P. aeruginosa in Cystic Fibrosis PatientsResists Host Defenses, AntibioticsOver many generations, this pathogen produces variants that resistdrugs, while adapting to host compartments and defenses

Niels Høiby

Cystic fibrosis (CF) patients have mu-tations in the cystic fibrosis trans-membrane conductance regulator(CFTR) gene that affect chloridechannels, decreasing paraciliary

fluid in the lower respiratory tract and impairingclearance there of inhaled microbes. This im-pairment leads to early recruitment of inflam-matory defense elements, such as polymorpho-nuclear leukocytes (PMN) and antibodies.

CF patients suffer from chronic and recurrentrespiratory tract infections, complicated byPMN inflammatory responses. In particular,persistent infections with Pseudomonas aerugi-nosa typically lead to lung failure, followed ei-ther by transplants or early death for CF pa-tients (Fig. 1).

Adaptive mechanisms help to explain how P.

aeruginosa so effectively persists for several de-cades in the respiratory tracts of CF patients,overcoming host defense mechanisms as well asintensive antibiotic therapy. For example, con-sider one 42-year-old male CF patient with achronic P. aeruginosa infection that lasted morethan 28 years. This patient developed antibodiesto this pathogen at the outset of the infectionand was treated at regular intervals with 114two-week courses of intravenous tobramycinand �-lactam antibiotics—and in the laterstages, on a daily basis with nebulized colistin.Nonetheless, a mucoid P. aeruginosa strain per-sisted as a biofilm in his lungs, which weredestroyed by his own activated PMNs.

P. aeruginosa Survives by Adapting to

Host Inflammatory Responses

P. aeruginosa adapts to the inflammatorydefense system of CF patients by formingmucoid biofilms. For example, newly diag-nosed CF infants with viral or bacterial lunginfections carry significantly increased num-bers of PMNs and alveolar macrophagescompared to uninfected CF infants or chil-dren without CF. When PMNs phagocytosebacteria, the host cells produce highly reac-tive oxygen species (ROS) that kill or inducemutations in the bacteria and also damagesurrounding host tissues.

We found that hydrogen peroxide by it-self or activated PMNs can induce muta-tions in the mucA gene of P. aeruginosastrain PAO1 that confers the mucoid phe-notype. Furthermore, 93% of mucoid P.aeruginosa from Scandinavian CF patientscarry mutations in that gene, directing thebacteria to produce alginate, which is an

Summary

• Cystic fibrosis (CF) patients typically developpersistent Pseudomonas aeruginosa lung infec-tions that lead to lung failure, attributable inpart to chronic host inflammatory responses.

• P. aeruginosa adapts to the inflammatory de-fense system of CF patients and occupies spe-cific anatomic niches, forming mucoid biofilmsor microcolonies that reflect particular condi-tions.

• Although P. aeruginosa biofilms are the mainreason for persistent infections in CF patients,conventional antibiotic-resistance mechanismsalso play a role; the eventual appearance ofmutator variants further helps to explain thehigh frequency of multiply antibiotic-resistantgenes in strains infecting CF patients.

Niels Høiby is Chairof the Departmentof Clinical Microbi-ology and Professorof Medical Microbi-ology at the Univer-sity of Copenhagen,Copenhagen, Den-mark.

Volume 1, Number 12, 2006 / Microbe Y 571

oxygen radical scavenger. The change also pro-tects P. aeruginosa against phagocytosis andbeing cleared from host lungs. Thus, the mucoidphenotype helps to protect this pathogen againstinflammatory defense mechanisms of the host.

Gram stains of mucoid P. aeruginosa from

sputum of CF patients show these bac-teria to be biofilms (Fig. 2). When thosebiofilms are grown in flow-cells or ani-mals, their resistance to PMNs and theantibiotic tobramycin depends on quo-rum sensing (QS)-regulated virulencefactors. Thus, QS mutants or QS inhib-itors (QSI) such as furanones, garlicextract, and ginseng render the biofilmssusceptible to PMNs and sensitive totobramycin.

Possible measures to prevent theseeffects include use of anti-inflammatorydrugs or antioxidants to prevent ROS-induced mutations in the mucA gene,macrolides such as azithromycin to in-hibit alginate synthesis, or QSIs to stopbiofilms from forming. Currently, how-ever, CF patients are treated early andaggressively in an effort to keep nonmu-coid P. aeruginosa strains from coloniz-ing their lungs. This approach, used inthe Danish CF Centre since 1989, pre-vents about 80% of chronic P. aerugi-nosa lung infections in CF patients.However, once chronic biofilms are es-tablished, systemic and nebulized anti-biotics are needed to maintain lungfunction.

The Conductive and Respiratory

Zones of Lungs

The humans lungs contain a smallerconductive zone and a larger respira-tory zone (Fig. 3). The respiratory zone,which is about 3,000 ml and accountsfor 95% of the lung volume, containsrespiratory bronchioles and alveolarducts and sacs. However, this part ofthe lungs contains no cilia and no sub-mucosal glands. Its defense system con-sists of several elements, such as alveo-lar macrophages and defensins. All thevenous blood of the body passesthrough the capillaries of the alveoles,forming a nearly continuous sheet of

blood and only a very thin barrier between itand air.

The smaller conducting zone, which is about150 ml or 5% of the lung volume, includes thetrachea, bronchi, and terminal bronchioles. Thispart of the lungs has cilia, goblet cells, submucal

F I G U R E 1

Cells from an autopsy of a Danish girl with CF who died due to chronic P. aeruginosa lunginfection. She had 21 precipitating antibodies in serum against P. aeruginosa. (A)Hematoxylin-eosin stain; magnification, x100, showing mucoid biofilms of P. aeruginosasurrounded by pronounced inflammation in the lung tissue. (B) Comparison of P.aeruginosa growing as an in vitro biofilm in a flow cell (upper left), growing as chronicalginate biofilm in a CF mouse model (lower right; Hoffmann et al., 2005), and in a CFpatient. Obvious morphological similarities are seen.

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Høiby Focuses on Cystic Fibrosis, but Faced down Smallpox and other Scourges

With the worldwide scourge ofsmallpox nearly complete in 1970,Niels Høiby found himself on thesecond day of his medical resi-dency caring for a student withthis disease. Høiby, the rest of thehospital staff, and 500 studentswere quarantined for 14 days. Al-though the patient died, nobodyelse, even those who previouslywere not vaccinated, became sick.“This success, and the excitingexperience, forever changed mylife,” Høiby recalls. “I wouldnever leave microbiology and in-fectious disease medicine.”

True to his word, Høiby de-voted his career to microbiologyand infectious diseases, focusingon Pseudomonas aeruginosa andother pathogens that provoke fre-quent and chronic lung infectionsin patients with cystic fibrosis(CF). His approach to aggres-sively treating infections with thisbacterium in CF patients in Den-mark has helped increase their av-erage lifespan from age 20 in1975 to age 50 in 2004. By com-parison, CF patients in the UnitedStates typically live only into theirlate 30s, according to the CysticFibrosis Foundation.

Høiby, 65, chairs the depart-ment of clinical microbiology atRigshospitalet and is professor ofmedical microbiology at the Uni-versity of Copenhagen. In addi-tion to his CF research, he runs alarge diagnostic clinical microbi-ology lab in a 1,100-bed hospitalthat covers all medical specialties.

Høiby was born when Germansoldiers occupied Denmark dur-ing World War II. “My father,who was a stockbroker, was ac-tive in the resistance against theGermans right from the begin-ning,” he says. His mother, edu-cated as a classical singer and

pianist at the Royal Danish Con-servatory, gave music lessonswhen he was a child. He has ayounger sister.

“There was no scientific tradi-tion in my family, and I thought,when I was in high school, that Iwould be a stockbroker like myfather,” he says. “I was perform-ing very well in mathematics andphysics in school, but I was alsovery interested in gymnastics andmusic.” He nearly followed hismother’s lead by pursuing a musi-cal career, he adds, noting: “Iplayed jazz trumpet for someyears, and Louis Armstrong wasmy favorite. I have his autographon a photo I took myself.”

However, Høiby changed hismind about a career in music dur-ing his last year of high school. “Ithought that I had to work withhuman beings, and with some sci-ence, and therefore I decided tostudy medicine,” he says. Later, hereceived his medical degree fromthe University of Copenhagen.

Besides his work in microbiol-ogy and infectious diseases, Høibywrites books and articles aboutmedical politics, a subject that cangive rise to debate. He alsofounded and chairs the DanishSociety to Assure the Doctor’sRight to Speak Freely, an organi-zation that arose from the experi-ence of a good friend and formerprofessor of pediatrics, who “wasforced to quit his job because hecriticized the administration’s re-striction on the economy of thehospital, which gave severe prob-lems for pediatric patients,” hesays.

Høiby coauthored a bookabout an SS doctor, Carl Vaer-net, who conducted medicalexperiments on homosexuals atthe Buchenwald concentration

camp duringWorld War II.“I have al-w a y s w o n -d e r e d w h ydoctors couldt r a n s f o r mfrom healersto ki l lers ,”Høiby says. “Vaernet escaped toArgentina, where he died 40 yearsago. His son was a famous neuro-surgeon in my hospital, whom Iknew very well, but the storyabout his father was not knownby the public before we startedlooking in the archives in Den-mark and Germany.”

Høiby visited Vaernet’s gravewhen he traveled to Buenos Airesto attend a CF meeting. “Thebook was received very well, and Iwas invited to give several lecturesat congresses for sexuality disor-ders in Denmark and Scandina-via—a very unusual experiencefor a microbiologist,” he says.

Høiby and his wife Birgit, anart historian, have three children.Their daughter is a veterinarian,one son a physiotherapist, andtheir other son a painter. “Mywife and I, together with all ourchildren and grandchildren, al-ways spend a week together inSwitzerland in the skiing season,”he says. “We love this week sinceit brings the whole family to-gether, [and] this tradition wasstarted by my parents 45 yearsago. We also have a summerhouseat the beach. Our children andgrandchildren use this house veryfrequently, so we have a goodfamily life.”

Marlene Cimons

Marlene Cimons is a freelance writerin Bethesda, Md.

Volume 1, Number 12, 2006 / Microbe Y 573

glands, and an arterial blood supplyfrom the aorta. The mucus is producedin the respiratory zone, and the defensesystem consists of elements such as themucociliary escalator.

When nebulized tobramycin, colis-tin, or other antibiotics are used to treatP. aeruginosa lung infections, the drugsreach very high concentrations in theconductive zone, but remain very low inthe respiratory zone. However, whenantibiotics are administered systemi-cally, they are found in very low con-centrations in sputum, but in high con-centrations in respiratory tissuesbecause antibiotics move through theblood directly to alveolar capillaries be-fore being distributed elsewhere in thebody. Because both the respiratory andconductive zones of CF patients typi-cally are infected with P. aeruginosa,combined systemic and nebulized anti-biotic treatments are indicated.

P. aeruginosa Cells Adapt To

Survive in the Lung Respiratory

Zone

The airspace of the respiratory zone isaerobic, containing 13% O2 and 5%CO2, so long as the air and blood sup-ply remain normal. If mucus plugs theairflow, this zone can become mi-croaerophilic, with oxygen levels drop-ping to 5%, and CO2 rising to 6%. Ifabscesses form, sites within this zonebecome anaerobic, and then localizedcells and tissues become necrotic. Al-though P. aeruginosa can grow underall these conditions, generation timesare shorter when this zone is aerobic.Neither macrophages nor PNMs sur-vive in anaerobic niches.

Mucoid P. aeruginosa biofilms typi-cally concentrate in the respiratory zoneof CF lungs, according to autopsy find-ings (Fig. 1). Such patients typicallyproduce antibodies against alginate,and there is pronounced PMN-domi-nated inflammation that surrounds bio-film-containing alveoles, damaging thetissues and impairing lung functions.

F I G U R E 2

Gram-stained sputum from four CF patients. A-B. P. aeruginosa biofilms and PMNs areseen. C. A P. aeruginosa biofilm with two distinct morphologies is seem mayberepresenting release of nonmucoid revertant from the mucoid biofilm. D. Sputum froma CF female, 44 years old, growth of mucoid and nonmucoid P. aeruginosa in sputum. P.aeruginosa since 1970, chronic mucoid P. aeruginosa since 1971. She has had 35–44precipitating antibodies in serum against P. aeruginosa for many years. A detachedalveolus with a P. aeruginosa biofilm is seen (double arrow, x100 & x100).

574 Y Microbe / Volume 1, Number 12, 2006

Detached biofilm-containing alveolessometimes are also found in sputum(Fig. 2D), and degradation productsfrom elastin and collagen can be de-tected in urine.

Healthy lungs contain 300,000,000alveoles, and they typically decline by1–2% per year, or by 2,700–5,400 perday. P. aeruginosa biofilms are focal,and localized tissue damage can be de-tected by high-resolution CT scans.Gradually, however, those focal infec-tions spread, impairing respiratoryfunction despite intensive antibiotictherapy.

Although many PMNs are found intissue surrounding mucoid P. aerugi-nosa biofilms, alginate apparentlykeeps PMNs from entering the biofilms(Fig. 1 and 2). Thus, P. aeruginosaadapt to the respiratory zone by form-ing mucoid biofilms that enable the bac-teria to withstand the host inflamma-tory response, which, however, helps todestroy localized lung tissue.

P. aeruginosa Cells also Adapt To

Survive in the Lung Conductive Zone

The lung conductive zone consists of 16 sets ofbronchi, extending from the trachea to the deep-set, terminal bronchioles. Only the uppermostfour or five sets, or “generations,” can be di-rectly observed by bronchoscopy, making it nec-essary to examine the the distal sets by lavage (orby examining lungs from transplants or at au-topsy).

In the conductive zone, P. aeruginosa micro-colonies are found mainly inside sputum, whereconditions are anaerobic. Thus, few bacteria arefound along the epithelial surface of the bronchi.Gram-stained smears of sputum from CF pa-tients contain not only mucoid biofilms but alsoplanktonic single cells. When cultured, suchsputum samples typically grow mucoid andnonmucoid cells, but sometimes also growsmall-colony-variant phenotypes.

Recent experiments suggest that such pheno-typic variation occurs in response to differencesin growth conditions that lead to mutations orother changes, such as the bacterial SOS re-sponse. For example, while growing a clinical

mucoid P. aeruginosa CF strain, we found non-mucoid cells and sometimes small-colony vari-ants (Fig. 2 A and C, Fig. 4). The mucoid phe-notype is unstable under anaerobic conditions.Meanwhile, the flagella-mediated motility of thenonmucoid phenotype is negatively regulated bythe sigma factor algT, which positively regulatesalginate biosynthesis. Thus, nonmucoid P.aeruginosa cells presumably move to the aerobicsurface, giving them a selective advantage aris-ing from a faster growth rate.

Mucoid P. aeruginosa strains from CF patientshave mutation insertions in mucA, as do many ofthe nonmucoid revertants from these patients.Furthermore, many of the nonmucoid revertantsare QS deficient and nonvirulent. Nonmucoidphenotypes generally do not give rise to a pro-nounced antibody response in the few patientswho are colonized with such phenotypes, perhapsreflecting their being in sputum and away fromkey cells of the immune system.

In general, mucoid biofilms from the aerobicrespiratory zone are transported to the anaero-bic sputum in the conductive zone, where thenonmucoid phenotypes split off. The anaerobic

F I G U R E 3

The conductive and respiratory zone of the lungs.

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conditions of sputum probably account for theabundance of dead PMNs found there—alongwith DNA, elastase, and myeloperoxidase fromdead PMNs. The P. aeruginosa nonmucoid re-vertants probably do not contribute much to

tissue damage and, therefore, do notrequire special therapeutic or prophy-lactic attention.

P. aeruginosa Cells Also Survive

by Adapting to Antibiotic Therapy

Despite being subject to intensive anti-biotic therapy, chronic P. aeruginosalung infections rarely, if ever, are erad-icated. Although P. aeruginosa biofilmsare the main reason for this persistence,the appearance of multiple drug-resis-tant isolates of P. aeruginosa in CF pa-tients suggests that conventional resis-tance mechanisms also play a role. Drugsto which such P. aeruginosa strains be-come resistant include �-lactams, cipro-floxacin, tobramycin, and colistin.

Biofilms of P. aeruginosa respond to�-lactam antibiotics by producing chro-mosomal �-lactamase. Nonmucoidphenotypes are more resistant to suchantibiotics and have higher oxygen rad-ical damage than do mucoid pheno-

types from the same patients. One countermea-sure that improves clinical outcomes involvesinducing antibodies in CF patients that are di-rected against chromosomal �-lactamases pro-duced by P. aeruginosa—and thus prolongingthe efficacy of this class of antibiotics.

Mutator strains help to explain thehigh frequency of multiply antibiotic-resistant P. aeruginosa strains from CFpatients (Fig. 5). Mutator strains typi-cally carry defects of their DNA repairenzymes, accounting for their muchhigher frequencies of mutations com-pared to wild-type strains. Mutatorstrains of P. aeruginosa from CF pa-tients often have increased levels ofDNA damage that may be due to en-counters with activated PMNs duringchronic infections. In general, we findno mutator strains during the first fewyears of such chronic infections. How-ever, the numbers of such strains after 5years increase steadily, reflecting howthis pathogen survives for decades insuch patients despite being exposedover many years to intensive antibiotictherapy.

F I G U R E 4

Adaptive divergence in vitro of Pseudomonas aeruginosa from a CF patient (Hoffmann etal., 2005). P. aeruginosa CF 57388A (mucoid) after 14 days shaken (left) or nonshakenstatic (right) aerobic culture in a flask, notice the appearance of 3 phenotypes: mucoid,nonmucoid, and small colony variants (SCV). Shaken: Mucoid colonies dominates (after1 week there was only mucoid colonies), Static: Nonmucoid and SCV colonies dominate(similar results after 1 week). CF 57388A (mucoid) has in mucA: at 170 deletion C, frameshift, stop codon 282 and at 293 insertion 105 bp. CF 57388B (nonmucoid) hasadditionally in algT at 147 insertion AGCCCAGGA, frame shift.

F I G U R E 5

Mutation frequency after exposure to rifampicin of 141 P. aeruginosa isolates collectedin five periods of chronic lung infection (0–5, 5–10, 10–15, 15–20, and 20–25 years) from11 patients with CF (Ciofu et al. 2005). The results for hypermutable (hp: filled triangles)and nonhypermutable (open circles) P. aeruginosa isolates are presented. The mutationfrequency after exposure to rifampicin of reference strains PAO1 is shown.

576 Y Microbe / Volume 1, Number 12, 2006

Evolutionary Implications of the

Adaptability of P. aeruginosa

The genome of the P. aeruginosa PAO1 straincontains 6.2 million base pairs and 5,570 genes,including a high proportion of regulatory, cata-bolic, transport, efflux, and chemotaxis genes,that help to explain its adaptibility. Strains fromCF patients with chronic lung infections showsome obvious differences when compared to thePAO1 strain. For instance, the strains from suchpatients typically are alginate producers, growin biofilms, split off nonmucoid and small col-ony variant phenotypes, and often are mutators

with many accumulated mutations that makethem auxotrophs.

Søren Molin from Danish Technical Univer-sity, other collaborators, and I find that clinicalCF strains grow much slower than PAO1, with amean generation time of 4 hours. After survivingin CF lungs for 30 years, such strains typicallyundergo 65,000 divisions, enough time to en-able them to adapt in many ways to life in CFlungs but perhaps no longer to survive else-where. Genomic analysis of such isolates couldhelp to explain more precisely the extent of thisadaptive process.

SUGGESTED READING

Bagge, N., M. Hentzer, J. B. Andersen, O. Ciofu, M. Givskov, and N. Høiby. 2004. Dynamics and spatial distribution ofbeta-lactamase expression in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 48:1168–1174.Bjarnsholt, T., P-.Ø. Jensen, M. Burmølle, M. Hentzer, J. A. J. Haagensen, H. P. Hougen, H. Calum, K. G. Madsen, C. Moser,S. Molin, N. Høiby, and M. Givskov. 2005. Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide andpolymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 151:373–383.Bjarnsholt, T., P.Ø. Jensen, T. B. Rasmussen, L. Christophersen, H. Calun, M. Hentzer, H.-P. Hougen, J. Rygaard, L. Eberl,N. Høiby, and M. Givskov. 2005. Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonasaeruginosa infections. Microbiology 151:3873–3880.Ciofu, O., B. Riis, T. Pressler, H. E. Poulsen, and N. Høiby. 2005. Occurrence of hypermutable P. aeruginosa in cystic fibrosispatients is associates with the oxidative stress caused by chronic lung inflammation. Antimicrob. Agents Chemother.49:2276–2282.Hoffmann, N., T. B. Rasmussen, P. Ø. Jensen, C. Stub, M. Hentzer, S. Molin, O. Ciofu, M. Givskov, H. K. Johansen, and N.Høiby. 2005. Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis. Infect. Immun.73:2504–2514.Høiby, N. 2002. Understanding bacterial biofilms in patients with cystic fibrosis: current and innovative approaches topotential therapies. J. Cystic Fibrosis 1:249–254.Lee, B., J. A. J. Haagensen, O. Ciofu, J. B. Andersen, N. Høiby, and S. Molin. 2005. Heterogeneity of biofilms formed bynon-mucoid Pseudomonas aeruginosa isolates from patients with cystic fibrosis. J. Clin. Microbiol. 43:5247–5255.Mathee, K., Ciofu, O., Sternberg, C., Lindum, P.W., Campbell, J.I.A., Jensen, et al. 1999. Mucoid conversion of Pseudomonasaeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology: 145:1349–1357.Song, Z. J., H. Wu, O. Ciofu, K. F. Kong, N. Hoiby, J. Rygaard, et al. 2003. Pseudomonas aeruginosa alginate is refractoryto Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection. J. Med. Microbiol.52:731–740.

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