Abstract

BackgroundPseudomonas aeruginosa (PA) strains with defective DNA mismatch repair genes generate numerous bacterial variants because of high mutation rates. In cystic fibrosis (CF), such mutator strains may lead to the rapid selection of survivors that are specifically adapted to the hostile environment of the inflamed CF lung

MethodsGenotypes and phenotypes of 111 PA variants descending from 3 distinct mutator strains obtained from 3 patients with CF were systematically characterized

ResultsWe demonstrated that PA mutS isolates accumulated in the CF lung during the observation period of 3–6 years, with dominance during the final stage of the disease. Mutator strains from the final stage of disease were multiresistant and had lost a set of established virulence-associated traits, including cytotoxicity for bronchial epithelial cells (Calu-3) and macrophages (J774). This pathoadaptation was associated with the loss of survival capacity in a typical environmental habitat, such as tap water. Strikingly, nonmutator strains that maintained their virulence potential persisted as a minority, probably with a preference for the lower airways

ConclusionsMutator strains may evolve from the initially infecting PA strain and generate numerous variants with a loss of destructive virulence factors, probably because of selection for improved survival in the deteriorated CF lung but at the expense of the ability to live freely

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. CFTR dysfunction leads to the secretion of a hyperosmolar, viscous mucus in the respiratory tract accompanied by an impaired anti-infective defense and the promotion of chronic lung infections, predominantly by Pseudomonas aeruginosa (PA) [1]. The ensuing pulmonary inflammation results in a progressive lung disease with ongoing tissue destruction caused by bronchitis, bronchiectasis, and exudate-plugged airways. During the end stage of lung disease, PA has been found in the most severely damaged airways attached to denuded membranes or in exudates as multicellular aggregates but rarely in deeper lung tissue [2]. The preceding lung disease is characterized by numerous focal inflammatory areas of different stages distributed all over the respiratory tract. The dynamics of physiological and histological changes in the CF lung appear to be closely related to the microbial colonization that typically begins with Staphylococcus aureus and Haemophilus influenzae and terminates with PA [3]

Recently, the occurrence of hypermutable PA, H. influenzae and S. aureus has been reported in patients with CF [4–7]. Various mutations in the PA mismatch repair system in the mutator genes mutS, mutL and uvrD have been identified [8]. A potential benefit of mutator strains during chronic lung infections is their enhanced adaptation rate to the ecological heterogeneity of the inflamed CF lung. Likewise, PA mutator strains have been reported in chronic respiratory diseases of non-CF origin [9]. In patients with CF, a frequency of PA mutator carriers of 36% has been found [4]. Strikingly, the impact of mutator strains on progressive CF lung disease has been poorly examined. This prompted us to retrospectively characterize sequential PA isolates from 3 patients with CF, each of whom harbored a PA mutS mutator with a distinct mutS frameshift mutation, during their last 3–6 years of life (the advanced stage of CF disease). The major aims of the present study were to (1) characterize the relationship of mutator status to genetic relatedness during ongoing colonization, by analyzing the clonality of sequential PA isolates and clone-specific mutS mutations in a patient; (2) phenotypically characterize mutator and nonmutator isolates with respect to a set of virulence-associated traits (VATs), amino acid auxothropy (Aux), and cytotoxicity; and (3) investigate the loss of traits required for survival in the environment, to better define the development of the microbial phenotype during colonization of the CF lung

Patients and Methods

PatientsIn patient M (born in 1967), chronic PA lung infection had been documented since 1997. He died at age 34 years, and during his last 7 months of life, coinfection with Stenotrophomonas maltophilia was reported. In patient V (born in 1972), PA colonization had been documented since 1992. Moreover, S. aureus was intermittently isolated from respiratory secretions during 1997–2000, and S. maltophilia had been cultured since 2001. This patient was on the wait list for lung transplantation but died before a donor lung was found, at age 30 years. In patient P (born in 1975), PA colonization had been documented since 1987. Patient P underwent lung transplantation in 2003. Besides PA, Achromobacter xylosoxidans had been repeatedly isolated since 1997. After transplantation, this patient’s clinical course was complicated, and she died in 2004. At that time, PA, A. xylosoxidans and S. aureus were cultured from respiratory secretions. The present study was performed in accordance with the guidelines of the ethics commission of the University of Munich

Bacterial strainsPA strains PAO1 (provided by L. Eberl, Department of Microbiology, Institute of Plant Biology, University of Zürich, Zürich, Switzerland) and PAN10 (lasB; provided by J. Thommassen, Department of Molecular Microbiology, Utrecht University, Utrecht, The Netherlands) were used as control strains. The carriage of PA mutS mutator strains of patients M, V, and P and PAO1 (mutS) has been described elsewhere [10]. PA isolates from these patients were collected during routine microbiological testing from 1998 to 2004, and distinct PA morphotypes from single specimens were collected and colony-forming units calculated from dilutions of 10−1, 10−4, and 10−5 plated on tryptone soy agar and MacConkey agar and stored at −80°C [11]

Genotypic assaysPulsed-field gel electrophoresis (PFGE) of PA using SpeI was performed as described elsewhere [12]. The relatedness of macrorestriction patterns was assessed visually using the Dice coefficient, SD [12]. Independent PFGE patterns (SD<0.75) representing different clones were assigned an arbitrary uppercase letter. Slightly differing PFGE patterns with an SD⩾0.75 were considered to be members of a clonal lineage and were assigned the same uppercase letter and different numbers in the order of enumeration. Random amplified polymorphic DNA (RAPD)–polymerase chain reaction (PCR) was performed using primer 208 [13]. RAPD patterns with an SD>0.8 (Quantity one 1-D software; Bio-Rad) were considered to be genetically related according to PFGE but were assigned numbers followed by lowercase letters. The identification of mutS frameshift mutations was performed by sequencing a mutS subfragment that included the mutS null mutation specific for the PA clone from patients M, V, and P (17-bp deletion, 8-bp insertion, and 1-bp insertion, respectively)

Phenotypic assaysAntimicrobial susceptibility testing was performed by agar dilution in accordance with British Society for Antimicrobial Chemotherapy break points: ⩽1, 2, and ⩾4 μg/mL for ciprofloxacin and ⩽32 and ⩾64 μg/mL for fosfomycin [14]. The determination of the frequency of rifampicin mutation has been described elsewhere, and, for mutator strains, a mutation frequency at least 20-fold higher than that of PAO1 was recommended [4]. The proteolytic phenotype (Prot) of PA was visualized on 10% skim-milk agar streaked with 5 μL of culture grown overnight in peptone trypticase soy broth (PTSB) and incubated for 18 h at 36°C and for 24 h at room temperature. Prot was scored according to proteolysis zone: ++, >5 mm; +, 2–5 mm; (+), <2 mm; and −, no proteolysis [15]. The elastolytic activity of PTSB culture supernatants was determined with elastin–Congo red (ECR; Sigma) [16]. The elastolytic phenotype (Ela) of PA strains with ECR solubilization less than that of PAN10 (lasB) was classified as negative

Pyocyanin extraction from supernatants of PA grown overnight was performed as described elsewhere [17]. The pyocyanin phenotype (Pyo) was reported as follows: +, OD520>0.8; (+), OD520=0.8–0.2; and −, OD520<0.2. The Cas phenotype was assessed by spotting 10 μL of PA culture onto chrome azurol S (CAS) agar; this indicated probable siderophore production by PA [18, 19]. A Cas phenotype was assumed if the ratio of halo diameter to colony diameter was <1. Cas+ phenotypes were scored by halo zone: +, ⩾5 mm; and (+), <5 mm. The pyoverdin phenotype (Pyv) was assessed on Pseudomonas agar F (Difco) incubated for 18 h at 36°C and for 24 h at room temperature

The swimming motility phenotype (Smo) was determined by inoculating 2 μL of PA culture onto 0.3% Luria-Bertani (LB) agar and estimating the swarm zone (+, >25 mm; −, <10 mm) [20]. PA isolates were checked for Aux for 20 common amino acids (Sigma), using M9 minimal agar, as described elsewhere [21]. The detection of the translocation component Pseudomonas outer protein (Pop)–D (anti-PopD was provided by A. M. Schreff, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich) and of exotoxin (Exo)–S of the PA type III secretion system (T3SS) from whole-cell lysates and supernatants was conducted by immunoblotting [22, 23]. ExoS- and PopD-immunostained bands were scored visually as +, (+), and −. Growth rates of PA in tap water were determined by inoculating PA into sterile-filtered tap water, incubating at 21°C (room temperature) and 37°C, and plating of serial dilutions of a 500-μL aliquot onto LB agar

Cytotoxicity assaysThe Calu-3 bronchial epithelial cell line (ATCC HTB-55; provided by R. Bals, Department of Internal Medicine, Division for Pulmonary Diseases, Hospital of the University of Marburg, Philipps-Universtat Marburg, Marburg, Germany) was maintained in MEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L sodium pyruvate, 1% nonessential amino acids, and 10% fetal calf serum (FCS). The J774 murine macrophage–like cell line (ATCC TIB-67) was cultivated in RPMI 1640 that contained 2 mmol/L l-glutamine, 10% FCS, and penicillin/streptomycin (Gibco-BRL Life Technologies). Cells were seeded in 96-well plates at 1×104 and 0.35×104 cells/well, respectively, and were infected with PA at an MOI of 100:1, centrifuged at 30 g for 5 min, and incubated for 4 h at 37°C and 5% CO2. Cells were washed once with PBS before the addition of culture medium with 400 μg/mL tobramycin. Cell viability was analyzed using the methyltiazole tetrazolium (Sigma) assay 5 and 20 h after infection to determine rapid cytotoxicity (r-Tox) and delayed cytotoxicity (d-Tox) [24]

Results

Study designIn the context of routine microbiological monitoring, respiratory samples were plated, and all PA colonies with a different morphotype (PA variants) were collected and counted. Recently, we screened PA isolates from 100 patients with CF for mutator phenotype using the rifampicin-resistance selection technique, and we identified mutators in 15 patients with CF, which were checked for mutations in the mutS gene [10]. Three patients (randomly termed M, V, and P) with 3 different PA mutS mutator strains and advanced-stage lung disease were chosen for the present retrospective study. During 1998–2004, we collected 111 PA isolates from these patients with CF—26 from patient M (table 1), 42 from patient V (table 2), and 43 from patient P (table 3)—and subjected them to genotypic and phenotypic analysis

Table 1

Pseudomonas aeruginosa isolates from patient M

Table 1

Pseudomonas aeruginosa isolates from patient M

Table 2

Pseudomonas aeruginosa isolates from patient V

Table 2

Pseudomonas aeruginosa isolates from patient V

Table 3

Pseudomonas aeruginosa isolates from patient P

Table 3

Pseudomonas aeruginosa isolates from patient P

Genotyping of PAThe clonality of these 111 isolates was checked by PFGE and RAPD-PCR (figure 1A–1C and tables 1–3). PA isolates from all 3 patients exhibited distinct PFGE and RAPD typing profiles, which indicates that these strains were epidemiologically unrelated. Four major PFGE patterns (clones A–D) were identified. PA clones A and B were isolated from patient M, whereas patient V and P each carried a single clone denoted as clone C and D, respectively. Strikingly, clone B from patient M disappeared during the observation period, whereas clone A, which represented the mutator lineage, persisted. RAPD revealed 4 major patterns assigned to RAPD types 1–4 that corresponded to clones A–D

Figure 1

Pulsed-field gel electrophoresis analysis of sequential Pseudomonas aeruginosa (PA) isolates from patients M (A) V (B) and P (C). SpeI macrorestriction patterns of isolates M2 (belonging to clone B), M1, M4, M5, M11, M13, M19, M20, and M21 (belonging to clone A) of patient M; isolates V1, V2, V8, and V34 of patient V; and isolates P1, P2, P3, P5, P7, and P12 of patient P belonging to clone C and D, respectively, are shown. The denotation of different PA subclones of clones A, C, and D is indicated above. Molecular weight marker (MW), λ-ladder, size 1018.5–48.5 kb (Bio-Rad)

Figure 1

Pulsed-field gel electrophoresis analysis of sequential Pseudomonas aeruginosa (PA) isolates from patients M (A) V (B) and P (C). SpeI macrorestriction patterns of isolates M2 (belonging to clone B), M1, M4, M5, M11, M13, M19, M20, and M21 (belonging to clone A) of patient M; isolates V1, V2, V8, and V34 of patient V; and isolates P1, P2, P3, P5, P7, and P12 of patient P belonging to clone C and D, respectively, are shown. The denotation of different PA subclones of clones A, C, and D is indicated above. Molecular weight marker (MW), λ-ladder, size 1018.5–48.5 kb (Bio-Rad)

Moreover, all mutator isolates from patients M, V, and P carried the clone-specific mutS null mutation (tables 1–3). In addition, we checked the percentage of mutator phenotypes in a single sputum sample from each patient (from patient M in September 2001, from patient V in November 2002, and from patient P in August 2003). From each sputum sample, 30 single PA colonies were randomly isolated and checked for the clone-specific mutS mutation. All PA colonies isolated belonged to the respective mutS mutator group, which indicates that PA mutators predominated in these sputum samples

In summary, genomic fingerprinting and mutS gene analysis indicated that, during their last years of life, all 3 patients were chronically infected with subclonal variants descended from 1 respective clone. Because nonmutator strains preceded the mutator strains in patients M and V and mutator and nonmutator isolates were concomitantly detected in patient P, we conclude that mutator strains evolved from a clonally related nonmutator strain within CF airways. The increase in the frequency of mutator isolates in subsequent samples showed that mutator variants persisted and accumulated in the lung during ongoing disease. During end-stage lung disease, PA isolates with a nonmutator phenotype were a minority in sputum samples (tables 1–3); however, nonmutator isolates M23, V17, V18, P34, P37, P40, P41, and P42 were recovered from bronchoalveolar lavage (BAL) samples

Phenotypic characterization of PAWe characterized PA isolates from the 3 patients with respect to antibiotic resistance, mucoidy, a set of established VATs, Aux, cytotoxicity, and survival in tap water. As to be expected, the rates of antibiotic resistances of PA isolates tended to increase with ongoing lung disease. Surprisingly, the majority of PA isolates were found to be nonmucoid, and there were no significant differences in mucoidity between mutator and nonmutator strains for clones A, C, and D (tables 1–3)

VATsThe PA isolates were characterized according to 6 well-established VATs: Ela, Prot, Pyo, Pyv, Smo, and the functionality of T3SS (secretion of ExoS) were assessed for (1) all PA clone A isolates from patient M; (2) selected nonmutator and isogenic mutators from patients V and P, respectively; and (3) PA isolates from patient P cultured from the last respiratory sample obtained after lung transplantation (table 4)

Table 4

Phenotypic characteristics of Pseudomonas aeruginosa (PA) nonmutator (NM) and mutator (M) isolates from patients with cystic fibrosis

Table 4

Phenotypic characteristics of Pseudomonas aeruginosa (PA) nonmutator (NM) and mutator (M) isolates from patients with cystic fibrosis

In summary, mutS isolates from patients M, V, and P recovered during the final stage of CF lung disease (late mutators) showed a distinct down-regulation or even loss of most VATs: the CAS phenotype was substantially reduced or negative in all late mutator isolates from the 3 patients and correlated with the decrease in fluorescence on Pseudomonas agar F, which indicates impaired pyoverdin production. ExoS production and secretion were impaired in almost all mutators and decreased gradually among isolates from patient M that were high, low, and non-ExoS producers (figure 2). PA isolates from patient M with reduced ExoS production also showed decreased PopD expression, which suggests a down-regulation of the entire ExoS-regulon probably that was caused by defects in the T3SS regulatory loop

Figure 2

Exotoxin (Exo)–S and Pseudomonas outer protein (Pop)–D immunoblot analysis in whole cell lysate (CL) and supernatant (SN) of PA clone A isolates of patient M using monoclonal anti-ExoS or anti-PopD. The blots were developed using enhanced chemiluminescence (Amersham)

Figure 2

Exotoxin (Exo)–S and Pseudomonas outer protein (Pop)–D immunoblot analysis in whole cell lysate (CL) and supernatant (SN) of PA clone A isolates of patient M using monoclonal anti-ExoS or anti-PopD. The blots were developed using enhanced chemiluminescence (Amersham)

Strikingly, nonmutator isolates from the 3 patients with chronic CF disease also lost various VATs but to a lesser extent than their isogenic mutator descendants: the 11 nonmutators from patient P were Pyo but showed high protease activity and high pyoverdin production (Prot+, Pyv+; data shown for P3, P4, P9, and P40–42). The 8 nonmutator isolates from patient M (clone A) were Ela but Prot+, Pyo+, and Pyv+. Among the 12 nonmutator isolates from patient V, the expression of VATs was variable, but each nonmutator maintained at least 3 of 6 VATs (data shown for V4, V5, V11, and V35). In conclusion, during chronic CF lung disease, different variants of PA expressing distinct sets of VATs occur, whereas late mutS mutators appear to converge to an avirulent phenotype because of the loss of larger sets of VATs

Cytotoxicity of PAThe cytotoxicity of PA is a complex phenotype that involves different virulence factors. We investigated the cytotoxic effects of early nonmutators and late mutators on Calu-3 and J774 cells (table 4). J774 macrophages were more susceptible to PA infection than were Calu-3 cells, which allowed the differentiation between r-Tox+ and d-Tox+ strains. By contrast, the toxicity phenotypes of r-Tox and d-Tox for Calu-3 cells were not significantly changed (only r-Tox Calu-3 data are shown)

In patient M, with the exception of 1 nonmutator isolate (M23), nonmutator and mutator isolates did not show r-Tox for Calu-3 cells. Mutator strains showed no d-Tox for J774 macrophages, whereas nonmutator strains did. In patient V, mutator strains subsequently lost r-Tox for Calu-3 cells and diminished d-Tox for J774 macrophages during the time of investigation, whereas nonmutator strains remained cytotoxic for both cell types. In patient P, mutator isolates were less cytotoxic with respect to r-Tox for Calu-3 cells and d-Tox for J774 macrophages than were nonmutator isolates at the early and late time points that were investigated. Thus, we were able to distinguish among 3 major cytotoxic groups of CF isolates: (1) the rapid cytotoxic group: strains causing a cytotoxic effect on Calu-3 and J774 cells (r-ToxCalu-3); (2) the delayed cytotoxic group: strains with a noncytotoxic effect for Calu-3 cells but delayed cytotoxicity for J774 cells (d-ToxJ774); and (3) the noncytotoxic group (n-Tox): final mutator isolates from patients M, V, and P

Survival of PA in tap waterWe assessed whether PA late mutator isolates were able to survive as free-living bacteria in an environment such as tap water (figure 3). After the transfer of PA from LB medium to tap water, the colony-forming units of all strains decreased during the first 12 h, probably as a consequence of the challenging nutritional deprivation and hypo-osmolarity of tap water. Afterward, PA PAO1 and PAO1 (mutS) showed an exponential increase in colony-forming units and entered the stationary phase at ∼50 h (figure 3A and 3B). As expected, PAO1 (mutS) and the PAO1 wild type exhibited similar growth curves. The PA CF isolate M1 (nonmutator), which was cultivated in parallel, showed a pronounced initial decrease in colony-forming units and a prolonged lag phase before colony-forming units exponentially increased. By contrast, mutator isolates M24, M25, and M26 (results are shown for M25 and M26) exhibited a continuous decrease in colony-forming units after inoculation into tap water; after 145 h, no cultivable bacteria were detected. The Aux of strains M24 to M26 might be responsible for their inability to grow and survive in tap water (table 4). Strikingly, comparable results were found for isogenic nonmutator and mutator isolates from patient V (V11 vs. V37 and V43) and patient P (P9 and P42 vs. P30). No cultivable bacteria were obtained from mutator isolates V43 and P30 after 144 h, although both exhibited a prototrophic phenotype according to the 20 amino acids tested. The prototrophic late mutator isolate P39 was able to survive in tap water for up to 200 h but at lower colony-forming units than its nonmutator variants, P9 and P42. These data suggest that, in contrast to PA nonmutators, mutators are less able to efficiently survive in tap water as cultivable bacteria independent of Aux, probably because of an extensive metabolic adaptation of mutators to the unique habitat of the CF lung and counterselection for the ability to be free-living

Figure 3

Survival of isogenic Pseudomonas aeruginosa (PA) mutator and nonmutator isolates in sterile-filtered tap water at 37°C (A) and room temperature (RT) (B–D). Luria-Bertani–grown bacterial cells were harvested by centrifugation, washed with PBS, inoculated in 200 mL of tap water, and slightly shaken at 50 rpm in 500-mL Erlenmeyer flasks. A and B PAO1 (black diamonds) and PAO1 (mutS) (black squares) as controls and CF isolates from patient M: M1 (white triangles) M25 (mutS) (white circles) and M26 (mutS) (asterisks). C Isolates from patient V: V11 (white triangles) V37 (mutS) (asterisks) and V43 (mutS) (white circles). D Isolates from patient P: P9 (white triangles) P30 (mutS) (white circles) P39 (mutS) (asterisks) and P42 (black triangles)

Figure 3

Survival of isogenic Pseudomonas aeruginosa (PA) mutator and nonmutator isolates in sterile-filtered tap water at 37°C (A) and room temperature (RT) (B–D). Luria-Bertani–grown bacterial cells were harvested by centrifugation, washed with PBS, inoculated in 200 mL of tap water, and slightly shaken at 50 rpm in 500-mL Erlenmeyer flasks. A and B PAO1 (black diamonds) and PAO1 (mutS) (black squares) as controls and CF isolates from patient M: M1 (white triangles) M25 (mutS) (white circles) and M26 (mutS) (asterisks). C Isolates from patient V: V11 (white triangles) V37 (mutS) (asterisks) and V43 (mutS) (white circles). D Isolates from patient P: P9 (white triangles) P30 (mutS) (white circles) P39 (mutS) (asterisks) and P42 (black triangles)

Discussion

The increase in the occurrence of hypermutable PA isolates among patients with CF implies that genetic and phenotypic diversification plays an important role in the rapid adaptation of PA by selection to the hostile and heterogeneous environment of the CF lung [4, 5, 25]. This could be also demonstrated when an in vitro–constructed PAO1 mutS mutant was used [26]. To systematically elucidate the phenotypic diversification of PA mutS mutator strains during chronic CF disease, we characterized, morphologically and phenotypically, PA isolates that had been recovered from the airways of 3 patients with CF during their last 3–6 years of life. These patients were found to carry different clones of PA mutator strains with distinct mutS frameshift mutations [10]

Genetic typing of sequential PA strains was performed using both RAPD-PCR and PFGE, and results showed that, during the late stage of disease, each patient was persistently infected by 1 PA clone (clone A, C, and D, respectively). Sequential PA isolates exhibited minor genomic variations displayed by restriction fragment–length polymorphism and RAPD subtypes, which suggested slight genomic diversification. Moreover, we detected isogenic PA mutS mutator and nonmutator isolates from all 3 patients and demonstrated an increase in the proportion of PA mutator isolates in CF sputum accompanying prolonged lung colonization by this pathogen. This suggested that the mutator strains evolved within CF airways and argued for a selective advantage of mutator strains during end-stage chronic lung infection. Nevertheless, among all 3 patients, a low proportion of PA nonmutator strains was found in sputum, whereas a higher ratio seemed to be recovered from BAL samples. This suggests that nonmutator and mutator strains coexist in the lung for long periods during advanced CF disease, possibly with preference in the lower respiratory tract

The phenotypic characterization of 102 PA isolates of clones A, C, and D (mutator lineages) demonstrated a broad phenotypic variability that was consistently associated with a reduction in VATs. With the progression of lung deterioration, the phenotypic diversification of mutS strains in all 3 patients converge on the following pattern of strongly attenuated phenotypes (reductive adaptation): Prot, Pyo, Pyv, CAS, Smo, T3SS, r-Tox, and d-Tox (or distinctly reduced d-Tox). Multiple antibiotic resistance emerged mainly with late mutator isolates but was also found for nonmutator isolates P34, P37, and P41–P43 and was thus independent of the mutator state. Genetic and phenotypic adaptation of PA chronically colonizing the CF lungs also occur independently of a constitutive mutator phenotype caused by mutations and, when possible, by recombination [27, 28]. Transient hypermutability is conceivable under stressful environments, possibly because of a RpoS-dependent down-regulation of mutS expression [29–31]. In addition, large-scale chromosomal rearrangements have been found in PA isolates from patients with CF

Interestingly, the in vitro infection of bronchial epithelial cells and macrophages with PA allowed us to discriminate among different cytotoxic variants of PA. The r-ToxCalu-3 pathotype with rapid cytotoxicity on Calu-3 and J774 cells was found mainly in nonmutator isolates and in the early mutator V7. By contrast, all other isolates listed in table 4 were impaired in their cytotoxicity, showing either a d-ToxJ774 or n-Tox trait. In conclusion, the losses of VATs and of in vitro cytotoxicity among mutator isolates were closely associated and probably demonstrate causality. Of note, only d-ToxJ774–positive isolates all produced remarkable amounts of pyocyanin, which suggests a responsibility of this agent for this phenotype, as has been reported for macrophages and neutrophils [32]

The loss of VATs of mutS strains was found to be associated with a failure to survive in tap water. This could be partially correlated with the Aux identified in some mutator isolates. The delay in the increase in numbers of cells in tap water of nonmutator isolate M1, compared with PAO1 and PAO1 (mutS) suggests that this strain also partially adapted to the CF lung at the cost of its ability to live freely. This kind of pathoadaptation may prevent the transmission of airway-selected variants through aqueous environments such as showers or swimming pools, which could be the only benefit for patients with CF

In summary, our study of the phenotypic diversification of mutator and nonmutator isolates of PA obtained from 3 different patients with CF infected with different clones that had distinct mutS defects provides evidence for the rapid adaptation of PA to the CF airways by the rapid generation of phenotypic and cytotoxic variants caused by hypermutability of the mutator phenotype. The intrapulmonary pathoadaptation leads to PA strains during the final stage of CF that have consistently lost typical VATs and the cytotoxicity of parental PA strains. In agreement with these results, 2 more recently reported mutS mutator strains, HM7 and HM11, showed a pathoadapted phenotype with reduced cytotoxicity (cell viability for r-ToxCalu-3, 78.6% and 89.6%; and for d-ToxJ774, 71.4% and 57.5%, respectively) (not included in table 4) [10]. The impact on the adaptation of PA in lungs of other CF pathogens, such as S. aureus, S. maltophilia and A. xylosoxidans that were recovered from respiratory specimens at lower proportions than PA but that still constitute part of the selective lung environment, is unclear. The set of sequential isolates of nonmutator and mutator strains from the 3 patients with CF provides us with well-characterized strains for studying changes in transcriptomes, proteomes, and metabolomes to elucidate the adaptation process and the characteristics of surviving PA isolates on a molecular level. Such investigations are currently under way in our laboratory

Acknowledgments

We thank K. Adler and M. Götzfried, for expert technical assistance; G. Maydl, for helpful technical advice on restriction fragment–length polymorphism; and our clinical colleagues, particularly M. Kappler and P. Latzin (Dr. von Haunersche Kinderklinik, Munich, Germany) and R. Fischer (Medizinische Klinik-Innenstadt, Munich, Germany), for providing cystic fibrosis specimens and clinical data

References

1
Lyczak
JB
Cannon
CL
Pier
GB
Lung infections associated with cystic fibrosis
Clin Microbiol Rev
 , 
2002
, vol. 
15
 (pg. 
194
-
222
)
2
Baltimore
RS
Christie
CD
Smith
GJ
Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis: implications for the pathogenesis of progressive lung deterioration
Am Rev Respir Dis
 , 
1989
, vol. 
140
 (pg. 
1650
-
61
)
3
Gilligan
PH
Microbiology of airway disease in patients with cystic fibrosis
Clin Microbiol Rev
 , 
1991
, vol. 
4
 (pg. 
35
-
51
)
4
Oliver
A
Canton
R
Campo
P
Baquero
F
Blazquez
J
High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection
Science
 , 
2000
, vol. 
288
 (pg. 
1251
-
4
)
5
Ciofu
O
Riis
B
Pressler
T
Poulsen
HE
Hoiby
N
Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation
Antimicrob Agents Chemother
 , 
2005
, vol. 
49
 (pg. 
2276
-
82
)
6
Watson
ME
Jr
Burns
JL
Smith
AL
Hypermutable Haemophilus influenzae with mutations in mutS are found in cystic fibrosis sputum
Microbiology
 , 
2004
, vol. 
150
 (pg. 
2947
-
58
)
7
Prunier A-L
Malbruny
B
Laurans
M
Brouard
J
Duhamel J-F
High rate of macrolide resistance in Staphylococcus aureus strains from patients with cystic fibrosis reveals high proportions of hypermutable strains
J Infect Dis
 , 
2003
, vol. 
187
 (pg. 
1709
-
16
)
8
Oliver
A
Baquero
F
Blazquez
J
The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants
Mol Microbiol
 , 
2002
, vol. 
43
 (pg. 
1641
-
50
)
9
Macia
MD
Blanquer
D
Togores
B
Sauleda
J
Perez
JL
Hypermutation is a key factor in development of multiple-antimicrobial resistance in Pseudomonas aeruginosa strains causing chronic lung infections
Antimicrob Agents Chemother
 , 
2005
, vol. 
49
 (pg. 
3382
-
6
)
10
Hogardt
M
Schubert
S
Adler
K
Gotzfried
M
Heeseman
J
Sequence variability and functional analysis of MutS of hypermutable Pseudomonas aeruginosa cystic fibrosis isolates
Int J Med Microbiol
 , 
2006
, vol. 
296
 (pg. 
313
-
20
)
11
Hogardt
M
Trebesius
K
Geiger
AM
Hornef
M
Rosenecker
J
Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients
J Clin Microbiol
 , 
2000
, vol. 
38
 (pg. 
818
-
25
)
12
Romling
U
Fiedler
B
Bosshammer
J
, et al.  . 
Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis
J Infect Dis
 , 
1994
, vol. 
170
 (pg. 
1616
-
21
)
13
Mahenthiralingam
E
Campbell
ME
Foster
J
Lam
JS
Speert
DP
Random amplified polymorphic DNA typing of Pseudomonas aeruginosa isolates recovered from patients with cystic fibrosis
J Clin Microbiol
 , 
1996
, vol. 
34
 (pg. 
1129
-
35
)
14
Andrews
JM
BSAC Working Party on Susceptibility Testing
BSAC standardized disc susceptibility testing method (version 5)
J Antimicrob Chemother
 , 
2006
, vol. 
58
 (pg. 
511
-
29
)
15
Kiratisin
P
Tucker
KD
Passador
L
LasR, a transcriptional activator of Pseudomonas aeruginosa virulence genes, functions as a multimer
J Bacteriol
 , 
2002
, vol. 
184
 (pg. 
4912
-
9
)
16
Ohman
DE
Cryz
SJ
Iglewski
BH
Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase
J Bacteriol
 , 
1980
, vol. 
142
 (pg. 
836
-
42
)
17
Yorgey
P
Rahme
LG
Tan
MW
Ausubel
FM
The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice
Mol Microbiol
 , 
2001
, vol. 
41
 (pg. 
1063
-
76
)
18
Schwyn
B
Neilands
JB
Universal chemical assay for the detection and determination of siderophores
Anal Biochem
 , 
1987
, vol. 
160
 (pg. 
47
-
56
)
19
Heesemann
J
Chromosomal-encoded siderophores are required for mouse virulence of enteropathogenic Yersinia species
FEMS Microbiol Lett
 , 
1987
, vol. 
48
 (pg. 
229
-
33
)
20
Fleiszig
SM
Arora
SK
Van
R
Ramphal
R
FlhA, a component of the flagellum assembly apparatus of Pseudomonas aeruginosa plays a role in internalization by corneal epithelial cells
Infect Immun
 , 
2001
, vol. 
69
 (pg. 
4931
-
7
)
21
Barth
AL
Pitt
TL
Auxotrophic variants of Pseudomonas aeruginosa are selected from prototrophic wild-type strains in respiratory infections in patients with cystic fibrosis
J Clin Microbiol
 , 
1995
, vol. 
33
 (pg. 
37
-
40
)
22
Hornef
MW
Roggenkamp
A
Geiger
AM
Hogardt
M
Jacobi
CA
Triggering the ExoS regulon of Pseudomonas aeruginosa: a GFP-reporter analysis of exoenzyme (Exo) S, ExoT and ExoU synthesis
Microb Pathog
 , 
2000
, vol. 
29
 (pg. 
329
-
43
)
23
Hogardt
M
Roeder
M
Schreff
AM
Eberl
L
Heesemann
J
Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS
Microbiology
 , 
2004
, vol. 
150
 (pg. 
843
-
51
)
24
Mosmann
T
Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays
J Immunol Methods
 , 
1983
, vol. 
65
 (pg. 
55
-
63
)
25
Kresse
AU
Dinesh
SD
Larbig
K
Romling
U
Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs
Mol Microbiol
 , 
2003
, vol. 
47
 (pg. 
145
-
58
)
26
Smania
AM
Segura
I
Pezza
RJ
Becerra
C
Albesa
I
Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa
Microbiology
 , 
2004
, vol. 
150
 (pg. 
1327
-
38
)
27
Smith
EE
Buckley
DG
Wu
Z
, et al.  . 
Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients
Proc Natl Acad Sci USA
 , 
2006
, vol. 
103
 (pg. 
8487
-
92
)
28
Romling
U
Schmidt
KD
Tummler
B
Large genome rearrangements discovered by the detailed analysis of 21 Pseudomonas aeruginosa clone C isolates found in environment and disease habitats
J Mol Biol
 , 
1997
, vol. 
271
 (pg. 
386
-
404
)
29
Bjedov
I
Tenaillon
O
Gerard
B
, et al.  . 
Stress-induced mutagenesis in bacteria
Science
 , 
2003
, vol. 
300
 (pg. 
1404
-
9
)
30
Ferenci
T
What is driving the acquisition of mutS and rpoS polymorphisms in Escherichia coli?
Trends Microbiol
 , 
2003
, vol. 
11
 (pg. 
457
-
61
)
31
van den
BD
Chin
AWT
Bloemberg
GV
Lugtenberg
BJ
Role of RpoS and MutS in phase variation of Pseudomonas sp. PCL1171
Microbiology
 , 
2005
, vol. 
151
 (pg. 
1403
-
8
)
32
Lau
GW
Hassett
DJ
Ran
H
Kong
F
The role of pyocyanin in Pseudomonas aeruginosa infection
Trends Mol Med
 , 
2004
, vol. 
10
 (pg. 
599
-
606
)
Presented in part: 10th International Congress on Pseudomonas, 27–31 August 2005, Marseille, France (talk S45)
Potential conflicts of interest: none reported
Financial support: German Cystic Fibrosis Foundation (grant F06/03)