In vitro efficacy of ciprofloxacin and gentamicin against a biofilm of Pseudomonas aeruginosa and its free-living forms.

BACKGROUND

Infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism acquires a mucoid phenotype and grows as microcolonies in a biofilm in the respiratory passages of the host. This acts as a protective niche and helps the organism to evade the host immune response. In the biofilm the organism has a high resistance to antibiotics, leading to problems in eradication, and poses a therapeutic challenge. We studied the antimicrobial susceptibility of P. aeruginosa ATCC 27853 in a biofilm and as free-living forms against ciprofloxacin and gentamicin, the commonly used antibiotics in patients with CF.

METHODS

Biofilm formation of P. aeruginosa ATCC 27853 was characterized by in vitro biofilm formation assay. The biofilm was detected by light microscopy and quantitated by measuring the absorbance at 575 nm and by viable bacterial counts. After the maximal biofilm was established, the effect of various concentrations of ciprofloxacin (1, 2 and 4 microg/ml) and gentamicin (4, 8, 16, 32 and 64 microg/ml) was observed and the minimal biofilm eradication concentration (MBEC) determined. The minimal bactericidal concentration (MBC) of both the antibiotics was determined against the free-living forms of the organism. The MBEC of the two antibiotics was further compared with the MBC.

RESULTS

On microscopic examination, the maximal biofilm of P. aeruginosa was established on a coverslip at 12 hours, the maximum absorbance was at 575 nm and viable counts were observed at 12 hours, which corresponded to the maximal biofilm production. The organisms in the biofilm showed a 4-fold greater resistance against ciprofloxacin and gentamicin as compared to the free-living forms.

CONCLUSION

In biofilm, P. aeruginosa shows greater resistance against antibiotics. This renders these antibiotics ineffective, leading to chronic and persistent infections.