Disk diffusion and ETEST (gradient diffusion) may produce false susceptible results for Pseudomonas aeruginosa from cystic fibrosis patients due to inducible colistin tolerance

Disk diffusion and ETEST may produce false susceptibility to colistin of P. aeruginosa. Delayed regrowth in the inhibition zone may be observed after 24h incubation in P. aeruginosa from cystic fibrosis patients (CF). Early reading may therefore misclassify P. aeruginosa as colistin susceptible. Our hypothesis is, that the delayed growth is due to inducible tolerance caused by changed LPS and this was tested by employing P. aeruginosa PAO1 and 10 Danish CF isolates for broth micro dilution (BMD), disk diffusion, ETEST, calculation of time-dependent concentration of colistin in blood agar, minimum biofilm eradication concentration, inducible tolerance of P. aeruginosa in liquid media containing colistin, gene expression analysis and whole genome sequencing. Population analysis was done to detect heteroresistance. PAO1 and 8 CF isolates gave same categorical susceptibility by BMD and ETEST. Two CF isolates were colistin resistant by ETEST but susceptible by BMD (major error). PAO1 and one colistin susceptible CF isolate did not show delayed growth in the inhibition zone although inducible tolerance of PAO1 was observed in liquid media since the diffusing colistin killed the bacteria in the inhibition zone. The other CF isolates showed delayed regrowth in the inhibition zone, and the colistin regrowth-ETEST-MIC was ≥64µg/ml. Five of 6 CF isolates had inducible tolerance in liquid media containing colistin whereas the sixth was completely resistant. They had mutations in various genes involved with inducible tolerance. In conclusion, inducible tolerance combined with mutations in the corresponding genes is the reason for the delayed growth and false susceptibility to colistin.