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“Detection and identification of specific bacteria in wound

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					“Detection and identification of
   specific bacteria in wound
 biofilms using peptide nucleic
     acid fluorescent in situ
   hybridisation (PNA FISH)”
    Malic S, Hill, KE, Hayes A, Percival SL,
          Thomas DW, Williams DW
     Microbiology (2009), 155, 2603-2611

                Jet-Janneke Grömmel,
            BS962, MSc Medical Microbiology
What this study was about:
Peptide nucleic acid fluorescent in situ
  hybridisation (PNA FISH) and confocal laser
  scanning microscopy (CLSM) were used to
  examine biofilms generated in vitro and on
  biopsies of chronic venous leg ulcers (CVLU).
Aims:
 To determine the spatial distribution of biofilms
  consisting of common wound bacteria.
 To demonstrate the use of PNA FISH and CLSM in the
  detection and examination of wound biofilms, in order to
  aid biofilm management in future.
Why?
Biofilms are aggregates of microorganisms
 in which cells adhere to each other and/or
 a surface.
Extracellular polymeric substance (EPS)
 formation, activation of resistance genes,
 and quorum sensing make biofilms very
 resistant to the immune response and
 antibiotics, often resulting in increased
 morbidity and treatment failure.
What they did:
• 4 bacterial strains were isolated from non-infected
  chronic leg ulcers: Staphylococcus aureus,
  Pseudomonas aeruginosa, Streptococcus oralis and
  Micrococcus luteus.
• Biofilms were constructed in constant-depth film
  fermenter (CDFF) and on reconstituted human epidermis
  (RHE).
• Biofilms and CVLU biopsies were imaged by CLSM after
  hybridisation with PNA probes with different fluorescent
  labels: Psaer-FITC for P.aeruginosa, Sta-CY5, for
  S.aureus and Bac-Unil-CY3 for all bacteria.
• Quantitative analysis of bacterial population of tissue
  culture Z stack using Image J software
Results – CLSM of biofilm in CDFF
      Biofilms were
       heterogenous
      P.aeruginosa (green)
       dominates throughout
       the biofilm (e)
      S.aureus (blue)
       concentrates towards
       the surface of the
       biofilm (f)
      Other 2 species
       concentrate towards the
       middle and lower part of
       the biofilm

Figure 1: d: Bac-Unil probe with CY3 label
       shows all bacteria as red e: Psaer
       probe with FITC label shows
       P.aeruginosa green f: Sta probe
       with CY5 label show S.aureus blue
       g: multiplex PNA staining
Results – CLSM of biofilm on RHE
Multilayered biofilms
and tissue invasion was
seen on RHE which was
deliberately cut
Only superficial growth
was seen on in tact RHE




                                     Figure 3: P. aeruginosa-specific (green
                                     bacteria) and the Staph. aureus specific probes
                                     (red bacteria) and with the epithelial cell nuclei
Figure 2: Gram-stained RHE showing
a: in tact skin, b: broken skin      counterstained with Hoechst dye (blue).
Results – CLSM on chronic leg ulcers

 Colonisation of CVLU was
  seen both with planktonic and
  aggregates of bacteria




Figure 4: A section from a CVLU using
PNA FISH and showing total bacteria
labelled with the universal bacterial PNA
probe (red bacteria) and Hoechst dye for
epithelial cell nuclei (blue).
Conclusions of the study
• PNA FISH and CLSM are effective in
  demonstrating the characteristics of
  biofilms
  Enables examination without disrupting the
   biofilm
  Images and identifies bacterial species
   quickly without traditional microbiology
  May aid in study of biofilm response to
   treatment strategies and diagnosis using
   clinical samples
Discussion
When considering the usefulness of this study the
  following questions need to be raised:
 How do we know biofilms are present, as
  opposed to colonisation?
 How does this study reflect wound biofilm
  formation in vivo, especially regarding variety of
  bacteria and external conditions?
 Is there a clinical and diagnostic advantage
  using these techniques compared to current
  methods
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