Phenotypic characterization of clonal and nonclonal Pseudomonas aeruginosa strains isolated from lungs of adults with cystic fibrosis

Abstract

The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P = 0.02), to show elastase and alkaline protease activity by zymography (P = 0.04 and P = 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P = 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P = 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.

FIG. 1.

DNA banding pattern following macrorestriction using a rare-cutting restriction enzyme, SpeI, and PFGE analysis using our previously described methods (1). Lanes 1, 2, and 3 are PAO1, AES-1, and AES-2 controls, respectively. Lanes 4 to 10 represent P. aeruginosa strains isolated from individual patients (Table 1). Lanes 5 to 8 have unique banding patterns and hence represent nonclonal strains. Isolates in lanes 9 and 10 (isolates 2 and 12) have a macrorestriction pattern identical to that of AES-1, and lane 4 (isolate 15) is identical to AES-2.

FIG. 2.

Protease activity on 1% gelatin zymograms using 10% SDS-PAGE for AES-1 (isolates 9, 12, 14, and 2), AES-2 (isolates 15, 17, 18, and 19), and nonclonal strains (isolates 29, 30, 36, and 41) (Table 1). Up to three bands were detected, corresponding to molecular masses of 51, 98, and 120 kDa. Bands at 98 and 120 kDa represented aggregate activity of elastase and staphylolysin, while the band at 51 kDa represented alkaline protease. Isolates included in the figure are representative of isolates in each group. PAO1 was included as a control.

FIG. 3.

Representative TLC analyses of AHLs produced by PAO1, AES-1 (isolates 6, 12, 4, and 14), AES-2 (isolates 16, 17, 15, and 18), and nonclonal (isolates 29, 30, 36, and 39) (Table 1) P. aeruginosa isolates from the lungs of CF patients and purified BHL. AHLs were resolved from unconcentrated supernatants using methanol-water, and representative spots were visualized with the biosensor C. violaceum (CV026). Up to two dots were noted and identified as being BHL and HHL as shown. Isolates included in the figure are representative of all isolates in each group. PAO1 and purified BHL were included as controls.