Polymorphonuclear leukocytes restrict growth of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients

Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo
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  Polymorphonuclear Leukocytes Restrict Growth of   Pseudomonasaeruginosa  in the Lungs of Cystic Fibrosis Patients Kasper N. Kragh, a Morten Alhede, a,b Peter Ø. Jensen, b Claus Moser, b Thomas Scheike, c Carsten S. Jacobsen, d Steen Seier Poulsen, f  Steffen Robert Eickhardt-Sørensen, a Hannah Trøstrup, b Lars Christoffersen, b Hans-Petter Hougen, g Lars F. Rickelt, e Michael Kühl, e,h,i Niels Høiby, a,b Thomas Bjarnsholt a,b Department of International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark  a ; Department of Clinical Microbiology,Rigshospitalet, Copenhagen, Denmark  b ; Department of Public Health, Section of Biostatistics, University of Copenhagen, Copenhagen, Denmark  c ; Geological Survey of Denmark and Greenland, Copenhagen, Denmark  d ; Marine Biological Section, Department of Biology, University of Copenhagen, Helsingør, Denmark  e ; Department of Biomedical Science, University of Copenhagen, Copenhagen, Denmark  f  ; Department of Forensic Medicine, University of Copenhagen, Copenhagen, Denmark  g ; PlantFunctional Biology and Climate Change Cluster, University of Technology Sydney, New South Wales, Sydney, Australia h ; Singapore Centre on Environmental Life ScienceEngineering, School of Biological Science, Nanyang Technological University, Singapore i Cysticfibrosis(CF)patientshaveincreasedsusceptibilitytochroniclunginfectionsby  Pseudomonas aeruginosa ,buttheecophysiologywithintheCFlungduringinfectionsispoorlyunderstood.Theaimofthisstudywastoelucidatethe in vivo growthphysiologyof  P. aeruginosa  withinlungsofchronicallyinfectedCFpatients.Anovel,quantitativepeptidenucleicacid(PNA) fluorescence  in situ  hybridization (PNA-FISH)-based method was used to estimate the  in vivo  growth rates of   P. aerugi-nosa directlyinlungtissuesamplesfromCFpatientsandthegrowthratesof  P. aeruginosa ininfectedlungsinamousemodel.Thegrowthrateof  P. aeruginosa  withinCFlungsdidnotcorrelatewiththedimensionsofbacterialaggregatesbutshowedaninversecorrelationtotheconcentrationofpolymorphonuclearleukocytes(PMNs)surroundingthebacteria.Agrowth-limiting effecton P. aeruginosa byPMNswasalsoobserved in vitro ,wherethislimitationwasalleviatedinthepresenceofthealternativeelectronacceptornitrate.Thefindingthat P. aeruginosa growthpatternscorrelatewiththenumberofsurroundingPMNspointstoabacteriostaticeffectbyPMNsviatheirstrongO 2 consumption,whichslowsthegrowthof  P. aeruginosa ininfectedCFlungs.Insupportofthis,thegrowthof  P. aeruginosa  wassignificantlyhigherintherespiratoryairwaysthanintheconduct-ingairwaysofmice.Theseresultsindicateacomplexhost-pathogeninteractioninchronic P. aeruginosa infectionoftheCFlung  wherebyPMNsslowthegrowthofthebacteriaandrenderthemlesssusceptibletoantibiotictreatmentwhileenablingthemtopersistbyanaerobicrespiration. P atients with the genetic disorder cystic fibrosis (CF) havehighlyviscousendobronchialmucusanddecreasedmucocili-ary clearance of the airways, which render them susceptible tochronic bacterial lung infections. Severe chronic  Pseudomonasaeruginosa lunginfectionsarethemostcommoncauseofmorbid-ity and mortality in CF patients (1, 2). Lungs of CF patients with chronic  P. aeruginosa  infections are characterized by intrabron-chialmucus-imbeddedaggregatesofbacterialcells(biofilms)sur-rounded by high numbers of polymorphonuclear leukocytes(PMNs) (3, 4). Such PMN-surrounded biofilms can persist over the lifetime of CF patients, despite an extensive inflammatory re-sponse and aggressive antibiotic treatment (5). Slow growth within bacterial biofilms is recognized as a majorcontributor to high antibiotic tolerance because the effectivenessof the majority of antibiotics in clinical use decreases with low bacterial metabolism (6, 7). Limited molecular oxygen (O 2 ) canfurther increase the tolerance of   P. aeruginosa  biofilms for antibi-otics  in vitro  (8). Mucus in the conducting airways of chronically infected CF patients is characterized by steep O 2  concentrationgradients ranging from normoxic to anoxic conditions, and thecombinationofslowdiffusivetransportandintenseO 2 consump-tionwithinthemucusleadstoanoxia(9).Thisisaccompaniedby  ongoingdenitrification,asevidencedbyN 2 Oproduction(10),thedenitrification biomarker OprF in sputum (11), antibodiesagainstnitratereductaseinserum(12),andupregulationofgenesfor denitrification (13, 14). O 2  gradients in the endobronchialsecretions are primarily a result of the O 2  consumed by PMNs forthe formation of reactive oxygen species (15, 16) during respira- tory burst (15, 16) and for the production of nitric oxide (10) by  nitric oxide synthase (54, 55). In addition, the fraction of O 2  con-sumed by PMNs for aerobic respiration in endobronchial secre-tions from chronically infected CF patients is negligible (15, 16). The growth rate of   P. aeruginosa  is diminished by the low avail-ability of O 2  (17); therefore, depletion of O 2  within the mucus of CF patients could serve as a limiting factor for the growth of   P.aeruginosa andmaycontributetotheslowgrowthof  P.aeruginosa in the sputum of chronically infected CF patients (18). Alterna-tively,ithasbeensuggestedthatisolatesfromchronicallyinfectedCF patients may develop genetic adaptations that reduce thegrowth rate of the bacteria (19). Under these conditions, theabove-mentioned denitrification indicators point to anaerobicrespiration using nitrate as an alternative metabolic mode of   P. Received  24 April 2014  Returned for modification  11 July 2014 Accepted  5 August 2014 Published ahead of print  11 August 2014 Editor:  B. A. McCormick Address correspondence to Thomas Bjarnsholt, material for this article may be found at /IAI.01969-14.Copyright © 2014, American Society for Microbiology. All Rights Reserved.doi:10.1128/IAI.01969-14 November 2014 Volume 82 Number 11 Infection and Immunity p. 4477–4486  4477   onA  pr i  l  1  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   aeruginosa , resulting in lower energy yields and possibly lowergrowthratesinbiofilmsintheCFlung.However,onlythe in vitro growthofbacteriaisolatedfromsputumsampleshasbeenstudied(18), and the actual growth rates of   P. aeruginosa  within CF lungshave neither been mapped nor correlated to growth limitation  invivo .In this study, we developed a new quantitative peptide nucleicacid fluorescence  in situ  hybridization (PNA-FISH)-basedmethod that enabled mapping of the  in vivo  growth rates of   P.aeruginosa  for the first time. This method was used to investigatethegrowthof  P.aeruginosa inchronicallyinfectedCFlungsandinthe conducting and respiratory airways of   P. aeruginosa -infectedmice. A significant negative correlation was observed between thegrowth rate and the abundance of PMNs surrounding the bacte-rialbiofilmaggregates.AstrongPMN-inducedO 2 limitationon P.aeruginosa  growth was confirmed  in vitro , while the bacterialgrowth limitation was alleviated in the presence of an alternativeelectron acceptor (nitrate) that enabled denitrification. MATERIALS AND METHODS Bacterial strains.  The  P. aeruginosa  PAO1 wild-type strain used in all  invitro  experiments was obtained from the  Pseudomonas  Genetic Stock Center (strain PAO0001 []). The Escherichia coli  laboratory strain MG1655 was used for production of spike-in DNA (20).  Ex vivo  CF patient samples.  Samples were obtained from explantedlungs of three CF patients chronically infected with  P. aeruginosa  (onemale and two females ranging from 30 to 42 years old). Tissue was col-lected following approval (KF-01278432) from the Danish Scientific Eth-icalBoard.Allthreepatientshadundergonedouble-sidedlungtransplan-tationattheCopenhagenUniversityHospital,Rigshospitalet.Lungtissuesamples ( n  6 to 7 from each patient) were removed immediately afterextraction. Samples ( n    20) were fixed in phosphate-buffered salinecontaining 4% paraformaldehyde and embedded in paraffin. Sections (4  m thick) were cut using a standard microtome and fixed on glass slides.The slides were stored at 4°C until further analysis. In total, 59 bacterialbiofilms were analyzed. Mouse model.  To examine differences in bacterial growth rate as afunction of O 2  partial pressure, we used a recently described model basedontheinstillationofbacteriaimmobilizedonsmallorlargealginatebeadsinto the respiratory or conducting zone of the lungs (21). Briefly, the  P.aeruginosa  strain PAO579 was propagated overnight at 37°C in ox broth(Statens Serum Institute, Denmark). The overnight culture was centri-fuged at 4°C and 4,400   g   and resuspended in 5 ml of serum bouillon(Department of Clinical Microbiology, Herlev Hospital, Denmark). Alg-inate (Protanal LF 10/60; FMC BioPolymer, Norway) was dissolved in0.9% NaCl to a concentration of 1% and sterile filtered. The bacterialculture was diluted 1:20 in the alginate solution. The solution was trans-ferred to a 10-ml syringe and placed in a syringe pump (model 3100;Graseby, United Kingdom), which fed the alginate to the encapsulationunit (Var J30; Nisco Engineering, Zurich, Switzerland). The alginate waspumpedintoagellingsolutionof0.1MCaCl 2 preparedin0.1MTris-HClbuffer (pH 7.0), which was agitated with a magnetic stirrer (RCT Basic;IKA, Germany). The beads stabilized under continuous stirring in thegelling bath for 1 h and were then washed twice with 0.9% NaCl contain-ing 0.1 M CaCl 2  before being transferred to 20 ml of 0.9% NaCl contain-ing0.1MCaCl 2 .Fivemillilitersofalginatebeadswasprepared,withmeanbead diameters of 136  m (range, 74 to 205  m;  n  72) and 40  m(range, 15 to 85  m;  n  72) for large and small beads, respectively.The number of bacteria in the alginate beads was determined by dis-solving the beads in 0.1 M citric acid buffer (pH 5.0) and plating thesupernatant for CFU counts.BALB/c female mice (11 weeks old; Taconic Europe A/S, Denmark)wereallowedtoacclimatizefor1weekbeforeuse.Themicehadfreeaccessto chow and water and were handled by trained personnel. All animalexperiments were authorized by the National Animal Ethics Committee,Denmark.Mice were anesthetized subcutaneously with a 1:1 mixture of etomi-date (Janssen, Denmark) and midazolam (Roche, Switzerland) (10 ml/kgbody weight) and then tracheotomized. Alginate beads embedded with  P.aeruginosa  PAO579 were installed in the left lung of BALB/c mice using abead-tipped needle. All mice received similar amounts of alginate beadsand  P. aeruginosa  cells (1  10 8 CFU/ml for both groups).Eight mice (four with each size of aggregate beads) were examinedeachday;twomicefrombothgroupswereeuthanizedat0,1,3,and5daysafter bacterial inoculation. The left lungs were fixed in a 4% (wt/vol)formaldehydesolution(VWR,Denmark).Bacterialgrowthwasmeasuredin34aggregates(14aggregatesfromrespiratoryairwaysand20aggregatesfrom conducting airways). Quantitative PNA-FISH.  Paraffin-embedded samples were deparaf-finizedbytreatmentwithxylene(twicefor5min),99.9%ethanol(EtOH;twice for 3 min), and 96% EtOH (twice for 3 min) and were then washedin MilliQ water three times for 3 min. A drop of a Texas Red-conjugatedPNA-FISH probe specific for  P. aeruginosa  16S rRNA (AdvanDx, USA)was applied to the tissue section and then covered with a coverslip (22). Samples were incubated for 90 min at 55°C (AdvanDx Workstation, Ad-vanDx, USA). The coverslip was removed, and the slides were washed inwarm washing buffer at 55°C (AdvanDx, USA) for 30 min and then airdried in the dark. A drop of Vectashield mounting medium with 4 = ,6 = -diamidino-2-phenylindole(DAPI;Vector,USA)wasplacedontopoftheslide,whichwasthencoveredwithacoverslip(Menzel-Glaser,Germany)and air dried for 15 min.Mounted slides were scanned using a confocal laser scanning micro-scope (CLSM) (Axio Imager.Z2, LSM710 CLSM; Zeiss) and the accom-panyingsoftware(Zen2010,version6.0;Zeiss,Germany).Extremelyhighresolution and color depth are required for precise quantification. There-fore,fluorescenceimageswererecordedatanemissionwavelengthof615nmwitharesolutionof6,144by6,144pixelsandatacolordepthof16bitswitha63  /1.4(numericalaperture)oilobjectiveusinglaserexcitationat594 nm. Each pixel was scanned twice. Images were stored in 16-bit TIFformat. Fluorescence in individual cells was quantified using the freewareprogramImageJ(NationalInstitutesofHealth,Bethesda,MD,USA).Thebackground signal was defined by a threshold value using the automatedMultiThresholder macro for ImageJ (K. Baler, G. Landini, and W. Ras-band, NIH, Bethesda, MD). For quantification, the ImageJ function “an-alyze particles” was used. The fluorescence intensity was calculated influorescence units (FU) as the mean of gray-scale units over a range from0 to 65,535. The correlation between FU and growth rate was used toestimate the growth rate (see Fig. 2; see also Supplemental Materials and Methods in the supplemental material). Using a correlation can result inthe prediction of a negative growth rate. Growth rates cannot be less thanzero; therefore, in these cases, the growth rate was considered to be slow.The length, width, and cross-sectional area of biofilm aggregates inlung tissue samples from CF patients, as well as the distances from indi-vidual biofilms to the edge of their mucus clumps in the lung tissue, weremeasured using Zeiss Zen 2010, version 6.0. A proxy for the level of in-flammation and PMN activity around each biofilm aggregate was ob-tained by counting all PMNs stained with DAPI within a distance of 20  m from the edge of the biofilm using Zeiss Zen 2010, version 6.0. PMNs on  P. aeruginosa in vitro .  One hundred milliliters of Krebs-Ringerbuffer(KRB)(PanumInstitute,Denmark)supplementedwith1%glucose was inoculated with 100  l of PAO1 and incubated overnight at37°C in a shaker. When the culture reached an optical density at 450 nm(OD 450 ) of 0.2, it was diluted to an OD 450  of 0.1 using KRB containingglucose at 37°C, after which 500  l was added to the airtight lower cham-ber of a 0.2-  m single-step filter vial (Thomson, USA) (see Fig. 7). Hu-man blood was collected from healthy volunteers with the approval (H-3-2011-117)oftheDanishScientificEthicalBoard.PMNswereisolatedasdescribedelsewhere(23).ExtractedPMNswereresuspendedinKRBcon- Kragh et al. 4478 Infection and Immunity   onA  pr i  l  1  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   taining glucose at 37°C to a final density of 2.5  10 7 PMNs/ml. In total,200  l of the PMN suspension was added to the chamber above the filter,while the chamber below the filter received 200   l of KRB containingglucose. Half of both the PMN-treated and untreated vials was supple-mented with 10 mM KNO 3 , and the vials were incubated at 37°C. After 0,2, and 4 h, 20  l of bacterial suspension from the airtight chamber wasfixed on Super Frost Plus slides (Thermo Scientific, USA) with GN Fixa-tionSolution(AdvanDx,USA)at65°Cfor20min.Slideswereanalyzedby quantitative PNA-FISH as described above.O 2  levels in the lower, airtight chamber containing the bacteria and inthe upper chamber containing the PMNs were measured using O 2 -sensi-tive sensor spots mounted inside the vials and monitored with a fiber-opticO 2 meter(Fibox3;PreSens,Germany)equippedwitha2-mmfiber-optic cable (24, 25). PMN activation was induced by 10   M phorbol12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA). Statistical analysis.  Statistical significance was evaluated using aMann-Whitney test. Multiple regressions were used to evaluate multifac-tormodelsofdata.Toevaluaterelationshipswithoutparametricassump-tions, Spearman’s rank correlation was used.  P   values of   0.05 were con-sidered to be significant. All tests were performed using GraphPad Prism,version 5 (GraphPad Software, USA) and InStat, version 3 (GraphPadSoftware). RESULTS Schaechter et al. defined a proportional relationship between therate of growth and the ribosomal content in  Salmonella enterica serovar Typhimurium cells (26), enabling estimates of the bacte-rial growth rate from the number of ribosomes. Fluorescently conjugatedPNAwashybridizedtotheRNAofintactribosomesin P. aeruginosa cells,andthefluorescentsignalwascorrelatedtothegrowth rate of the bacteria. Based on the use of quantitative PNA-FISH and real-time PCR (RT-PCR) specific for  P. aeruginosa  16SrRNA, the ribosomal content of   in vitro  pure culture samplestaken at different growth phases was determined. The specificgrowth rate was calculated at the exact sampling time based onopticaldensity(OD)measurements.Foreachsample,theaveragenumber of fluorescence intensity units (FU) emitted by the PNA-FISH-treated cells was quantified. Between 10 and 200 cells weremeasured at each time point, and the number of rRNA moleculesper rRNA gene molecule (i.e., the number of ribosomes per ribo-some/protein-encoding gene, or the number of ribosomes) wasquantified by RT-PCR. Fluorescence microscopy showed a cleardifferenceinthefluorescenceintensityofcellssampledatdifferentgrowth rates (Fig. 1). Correlatingfluorescencetogrowthrate. Themeannumberof FU emitted in pixels, within a threshold that discriminates back-ground fluorescence, was plotted as a function of the number of ribosomes in each sample. Samples were taken to represent cul-tures in exponential growth, decreasing growth, and stationary phase (labeled green, yellow, and red, respectively, in Fig. 2). FIG 1  Pseudomonas aeruginosa  at different growth rates. The cells were treated with PNA-FISH probes targeting  P. aeruginosa  16S rRNA. The specific growthrates, in divisions (div) per hour, are indicated on the panels. FIG2  CorrelationsbetweenfluorescenceintensityandrRNAorgrowthrate.(A)Correlationbetweentheaveragefluorescenceintensityin P.aeruginosa cellsandthenumberofrRNAmoleculesperrRNAgenemolecule,asmeasuredbyRT-PCR.Theblacklineshowsthecalculatedcorrelation,andthetwodottedlinesshow the95%confidenceinterval.Thecorrelationhasan R 2 fitat0.7222.Therelationshipisdescribedby   y   0.0447   x   46.3.(B)Correlationbetweentheaveragemeasuredfluorescenceintensity(FU)in P.aeruginosa andthegrowthratedeterminedfromODmeasurementsofbacterialculturesamplesasafunctionoftime.The black line shows the calculated correlation, and the two dotted lines show the 95% confidence interval. The correlation has an  R 2 fit at 0.7627. Therelationship is described by   y   (1.146  10  4 )   x   1.031. PMNs Restrict  P. aeruginosa  Growth in CF LungsNovember 2014 Volume 82 Number 11  4479   onA  pr i  l  1  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om   There was a significant ( P   0.0001) linear correlation betweenFU values and rRNA: number of rRNA molecules per rRNA genemolecule  0.0447  FU  46.3 ( R 2  0.722) (Fig. 2A). The lack  of a linear relationship and normally distributed residuals for low levels of FU does not invalidate our conclusion of a significantly positive relationship, as shown by the computed Spearman’s cor-relation (  0.7936,  P   0.0001). As the specific growth rateswere known for each sample, the fluorescence intensity and therRNA content could be expressed as a function of the growth rateand viceversa .Therewasalsoasignificant( P   0.0001)linearcorre-lation between FU and the specific growth rate: growth rate   (1.146    10  4 )    FU    1.031 ( R 2   0.763) (Fig. 2B). Theserelationships enabled us to estimate the growth rate based on flu-orescence intensity measurements. The few outliers that prevent anormal distribution of the data did not alter the statistically signifi-cant positive relationship (Spearman’s correlation,  0.9104;  P   0.0001). Environmental regulation of ribosomal activity.  Quantita-tive PNA-FISH was used in two starvation experiments to inves-tigate ribosomal content in  P. aeruginosa  in response to suddencarbon/nitrate starvation and O 2  depletion. When the exponen-tially growing culture was deprived of all carbon or nitrogensources, a decline in the FU value was observed that could bedescribed as a mono-exponential decay (see Fig. S1A in the sup-plemental material) ( R 2  0.93), with an FU decay constant of 58.9%h  1 ,whichreachedanasymptoticvalueof8,558FUafter6to 7 h. This correlated well with findings from the growth phasestudy showing a baseline of 8,800  932 FU (mean  standarddeviation [SD]) in cells from a very late stationary phase (  24 hafterinoculation).Whencellswereexposedtoasuddenshiftfromoxic to anoxic conditions, resulting in O 2  depletion, a similar ex-ponentialdecayofFU( R 2  0.91andFUdecayconstantof51.4%h  1 )wasobserved,whichreachedanasymptoticlevelof8,267FUafter 7 h of anoxia (see Fig. S1B). Growth rates in clinical samples.  To directly estimate thegrowth of   P. aeruginosa  in the lungs of end-stage CF patients, thequantitative PNA-FISH method was used on explanted lungsfrom three CF patients. Tissue samples ( n  20) were collected torepresent all regions of the infected lungs. Many biofilm aggre-gates were embedded in mucus in the conducting zone and weresurrounded by PMNs, consistent with earlier observations (4)(Fig. 3). Using quantitative PNA-FISH, the mean specific growthrate was estimated in each of the biofilm aggregates ( n  59). Ahighvariabilityingrowthratewasfoundamongthesamplesfromall three patients, ranging from 0 to 0.90 divisions per hour (Fig.4).Similarheterogeneitywasalsoobservedwithineachtissuesec-tion. In a 1-cm 2 section, growth rates ranging from 0 to 0.65 divi-sions per hour were observed. Interestingly, bacteria isolated justprior to lung transplantation were not growth limited  in vitro  asthemediangrowthratewasestimatedtobe1.2divisionsperhour(range, 1.15 to 1.60), which is significantly ( P   0.0058) higherthan the  in situ  growth rate of 0.217 divisions per hour (range,  0.10 to 0.67). Effects of biofilm aggregate size, depth, and diameter ongrowth rate.  The size, depth within the mucus, and diameter of eachbiofilmaggregateintheCFlungsamplesweremeasured,andpossible synergistic correlations to the  in vivo  growth rates of   P.aeruginosa  were investigated by multiple regression analysis. Nosignificant synergistic interactions were found that could explainthe observed heterogeneity of the bacterial growths rates, such assize, depth, and diameter ( P     0.3665), size and depth ( P    0.2413), size and diameter ( P   0.4513), or depth and diameter( P   0.6841). The average size of the biofilm aggregates was 520 FIG 3  Micrograph of   P. aeruginosa -infected lung tissue. Light and fluorescence microscopy images (magnification,  170) of periodic acid-Schiff- and hema-toxylin-stained sections (A and B) and PNA-FISH-stained sections (C and D) containing luminal and mucosal accumulations of inflammatory cells. The  P.aeruginosa -positive areas are seen as well-defined lobulated clarifications surrounded by inflammatory cells. Red arrows indicate PMNs, and green arrowsindicate  P. aeruginosa  biofilm aggregates. Kragh et al. 4480 Infection and Immunity   onA  pr i  l  1  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om    m 2 (range, 4 to 3,227   m 2 ). The lack of correlation betweengrowth rate and size is depicted in Fig. 5. PMN counts and effects on  P. aeruginosa  growth.  While ag-gregate size and location do not affect growth rate in the CF lung,an alternative explanation is that slower-growing aggregates may be limited for an important nutrient. Previous observations show thatPMNsincreasetheirO 2 consumptionuponcontactwithbac-teria  in vitro  (11, 27); we hypothesized that slow-growing aggre- gates may be surrounded by significantly higher levels of PMNsthan aggregates with a higher growth rate. The number of PMNswas counted within 20  m around each biofilm aggregate, and asignificant inverse correlation was found (    0.4471,  P    0.0004) between the PMN count and the  in vivo  growth rate of   P.aeruginosa  (Fig. 6). Invitro confirmationofabiostaticfunctionofPMNs. Totestwhether PMNs can limit the growth of   P. aeruginosa , bacterial FIG 4  Growth rates measured in lung tissue and peak growth rate achieved by isolates. The specific growth rate of   Pseudomonas aeruginosa  was estimated by quantitativePNA-FISHin59biofilmaggregatesin20tissuesectionsfromexplantedlungsfromthreeCFpatients(solidsymbols).Thehighestexponential(exp)growth measurements from isolates are shown as open symbols. The horizontal line represents the median rate in each patient. Dates of sampling are, patient; **,  P   0.01; ***,  P   0.001. FIG 5  Growth rates versus biofilm aggregate size. Growth rates measured inbiofilm clusters in  ex vivo  CF lung tissue are shown as a function of size. Therewas no significant correlation between size and growth ( P   0.1891). FIG 6  Growth rates measured in lung tissue as a function of the number of surrounding PMNs. The specific  in vivo  growth rate of   Pseudomonas aerugi-nosa  was estimated by quantitative PNA-FISH as a function of the total num-ber of PMNs within a 20-  m radius from the edge of the 59 measured biofilmaggregates in 20 tissue sections of explanted lungs from three CF patients.There was a significant negative Spearmen’s correlation (  0.4471,  P   0.0004) between the specific growth rate and the number of PMNs in thesurrounding mucus. PMNs Restrict  P. aeruginosa  Growth in CF LungsNovember 2014 Volume 82 Number 11  4481   onA  pr i  l  1  ,2  0 1  6  b  y  g u e s  t  h  t   t   p:  /   /  i   ai  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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