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.

Phenotypic & genotypic variants of Pseudomonas aeruginosa isolated from children with cystic fibrosis in India.

BACKGROUND & OBJECTIVES: Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With increase in the chronicity of the disease, there is a diversification of the organism into different colony morphological types. The antimicrobial susceptibility of the organism varies with its colony morphology. The present work was carried out to study the different morphotypes of P. aeruginosa isolated from patients of cystic fibrosis. METHODS: We studied 38 children with CF attending the Paediatric Chest Clinic at the All India Institute of Medical Sciences, New Delhi, India during October 2000-January 2001 who were regularly followed up at the clinic. Patients were divided into 2 groups, Group 1 included all patients chronically infected with P. aeruginosa and Group 2 included patients who were infrequently colonized with this organism. Different colony morphological types of P. aeruginosa on culture media were identified. They were characterized by phenotypic methods using antibiograms and genotypic methods using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction and PCR-ribotyping. RESULTS: Fourteen of the 38 patients were colonized at least once with P. aeruginosa. Eight patients belonged to Group 1 and 42 isolates were obtained from these patients. Group 2 had 6 patients and 9 isolates were obtained from them. All patients in Group 1 harboured different colony morphotypes (Types 1-6) while all 6 patients in Group 2 showed a single type of colony morphology (Type 1). The isolates from Group 1 patients showed higher antimicrobial resistance as compared to Group 2 patients. Molecular typing of the isolates revealed 10 ERIC-PCR patterns and 2 PCR-ribotyping patterns among Group 1 and 2 ERIC-PCR and 1 PCR-ribotyping pattern among patients of Group 2. INTERPRETATION & CONCLUSION: The frequency of different morphotypes of P. aeruginosa and antibiotic resistance was higher among Group 1 patients. On molecular typing, more than one genotype was isolated from Group 1 patients while only one genotype was isolated from patients in Group 2. We conclude that at a given time, chronically infected patients can be colonized by phenotypically and genotypically distinct strains of P. aeruginosa which has an implication in the management of these patients.