Characterization of Pseudomonas aeruginosa isolated from chronically infected children with cystic fibrosis in India

Abstract

Background: Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis (CF). With chronicity of infection, the organism resides as a biofilm, shows multi-drug resistance, diversifies its colony morphology and becomes auxotrophic. The patients have been found to be colonized with multiple genotypes. The present work was carried out to characterize P. aeruginosa isolated from children with cystic fibrosis using phenotypic and genotypic methods.

Results: We studied 56 patients with CF attending the Pediatric Chest clinic at All India Institute of Medical Sciences, New Delhi, India during August 1998-August 2001. These patients were regularly followed up at the clinic. Out of 56 patients, 27 were culture positive for P. aeruginosa where 8 were chronically infected (Group1) and 19 were intermittently colonized with the organism (Group2). Patients under Group1 had significantly higher rates of hospitalization, death and colonization with different colony morphotypes (p < 0.05). The isolates from Group1 patients were the positive producers of extended spectrum beta lactamase. A total of 5 auxotrophs were recovered from 2 patients where one was chronically infected with P. aeruginosa and the other was a recently enrolled patient. The auxotrophs had the specific requirement for methionine and arginine. Molecular typing revealed 33 ERIC-PCR (E1-E33) and 5 PCR-ribotyping (P1-P5) patterns. By ERIC-PCR, 4 patients were colonized with 2-4 genotypes and the remaining 23 patients were colonized with the single genotype.

Conclusion: With chronicity of infection, P. aeruginosa becomes multidrug resistant, diversifies its colony morphology, acquires mucoidity and shows auxotrophy for amino acids. The chronically infected patients can be colonized with multiple genotypes. Thus in a particular clinical set up, high index of suspicion should be there for diagnosis of CF patients so as to prevent the delay in diagnosis and management of CF patients.

Figure 1

Detection of Extended spectrum β lactamase in P. aeruginosa isolate from a patient (180/95) by:A. Disc Diffusion method (Amika: Amikacin; Cipro: Ciprofloxacin; Pipra: Piperacillin; Genta: Gentamicin; CAZ: Ceftazidime). B. Etest method (AB Biodisk, Sweden).

Figure 2

Detection of Auxotrophy of P. aeruginosa. Isolate No: Patient PCC.No. 1: PCC.No. 499/2000; 2–5: PCC.No. 180/95, A: P. aeruginosa ATCC 27853; N: P. aeruginosa NCTC 50184(meth^-). Plate A: Growth of P. aeruginosa on MAM supplemented with arginine only, Plate B: Growth of P. aeruginosa on MAM supplemented with all 22 amino acids Plate C: Growth of P. aeruginosa on MAM supplemented with all 22 amino acids except arginine,

Figure 3

ERIC-PCR patterns of P. aeruginosa isolates from the patient A. PCC. No. 180/95. • Lane M : 100 bp ladder, • Lane 1 : Pattern E1, • Lane 2 : Pattern E3, • Lane 3–5 : Pattern E1, • Lane 6–11 : Pattern E2. B. PCC.No. 180/95 and 179/93 • Lane M : 100 bp ladder, • Lane 1–7 (PCC.No.180/95) : Pattern E4, • Lane 8 (PCC. No. 179/93) : Pattern E5, • Lane 9 (PCC.No.180/95) : Pattern E4, • Lane 10 : P. aeruginosa ATCC 27853 • Lane 11 : Negative control

Figure 4

PCR-ribotyping of P. aeruginosa isolates from the patient A. PCC.No. 180/95 • Lane M : 100 bp ladder, • Lane1, 3, 7 and 9 : Pattern P1, • Lane 2, 4 and 8 : Pattern P2, • Lane 5, 10–14 : Pattern P3, • Lane 6 : Pattern P4B. PCC.No. 180/95 and 179/93 • Lane M : 100 bp ladder, • Lane 1,3 and10 (PCC.No.180/95) : Pattern P2, • Lane 2,4 (PCC.No.180/95) : Pattern P4, • Lane 5–7 and 9 (PCC.No.180/95) : Pattern P1, • Lane 8 (PCC.No.179/93 : Pattern P5, • Lane 11 : Negative control