Bacteria associated with Bacterial Vaginosis

Document Sample
Bacteria associated with Bacterial Vaginosis Powered By Docstoc
					            The Complexity of
            Bacterial Vaginosis:
       Do Gardnerella vaginalis biofilms
      contribute to colonization by other
       anaerobes in bacterial vaginosis?
                    August 5, 2008
Annica Stull-Lane       VCU Mentor: Kimberly Jefferson
BBSI Summer 2008            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
Complications with BV
  Preterm delivery
  Increased risk of PID
  Increased acquisition
  & transmission of HIV
  Recurring infections!
      Treatment


      http://the-half-decent-pharmaceutical-chemistry-blog.chemblogs.org/archives/2006/11/23/drug-of-the-week-metronidazole
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, is the
presence of a certain species necessary for BV??
                    Wilson, J. Managing recurrent bacterial vaginosis. Sex Transm Infect 2004;80:8-11.
Bacteria Associated with BV
 Gardnerella vaginalis --- most prevalent
    Gram-positive or variable rod-shaped anaerobe
    Present in up to 95% of BV cases, but not sole factor, 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, Mendling W, Loening-Baucke V, et al. Adherent biofilms in bacterial vaginosis. Obstet
              Gynecol 2005;106.5;1012-23.
    Bacterial Biofilms
            Complex matrix of polysaccharides, proteins
           and/or nucleic acids
       Resistance to chemicals, pH extremes, host
     immune defenses, and antibiotics
                Related to recurrence of BV?




                            S. aureus biofilm on a catheter
http://phil.cdc.gov/phil/details.asp
 Succession in Biofilms
  initial colonizers and
 secondary colonizers
     For example, 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.nature.com.proxy.library.vcu.edu/nrmicro/journal/v2/n7/fig_tab/nrmicro928_F3.html
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, lactic acid, H2O2 etc…
  Experiments
       Adherence
          Do the anaerobes stick better to vaginal
           epithelial cells in the presence of Gv?
       Growth
          Does the presence of a Gv biofilm facilitate
           the growth of strict anaerobes?


• For the three anaerobes, strict anaerobic conditions
required.
Maintaining Anaerobic Conditions

Anaerobic air mix:
• 5% CO2
• 5% H2
• 90% N2
Adherence Assay:                       • Single bacterial
      Confocal Microscopy              strain on ME180
                                       vaginal epithelial cells
  BacLight Green Bacterial Stain
  Red Vibrant Cell Membrane Stain            • Multi-species?


                                              • How can one
                                              measure
                                              adherence?
       Control              G. vaginalis




P. bivia           A. vaginae       M. mulieris
What is qPCR?
• SYBR Green as a fluorescent dye
                                    Repeat 40X,
DNA template                        measuring
                                    fluorescence
                                    after each
 Denaturation
                                    cycle
Primers anneal


 Extension
What is qPCR?
• 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, so
                                use equation:
                                      (- cycle threshold value)
                                    2^

                                    Acquiring DNA…
Adherence in Co-culture: Methods
      Gardnerella vaginalis (Gv)
         Mobiluncus mulieris (Mm)
         Prevotella bivia (Pb)         • Centrifuge bacteria
         Atopobium vaginae (Av) not    onto epithelial cells
          tested yet                    and allow to adhere
                                        30 min again
ME180 Vaginal Epithelial Cells


Pb            Pb       Mm          Mm    • Wash with PBS to
                                         remove bacteria
Pb            Pb       Mm          Mm    that have not
                                         adhered
Adherence Assay: Data
  Extract bacterial DNA from wells with Qiagen
 DNeasy Kit
 qPCR to determine relative amounts of DNA,
 i.e. relative amount of adherence
  Use different 16s primers to differentiate
 between bacteria strains
  DNA amount corresponds to cell amount, but
 qPCR does not discriminate if cell is dead or
 alive
     CFU’s to determine initial amount of inoculated cells
Adherence Assay: Data
               Adherence of M. mulieris to epithelial cells
                     with and without G. vaginalis
                     0.9
                                      p=0.027
Relative amount of




                     0.8

                     0.7
  Mm DNA




                     0.6

                     0.5

                     0.4

                     0.3

                     0.2

                     0.1

                      0

                           Mm                   GvMm
                                    Bacteria
Adherence Assay: Data
                               Adherence of P. bivia to epithelial cells
                                   with and without G. vaginalis
   Relative amount of Pb DNA



                               6



                               5
                                                p=0.022

                               4



                               3


                               2



                               1



                               0


                                      Pb only              GvPb
                                                Bacteria
 Conclusion: Data suggests that G.
vaginalis biofilms decrease adherence.
   Anaerobes adhere better without biofilm
   Also, no detectable co-aggregation between Gv and
    each anaerobe
      Implication: Perhaps competing for a receptor
       on the cell?


 Next Question: Does a G. vaginalis
biofilm aid growth?
 Growth Assay
Experiment both in aerobic and anaerobic conditions


     Pb                          Incubate 3-5 days
     Pb


     Pb   Gv   BHIG
                               Qiagen DNeasy Kit to
                              extract DNA for qPCR
     Pb   Gv   BHIG




 Gv biofilm:
Pb anaerobe: Pb       (repeated for all 3 anaerobes)
Growth Assay: qPCR Data
         Preliminary results for Mobiluncus mulieris

                         Growth Assay: aerobic and anaerobic growth of M.
                          mulieris with and without a G. vaginalis biofilm
                1.2

                                                                p=0.0003
Relative amount of




                     1

                                 p=0.0002
                0.8
  Mm DNA




                0.6



                0.4



                0.2



                     0


                             Mm only        GvMm            Mm only         GvMm
                                  Aerobic                       Anaerobic
 Growth Assay: qPCR Data
                               Preliminary results for Prevotella bivia

                                      Growth Assay: aerobic and anaerobic growth of P.
                                         bivia with and without a G. vaginalis biofilm
                      Pb DNA




                               0.45                                          p=0.0001
   Relative amount of DNA




                                0.4

                               0.35

                                0.3
                                           p=0.0001
                               0.25

                                0.2

                               0.15

                                0.1

                               0.05

                                 0


                                          Pb only     GvPb                Pb only   GvPb
                                            Aerobic                           Anaerobic
• Av and Mm grow much slower than Pb
Growth Assay: qPCR Data
             Preliminary results for Atopobium vaginae

                           Growth Assay: aerobic and anaerobic growth of A.
                            vaginae with and without a G. vaginalis biofilm
                     1.6
                                                                   p=0.62
                                  p=0.44
Relative amount of




                     1.4


                     1.2


                      1
  Av DNA




                     0.8


                     0.6


                     0.4


                     0.2


                      0


                               Av only        GvAv           Av only       GvAv
                                         Aerobic               Anaerobic
Implications
   Growth was condition-specific for Pb, 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,
     Competition with Gardnerella?
     Gardnerella produces acid
     Does Gardnerella produce bacteriocins?
        Preliminary inhibition assay
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

                       • suggests that
  Av             Pb    inhibition is nonspecific,
                       or the concentration
                       was too low
Conclusions
 Adherence assay
    Confocal: Gv and Mm adhere strongly to epithelial cells,
     Pb and Av don’t adhere as well
    Gv negatively affects adherence of Mm and Pb
 Growth assay
    Av may not be affected by Gv biofilm
    Mm may grow better alone
    Anaerobically, Pb may grow better alone,
     aerobically, Pb grows better with a Gv biofilm
  Unclear whether Gv contributes to growth of
 anaerobes in BV. Other conditions? Other factors?
    Host factors              Loss of lactobacillus
  Future Directions…
       Academic Year: qPCR analysis
          DNA from additional adherence and growth assays
          Correlate cycle threshold value with CFU counts
          Also, literature search for physiological conditions of
           the vagina

        BBSI Summer 2009:
  Repeat preliminary experiments, gather more data
     qPCR analysis for adherence and growth assays using CFU’s
  Growth assays, in presence of H2O2, lactic acid, as
well as other vaginal conditions
   How does Gv decrease growth of other anaerobes?
Acknowledgements
 The Jefferson Lab
    Dr. Kimberly Jefferson
    Jennifer Patterson
 Dr. Philippe Girerd
 VCU BBSI and Jeff Elhai
 BBSI Friends
 NSF & NIH!
Questions?
References
1)    Shin LY, Kaul R. Stay it with flora: maintaining vaginal health as a possible avenue for prevention
      of human immunodeficiency virus acquisition. J Infect Dis 2008;197.10:1355-7.
2)     Fredricks DN, Fieldler TL, Thomas KK, Oakley BB, Marrazzo JM. Targeted PCR for detection of
      vaginal bacteria associated with bacterial vaginosis. J Clin Microbiol 2007;45.10:3270-6.
3)    Sha BE, Chen HY, Wang QJ, Zariffard MR, Cohen MH, Spear GT. Utility of Amsel criteria, Nugent
      score, and quantitative PCR for Gardnerella vaginalis, Mycoplasma hominis, and Lactobacillus spp.
      for diagnosis of bacterial vaginosis in human immunodeficiency virus-infected women. J Clin
      Microbiol 2005;43.9:4607-12.
4)     Simhan HN, Caritis SN, Krohn MA, Hillier SL. The vaginal inflammatory milieu and the risk of early
      premature preterm rupture of membranes. Am J Obstet Gynecol 2005;192:213-8.
5)    Patterson JL, Girerd PH, Karjane NW, Jefferson KK. Effect of biofilm phenotype on resistance of
      Garderella vaginalis to hydrogen peroxide and lactic acid. Am J Obstet Gynecol 2007;197:170.e1-
      170.e7.
6)    Wilson, J. Managing recurrent bacterial vaginosis. Sex Transm Infect 2004;80:8-11.
7)     Bradshaw CS, Morton AN, Hocking J, Garland SM, Morris MB, Moss LM, Horvath LB, Kuzevska I,
      Fairley CK. High recurrence rates of bacterial vaginosis over the course of 12 months after oral
      metronidazole therapy and factors associated with recurrence. J Infect Dis 2006;193:1478-86.
8)    Swidsinski A, Mendling W, Loening-Baucke V, Ladhoff A, Swidsinski S, Hale LP, Lochs H. Adherent
      biofilms in bacterial vaginosis. Obstet Gynecol 2005;106.5;1012-23.
9)    Swidsinski A, Mendling W, Loening-Baucke V, Swidsinski S, Dorffel Y, Scholze J, Lochs H,
      Verstraelen H. An adherent Gardnerella vaginalis biofilm persists on the vaginal epithelium after
      standard therapy with oral metronidazole. Am J Obstet Gynecol 2008;198:97-99.
10)   Monroe D. Looking for chinks in the armor of bacterial biofilms. PLoS Biology 2007;5.11:2458-61.

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:12
posted:7/27/2012
language:
pages:29