V-antigen genotype and phenotype analyses of clinical isolates of Pseudomonas aeruginosa

Abstract

The pcrV genotype was analyzed in clinical isolates of Pseudomonas aeruginosa which showed a negative phenotype for secretion of V-antigen PcrV. The suppression of PcrV secretion in these isolates was due not to a lack of the pcrV gene but rather to suppression of PcrV expression.

FIG. 1.

Detection of pcrV in clinical isolates of P. aeruginosa by PCR. pcrV genes were amplified by PCR using genomic DNA from clinical isolates of P. aeruginosa as template DNA and a specific primer set outside of the coding region (Table 2). The PCR products were analyzed by 0.9% agarose gel electrophoresis and ethidium bromide staining. PCR genotyping analysis showed that all isolates possessed pcrV. The figure shows data for 20 clinical isolates and a laboratory strain, PA103.

FIG. 2.

Phenotype analysis of PcrV expression and secretion in clinical isolates of P. aeruginosa. Immunoblot analysis by a highly sensitive chemiluminescence method was used to determine the phenotype of PcrV secretion in selected P. aeruginosa isolates. PcrV proteins from P. aeruginosa PA103 (a laboratory strain) and PA1027 (a clinical isolate from an acutely infected patient) were used as positive controls for detection. Isolates were cultured in Luria-Bertani medium overnight, and the next day, 10% of the volume of the overnight culture was transferred into Ca²⁺-chelating deferrated tryptic soy broth medium and cultured for 8 h at 32°C. (Top panels) PcrV secreted into the inducing culture medium. (Bottom panels) PcrV in cell-associated fractions. PcrV was recognized as a 34-kDa band.

FIG. 3.

In vivo PcrV expression in clinical isolates of P. aeruginosa. Immunoblot analysis by a chemiluminescence method was used to determine the expression of PcrV in P. aeruginosa isolates instilled in mice. Bacteria were recovered by BAL 4 h after instillation into the lungs. The bacterial density among the samples was adjusted by quantitative culture of BAL fluid on agar plates. The secreted protein from P. aeruginosa PA103 (a laboratory strain) was used as a positive control for PcrV detection. PcrV was recognized as a 34-kDa band. PA1065 and PA1079 were isolates from cystic fibrosis patients and did not express PcrV in vivo.