The role of biofilm in wounds

Biofilms  have  long  been  implicated  in  persistent  infections  and  have  recently  been associated  with  chronic  wounds.  The  role  of  bacteria  in  wounds  is  not  yet  fully understood  and  their  ability  to  form  biofilm  is  yet  to  be  fully  elucidated.    Biofilms  are associated  with  phenotypic  features  such  as  the  signalling  molecules  for  regulation  of activities within biofilm and secretion of extracellular polymeric substances (EPS). The presence  of  biofilm  may  be  confirmed  by  specialised  microscopy  techniques  or  by detecting biofilm markers. Routine methods are not yet availablefor detecting biofilms in wounds. The aims of this project were to investigate the role of biofilm in wounds, by examining  the  ability  of  wound  isolates  to  form  biofilm  and  produce  signalling molecules and by developing a wound model; to relate laboratoryfindings with in vivoactivity by  exploring the possibility of detecting  biofilm markers in dressings removed from chronic wounds.Biofilm  characteristics  of  97 Pseudomonas  aeruginosastrains  isolated  from  wounds were  investigated.  Their  antibiotic  susceptibility  to  commonly  used  antibiotics  was determined.The isolates were examined for ability to form biofilm and to produce acyl-homoserine lactone (AHL) signalling molecules in batch culture and were characterised using PCR.A wound model was developed for biofilm continuous culture using gauze as  the  substratum  for  biofilm attachmentand  culture  effluent  was  examined  for  AHL production  and  detached  fragments  of  biofilm.Gauze  removed  from  the  culture  at  30 hours was  examined  with  scanning  electron  microscopy  (SEM).    Thirty  five  dressings removed  from  chronic  wounds  were  investigated  for  the  presence  of  biofilm  markers including  AHL  and  EPS.  Organisms  from  the  wound  dressings  were  isolated  and examined  for  biofilm  formation,  AHL  and  EPS.Thirty  hour  biofilm  of P.  aeruginosawound  isolate  was  exposed  to  40  %  (w/v)  honey  in  wound  model continuous culture.The  potential  of  some  honeys  to  inhibit  the  growth  and  quorum  sensing  (QS)  of  a biofilm forming organism, Chromobacterium violaceumwas investigated.Over 90%  of  the  97  cultures  of P.aeruginosawere  resistant  to  3  antibiotics  while resistance  to  4  others  ranged  between  3 –19%.  Eighty  eight  of  the  isolates  (90.72%) formed  biofilm  while  78  (81.4%)  produced  AHL.  PCR  characterisation  of  the  isolates showed that 82 (84.53%) have 100% genetic similarity linkage to the cohort, 3 (3.09%) have  75 -99%  while  12  (12.37%)  are  of  50 -75% linkage.  Examination  of  stained culture effluent smearfrom wound model revealed biofilmembedded in EPS and AHL was  detected  in  sterilised culture effluent.  SEM  examination  of  gauze  removed  from wound  model  after  30  hours  culture  confirmed  biofilm  structures.  Eleven  (31.4%) wound  dressings  tested  positive  for  AHL,  28  (80%)  contained  EPS (4  not  tested  for EPS).  Organisms  that  formed  biofilm  were  isolated  from  32  (91.4%) dressings while Gram  negative  bacteria  that  produced  AHL  were  isolated  from  13  dressings.    Two selected  Gram  negative  bacteria  from  wound  dressing  that  were  cultured  in  wound model showed progressive biofilm formation with EPS and AHL production. The 30 hr biofilm  exposed  to  honey  was  dislodged  within  6  hours  and  no  viable  organism  was recovered  from  culture.  Honey  inhibited  the  growth  and  QS  of C.  violaceum in  a  dose dependent  manner.  Of  the  10  honeys  examined,  8 (80%)inhibited  the  growth  and quorum sensing of C. violaceum, 1 slightly inhibited quorum sensing while 1 showed no inhibitory effect.Indicators of biofilms detected in used wound dressings have the potential to be used in the  diagnosis  of  biofilms  in  chronic  wounds.  The  antimicrobial  effect  of  honey  on biofilm and quorum sensing as shown in this study suggests that application of honey in wound management will provide effective treatment for wounds with biofilm.