proxy.edu/nrmicro/journal/v2/n7/fig_tab/nrmicro928_F3.com.Succession in Biofilms initial colonizers and secondary colonizers  For example.nature.h .vcu. dental plaque  Streptococcus and Actinomyces  Fusobacterium Later colonizers depend on earlier colonizers for adherence and growth in an otherwise unsuitable environment  Succession in BV? http://www.library.

lactic acid.Initial Hypothesis: Gardnerella vaginalis biofilm acts as an initial colonizer  This allows for other BV-associated anaerobes to adhere to the vaginal epithelium and survive in the presence of O2. H2O2 etc… .

strict anaerobic conditions required.Experiments Adherence  Do the anaerobes stick better to vaginal epithelial cells in the presence of Gv? Does the presence of a Gv biofilm facilitate the growth of strict anaerobes? Growth  • For the three anaerobes. .

Maintaining Anaerobic Conditions Anaerobic air mix: • 5% CO2 • 5% H2 • 90% N2 .

bivia A. mulieris . vaginae M.Adherence Assay: BacLight Green Bacterial Stain Red Vibrant Cell Membrane Stain Confocal Microscopy • Single bacterial strain on ME180 vaginal epithelial cells • Multi-species? • How can one measure adherence? Control G. vaginalis P.

measuring fluorescence after each cycle .• SYBR Green as a fluorescent dye DNA template Denaturation Primers anneal Extension What is qPCR? Repeat 40X.

so use equation: What is qPCR? 2^ (.cycle threshold value) Acquiring DNA… .• SYBR Green as a fluorescent dye DNA amount and fluorescence will reach a threshold value after a number of cycles Cycle threshold value: lower number = reached threshold in fewer cycles = started with more DNA • Logarithmic scale.

Adherence in Co-culture: Methods Gardnerella vaginalis (Gv)    Mobiluncus mulieris (Mm) Prevotella bivia (Pb) Atopobium vaginae (Av) not tested yet ME180 Vaginal Epithelial Cells Pb Pb Pb Pb Mm Mm Mm Mm • Centrifuge bacteria onto epithelial cells and allow toagain 30 adhere min • Wash with PBS to remove bacteria that have not adhered .

but qPCR does not discriminate if cell is dead or alive  CFU’s to determine initial amount of inoculated cells .e. relative amount of adherence Use different 16s primers to differentiate between bacteria strains DNA amount corresponds to cell amount.Adherence Assay: Data Extract bacterial DNA from wells with Qiagen DNeasy Kit qPCR to determine relative amounts of DNA. i.

vaginalis 0.4 0.027 Mm Bacteria GvMm .Adherence Assay: Data Adherence of M.1 0 p=0.8 0.9 Relative amount of MmDNA 0.3 0.5 0. mulieris to epithelial cells with and without G.2 0.6 0.7 0.

bivia to epithelial cells with and without G. vaginalis Relative amount of Pb DNA 6 p=0.Adherence Assay: Data Adherence of P.022 5 4 3 2 1 0 Pb only GvPb .

Conclusion: Data suggests that G. vaginalis biofilm aid growth? .   Anaerobes adhere better without biofilm Also. vaginalis biofilms decrease adherence. no detectable co-aggregation between Gv and each anaerobe  Implication: Perhaps competing for a receptor on the cell? Next Question: Does a G.

Growth Assay Experiment both in aerobic and anaerobic conditions Pb Pb Pb Pb Gv Gv Incubate 3-5 days BHIG BHIG  Qiagen DNeasy Kit to extract DNA for qPCR Gv biofilm: b anaerobe: Pb (repeated for all 3 anaerobe .

4 0 .2 1 p=0.8 0 .6 0 .Growth Assay: qPCR Data Preliminary results for Mobiluncus mulieris G ro w th A s s a y: a e ro b ic a n d a n a e ro b ic g ro w th o f M.0002 Relative amount of Mm DNA 0 . av a g in a lisio film G 1 .0003 p=0.2 0 M m o n ly A e ro b ic G vM m M m o n ly A n a e ro b ic G . w b m u lie ris ith a n d w ith o u t .

15 0.1 0. vaginalis 0. biofilm bivia w ith and w ithoutGa.35 0.Growth Assay: qPCR Data Preliminary results for Prevotella bivia Relative amount of DNA DNA Pb G row th A ssay: aerobic and anaerobic grow th of P.3 0.05 0 p=0.0001 p=0.25 0.45 0.0001 Pb only G vPb Pb only G vPb A erobic A naerobic • Av and Mm grow much slower than Pb .2 0.4 0.

4 0.2 1 0.2 0 Av only GvAv Av only GvAv Aerobic Anaerobic .Growth Assay: qPCR Data Preliminary results for Atopobium vaginae Growth Assay: aerobic and anaerobic growth of A.8 0.44 p=0.62 1.4 Relative amount of Av DNA p=0.6 1. a vaginae with and without G. vaginalisbiofilm 1.6 0.

Implications Growth was condition-specific for Pb.    Competition with Gardnerella? Gardnerella produces acid Does Gardnerella produce bacteriocins?  Preliminary inhibition assay . so next step is to find oxygen level of vagina Seems that some anaerobes may actually grow worse in the presence of a Gv biofilm In that case.

or the concentration was .Disk Diffusion Assay • a filter disk was soaked in sterile Gv culture supernatant and placed on a lawn of anaerobic bacteria • no zone of inhibition Av Pb • suggests that inhibition is nonspecific.

Pb and Av don’t adhere as well Gv negatively affects adherence of Mm and Pb Av may not be affected by Gv biofilm Mm may grow better alone Anaerobically. aerobically. Pb grows better with a Gv biofilm Growth assay    Unclear whether Gv contributes to growth of anaerobes in BV.Conclusions Adherence assay   Confocal: Gv and Mm adhere strongly to epithelial cells. Other conditions? Other factors?  Host factors  Loss of lactobacillus . Pb may grow better alone.

gather more data  qPCR analysis for adherence and growth assays using CFU’s Growth assays. literature search for physiological conditions of the vagina BBSI Summer 2009: Repeat preliminary experiments. as well as other vaginal conditions .Future Directions… Academic Year: qPCR analysis    DNA from additional adherence and growth assays Correlate cycle threshold value with CFU counts Also. lactic acid. in presence of H2O2.

Kimberly Jefferson Jennifer Patterson Dr.Acknowledgements The Jefferson Lab   Dr. Philippe Girerd VCU BBSI and Jeff Elhai BBSI Friends NSF & NIH! .

Questions? .

Oakley BB. Fieldler TL. Kuzevska I. Loening-Baucke V.e7. Patterson JL. Thomas KK. Adherent biofilms in bacterial vaginosis.11:2458-61. J Clin Microbiol 2007. and quantitative PCR for Gardnerella vaginalis.192:213-8.5.5. Nugent score. J Clin Microbiol 2005. Loening-Baucke V. Dorffel Y. Hale LP. Mycoplasma hominis. Hillier SL.10:3270-6. Fredricks DN. Sex Transm Infect 2004. Targeted PCR for detection of vaginal bacteria associated with bacterial vaginosis. Lochs H. Garland SM. Bradshaw CS.106. Caritis SN.9:4607-12. Mendling W. Am J Obstet Gynecol 2005. Ladhoff A. Marrazzo JM. Kaul R.43. Krohn MA. Horvath LB. Swidsinski S. The vaginal inflammatory milieu and the risk of early premature preterm rupture of membranes. Obstet Gynecol 2005. J Infect Dis 2006. High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence. Am J Obstet Gynecol 2008. Hocking J. Monroe D. Sha BE.e1-170.1012-23. J Infect Dis 2008. Karjane NW. Morris MB. Effect of biofilm phenotype on resistance of Garderella vaginalis to hydrogen peroxide and lactic acid. An adherent Gardnerella vaginalis biofilm persists on the vaginal epithelium after standard therapy with oral metronidazole. Lochs H. Managing recurrent bacterial vaginosis.45.80:8-11. Morton AN. Am J Obstet Gynecol 2007. Chen HY. Swidsinski A. Fairley CK. Girerd PH.References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) Shin LY.193:1478-86. PLoS Biology 2007.197:170. . Jefferson KK.197. Wilson. Utility of Amsel criteria. J. Swidsinski A.10:1355-7. Spear GT. Moss LM. Scholze J. Mendling W. Stay it with flora: maintaining vaginal health as a possible avenue for prevention of human immunodeficiency virus acquisition. Simhan HN. Swidsinski S. Zariffard MR. Looking for chinks in the armor of bacterial biofilms. and Lactobacillus spp. Verstraelen H. for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women. Wang QJ. Cohen MH.198:97-99.

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