PSEUDOMONAS AERUGINOSA BIOFILM FORMATION IN CYSTIC FIBROSIS AND THE EFFECT OF MUCOIDY AND AZITHROMYCIN

Chronic lung infection with P. aeruginosa is the major cause of morbidity and mortality in cystic fibrosis (CF). Most CF patients initially acquire a unique nonmucoid strain of P. aeruginosa. The subsequent conversion to a mucoid phenotype is associated with an accelerated decline in lung function. This thesis explored the role of mucoid and non-mucoid P. aeruginosa biofilm formation in CF lung disease. The initial objective was to find a reliable way of quantifying biofilm formation in vitro to assess P. aeruginosa clinical isolates. A microtitre assay was modified to study biofilm formation by isogenic mucoid and non-mucoid strains from clinical isolates. The CF isolates were slow to form biofilm resulting in the assay being extended to 72 hours. A group of genomically diverse P. aeruginosa isolates formed different amounts of biofilm in vitro. Genotype did not predict biofilm morphotype suggesting factors such as differential gene expression are important. Mucoid conversion and biofilm formation are both important in the pathogenesis of CF bronchiectasis, however the relationship between the two is not clear. Recent studies comparing genetically manipulated isogenic mucoid and non-mucoid P. aeruginosa stains reached conflicting conclusions. Multiple clinical isolates of P. aeruginosa have not been examined before. Biofilm produced by isogenic mucoid and non-mucoid P. aeruginosa strains isolated from CF patients (n=10) was quantified to determine if: (1) CF isolates produced different amounts of biofilm; and (2) mucoid clinical isolates form more biofilm than their isogenic non-mucoid parent strain. Factors with a role in biofilm formation: flagellar motility; twitching motility and growth were assessed. The reproducibility of the modified microtitre assay was confirmed (Spearmans correlation coefficient 0.884 p24 hours to form significant amounts. CF P. aeruginosa isolates showed reduced motility and planktonic growth compared to the laboratory reference strain PAO1. Isolates from three patients did not form any biofilm at 72hours. These isolates were not deficient in growth, flagellar activity or twitching motility. There was no difference in biofilm formation between isogenic mucoid and non-mucoid isolates (p= 0.80 at 24 hours, 0.96 at 48 hours and 0.74 at 72 hours). Mucoid P. aeruginosa CF isolates did not produce more biofilm than isogenic non-mucoid isolates. Long term Azithromycin (AZM), a macrolide antibiotic, improves outcomes in CF patients. The mechanisms remain unclear but may include antibiofilm effects on P. aeruginosa. The effect of sub-MIC AZM (2Gg/ml, 8Gg/ml and 32Gg/ml) on 72 hour biofilm formation by seven isogenic mucoid and non-mucoid P. aeruginosa isolates from CF patients was studied. Biomass, as indicated by crystal violet (CV) staining, was determined by the modified microtitre assay. AZM resulted in increased P. aeruginosa biofilm biomass. There was a 150% increase in non-mucoid isolate biomass with exposure to AZM 32Gg/ml (95% CI for difference 0.10 to 0.66, p=0.005). Mucoid isolates showed a similar (91%) increase in biomass with AZM although this did not achieve statistical significance (95% CI -0.039 to 0.94, p=0.079). Active metabolism by biofilm bacteria was measured using the respiratory indicator 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Overall AZM had no significant effect on CTC staining (p=0.141). However, when CTC activity was considered per unit of biomass (CTC/CV), there was a significant reduction in activity seen with increasing AZM concentration in mucoid and non-mucoid isolates (p=0.007). Although AZM caused an increase in biomass, there was reduction in the metabolic activity of the biofilm. There was no difference between isogenic mucoid and nonmucoid isolates in response to AZM (p=0.514), suggesting the effect of Azithromycin on biofilms is independent of mucoidy. Using a reliable and reproducible modified microtitre plate assay the above data show that mucoid clinical P. aeruginosa isolates did not form more biofilm than the isogenic non-mucoid parent strain. Clinical P. aeruginosa isolates exposed to AZM produced thicker biofilms with reduced metabolic activity. The effects were similar in both mucoid and non-mucoid isogenic isolates suggesting AZM activity is independent of mucoidy. Enhanced biofilm formation does not appear to explain the accelerated lung disease seen in CF patients after mucoid conversion of P. aeruginosa. The above studies highlight the complexity of biofilm formation by P. aeruginosa and the need to evaluate clinical isolates