INFECrION AND IMMUNITY, Feb. 1994, p. 596-605 Vol. 62, No. 20019-9567/94/$04.00+0Copyright C 1994, American Society for Microbiology

Nonmotility and Phagocytic Resistance of Pseudomonas aeruginosaIsolates from Chronically Colonized Patients

with Cystic FibrosisESHWAR MAHENTHIRALINGAM,1* MAUREEN E. CAMPBELL,' AND DAVID P. SPEERT 2'3

Department of Paediatrics,' Department of Microbiology and Immunology,2 and Canadian Bacterial DiseasesNetwork,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), bacterialfeatures responsible for its persistence are understood poorly. We observed that many P. aeruginosa isolatesfrom chronically colonized patients were nonmotile and resistant to phagocytosis by macrophages. P. aeruginosaisolates were collected from 20 CF patients for up to 10 years. Isolates from early colonization were highly motileand expressed both flagellin and pilin. However, many isolates from chronically colonized patients lackedflagellin expression and were nonmotile; a total of 1,030 P. aeruginosa CF isolates were examined, of which 39%Owere nonmotile. Moreover, sequential isolates recovered from several of the CF patients were consistentlynonmotile for up to 10 years. Lack of motility was rare among environmental isolates (1.4%) and other clinicalisolates (3.7%) of P. aeruginosa examined. Partial complementation of motility in nonmotile P. aeruginosaisolates was achieved by introduction of extra copies of the rpoN locus carried on plasmid pPT212, indicatingthat the alternate sigma factor, RpoN, may be involved in the coordinate regulation of virulence factors duringCF infection. We hypothesize that the nonmotile phenotype may provide P. aeruginosa a survival advantage inchronic CF infection by enabling it to resist phagocytosis and conserve energy.

Pseudomonas aeruginosa is the predominant respiratorypathogen in patients with cystic fibrosis (CF). P. aeruginosastrains recovered from chronic CF infection are phenotypicallydifferent from wild-type environmental isolates: they are serumsensitive and endowed with a rough lipopolysaccharide (9);they are mucoid (12); and they show decreased motility and arechemotactically deficient (19). Deficiency in the flagellation ofP. aeruginosa strains isolated from CF patients has beenobserved in patients in poor clinical condition (19), and thesenonmotile isolates demonstrated many avirulent properties(18). Burke et al. (3) also noted that CF isolates may becomeless motile during CF infection, but the prevalence of isolatesattenuated in motility within chronic CF infection has not beenexamined in depth.

Flagellum-mediated motility has been shown in animalmodels and in vitro to be an important virulence factor for P.aeruginosa. Nonflagellated strains are attenuated in their vir-ulence in the mouse burn model (7) and demonstrate areduced ability to bind and colonize cell surfaces in vitro (11,25). Recently, it has been shown that the expression offlagellum synthesis and synthesis of a number of other P.aeruginosa virulence factors, which include pili and nonpilusadhesins, may be coordinately regulated by the alternate sigmafactor, RpoN (6, 27, 36). Pier et al. (24) demonstrated thatRpoN mutants of P. aeruginosa, lacking both flagella and pili,established colonization in a mouse mucosal model poorlywhile still surviving throughout infection. These data indicatethat RpoN-dependent products are important for P. aeruginosacolonization but not necessarily for survival.

P. aeruginosa also possesses a number of characteristicswhich may enable it to evade host phagocytic defenses during

* Corresponding author. Mailing address: Childrens Hospital Re-search Center, 950 West 28th Ave., Vancouver, B.C., Canada V5Z4H4. Phone: (604) 875 2466. Fax: (604) 875 2496. Electronic mailaddress: speert2@cbdn.ca.

chronic CF infection. Elaboration of mucoid exopolysaccha-ride reduces the susceptibility of P. aeruginosa to opsonic andnonopsonic phagocytosis by neutrophils and macrophages (1,4, 14), and P. aeruginosa also secretes a variety of exoproductsand proteases which may reduce the efficiency of both opsonicand nonopsonic phagocytosis by phagocytes (reviewed in ref-erence 30). However, the influence of motility on susceptibilityto phagocytosis has not been determined.We report that nonmotile P. aeruginosa was frequently

recovered from CF patients who have been colonized chroni-cally and that P. aeruginosa of a stable nonmotile phenotype,expressing neither flagellin nor pilin and possessing many traitscharacteristic of P. aeruginosa RpoN mutants, was maintainedduring CF infection. In addition, we show that nonmotile CFisolates, deficient in flagellar formation, were resistant toingestion by macrophages, a feature which may enable theorganisms to persist in the respiratory tracts of patients withCF once colonization is established.

MATERIALS AND METHODS

Bacterial strains and culture. Pseudomonas spp. were col-lected from the respiratory samples of patients with CF at theBritish Columbia's Children's Hospital and Shaughnessy Hos-pital, Vancouver, British Columbia, Canada. Sputum or throatsamples were plated onto Columbia agar containing 5% sheepblood, MacConkey agar, and chocolate agar. P. aeruginosa wasisolated by using conventional diagnostic techniques and fur-ther characterized by standard antimicrobial susceptibility testsby the hospital microbiology laboratories. These P. aeruginosaisolates were then processed in our laboratory as follows.Isolates were phenotypically separated on the basis of classic,enterobacter, mucoid, or dwarf morphology (38). P. aeruginosaspecies was confirmed by plating on selective medium contain-ing phenanthroline and 9-chloro-9[4-(diethylamino)phenyl]-9,10-dihydro-10-phenylacridinehydrochloride (5) and subcul-

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tured onto blood agar, and confluent growth was removed andresuspended in 1.5 ml of Mueller-Hinton broth containing 8%dimethyl sulfoxide prior to freezing at - 70°C. Overall, theselection procedures were designed to collect all phenotypi-cally different P. aeruginosa strains present in a given respira-tory sample.Non-CF clinical isolates of P. aeruginosa were obtained from

Children's Hospital and Vancouver General Hospital, BritishColumbia, and Edmonton General Hospital Edmonton, Al-berta, Canada. Environmental P. aeruginosa strains were de-rived from two sources: our own laboratory collection isolatedfrom various garden vegetables and a collection of riverisolates obtained from Robert Hancock (Department of Mi-crobiology, University of British Columbia, Vancouver, BritishColumbia, Canada). All bacterial culture media and reagentswere purchased from Difco Laboratories, Detroit, Mich. Fro-zen strains were revived on tryptic soy agar and grown inLuria-Bertani (L) broth for phagocytic assays as follows. Aloopful of confluent bacterial growth was inoculated into 5 mlof L broth contained in a 13-ml tube (Falcon) and grown withend-over-end rotation for 3 to 4 h at 37°C. Nonmotile strainsgrew very poorly under static conditions and required aerationby agitation. Minimal salts medium was prepared as describedpreviously (10) and supplemented with 0.2% individual aminoacids as required.

Motility measurement. The motility of P. aeruginosa isolateswas assessed by the diameter of colonial spreading in softL-broth agar (containing 0.3% agar). Confluent growth fromplates was picked and stabbed into the center of duplicate softagar plates with sterile toothpicks. After 24 h of growth at37°C, the diameter of bacterial spreading in each plate wasmeasured in millimeters, and the mean was determined; strainswith colonial growth of 5 mm or less in diameter wereconsidered nonmotile.

Pilus phage sensitivity assay. Presence of surface pili on P.aeruginosa strains was confirmed by plating a 5-,il drop ofculture supernatant containing 4 x 107 particles of bacteri-ophage P04 (2) onto a freshly spread lawn of bacteria made onL agar. Bacteria were grown overnight at 37°C. A zone ofclearing was seen with piliated P. aeruginosa, indicating celllysis and the assembly of nonretractile pili.Murine macrophage phagocytosis. All cell culture media

and reagents were purchased from GIBCO-BRL, Gaithers-burg, Md. Murine phagocytes were harvested from the perito-neal cavities of 6- to 8-week-old female BALB/c mice 3 daysafter elicitation by 2-ml intraperitoneal injection of 4% Brew-er's complete thioglycolate broth (Difco). Leukocytes (2 x 105cells in 1 ml of RPMI 1640 containing 10% [vol/vol] heat-inactivated fetal calf serum and 10 mM N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid [HEPES]) were addedto the wells of 24-well tissue culture plates containing 11-mm-diameter acid-washed glass coverslips and incubated for 1 to 2h at 37°C in 5% CO2. Adherent macrophages were washed byimmersion in phosphate-buffered saline, and the coverslip wasplaced in a fresh 24-well plate containing 450 RI of phosphate-buffered phagocytic medium (29) supplemented with 10 mMD-glucose, a critical requirement for avid ingestion of P.aeruginosa (32). After 30 min of equilibration at 37°C (airbuffered), 50 pI of bacterial culture (diluted in L broth to A600= 0.6) was added to the macrophages, and ingestion wasallowed to proceed for 1 h at 37°C. This dilution of bacteriaapproximated a ratio of 50 to 100 viable bacteria per macro-phage. Extracellular bacteria were lysed by washing the mono-layers with lysozyme and water as previously described (32).Fixed coverslips were stained with freshly diluted 3% Giemsastain in pH 6.8 phosphate buffer (BDH Chemicals, Toronto,

Ontario, Canada) for 30 min. Intracellular bacteria werecounted by light microscopy as described previously (34).SDS-PAGE and immunoblotting. Protein extracts from P.

aeruginosa isolates were prepared as follows. Overnight bacte-rial growth was scraped from a tryptic soy agar plate andresuspended in 1.5 ml of 20 mM Tris-Cl (pH 7.5); 1 ml of thissuspension was added to an equal volume of 0.1-mm-diameterglass beads in a 2-ml microcentrifuge tube, and the bacteriawere disrupted for 2 min on a Bead-Beater device (BiospecProducts, Bartlesville, Okla.). Bacterial debris was sedimentedby microcentrifugation at 17,000 x g for 5 min; 300 ptl of thecleared extract was mixed with an equal volume of sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) sample buffer (2% SDS, 100 mM dithiothreitol, 40%glycerol, 0.002% bromophenol blue, 125 mM Tris-HCl [pH6.8]), boiled for 5 min, and stored at 4°C. The proteinconcentration of the remaining extract was determined by thebicinchoninic acid procedure (28).

Solubilized protein extracts were fractionated on polyacryl-amide gels (1,6) and transferred to nitrocellulose filters bysemidry electrophoretic transfer (15). After transfer, the filterswere blocked with 4% nonfat milk powder in Tris-bufferedsaline. The blots were cut horizontally at the mobility of the27-kDa prestained protein molecular weight marker (Bio-RadLaboratories Ltd., Missisauga, Ontario, Canada); the largermolecular size range was probed with flagellin antiserum, andthe lower molecular size range was probed with pilin rabbitantiserum. Incubations with antisera and conjugated antibodywere performed in 2% milk powder in Tris-buffered salinecontaining 0.05% Tween 20. Blots were probed with alkalinephosphatase-labelled anti-rabbit antibody (Kirkegaard & PerryLaboratories Inc., Gaithersburg, Md.) and developed by usingthe bromochloroindolyl phosphate-nitroblue tetrazolium sub-strate system.

Polyclonal rabbit antiserum against the type a, 45-kDaflagellin of P. aeruginosa P1 (31) was prepared in our labora-tory by standard subcutaneous immunization of a New Zea-land White rabbit with 500 ,ug of fast protein liquid chroma-tography-purified flagellin in Freund's complete adjuvant;further injections of 100 jig of flagellin were subsequentlyadministered at 4-week intervals, and serum was collected 3weeks after the final boost. Polyclonal rabbit antibody againstpili from P. aeruginosa PAK (39), recognizing the 15-kDa pilinsubunit protein, was kindly provided by William Paranchych,Department of Microbiology, University of Alberta, Edmon-ton, Alberta, Canada. Both antisera were adsorbed with anRpoN mutant of P. aeruginosa PAK (36) in order to removecross-reactive components and used at a dilution of 1/1,000.Protein extracts from P. aeruginosa P1 and PAK were loadedon each gel as positive controls for the flagellin and pilinantisera, respectively.

Mobilization of plasmids. Plasmid pPT212, carrying theP. aeruginosa rpoN locus (36), was kindly provided by StephenLory, University of Washington, Seattle. The plasmid wasmobilized into CF strains by triparental matings with anEscherichia coli DH5a(pPT212) donor and E. coli HB101(pRK2073) as previously described (8). P. aeruginosa transcon-jugants were selected on Pseudomonas isolation agar (Difco)supplemented with gentamicin at 200 pug/ml. Transconjugantswere subsequently grown and maintained with L-broth me-dium containing 50 ,ig of gentamicin per ml.

RESULTS

Motility of sequential CF isolates. Sequential P. aeruginosaisolates from a total of 20 patients were studied, and the

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598 MAHENTHIRALINGAM ET AL.

TABLE 1. Motility of sequential P. aeruginosa isolates fromCF patients

)a

Age (yr) at Total no. No. No. () No.Patient (sex)" which iYsolates Toan- N. N.(% N.Px

wee collcted of isolates motile nonmotile mucoidwere collected

1 (F) 0.6b-38c 25 22 3 (12) 32 (F) 10-19.4c 55 52 3 (5) 193 (F) 7.4-14.6c 44 22 22 (50) 134 (M) 21.9-32.3 34 11 23 (67) 105 (M) 16.8-27.4 47 5 42 (89) 216 (F) 9.3-19-9C 174 101 73 (42) 557 (M) 22-32.4 22 13 9 (41) 78 (M) 10.7-15.2c 75 73 2 (3) 239 (F) 4.3-14.8 63 47 16 (25) 1410 (M) 10.8-16.6 37 0 37 (100) 2011 (F) 22.8-29.1 42 18 24 (57) 1812 (M) 7.9b-13.9 35 31 4 (11) 513 (M) 0.Sb_9.0 59 51 8 (13) 2914 (M) 13.9-22.4c 86 58 28 (32) 3015 (M) 5.1-15.2 46 15 31 (67) 3816 (F) 4.5b_10.3 40 39 1 (2) 1317 (F) 2.9b-8.7 34 3 31 (91) 318 (F) 20.2-26.9c 46 28 18 (39) 1919 (F) 3.1b_11.5 30 30 0 (0) 820 (F) 3.5-14.1 36 3 33 (91) 17

Total 1,030 622 408 365Percentage 60.4 39.5 35.4

a F, female; M, male.b Age at colonization.c Age at death.

motility data obtained are summarized in Table 1. NonmotileP. aeruginosa strains were initially observed in isolates col-lected from patients 3 and 4, and sequential isolates from thesepatients were the first to be characterized. The remainingpatients' isolates were chosen from our laboratory collectionon the basis of the following criteria: (i) sequential P. aerugi-nosa isolates had been collected from the patient for a

minimum period of 2 years; (ii) contiguity of collection wasmaintained over the period studied, and (iii) the study group ofpatients was balanced for sex. Overall, mean duration of straincollection for the 20 patients studied was 7.95 years, and 5 ofthe 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 werenot linked; both motile and nonmotile isolates were found tobe mucoid. The majority of nonmotile P. aeruginosa isolatesscreened were stable, and their inability to swarm in soft agarwas unaffected by reduction of growth temperature or in-creased duration of incubation (data not shown).

Motility of clinical and environmental P. aeruginosa isolates.The occurrence of nonmotile strains among non-CF clinicalisolates and environmental P. aeruginosa strains examined issummarized in Table 2. Only 1 of the 71 environmental strainsstudied was nonmotile (1.41%), while 6 of the 164 non-CFclinical isolates tested were nonmotile (3.66%). No relation-ship between source of P. aeruginosa and motility was appar-

ent; nonmotile P. aeruginosa were rarely found outside of CFrespiratory secretion.

Characterization of initial P. aeruginosa isolates. The iso-lates 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 ata 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-CFclinical strains and environmental isolates

Type of isolate No. of strains No. nonmotile % Nonmotile

ClinicalBacteremia 21 1Burn 10 0Ear 15 1Rectal 6 0Respiratory 39 2Urine 30 2Wound 20 0Other" 20 0

Total 161 6 3.7

EnvironmentalGarden vegetable 37 0River 34 1

Total 71 1 1.4

CF 1,030 408 39.5

aAppendix, cyst, eye, foot, groin, knee, placental, skin, umbilicus.

by immunoblotting was not conclusive: the primary isolatefrom patient 12 expressed PAK-type pilin, and extracts fromthe other five primary isolates cross-reacted weakly with theantiserum but did appear to show the presence of prepilin andpilin bands (Fig. 1). All six initial isolates were sensitive to thepilus-specific phage P04, confirming the presence of pili onthese bacteria (Table 3). None of the primary isolates exam-ined were mucoid, and they were all susceptible to ingestionby 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 patientstudied demonstrated three patterns of motility: (i) isolatesrecovered were predominantly motile over the period studied(Fig. 2A); (ii) motile and nonmotile P. aeruginosa isolates wererecovered simultaneously, with neither phenotype predominat-ing (Fig. 2B), and (iii) nonmotile isolates became the predom-inant phenotype collected (Fig. 2C). These divisions werebased on the percentage of nonmotile isolates recovered fromeach patient during the study; the percentages at whichdivisions were drawn were chosen arbitrarily and serve merelyto illustrate the P. aeruginosa phenotypes present among thepatients studied.

TABLE 3. Phenotypic features of primary P. aeruginosa isolatesand control strain PAK"

Motilit in soft agaPhagocytosis (mean no. of

Isolate (Motltyin softagma)r bacteria ingested/macrophage(meaniam [m]) +SEM, n = 4)

Primary isolate frompatient no:

1 67 12.02 ± 0.9612 47 6.3 ± 0.3913 80 3.4 ± 0.3316 75 3.8 ± 1.017 30 6.7 ± 0.4119 72 5.28 ± 0.38

PAK 70 5.37 ± 0.66

aAll isolates were susceptible to pilus phage P04 and had a nonmucoidcolonial morphology.

b Mean diameter of spread in soft agar after 24 h of incubation at 37°C(duplicate test).

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NONMOTILE P. AERUGINOSA IN CF INFECTION 599

Pl PAK M 1 12 13 16 17 19

F

FIG. 1. Immunoblot of initial isolates of P. aeruginosa collectedfrom patients 1, 12, 13, 16, 17, and 19. Protein extracts were separatedon 12 and 16% acrylamide gels for panel F and P, respectively. Aftertransfer to nitrocellulose, the extracts were probed for flagellin (F) andpilin (P) expression, using the sera described in Materials and Meth-ods. For the colonizing isolates, 15 ,ug of total protein was examined inpanel F and 30 ,ig was examined in panel P. Extracts from P.aeruginosa P1 (5 jig in panel F; 30 ,ug in panel P) and PAK (10 ,ug inpanel F; 3 ,ug in panel P) were run as positive controls for flagellin andpilin, respectively. Prestained protein molecular weight markers were

loaded in lane M.

Isolates collected from patients 1, 2, 8, 12, 13, 16, and 19remained predominantly motile; more than 85% of P. aerugi-nosa isolates recovered from each of these patients spread insoft agar (Table 1). Profiles of isolate motility versus age are

shown for patients 2, 8, 12, and 19 in Fig. 2A to illustrate themaintenance of motility in this group of CF patients. Theprevalence of nonmotile P. aeruginosa varied between 25 and60% for the isolates collected from patients 3, 6, 9, 11, 14, and18 (Table 1). Motile and nonmotile P. aeruginosa isolates wereharbored in parallel by these patients, and plots of isolatesmotility 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 (pa-tients 4, 5, 10, 15, 17, and 20) were predominantly nonmotile.More than 60% of the isolates recovered from the latter groupof patients were nonmotile; the predominance of nonmotile P.aeruginosa collected from patients 4, 5, 10, and 15 is shown inFig. 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 nonmotilebacteria may be approximated from patients who had beenstudied from primary infection. Initial P. aeruginosa isolateswere collected from patients 1, 12, 13, 16, 17, and 19; nonmo-tile 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; allisolates from patient 19 were motile. Thus, the mean (+standard error) duration of infection prior to appearance ofnonmotile P. aeruginosa was 3.07 (±1.98) years, excludingpatient 19. The mean duration of infection before the emer-

gence of mucoid P. aeruginosa in this group of six patients was

3.37 (± 1.76) years.

Flagellin and pilin expression of P. aeruginosa isolated fromchronically colonized CF patients. Immunoblot analysis offlagellin and pilin expression is shown in Fig. 3 for several ofthe sequential P. aeruginosa isolates from patients 3, 4, 5, 7, 12,and 15. Absence of flagellin expression correlated with absenceof motility in soft agar for the P. aeruginosa isolates examinedby 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 popula-tion. For the majority of the nonmotile P. aeruginosa isolatescharacterized by immunoblotting in Fig. 3, pilin but notflagellin was detected. Nonmotile P. aeruginosa isolates col-lected from patients 5, 7, 12, and 15 synthesized pilin and

assembled pili. Although reaction with the antipilin serum waspoor for many of these nonflagellated isolates (Fig. 3), mostwere susceptible to phage P04, indicating the presence of pili(data not shown). Nonmotile bacteria from patients 3 and 4were exceptions to this trend; these isolates appeared to lackexpression of both pilin and flagellin and were also resistant tothe pilus phage P04 (data shown in Table 4 for one represen-tative nonmotile isolate from each patient). Nonmotile bacte-ria from patient 4 (e.g., isolate 1277; Table 4) were also unableto grow on minimal medium without glutamine. The absenceof flagellin, pilin, and nitrogen assimilation gene expressionfrom isolate 1277 are characteristics of a P. aeruginosa RpoNmutant (36). These RpoN - P. aeruginosa isolates were thepredominant P. aeruginosa phenotype isolated from patient 4for a period of 7 years.The flagellin and pilin expression profile of sequential

isolates from patient 12 clearly indicates the transition frommotile primary isolates to the appearance of nonmotile P.aeruginosa during CF infection. P. aeruginosa was first col-lected from patient 12 at 7.9 years of age; this isolate wasmotile, piliated, and flagellated (Fig. 1 and 3; Table 3). Motileisolates continued to be collected for 5 years, at which pointthe 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 predominatedover the next year and was still being recovered from thispatient at 13.9 years of age.

Expression of flagellin did not correlate with expression ofmucoidy for the P. aeruginosa isolates examined by immuno-blotting. 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 frompatient 15 were all mucoid but lacked flagellin expression (Fig.3). In contrast, P. aeruginosa isolates recovered at 8.5 years ofage from patient 3 and 23.3 years of age from patient 4 weremucoid, expressed flagellin (Fig. 3), and were motile (Fig. 2).

Effect of motility on nonopsonic phagocytosis of P. aerugi-nosa. Motile and nonmotile P. aeruginosa isolates from patients3, 4, 7, and 12 were incubated with murine thioglycolate-elicited macrophages in the absence of serum opsonins (Fig.4). All motile isolates characterized were susceptible to inges-tion, whereas nonmotile bacteria recovered later from eachpatient (Fig. 2) were resistant to nonopsonic phagocytosis bymurine macrophages. Phagocytosis-resistant nonmotile bacte-ria all lacked expression of flagellin, but some expressed pilin.Immunoblot examination of flagellin and pilin expression inthese isolates is shown in Fig. 3, and susceptibility to phageP04 together with a summary of their phenotypic features isshown in Fig. 4. Nonmotile isolates 1608 and 1277 werenonpiliated, whereas nonmotile isolates 4492 and 4503 pos-sessed surface pili; however, all of these isolates were notingested by murine macrophages in the absence of serum.Isolate 511 (motile and phagocytic susceptible) was nonpili-ated, indicating that the presence of pili was not required forthe nonopsonic ingestion of the CF P. aeruginosa isolatesexamined.

Partial complementation of motility in nonmotile CF iso-lates. Plasmid pPT212, carrying the rpoN locus from P. aerugi-nosa PAK (36), was introduced into nonmotile P. aeruginosaCF strains by conjugation in order to assess the role that theregulatory sigma factor, encoded by rpoN, may have in theappearance of the nonmotile phenotype. Various phenotypicchanges resulted from the acquisition of extra copies of rpoNby representative nonmotile P. aeruginosa isolates collectedfrom patients 3 and 4 (summarized in Table 4); no changeswere seen in bacteria carrying the plasmid vector pSP329Galone (data not shown). These nonmotile P. aeruginosa iso-

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patients from whom motile and nonmotile isolates were collected concurrently; (C) patients from whom nonmotile isolates were recoveredpersistently. Strain numbers are indicated for the motile and nonmotile P. aeruginosa isolates collected from patients 3, 4, 7, and 11 which wereused in phagocytic assays (see Fig. 4).

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NONMOTILE P. AERUGINOSA IN CF INFECrION 601

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lates, which were originally RpoN- in phenotype, were par-tially complemented by the introduction of extra copies of therpoN locus (strains containing plasmid pPT212 were desig-nated by the suffix "RP"). Strains 1608RP and 1822RP (de-rived from nonmotile isolates recovered 3 months apart frompatient 3) synthesized flagellin (Fig. 5); however, strain1608RP did not swarm in soft agar and was not actively motilewhen visualized microscopically (Table 4). Pilin synthesis instrains 1608RP and 1822RP appeared to be enhanced over thatof the parental isolates; however, strain 1608RP was not highlysusceptible to phage P04, indicating that surface pili werelargely absent.Nonmotile isolates 1277 and 3000, recovered from patient 4

approximately 3 years apart, were able to synthesize flagellinafter acquisition of extra copies of the rpoN locus (Fig. 5). Themotility of strain 1277RP in soft agar was negligible; however,its colonial morphology was more diffuse than that of theparental isolates (Table 4). Microscopic examination of aculture of 1277RP showed that a small proportion of thebacteria were highly motile (Table 4) and indicated thatflagellin, detected by immunoblotting (Fig. 5), was beingassembled into a functional flagellum in a few bacteria. Strain1277RP was susceptible to phage P04, in contrast to itsparental strain (Table 4), even though no difference in pilinexpression was detected by immunoblotting (Fig. 5). Strain1277RP was also able to grow on minimal medium withoutglutamine supplementation (Table 4). Strain 1277RP was

susceptible to nonopsonic ingestion by murine macrophages, incontrast to its nonmotile parent; however, strain 1608RPremained resistant to nonopsonic phagocytosis.

No obvious phenotypic changes were observed when plas-mid pPT212 was introduced into CF strains lacking flagellinbut able to synthesize pilin expression (data not shown),indicating that the phenotype of these strains may have arisenfrom events affecting flagellum synthesis independently ofRpoN.

DISCUSSION

P. aeruginosa possesses a number of characteristics, such as

acquisition of a rough lipopolysaccharide and secretion ofmucoid exopolysaccharide, proteases, and exotoxins, whichmay aid colonization and survival in the CF lung. Thesevirulence factors have been well studied, and there is a greatdeal of literature about their role in pathogenesis. It has beensuggested that factors such as piliation and flagellation are

important in the colonization of the CF airway (25) and in theestablishment of other infections (7, 11). The presence ofnonmotile, nonflagellated P. aeruginosa in CF infection hasbeen described previously (19); however, the prevalence andmechanism of selection of this phenotype have not beenexplored adequately. In this report, we have presented dataindicating that nonmotile P. aeruginosa is prevalent in chronicCF infection and that P. aeruginosa may utilize specific regu-latory mechanisms to modulate the expression of this alteredphenotype during infection.

P. aeruginosa isolates from the environment and from otherclinical sources were predominantly motile. P. aeruginosaisolates recovered from CF patients early in the course ofinfection matched the highly motile phenotype of environmen-

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Patient 12 Patient 15FIG. 3. Immunoblot analysis of sequential P. aeruginosa isolates from patients 3, 4, 5, 7, 12, and 15. Whole cell lysates (30 ,ug of protein) of

representative isolates from each patient were fractionated on 16% acrylamide gels and transferred to nitrocellulose. Panel F was probed withflagellin antiserum, and panel P was probed with pilin antiserum (see Materials and Methods). Extracts from P. aeruginosa P1 (30 ,ug of protein)and PAK (3 ,ug of protein) were run as positive controls for flagellin and pilin, respectively. Prestained protein molecular weight markers were

loaded in lanes M. Strain numbers are indicated in parentheses for the motile and nonmotile P. aeruginosa isolates from patients 3, 4, 7, and 11which were used in phagocytic assays (see Fig. 4). Numbers above lanes indicate patient ages (in years) at time of isolate recovery.

tal isolates, expressing flagellin and pilin in a normal manner;these findings are in agreement with published data suggestingthe importance of these virulence factors for respiratoryadherence and colonization in CF (6, 22, 24, 25, 27). However,

our study indicates that once chronic colonization is estab-lished, expression of these virulence factors may not be neces-

sary for survival of P. aeruginosa in certain CF patients.Motility and mucoidy appeared to be regulated indepen-

TABLE 4. Summary of phenotypic features of representative nonmotile P. aeruginosa CF strains from patient 3 (strain 1608) and patient 4(strain 1277) and derivative strains carrying plasmid pPT2l2 (rpoN)

Swarming in Motility observed Susceptibility to pilus Growth on minimal Phagocytosis (mean no. ofStrain soft agar by microscopy phage P04 medium without bacteria ingested/macrophageStrainsoft agar by microscopy phage P04 glutamine

±SEM, n = 6)

Parental1608 - - -a + 0.25 ± 0.061277 0.36 ± 0.06

pPT212 derivative1608RP - - _a + 0.64 ± 0.091277RP _b + + + 5.26 ± 0.39a A faint zone of clearing was observed after addition of phage P04, but the majority of bacteria grew in the presence of phage.b Colony in soft agar was diffuse in appearance but had not spread more than 5 mm in diameter.

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NONMOTILE P. AERUGINOSA IN CF INFECTION 603

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FIG. 4. Nonopsonic phagocytosis by murirand nonmotile P. aeruginosa isolates. Motilcmotile later isolates from patients 3, 4, 7, an(murine macrophages in the absence of serufrom which each isolate was collected is iiBacteria ingested after 1 h of incubation ari

(mean plus standard error of the mean for siexpression (immunoblot data shown in Fig.(based on pilus phage susceptibility and imar3) are summarized at the bottom.

dently among the CF P. aeruginosa isexpression of mucoidy did not inhibit t:aeruginosa to swarm in soft agar. The insand mucoidy is in agreement with previ4vations (20, 36) which demonstrate th;phenotypes is achieved by separate menosa. Interestingly, the periods of infepearance of nonmotile and mucoid P. aesimilar (approximately 3 years), suggestadaptations may occur in response to si

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FIG. 5. Immunoblot demonstrating acqcpilin synthesis in nonmotile P. aeruginosapPT212. Protein extracts (40 jig of protSDS-PAGE (16% acrylamide gel), transferrprobed with antiflagellin (F) and antipilinnumbers are indicated, and pPT212-carryirnated by the suffix "RP." Extracts from P. ae

protein) and P1 (40 ,ug of protein) were r

controls, respectively. Prestained protein mwere loaded in lane M.

motile strain of disease that are found only after chronic colonization hasnon-motile strain been established.

We were unable to demonstrate any major differences in theclinical condition of patients harboring nonmotile or motile P.aeruginosa isolates (data not shown). In earlier studies, non-motile P. aeruginosa was recovered predominantly from pa-tients in poor clinical condition (19). We found that nonmotileP. aeruginosa was associated with patients who had beenchronically colonized for several years; it therefore follows thatthese patients generally may be in poor clinical condition.However, patients 4, 5, 10, and 11, who were infected withpredominantly nonmotile P. aeruginosa, were among the olderpatients studied (mean age at the time of preparation of thisreport was 26.3 years) and remained healthy over the periodstudied. These data suggest that nonmotile P. aeruginosa maysimply be markers of chronic disease and neither more nor lessvirulent than the motile variants.

_7)

IlThe majority of nonmotile isolates characterized by immu-(7) (12) noblotting lacked flagellin synthesis, but pilin expression and

5j 4492 2846 4503 assembly of pili was detectable. Very few isolates examined+ - + - were chemotactic mutants that were flagellated but unable to

spread in motility medium (data not shown). The absence of+ + + + flagellin but presence of pilin in some nonmotile P. aeruginosane macrophages of motile isolates suggests that regulation of the flagellar formatione early isolates and non- operon, which is independent of RpoN, may be responsible ford 12 were incubated with the appearance of such isolates during CF infection. Theim opsonins; the patient alternative sigma factor, FliA, that is responsible for transcrip-ndicated in parentheses. .. 'e shown for each isolate tion of the flagellin gene (35) may play a role in the appearanceix experiments). Flagellin of such isolates. Expression of fliA in P. aeruginosa has been3) and pilin expression proposed to be under the control of various regulatory ele-

nunoblot data shown Fig. ments and may be modulated in response to environmentalsignals (35). Indeed, a wide range of adverse conditions havebeen shown to cause lack of flagellum formation and motilityin E. coli (17), and repression of flagellin synthesis has beenproposed to result from a novel system for sensing harsh

,olates examined, and environments which is independent of catabolite repression orhe ability of motile P. chemotactic regulation (26). It is not unreasonable to assumedependence of motility that P. aeruginosa may possess similar sensing and regulatoryously published obser- systems which cause loss of flagellum formation under theat regulation of these conditions prevalent in the CF lung.chanisms in P. aerugi- Flagellation, piliation, and motility in P. aeruginosa may alsoction prior to the ap- be coordinately regulated by RpoN (36), and nonmotile P.eruginosa isolates were aeruginosa isolates recovered from two of the CF patientsting that these specific studied appeared to have derived from regulatory eventsimilar signals or states involving this alternate sigma factor. Although such CF isolates

possessed traits characteristic of P. aeruginosa RpoN mutants,they were complemented only partially for motility by theintroduction of plasmid-borne copies of the rpoN locus. Strains1277, 1608, 1822, and 3000, the RpoN- CF isolates, had notirreversibly lost the ability to express flagellin and pilin (Fig. 5).Isolate 1608, collected from patient 3, though lacking inexpression of flagella and pili, was able to grow on minimalmedium without the addition of glutamine (Table 4), whichalso indicates that this was not the RpoN - phenotype.Complementation of isolate 1277 with the rpoN locus restoredthe phagocytic susceptibility of this isolate; however, nonmotilestrain 1608RP, although synthesizing flagellin, was not motile

uisition of flagellin and or susceptible to nonopsonic phagocytosis. Overall, the intro-strains carrying plasmid duction of extra copies of rpoN into these nonmotile P.ein) were separated by aeruginosa isolates partially corrected their motility defects anded to nitrocellulose, and indicated that the regulatory events leading to the appearance(P) sera. Parental strainng derivatives are desig- of this phenotype are complex and not due exclusively to?ruginosa PAK (1.5 jig of mutation In the rpoN locus.un as pilin and flagellin Interestingly, the RpoN- isolates from patient 4 were theolecular weight markers predominant phenotype isolated over a 7-year period of infec-

tion, suggesting that these strains were at no survival disadvan-

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tage. The glutamine auxotrophy of nonmotile isolates collectedfrom patient 4 may not be a significant disadvantage under thecondition prevalent in the inflamed tissues of the CF lung,where cellular lysis may enable P. aeruginosa access to a widerange of nutrients. The ability for P. aeruginosa RpoN mutantsto survive in infection has been clearly demonstrated in amurine mucosal colonization model (24). Studies of pilinexpression by serial CF isolates of P. aeruginosa (23) haveshown that assembly of pili may be turned off during CFinfection. The nonmotile sequential isolates collected frompatient 4 clearly demonstrate that P. aeruginosa of an RpoN -phenotype, lacking both flagellin and pilin, may be selectednaturally and survive in vivo during CF infection.

Evasion of host defenses may be the major requirement forthe survival of P. aeruginosa in the CF lung once chroniccolonization is established. The inability of macrophages tophagocytose nonflagellated CF isolates, demonstrated herein,may be one reason for the selection of this phenotype in CF.Previous data (13) had implicated pili as the major ligand forthe nonopsonic ingestion of P. aeruginosa by macrophages;piliation and hydrophobicity of P. aeruginosa were also corre-lated with phagocytic susceptibility (33). Phagocytosis of CF P.aeruginosa isolates examined in this study was not solelydependent on the presence of pili; strain 511 (Fig. 5), lackeddetectable surface pili but was ingested by macrophages in theabsence of serum opsonins. Nonopsonic phagocytosis of the P.aeruginosa CF isolates examined in our study appeared to bedependent on strain motility and the presence of a functionalflagellum. The phagocytosis resistance of nonmotile bacteriacould not be attributed solely to lack of contact betweenbacteria and macrophages; performing the phagocytic assayson a rocking platform or forcing the bacteria down onto themonolayer by centrifugation both failed to enhance the non-opsonic ingestion of the nonmotile CF isolates examined (datanot shown). The ability of the nonflagellated and RpoN - CFisolates examined in this study to resist nonopsonic uptake bymacrophages is consistent with studies indicating that RpoN-dependent ligands play a major role in the recognition of P.aeruginosa by macrophages in the absence of serum opsonins(13, 21). The phagocytosis resistance of nonmotile bacteriatogether with the finding that macrophage phagocytosis of P.aeruginosa is critically dependent on the presence of glucose(32) may also help to explain why P. aeruginosa is such anefficient respiratory pathogen in CF. The primary isolates fromCF infection studied were all motile and susceptible to inges-tion in the presence of glucose; however, the low concentrationof glucose in bronchial fluid (37) may initially preclude ade-quate clearance of such motile isolates from the CF lung.

In conclusion, we have demonstrated that initial isolates ofP. aeruginosa from patients with CF are motile, flagellated, andpiliated, resembling phenotypically strains isolated from theenvironment or from clinical conditions characterized by acuteinfection. Isolates of P. aeruginosa recovered from CF patientsafter chronic colonization has been established are frequentlynonmotile and mucoid, two phenotypic characteristics whichmay aid P. aeruginosa in its evasion of host defenses. Loss offlagellum formation and the attenuation of expression ofnonessential genes, as occurs during chronic respiratory tractcolonization in CF, may reflect an adaptive response toconserve energy and enable bacterial survival under hostileconditions; this phenotypic conversion might coincidentallypromote the survival of P. aeruginosa by providing a means forresisting host defenses. The involvement of alternate sigmafactors in the coordinate control of virulence factor expressionduring chronic infection may also be fundamental to thepathogenesis of P. aeruginosa in CF.

ACKNOWLEDGMENTS

Excellent technical assistance was provided by Nicole Glenham, LisaThorson, and Spencer Matheson.We thank Stephen Lory, Sameer Barghouthi, Britt-Inger Marklund,

and Richard Stokes for helpful discussions during preparation of themanuscript.E.M. was supported by a fellowship from the Canadian Cystic

Fibrosis Foundation (CCFF). This work was supported with fundsfrom the CCFF, the Medical Research Council (Canada), and theCanadian Bacterial Diseases Network.

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