Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

Bacterial strains and culture conditions.Investigations were carried out using 43 P. aeruginosa isolates from the sputa of 43 patients attending the adult Royal Prince Alfred Hospital (RPAH) CF Clinic, Sydney, Australia, between 2001 and 2004. The male-to-female ratio of the 43 patients was 21:22. The mean age was 27 years, and the age range was 18 to 58 years (Table 1). These values are consistent with those for the RPAH CF Clinic population as a whole. The study was approved by the Institutional Ethics Committee (approval X02-0320), and subjects provided informed consent for the work. All patients had been chronically infected with P. aeruginosa for a minimum of 4 years. Eighteen of the 43 (42%) isolates were derived from sputa collected at the time of exacerbation, which is defined according to criteria established by Fuchs and colleagues (11), and the remaining samples were obtained during routine clinic visits.

Isolates were routinely identified as being P. aeruginosa by growth on cetrimide fucidin cephaloridine (Oxoid, Adelaide, Australia), acetamide, and Pseudosel agars (both BBL, Liverpool, Australia); a positive oxidase reaction (MedVet Science, Adelaide, Australia); growth on Columbia horse blood agar (Oxoid, Adelaide, Australia) at 42°C; and resistance to 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (Dutec Diagnostics, Croydon, Australia). The identification of isolates showing inconsistent results on these tests was confirmed using the API 20E system. Genotyping using a restriction enzyme, SpeI, followed by pulsed-field gel electrophoresis (PFGE) previously showed that two clones (designated AES-1 and AES-2) infected 40% and 6% of patients, respectively (P. Tingpej, B. Rose, and C. Harbor, unpublished data). In this study, a clonal strain was defined as one showing the same PFGE banding pattern (or a difference of three bands or less) in three or more patients (43). Figure 1 shows DNA banding patterns from AES-1 and AES-2 and nonclonal strains. DNA macrorestriction patterns of AES-1 and AES-2 were found to share 67% similarity. Isolates used in this study were randomly selected from the clonal and nonclonal pools in proportion to their prevalence in the RPAH Clinic (14 AES-1, 5 AES-2, and 24 nonclonal isolates). The well-characterized wound-derived P. aeruginosa PAO1 (17) was included as a control. The AHL reporter strains Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136 were used for AHL assays, and Staphylococcus aureus strain ATCC 12600 was used for the staphylolysin assay. Pseudomonas putida was used as a negative control for dot hybridization. For protease production, bacteria were grown in tryptone soya broth (TSB; Oxoid, Adelaide, Australia) to late exponential phase (optical density at 600 nm [OD₆₀₀] ≈ 1.6) at 37°C with rapid agitation. For ExoS Western blot assays, strains were grown in TSB with 1 mM EGTA to an OD₆₀₀ of 1.0 (20). For AHL analyses, isolates were grown in TSB to late exponential phase or in AB medium supplemented with 0.2% glucose to an OD₆₀₀ of 1.0 (4, 12). A. tumefaciens was grown in supplemented minimal A medium (A+) (29), and C. violaceum was grown in TSB, both at 30°C. Supernatants were collected and filtered through a 0.22-μm filter for protease, exoenzyme, and AHL detection. Cell pellets were collected for gene amplification and dot hybridization studies.

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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.

Gene detection by PCR.DNA was extracted from cell pellets using the microLYSIS kit (Microzone Ltd., Sussex, United Kingdom). PCRs were carried out for the presence of five QS genes, vfr (5′-TGTTCTTCCAGGAGCGTGG-3′/3′-TCGCAAAATCACATCGAC-5′), lasI, lasR, rhlI, and rhlR (45); three protease genes, lasA (26), lasB, and aprA (45); the rhamnolipid gene rhlAB (45); and two exotoxin genes, exoU and exoS (46), as previously described.

Dot blot hybridization.When PCR screening identified a strain that apparently did not possess the lasI or lasR gene, dot blot hybridization was performed to confirm the negative findings. Bacterial cell pellets were washed and resuspended in phosphate-buffered saline to an OD₆₆₀ of 0.2. Digoxigenin-labeled DNA probes for the lasI and lasR genes were generated from the PAO1 strain using the primers for the PCRs (described above) according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany). Bacterial cell suspensions were dot blotted onto a Hybond N⁺ membrane (General Electric Healthcare, Fairfield, CT). Blots were then hybridized using DIG EasyHyb buffer (Roche Diagnostics GmbH, Mannheim, Germany). Hybrids were visualized by standard alkaline phosphatase colorimetric detection. P. putida cells were used as a negative control.

Exoprotease assays. (i) Total protease activity.Total protease activity was determined as described previously (47). The protease activity was normalized to the densities (OD₆₀₀) of the cultures grown. Results were mean values ± standard deviations (SD) from three replicate experiments carried out on three separate occasions.

(ii) Zymography.Gelatin zymography for protease activity was carried out using 10% polyacrylamide gels containing 0.1% (wt/vol) gelatin (Bio-Rad, Regents Park, Australia). Unconcentrated supernatants were denatured in sodium dodecyl sulfate (SDS) sample buffer. Following electrophoresis, gels were washed in 2.5% Triton X-100 (Sigma-Aldrich, MO) to renature proteins, incubated overnight at 37°C to induce protease activity, and then stained with 0.25% Coomassie blue for 2 h with gentle shaking (10). Zymograms were imaged using an Olympus (Tokyo, Japan) C-3040Zoom digital image acquisition system. Protease activities were represented by translucent bands against a Coomassie blue background. All isolates were tested in triplicate on at least three occasions.

(iii) Elastase-specific activity. lasB-mediated elastolytic activity was determined by the elastin-Congo red (ECR) assay as described previously (35). Uninoculated TSB was included as a negative control. All isolates were tested in triplicate on at least two occasions. Results are represented as mean values ± SD.

(iv) Staphylolysin activity. lasA-mediated staphylolytic activity was determined as described previously (23). All isolates were tested in triplicate at least twice, and results shown are mean values ± SD.

Chitinase activity.Chitinase activity was determined using the carboxymethyl-chitin-remazol brilliant violet assay adapted from methods described by the manufacturer (Loewe Biochemica, Munich, Germany) and Folders et al. (9). Remazol brilliant violet liberated by chitinase activity was measured at 550 nm and normalized to the cell density. Uninoculated TSB was used as a negative control. All isolates were tested in triplicate on at least two occasions, and results are represented as mean values ± SD.

Rhamnolipid production.The presence or absence of rhamnolipids was assessed by the rhamnolipid-mediated alkane degradation method (49). PAO1 cultures grown under the same conditions as the test isolates and uninoculated TSB were used as positive and negative controls, respectively.

Western blots for exoenzyme S.Supernatants were boiled for 5 min prior to 12% (wt/vol) nonreducing SDS-polyacrylamide gel electrophoresis (PAGE) at 100 mA for 2 h. Separated protein was electrophoretically blotted at 30 V onto a Tris-glycine buffer-equilibrated polyvinylidene difluoride membrane (Immuno-blot; Bio-Rad, Regents Park, Australia) using the Bio-Rad Mini-Transblot system. Blots were blocked in 5% (wt/vol) bovine serum albumin in phosphate-buffered saline, subsequently incubated overnight with chicken polyclonal exoenzyme S antibody (Abcam, Cambridge, United Kingdom) at a 1:10,000 dilution, and developed in anti-chicken antibody alkaline phosphatase conjugate (1:1,000) (Sigma-Aldrich, MO) using BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate dipotassium-nitrotetrazolium blue chloride) solution (Sigma-Aldrich, MO). Tests were carried out in duplicate on two different occasions.

Thin-layer chromatography.AHLs were identified by analytical thin-layer chromatography (TLC) initially from bacteria grown in TSB as described previously for our studies of microbial keratitis (48). Chromatograms were developed using C₁₈ reversed-phase silica gel TLC plates (Whatmann, Clifton, NJ) with methanol-water (60:40, vol/vol). Plates were overlaid with a thin film of TSB or A+ agar seeded with either C. violaceum CV026 or A. tumefaciens A136 and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (40 μg/ml), respectively. Short-chain AHLs (BHL and HHL) were detected by the production of violacein by C. violaceum CV026, resulting in purple spots at the site of AHL migration. Long-chain AHLs (OdDHL, N-3-oxodecanoyl-l-homoserine lactone [ODHL], and N-3-oxohexanoyl-l-homoserine lactone [OHHL]) were detected by the traG:lacZ/traR reporter in A. tumefaciens A136, inducing β-galactosidase expression and the development of blue/green dots at the site of AHL migration. HHL was also detected using the A. tumefaciens reporter. The purified BHL, HHL, and OdDHL used as positive controls were kindly supplied by Naresh Kumar (Department of Chemistry, The University of New South Wales, Sydney, Australia). It was reported previously that P. aeruginosa strains from lungs of CF patients produce the highest amounts of long-chain AHL in AB medium containing 0.2% glucose (12). Thus, bacteria that tested negative for long-chain AHLs when grown in TSB medium were retested following culture in AB medium supplemented with 0.2% glucose. All samples were tested at least three times.

Statistical analyses.Pearson's chi-square test was used to analyze the secretion of the virulence factors and AHL molecules between clonal and nonclonal groups. P values of <0.05 were considered to be significant. Relative risk (RR) and 95% confidence intervals (CIs) were used to determine the strengths of the associations. The levels of virulence factor production were analyzed using a standard t test for normally distributed data and using the Mann-Whitney U test for nonparametric data. Relationships between protease, elastase activity, and patient age were calculated using Pearson's correlation coefficient (r).

Mucoidy.The degree of mucoidy of the isolates was assessed at isolation on horse blood agar by visual inspection and designated as negative, +(low), ++(medium), or +++(high). Forty-one of the 43 (95%) isolates were mucoid. Of these, nine (21%) showed + mucoidy, 25 (58%) showed ++ mucoidy, and seven (16%) showed +++ mucoidy (Table 1). All colonies of each mucoid isolate were mucoid.

Presence of virulence factor and QS genes.All 43 isolates produced bands of the predicted sizes in the vfr, rhlI, rhlR, lasA, lasB, aprA, and rhlAB PCRs. One isolate (nonclonal) was negative for both lasI and lasR. The absence/mutation of both genes was confirmed by dot hybridization. There was no evidence of large-scale insertions or deletions within the amplified regions of any of the genes. All 43 samples were positive for the exoS gene and negative for the exoU gene.

Exoprotease production.Thirty-seven of the 43 (86%) isolates produced positive results in the total protease assay. Levels were variable and in some cases very low (Table 1). SDS-10% PAGE zymography produced a protease profile of up to three bands for each sample (Fig. 2). Bands were detected at molecular masses of approximately 120 kDa, 98 kDa, and 51 kDa. Elastase and staphylolysin were represented by bands at 120 kDa and 98 kDa, respectively, and alkaline protease was represented by the band at 51 kDa (48). In some samples, there were two bands at 51 kDa. Although the protease IV enzyme has a molecular mass of 26 kDa, it aggregates under mild SDS denaturing conditions and is resolved at a molecular mass of 350 kDa (4). It was therefore barely detectable on the 10% zymograms used in this study. Twenty-four of the 43 (56%) isolates produced elastase/staphylolysin, and 22 of 43 (51%) isolates produced alkaline protease (Table 2). The amount of protease secreted by individual isolates as indicated by band intensity was variable. Thirty-two of the 43 (74%) isolates exhibited elastase activity using the ECR assay. Levels ranged from 3 mU/ml to 367 mU/ml (Table 1). All 24 isolates that produced elastase on zymography also showed elastase activity in the ECR assay. Eight isolates that were negative by zymography showed low levels of activity using the ECR assay. As expected, there was a strong correlation between total protease and elastase activities (r = 0.847; P < 0.001). Forty of the 43 (93%) isolates produced staphylolysin, but activities were generally low, ranging from 1 mU/ml to 91 mU/ml (Table 1).

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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.

Chitinase activity and rhamnolipid production.Thirty-four of the 43 (79%) isolates showed chitinase activity; levels ranged from 6 mU/ml to 168 mU/ml (Table 2). Thirty-five of the 43 (81%) isolates produced rhamnolipids (Table 2).

Exoenzyme S production.Thirty-three of the 43 (77%) isolates grown in type III secretion system-inducing low-calcium medium showed a single band of 36 kDa corresponding to the ExoS protein on the Western blots. The intensities of the bands were variable. Results for individual isolates are shown in Table 1.

AHL production.When results from the two AHL biosensors were combined, the number of spots varied from none to five. Thirty-two of the 43 (74%) samples produced spots corresponding to one or more AHLs (Table 2). Using the reporter strain C. violaceum CV026, two spots, most likely representing BHL and HHL (12, 47), were noted. Thirty-one of the 43 (72%) isolates produced rhl-associated AHLs. Of these isolates, 19 (44%) produced a spot corresponding to both HHL and BHL (Fig. 3), and 12 (28%) had a spot corresponding to HHL but not BHL. Spot densities and diameters varied among isolates. Using the A. tumefaciens A136 reporter strain that detects las-directed AHLs with a high degree of sensitivity (37), up to four spots were detected, most likely corresponding to OdDHL, ODHL, HHL, and OHHL (47). Following growth in the TSB medium used previously in our microbial keratitis study (47), at least one las-directed AHL was detected in 16 (37%) isolates. Of these isolates, OdDHL was detected in 8 isolates, ODHL was detected in 11 isolates, and OHHL was detected in 6 isolates. Three additional isolates yielded OHHL spots in assays using supplemented AB medium. The HHL results using the A. tumefaciens A136 reporter were consistent with those using the C. violaceum CV026 reporter. As expected, the isolate that tested negative for the lasI and lasR genes had undetectable levels of AHL.

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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.

Overall relationships between phenotypic and patient characteristics.Isolates with positive results for one or both rhl-associated AHLs were significantly more likely to show total protease (RR = 1.7 [CI = 1.0 to 2.7]; P = 0.004) and elastase (RR = 5.8 [CI = 1.6 to 20.6]; P = 0.006) activities than those testing negative for these AHLs. The levels in each case were also significantly higher (both P < 0.01). However, when the las-directed AHL data were included, the associations between AHL and protease production were no longer significant. There were no relationships between the production of proteases or AHLs and exoenzyme S, rhamnolipids, and chitinase and no association between mucoidy and other phenotypic characteristics. There was a trend for decreasing protease and elastase activity with increasing patient age, but the relationship reached statistical significance for elastase only (r = −0.34; P = 0.03). Patient age may provide some indication of the duration of infection. There were no relationships between exacerbation and any of the phenotypic characteristics.

Clonal versus nonclonal groups.Data for AES-1 and AES-2 were combined for these analyses because of small numbers. The male-to-female ratios in the combined clonal versus nonclonal groups were similar, 9:10 and 12:12, respectively, as were the mean ages (and age ranges), 25 (18 to 37) and 29 (19 to 58) years, respectively. A greater proportion of nonclonal isolates produced +++ mucoidy than clonal isolates (7 of 24 versus 0 of 19), but proportions of isolates with mucoidy at the + and ++ levels were similar between clonal and nonclonal groups, and there was no overall relationship between mucoidy and genotype. There was no significant difference in the probability of having a clonal strain among patients with exacerbation compared with those without (7 of 18 [39%] versus 12 of 25 [48%]).

As shown in Table 2, clonal isolates were significantly more likely than nonclonal isolates to produce proteases including elastase, alkaline protease, and total protease. Clonal isolates also tended to have greater total protease and elastase activities than nonclonal isolates, although the differences were not statistically significant in this relatively small sample.

There was no difference in relationships between patient age and total protease production in the clonal versus nonclonal groups. Elastase levels were more likely to decrease with increasing patient age in the nonclonal group (r = −0.38; P = 0.07) than in the clonal group (r = −0.08, P = 0.75), but this relationship did not reach statistical significance. Clonal isolates were also more likely to have at least one detectable AHL and to exhibit BHLs/HHLs, but these trends did not reach statistical significance. There were no relationships between genotype and the production of ExoS, rhamnolipids, or chitinase. Among the nonclonal group, there was a significant correlation between elastase production and mucoidy (r = 0.46; P = 0.03). This relationship was not evident in the clonal group, but these findings should be interpreted with caution because of the limited sample size and because mucoidy was evaluated visually rather than by quantitation.