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.