Dimitrov, B.

Humphreys, H.

Stevens, N.T.

Urbach, V.

Whitaker, G.

English

Southampton (e-Prints Soton)

  P. aeruginosa is a Gram-negative bacillus that causes chronic lung infections in CF  patients.  Continuous  activation  of  innate  immune  responses  in  the  lung results in progressive destruction of the lung .Treatment is complicated due to P. aeruginosa alginate production and biofilm formation. This is because biofilm cells have an innate resistance to antibiotics. Biofilm formation is influenced by local environmental conditions such as oxygen availability, osmolarity and pH. Mature biofilm formation is characteristic of chronic infection in the airway of CF  individuals.  Understanding  the  influence  of  such  factors  on  biofilm development  is  necessary  if  we  are  to  find  new  therapeutic  approaches  to treating biofilm-related infections in CF patients. Background & Aims Characterising the biofilm forming capabilities of Cystic Fibrosis Pseudomonas aeruginosa clinical isolates  G. Whitaker1, V. Urbach2,3, B. Dimitrov4 H. Humphreys1,5, N. T. Stevens1 Departments of 1Clinical Microbiology & 2Molecular Medicine, RCSI, Dublin 9. Ireland 3National Children’s Research Centre, Our Lady’s Children’s Hospital, Crumlin, Dublin 12. Ireland 4 Academic Unit of Primary Care & Population Sciences, University of Southampton, Southampton General Hospital, Southampton SO166YD, England, UK 5Department of Microbiology Beaumont Hospital, Dublin 9. Ireland Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis conductance regulator (CFTR) gene. In the lungs, CFTR dysfunction results in dehydration of  the  airway  surface  liquid  and  reduced  mucus  clearance.  This  supports colinisation of the airway with Pseudomonas aeruginosa and production of a biofilm.  Biofilm  formation  is  influenced  by  local  environmental  conditions such  as  oxygen  availability,  osmolarity  and  pH.  The  biofilm  forming capabilities of eight P. aeruginosa CF isolates and the biofilm-positive strain PAK  were  investigated  under  varying  in  vitro  growth  conditions  using  a microtitre plate biofilm assay.  Pulsed-field gel electrophoresis confirmed there was  clonal  variability  amongst  the  clinical  isolates.  Under  aerobic  growth conditions,  6 (75%) of the isolates and PAK were strongly adherent in Tryptic Soya Broth (TSB) supplemented with 0.5% NaCl.  The effect of pH on biofilm production  was  investigated  by  varying  the  concentration  of  glucose  in  the growth media. Using PAK, it was found that there was a significant increase in biofilm formation in TSB supplemented with 1% and 2% glucose compared to TSB alone (P ≤ 0.022 & P ≤ 0.019 respectively).  This was associated with a small but significant decrease in pH of the growth media from pH 8.45 in TSB alone, to pH 7.83 in TSB + 1% glucose and pH 6.77 in TSB + 2% glucose ( P ≤ 0.022 respectively). Five (62.5%) of the clinical isolates were strongly adherent when the media was supplemented with 1% glucose. Anaerobically, isolates were  either  weakly  adherent  or  non  adherent  in  all  conditions.  Biofilm formation  was  variable  but  most  isolates  became  more  adherent  with  the addition of NaCl or glucose alone. The effect of pH on biofilm appears to variable  in  these  isolates.  Understanding  the  factors  influencing  the pathogenesis of P. aeruginosa biofilm infections in the CF lung is necessary for better management of these patients.  Abstract Aims: To  investigate  the  biofilm  forming  capabilities  of  P.  aeruginosa  CF  clinical isolates under varying in vitro environmental conditions  To investigate the effect of pH on P. aeruginosa  in vitro biofilm development.   To determine the existence of clonal relationships between the clinical isolates Results 1 Stepanovic et al., J. Microbiol. Methods, 2000; 40:175-179 Methods Bacterial  strains  and  isolates:  Clinical  isolates  were  collected  from  sputum specimens obtained from CF patients attending Beaumont Hospital. Control strain, PAK, was obtained from Prof. Alain Filloux, Imperial College, London, UK.  Microtitre Plate Biofilm assay: Biofilm assays were performed in Tryptic soya broth (TSB) alone or TSB supplemented with NaCl (0.5-2%) and glucose (0.5-2%) under aerobic and anaerobic growth conditions for 48 h. Absorbance of adhered stained  cells  was  obtained  at  595  nm  and  biofilm  phenotypes  were  defined according to the criteria outlined by Stepanovic et al.1  Pulsed field gel electrophoresis: clonal variability amongst clinical isolates was investigated  using  PFGE.  Following  staining  with  Ethidium  bromide.  Banding patterns were analysed using GelCompar II® software. Results Figure 1. The dendrogram was constructed with GelCompar II ® software (Ver 6.5) by clustering analysis  determined  using  the  Dice  coefficient  (tolerance  1%).  Clonal  groups  were  based  on  a similarity of ≥80% (as indicated by the highlighted groups). Figure 2. Effect of NaCl on biofilm forming capabilities of CF P. aeruginosa isolates under aerobic  growth conditions. Graphs show mean Biofilm OD at 595 nm. Biofilm conditions were Tryptic soy broth (TSB), TSB + 0.5%, 1%, and 2% NaCl,. Isolates were identified as non-adherent (•), weakly adherent (ﾧ), moderately adherent (#) or strongly adherent (*). A B Figure 3. Effect of glucose on biofilm forming capabilities of CF P. aeruginosa isolates under aerobic  growth conditions (A) and on pH of growth media following  aerobic biofilm growth (B)  Graphs show mean Biofilm OD at 595 nm. Biofilm conditions were TSB, TSB + 1% glucose and TSB + 2%  glucose. Isolates were identified as non-adherent (•), weakly adherent (ﾧ), moderately adherent (#) or strongly adherent  (*).  There  was  a  significant  increase  biofilm  formation  in  CFPA5  and  CFPA11  with  the addition of 2% and 1% glucose respectively (P≤0.0001). There were significant decreases in pH of the media in which these biofilms formed (*) where P≤0.0001. For PAK (** and ***) denotes a significant decrease in pH compared to TSB alone where  P ≤0.022. Conclusions & Future Work Despite some clonal similarity, biofilm formation was variable and strain dependent but  NaCl  (0.5%  w/v)  and  glucose  (1%  &  2%  w/v)  induce  the  process  in  some aerobically.  Anaerobic conditions appear to reduce biofilm development. Some isolates became more adherent as pH decreased (e.g. CFPA11 in TSB + 1% glucose), while others favored neutral to slightly acidic or basic growth conditions. More investigations characterising a larger group of isolates may determine a true role of pH in influencing biofilm development of P. aeruginosa in the CF lung.  Anaerobically  isolates were non-adherent or weakly adherent  under all growth conditions(data not shown) Figure  4.  Correlation  between  biofilm forming  capabilities  of  CF  P.  aeruginosa isolates  and  pH  of  growth  media.  Graphs show the tendencies for a relationship for each isolate – some of them show negative relationship  (CFPA5,  CFPA7,  CFPA11, CFPA17 and PAK) while the others exhibit positive  correlation  (CFPA1,  CFPA4, CFPA12 and CFPA16).      The  correlation  analysis  also  estimates the  squared  correlation  coefficient  (R2) which  is  an  indicator  of  the  variance  in biofilm forming capabilities that might be explained (at least, theoretically) by the pH variance.  Despite  their  pilot  nature  the established  tendencies  warrant  further investigations TSB + 1% glucose TSB + 2% glucose TSB + 2% glucose Project was generously funded by the Royal College of Surgeons in Ireland (RCSI) Summer Research School , which provides opportunities for undergraduate health science students to obtain valuable laboratory experience. 

Characterising the biofilm forming capabilities of Cystic Fibrosis Pseudomonas aeruginosa clinical isolates

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