Vol. 62, No. 2
INFECrION AND IMMUNITY, Feb. 1994, p. 596-605 0019-9567/94/$04.00+0 Copyright C 1994, American Society for Microbiology
Nonmotility and Phagocytic Resistance of Pseudomonas aeruginosa Isolates from Chronically Colonized Patients with Cystic Fibrosis ESHWAR MAHENTHIRALINGAM,1* MAUREEN E. CAMPBELL,' AND DAVID P. SPEERT 2'3 Department of Paediatrics,' Department of Microbiology and Immunology,2 and Canadian Bacterial Diseases Network,3 University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4 Received 15 September 1993/Returned for modification 2 November 1993/Accepted 24 November 1993
Although Pseudomonas aeruginosa chronically colonizes most older patients with cystic fibrosis (CF), bacterial features responsible for its persistence are understood poorly. We observed that many P. aeruginosa isolates from chronically colonized patients were nonmotile and resistant to phagocytosis by macrophages. P. aeruginosa isolates were collected from 20 CF patients for up to 10 years. Isolates from early colonization were highly motile and expressed both flagellin and pilin. However, many isolates from chronically colonized patients lacked flagellin expression and were nonmotile; a total of 1,030 P. aeruginosa CF isolates were examined, of which 39%O were nonmotile. Moreover, sequential isolates recovered from several of the CF patients were consistently nonmotile for up to 10 years. Lack of motility was rare among environmental isolates (1.4%) and other clinical isolates (3.7%) of P. aeruginosa examined. Partial complementation of motility in nonmotile P. aeruginosa isolates was achieved by introduction of extra copies of the rpoN locus carried on plasmid pPT212, indicating that the alternate sigma factor, RpoN, may be involved in the coordinate regulation of virulence factors during CF infection. We hypothesize that the nonmotile phenotype may provide P. aeruginosa a survival advantage in chronic CF infection by enabling it to resist phagocytosis and conserve energy.
chronic CF infection. Elaboration of mucoid exopolysaccharide reduces the susceptibility of P. aeruginosa to opsonic and nonopsonic phagocytosis by neutrophils and macrophages (1, 4, 14), and P. aeruginosa also secretes a variety of exoproducts and proteases which may reduce the efficiency of both opsonic and nonopsonic phagocytosis by phagocytes (reviewed in reference 30). However, the influence of motility on susceptibility to phagocytosis has not been determined. We report that nonmotile P. aeruginosa was frequently recovered from CF patients who have been colonized chronically and that P. aeruginosa of a stable nonmotile phenotype, expressing neither flagellin nor pilin and possessing many traits characteristic of P. aeruginosa RpoN mutants, was maintained during CF infection. In addition, we show that nonmotile CF isolates, deficient in flagellar formation, were resistant to ingestion by macrophages, a feature which may enable the organisms to persist in the respiratory tracts of patients with CF once colonization is established.
Pseudomonas aeruginosa is the predominant respiratory pathogen in patients with cystic fibrosis (CF). P. aeruginosa strains recovered from chronic CF infection are phenotypically different from wild-type environmental isolates: they are serum sensitive and endowed with a rough lipopolysaccharide (9); they are mucoid (12); and they show decreased motility and are chemotactically deficient (19). Deficiency in the flagellation of P. aeruginosa strains isolated from CF patients has been observed in patients in poor clinical condition (19), and these nonmotile isolates demonstrated many avirulent properties (18). Burke et al. (3) also noted that CF isolates may become less motile during CF infection, but the prevalence of isolates attenuated in motility within chronic CF infection has not been examined in depth. Flagellum-mediated motility has been shown in animal models and in vitro to be an important virulence factor for P. aeruginosa. Nonflagellated strains are attenuated in their virulence in the mouse burn model (7) and demonstrate a reduced ability to bind and colonize cell surfaces in vitro (11, 25). Recently, it has been shown that the expression of flagellum synthesis and synthesis of a number of other P. aeruginosa virulence factors, which include pili and nonpilus adhesins, may be coordinately regulated by the alternate sigma factor, RpoN (6, 27, 36). Pier et al. (24) demonstrated that RpoN mutants of P. aeruginosa, lacking both flagella and pili, established colonization in a mouse mucosal model poorly while still surviving throughout infection. These data indicate that RpoN-dependent products are important for P. aeruginosa colonization but not necessarily for survival. P. aeruginosa also possesses a number of characteristics which may enable it to evade host phagocytic defenses during
MATERIALS AND METHODS Bacterial strains and culture. Pseudomonas spp. were collected from the respiratory samples of patients with CF at the British Columbia's Children's Hospital and Shaughnessy Hospital, Vancouver, British Columbia, Canada. Sputum or throat samples were plated onto Columbia agar containing 5% sheep blood, MacConkey agar, and chocolate agar. P. aeruginosa was isolated by using conventional diagnostic techniques and further characterized by standard antimicrobial susceptibility tests by the hospital microbiology laboratories. These P. aeruginosa isolates were then processed in our laboratory as follows. Isolates were phenotypically separated on the basis of classic, enterobacter, mucoid, or dwarf morphology (38). P. aeruginosa species was confirmed by plating on selective medium containing phenanthroline and 9-chloro-9[4-(diethylamino)phenyl]9,10-dihydro-10-phenylacridinehydrochloride (5) and subcul-
* Corresponding author. Mailing address: Childrens Hospital Research Center, 950 West 28th Ave., Vancouver, B.C., Canada V5Z 4H4. Phone: (604) 875 2466. Fax: (604) 875 2496. Electronic mail address:
[email protected].
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tured onto blood agar, and confluent growth was removed and resuspended in 1.5 ml of Mueller-Hinton broth containing 8% dimethyl sulfoxide prior to freezing at - 70°C. Overall, the selection procedures were designed to collect all phenotypically different P. aeruginosa strains present in a given respiratory sample. Non-CF clinical isolates of P. aeruginosa were obtained from Children's Hospital and Vancouver General Hospital, British Columbia, and Edmonton General Hospital Edmonton, Alberta, Canada. Environmental P. aeruginosa strains were derived from two sources: our own laboratory collection isolated from various garden vegetables and a collection of river isolates obtained from Robert Hancock (Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada). All bacterial culture media and reagents were purchased from Difco Laboratories, Detroit, Mich. Frozen strains were revived on tryptic soy agar and grown in Luria-Bertani (L) broth for phagocytic assays as follows. A loopful of confluent bacterial growth was inoculated into 5 ml of L broth contained in a 13-ml tube (Falcon) and grown with end-over-end rotation for 3 to 4 h at 37°C. Nonmotile strains grew very poorly under static conditions and required aeration by agitation. Minimal salts medium was prepared as described previously (10) and supplemented with 0.2% individual amino acids as required. Motility measurement. The motility of P. aeruginosa isolates was assessed by the diameter of colonial spreading in soft L-broth agar (containing 0.3% agar). Confluent growth from plates was picked and stabbed into the center of duplicate soft agar plates with sterile toothpicks. After 24 h of growth at 37°C, the diameter of bacterial spreading in each plate was measured in millimeters, and the mean was determined; strains with colonial growth of 5 mm or less in diameter were considered nonmotile. Pilus phage sensitivity assay. Presence of surface pili on P. aeruginosa strains was confirmed by plating a 5-,il drop of culture supernatant containing 4 x 107 particles of bacteriophage P04 (2) onto a freshly spread lawn of bacteria made on L agar. Bacteria were grown overnight at 37°C. A zone of clearing was seen with piliated P. aeruginosa, indicating cell lysis and the assembly of nonretractile pili. Murine macrophage phagocytosis. All cell culture media and reagents were purchased from GIBCO-BRL, Gaithersburg, Md. Murine phagocytes were harvested from the peritoneal cavities of 6- to 8-week-old female BALB/c mice 3 days after elicitation by 2-ml intraperitoneal injection of 4% Brewer's complete thioglycolate broth (Difco). Leukocytes (2 x 105 cells in 1 ml of RPMI 1640 containing 10% [vol/vol] heatinactivated fetal calf serum and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES]) were added to the wells of 24-well tissue culture plates containing 11-mmdiameter acid-washed glass coverslips and incubated for 1 to 2 h at 37°C in 5% CO2. Adherent macrophages were washed by immersion in phosphate-buffered saline, and the coverslip was placed in a fresh 24-well plate containing 450 RI of phosphatebuffered phagocytic medium (29) supplemented with 10 mM D-glucose, a critical requirement for avid ingestion of P. aeruginosa (32). After 30 min of equilibration at 37°C (air buffered), 50 pI of bacterial culture (diluted in L broth to A600 = 0.6) was added to the macrophages, and ingestion was allowed to proceed for 1 h at 37°C. This dilution of bacteria approximated a ratio of 50 to 100 viable bacteria per macrophage. Extracellular bacteria were lysed by washing the monolayers with lysozyme and water as previously described (32). Fixed coverslips were stained with freshly diluted 3% Giemsa stain in pH 6.8 phosphate buffer (BDH Chemicals, Toronto,
NONMOTILE P. AERUGINOSA IN CF INFECTION
597
Ontario, Canada) for 30 min. Intracellular bacteria were counted by light microscopy as described previously (34). SDS-PAGE and immunoblotting. Protein extracts from P. aeruginosa isolates were prepared as follows. Overnight bacterial growth was scraped from a tryptic soy agar plate and resuspended in 1.5 ml of 20 mM Tris-Cl (pH 7.5); 1 ml of this suspension was added to an equal volume of 0.1-mm-diameter glass beads in a 2-ml microcentrifuge tube, and the bacteria were disrupted for 2 min on a Bead-Beater device (Biospec Products, Bartlesville, Okla.). Bacterial debris was sedimented by microcentrifugation at 17,000 x g for 5 min; 300 ptl of the cleared extract was mixed with an equal volume of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (2% SDS, 100 mM dithiothreitol, 40% glycerol, 0.002% bromophenol blue, 125 mM Tris-HCl [pH 6.8]), boiled for 5 min, and stored at 4°C. The protein concentration of the remaining extract was determined by the bicinchoninic acid procedure (28). Solubilized protein extracts were fractionated on polyacrylamide gels (1,6) and transferred to nitrocellulose filters by semidry electrophoretic transfer (15). After transfer, the filters were blocked with 4% nonfat milk powder in Tris-buffered saline. The blots were cut horizontally at the mobility of the 27-kDa prestained protein molecular weight marker (Bio-Rad Laboratories Ltd., Missisauga, Ontario, Canada); the larger molecular size range was probed with flagellin antiserum, and the lower molecular size range was probed with pilin rabbit antiserum. Incubations with antisera and conjugated antibody were performed in 2% milk powder in Tris-buffered saline containing 0.05% Tween 20. Blots were probed with alkaline phosphatase-labelled anti-rabbit antibody (Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) and developed by using the bromochloroindolyl phosphate-nitroblue tetrazolium substrate system. Polyclonal rabbit antiserum against the type a, 45-kDa flagellin of P. aeruginosa P1 (31) was prepared in our laboratory by standard subcutaneous immunization of a New Zealand White rabbit with 500 ,ug of fast protein liquid chromatography-purified flagellin in Freund's complete adjuvant; further injections of 100 jig of flagellin were subsequently administered at 4-week intervals, and serum was collected 3 weeks after the final boost. Polyclonal rabbit antibody against pili from P. aeruginosa PAK (39), recognizing the 15-kDa pilin subunit protein, was kindly provided by William Paranchych, Department of Microbiology, University of Alberta, Edmonton, Alberta, Canada. Both antisera were adsorbed with an RpoN mutant of P. aeruginosa PAK (36) in order to remove cross-reactive components and used at a dilution of 1/1,000. Protein extracts from P. aeruginosa P1 and PAK were loaded on each gel as positive controls for the flagellin and pilin antisera, respectively. Mobilization of plasmids. Plasmid pPT212, carrying the P. aeruginosa rpoN locus (36), was kindly provided by Stephen Lory, University of Washington, Seattle. The plasmid was mobilized into CF strains by triparental matings with an Escherichia coli DH5a(pPT212) donor and E. coli HB101 (pRK2073) as previously described (8). P. aeruginosa transconjugants were selected on Pseudomonas isolation agar (Difco) supplemented with gentamicin at 200 pug/ml. Transconjugants were subsequently grown and maintained with L-broth medium containing 50 ,ig of gentamicin per ml. RESULTS of CF isolates. Sequential P. aeruginosa sequential Motility isolates from a total of 20 patients were studied, and the
598
INFECT. IMMUN.
MAHENTHIRALINGAM ET AL.
TABLE 1.
Motility of sequential P. aeruginosa isolates from CF patients Age (yr) at
Total no. iYsolates ofToanisolates
Patient (sex)"xwhich P wee collected collcted were )a
1 (F) 2 (F) 3 (F) 4 (M) 5 (M) 6 (F) 7 (M) 8 (M) 9 (F) 10 (M) 11 (F) 12 (M) 13 (M) 14 (M) 15 (M) 16 (F) 17 (F) 18 (F) 19 (F) 20 (F)
0.6b-38c 10-19.4c 7.4-14.6c 21.9-32.3 16.8-27.4 9.3-19-9C 22-32.4 10.7-15.2c 4.3-14.8 10.8-16.6 22.8-29.1
7.9b-13.9 0.Sb_9.0 13.9-22.4c 5.1-15.2
4.5b_10.3 2.9b-8.7 20.2-26.9c
3.1b_11.5 3.5-14.1
Total Percentage
25 55 44 34 47 174 22 75 63 37 42 35 59 86 46 40 34 46 30 36
1,030
No. N. motile
22 52 22 11 5 101 13 73 47 0 18 31 51 58 15 39 3 28 30 3 622 60.4
() No. N.(%
nonmotile
No. N. mucoid
3 (12) 3 (5) 22 (50) 23 (67) 42 (89) 73 (42) 9 (41) 2 (3) 16 (25) 37 (100) 24 (57) 4 (11) 8 (13) 28 (32) 31 (67) 1 (2) 31 (91) 18 (39) 0 (0) 33 (91)
3 19 13 10 21 55 7 23 14 20 18 5 29 30 38 13 3 19 8 17
408 39.5
365 35.4
F, female; M, male. Age at colonization. c Age at death.
a
b
motility data obtained are summarized in Table 1. Nonmotile P. aeruginosa strains were initially observed in isolates collected from patients 3 and 4, and sequential isolates from these patients were the first to be characterized. The remaining patients' isolates were chosen from our laboratory collection on the basis of the following criteria: (i) sequential P. aeruginosa isolates had been collected from the patient for a minimum period of 2 years; (ii) contiguity of collection was maintained over the period studied, and (iii) the study group of patients was balanced for sex. Overall, mean duration of strain collection for the 20 patients studied was 7.95 years, and 5 of the patients were followed for 10 years or more. Of 1,030 P. aeruginosa CF isolates studied, 39.5% were nonmotile and 35.4% were mucoid. Motility and mucoidy were not linked; both motile and nonmotile isolates were found to be mucoid. The majority of nonmotile P. aeruginosa isolates screened were stable, and their inability to swarm in soft agar was unaffected by reduction of growth temperature or increased duration of incubation (data not shown). Motility of clinical and environmental P. aeruginosa isolates. The occurrence of nonmotile strains among non-CF clinical isolates and environmental P. aeruginosa strains examined is summarized in Table 2. Only 1 of the 71 environmental strains studied was nonmotile (1.41%), while 6 of the 164 non-CF clinical isolates tested were nonmotile (3.66%). No relationship between source of P. aeruginosa and motility was apparent; nonmotile P. aeruginosa were rarely found outside of CF respiratory secretion. Characterization of initial P. aeruginosa isolates. The isolates first collected from patients 1, 12, 13, 16, 17, and 19 were highly motile in soft agar (Table 3). Flagellin expression was detected by immunoblotting in all of these isolates and was at a level equivalent to that of the motile control strains P. aeruginosa P1 and PAK (Fig. 1). Detection of pilin expression
TABLE 2. Occurrence of nonmotile P. aeruginosa among non-CF clinical strains and environmental isolates Type of isolate
Clinical Bacteremia Burn Ear Rectal
Respiratory Urine Wound Other"
Total Environmental Garden vegetable River Total CF
No. of strains
% Nonmotile
No. nonmotile
21 10 15 39 30
1 0 1 0 2 2
20 20
0 0
161
6
3.7
37 34 71
0 1 1
1.4
1,030
408
39.5
6
aAppendix, cyst, eye, foot, groin, knee, placental, skin, umbilicus. by immunoblotting was not conclusive: the primary isolate from patient 12 expressed PAK-type pilin, and extracts from the other five primary isolates cross-reacted weakly with the antiserum but did appear to show the presence of prepilin and pilin bands (Fig. 1). All six initial isolates were sensitive to the pilus-specific phage P04, confirming the presence of pili on these bacteria (Table 3). None of the primary isolates examined were mucoid, and they were all susceptible to ingestion by murine macrophages in the absence of serum opsonins (Table 3). Loss of motility by P. aeruginosa during CF infection. Sequential P. aeruginosa isolates collected from each patient studied demonstrated three patterns of motility: (i) isolates recovered were predominantly motile over the period studied (Fig. 2A); (ii) motile and nonmotile P. aeruginosa isolates were recovered simultaneously, with neither phenotype predominating (Fig. 2B), and (iii) nonmotile isolates became the predominant phenotype collected (Fig. 2C). These divisions were based on the percentage of nonmotile isolates recovered from each patient during the study; the percentages at which divisions were drawn were chosen arbitrarily and serve merely to illustrate the P. aeruginosa phenotypes present among the patients studied. TABLE 3. Phenotypic features of primary P. aeruginosa isolates and control strain PAK" Isolate
Motilit in soft soft (Motltyin (mean iamagma)r [m]) aga
Phagocytosis (mean no. of bacteria ingested/macrophage
+SEM, n = 4)
Primary isolate from patient no: 1 12 13 16 17 19 PAK
67 47 80 75 30 72 70
12.02 6.3 3.4 3.8 6.7 5.28 5.37
± ± ± ± ± ± ±
0.96 0.39 0.33 1.0 0.41 0.38 0.66
aAll isolates were susceptible to pilus phage P04 and had a nonmucoid colonial morphology. b Mean diameter of spread in soft agar after 24 h of incubation at 37°C (duplicate test).
VOL. 62, 1994
NONMOTILE P. AERUGINOSA IN CF INFECTION Pl PAK
M
1
12
13
16
17
19
F
FIG. 1. Immunoblot of initial isolates of P. aeruginosa collected from patients 1, 12, 13, 16, 17, and 19. Protein extracts were separated on 12 and 16% acrylamide gels for panel F and P, respectively. After transfer to nitrocellulose, the extracts were probed for flagellin (F) and pilin (P) expression, using the sera described in Materials and Methof total protein was examined in ods. For the colonizing isolates, 15 was examined in panel P. Extracts from P. panel F and 30 in panel P) and PAK (10 ,ug in aeruginosa P1 (5 jig in panel F; 30 panel F; 3 in panel P) were run as positive controls for flagellin and pilin, respectively. Prestained protein molecular weight markers were
,ig
,ug
,ug ,ug
loaded in lane M.
Isolates collected from patients 1, 2, 8, 12, 13, 16, and 19 remained predominantly motile; more than 85% of P. aeruginosa isolates recovered from each of these patients spread in soft agar (Table 1). Profiles of isolate motility versus age are shown for patients 2, 8, 12, and 19 in Fig. 2A to illustrate the maintenance of motility in this group of CF patients. The prevalence of nonmotile P. aeruginosa varied between 25 and 60% for the isolates collected from patients 3, 6, 9, 11, 14, and 18 (Table 1). Motile and nonmotile P. aeruginosa isolates were harbored in parallel by these patients, and plots of isolates motility versus age are shown for patients 3, 6, 7, and 11 in Fig. 2B. During the course of chronic respiratory infection, P. aeruginosa isolates collected from the remaining patients (patients 4, 5, 10, 15, 17, and 20) were predominantly nonmotile. More than 60% of the isolates recovered from the latter group of patients were nonmotile; the predominance of nonmotile P. aeruginosa collected from patients 4, 5, 10, and 15 is shown in Fig. 2C. The age at which nonmotile P. aeruginosa isolates were first collected varied among the 20 patients studied. The mean duration of colonization prior to appearance of nonmotile bacteria may be approximated from patients who had been studied from primary infection. Initial P. aeruginosa isolates were collected from patients 1, 12, 13, 16, 17, and 19; nonmotile isolates first appeared after 2.3, 5.2, 2.9, 4.6, and 0.2 years of infection, respectively, for the first five of these patients; all isolates from patient 19 were motile. Thus, the mean (+ standard error) duration of infection prior to appearance of nonmotile P. aeruginosa was 3.07 (±1.98) years, excluding patient 19. The mean duration of infection before the emergence of mucoid P. aeruginosa in this group of six patients was 3.37 (± 1.76) years. Flagellin and pilin expression of P. aeruginosa isolated from chronically colonized CF patients. Immunoblot analysis of flagellin and pilin expression is shown in Fig. 3 for several of the sequential P. aeruginosa isolates from patients 3, 4, 5, 7, 12, and 15. Absence of flagellin expression correlated with absence of motility in soft agar for the P. aeruginosa isolates examined by immunoblotting, except for three isolates from patient 5 (collected at ages 16.9, 18, and 19 years). These isolates, although nonmotile in agar, produced traces of flagellin (Fig. 3) which may represent a minority of bacteria expressing a motile phenotype within a predominantly nonmotile population. For the majority of the nonmotile P. aeruginosa isolates characterized by immunoblotting in Fig. 3, pilin but not flagellin was detected. Nonmotile P. aeruginosa isolates collected from patients 5, 7, 12, and 15 synthesized pilin and
599
assembled pili. Although reaction with the antipilin serum was poor for many of these nonflagellated isolates (Fig. 3), most were susceptible to phage P04, indicating the presence of pili (data not shown). Nonmotile bacteria from patients 3 and 4 were exceptions to this trend; these isolates appeared to lack expression of both pilin and flagellin and were also resistant to the pilus phage P04 (data shown in Table 4 for one representative nonmotile isolate from each patient). Nonmotile bacteria from patient 4 (e.g., isolate 1277; Table 4) were also unable to grow on minimal medium without glutamine. The absence of flagellin, pilin, and nitrogen assimilation gene expression from isolate 1277 are characteristics of a P. aeruginosa RpoN mutant (36). These RpoN - P. aeruginosa isolates were the predominant P. aeruginosa phenotype isolated from patient 4 for a period of 7 years. The flagellin and pilin expression profile of sequential isolates from patient 12 clearly indicates the transition from motile primary isolates to the appearance of nonmotile P. aeruginosa during CF infection. P. aeruginosa was first collected from patient 12 at 7.9 years of age; this isolate was motile, piliated, and flagellated (Fig. 1 and 3; Table 3). Motile isolates continued to be collected for 5 years, at which point the first nonmotile isolate, isolate 4503, was recovered (Fig. 2A); this isolate did not express flagellin but did produce pilin (Fig. 3). Nonmotile P. aeruginosa subsequently predominated over the next year and was still being recovered from this patient at 13.9 years of age. Expression of flagellin did not correlate with expression of mucoidy for the P. aeruginosa isolates examined by immunoblotting. Isolates recovered at age 26.9 years from patient 7, 13.9 years from patient 12, and 10.3, 12.2, and 15.2 years from patient 15 were all mucoid but lacked flagellin expression (Fig. 3). In contrast, P. aeruginosa isolates recovered at 8.5 years of age from patient 3 and 23.3 years of age from patient 4 were mucoid, expressed flagellin (Fig. 3), and were motile (Fig. 2). Effect of motility on nonopsonic phagocytosis of P. aeruginosa. Motile and nonmotile P. aeruginosa isolates from patients 3, 4, 7, and 12 were incubated with murine thioglycolateelicited macrophages in the absence of serum opsonins (Fig. 4). All motile isolates characterized were susceptible to ingestion, whereas nonmotile bacteria recovered later from each patient (Fig. 2) were resistant to nonopsonic phagocytosis by murine macrophages. Phagocytosis-resistant nonmotile bacteria all lacked expression of flagellin, but some expressed pilin. Immunoblot examination of flagellin and pilin expression in these isolates is shown in Fig. 3, and susceptibility to phage P04 together with a summary of their phenotypic features is shown in Fig. 4. Nonmotile isolates 1608 and 1277 were nonpiliated, whereas nonmotile isolates 4492 and 4503 possessed surface pili; however, all of these isolates were not ingested by murine macrophages in the absence of serum. Isolate 511 (motile and phagocytic susceptible) was nonpiliated, indicating that the presence of pili was not required for the nonopsonic ingestion of the CF P. aeruginosa isolates examined. Partial complementation of motility in nonmotile CF isolates. Plasmid pPT212, carrying the rpoN locus from P. aeruginosa PAK (36), was introduced into nonmotile P. aeruginosa CF strains by conjugation in order to assess the role that the regulatory sigma factor, encoded by rpoN, may have in the appearance of the nonmotile phenotype. Various phenotypic changes resulted from the acquisition of extra copies of rpoN by representative nonmotile P. aeruginosa isolates collected from patients 3 and 4 (summarized in Table 4); no changes were seen in bacteria carrying the plasmid vector pSP329G alone (data not shown). These nonmotile P. aeruginosa iso-
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Age of patient (years) FIG. 2. Plots of P. aeruginosa isolate motility versus age of patients. (A) Patients from whom predominantly motile isolates were collected; (B) patients from whom motile and nonmotile isolates were collected concurrently; (C) patients from whom nonmotile isolates were recovered persistently. Strain numbers are indicated for the motile and nonmotile P. aeruginosa isolates collected from patients 3, 4, 7, and 11 which were used in phagocytic assays (see Fig. 4). 600
NONMOTILE P. AERUGINOSA IN CF INFECrION
VOL. 62, 1994
601
(C) Non-motile Patient 4
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lates, which were originally RpoN- in phenotype, were partially complemented by the introduction of extra copies of the rpoN locus (strains containing plasmid pPT212 were designated by the suffix "RP"). Strains 1608RP and 1822RP (derived from nonmotile isolates recovered 3 months apart from patient 3) synthesized flagellin (Fig. 5); however, strain 1608RP did not swarm in soft agar and was not actively motile when visualized microscopically (Table 4). Pilin synthesis in strains 1608RP and 1822RP appeared to be enhanced over that of the parental isolates; however, strain 1608RP was not highly susceptible to phage P04, indicating that surface pili were largely absent. Nonmotile isolates 1277 and 3000, recovered from patient 4 approximately 3 years apart, were able to synthesize flagellin after acquisition of extra copies of the rpoN locus (Fig. 5). The motility of strain 1277RP in soft agar was negligible; however, its colonial morphology was more diffuse than that of the parental isolates (Table 4). Microscopic examination of a culture of 1277RP showed that a small proportion of the bacteria were highly motile (Table 4) and indicated that flagellin, detected by immunoblotting (Fig. 5), was being assembled into a functional flagellum in a few bacteria. Strain 1277RP was susceptible to phage P04, in contrast to its parental strain (Table 4), even though no difference in pilin expression was detected by immunoblotting (Fig. 5). Strain 1277RP was also able to grow on minimal medium without glutamine supplementation (Table 4). Strain 1277RP was susceptible to nonopsonic ingestion by murine macrophages, in contrast to its nonmotile parent; however, strain 1608RP remained resistant to nonopsonic phagocytosis.
No obvious phenotypic changes were observed when plasmid pPT212 was introduced into CF strains lacking flagellin but able to synthesize pilin expression (data not shown), indicating that the phenotype of these strains may have arisen from events affecting flagellum synthesis independently of RpoN.
DISCUSSION P. aeruginosa possesses a number of characteristics, such as acquisition of a rough lipopolysaccharide and secretion of mucoid exopolysaccharide, proteases, and exotoxins, which may aid colonization and survival in the CF lung. These virulence factors have been well studied, and there is a great deal of literature about their role in pathogenesis. It has been suggested that factors such as piliation and flagellation are important in the colonization of the CF airway (25) and in the establishment of other infections (7, 11). The presence of nonmotile, nonflagellated P. aeruginosa in CF infection has been described previously (19); however, the prevalence and mechanism of selection of this phenotype have not been explored adequately. In this report, we have presented data indicating that nonmotile P. aeruginosa is prevalent in chronic CF infection and that P. aeruginosa may utilize specific regulatory mechanisms to modulate the expression of this altered phenotype during infection. P. aeruginosa isolates from the environment and from other clinical sources were predominantly motile. P. aeruginosa isolates recovered from CF patients early in the course of infection matched the highly motile phenotype of environmen-
INFEC-F. IMMUN.
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