Staphylococcus Aureus Alters Growth Activity, Autolysis, And Antibiotic Tolerance

Staphylococcus aureus Alters Growth Activity, Autolysis, andAntibiotic Tolerance in a Human Host-Adapted Pseudomonasaeruginosa Lineage

Charlotte Frydenlund Michelsen,a Anne-Mette Juel Christensen,a Martin Saxtorph Bojer,b Niels Hiby,c Hanne Ingmer,b Lars Jelsbaka

Department of Systems Biology, Technical University of Denmark, Lyngby, Denmarka; Department of Veterinary Disease Biology, Food Safety and Zoonoses, University ofCopenhagen, Frederiksberg, Denmarkb; Institute for International Health, Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmarkc

Interactions amongmembers of polymicrobial infections or between pathogens and the commensal flora may determine diseaseoutcomes. Pseudomonas aeruginosa and Staphylococcus aureus are important opportunistic human pathogens and are bothpart of the polymicrobial infection communities in human hosts. In this study, we analyzed the in vitro interaction between S.aureus and a collection of P. aeruginosa isolates representing different evolutionary steps of a dominant lineage, DK2, that haveevolved through decades of growth in chronically infected patients. While the early adapted P. aeruginosaDK2 strains outcom-peted S. aureus during coculture on agar plates, we found that later P. aeruginosaDK2 strains showed a commensal-like interac-tion, where S. aureus was not inhibited by P. aeruginosa and the growth activity of P. aeruginosa was enhanced in the presenceof S. aureus. This effect is mediated by one or more extracellular S. aureus proteins greater than 10 kDa, which also suppressedP. aeruginosa autolysis and prevented killing by clinically relevant antibiotics through promoting small-colony variant (SCV)formation. The commensal interaction was abolished with S. aureus strains mutated in the agr quorum sensing system or in theSarA transcriptional virulence regulator, as well as with strains lacking the proteolytic subunit, ClpP, of the Clp protease. Ourresults show that during evolution of a dominant cystic fibrosis lineage of P. aeruginosa, a commensal interaction potential withS. aureus has developed.

Most microbial species are embedded within mixed-speciescommunities where mutualistic, antagonistic, and neutralinteractions within the community control behaviors and activi-ties of the individual species. In relation to microbial infections, itis becoming clear that interactions between bacterial pathogensand other microbial species present at the infection site (eithercoinfecting pathogens or commensal bacteria) can result in al-tered pathogen behaviors such as enhanced virulence (1, 2), bio-film formation (3), and antibiotic tolerance (4), which may influ-ence disease progression and clinical outcome of the infection.Despite advances in elucidating the molecular details underlyingmicrobial interactive processes, the extent to which evolutionaryprocesses remodel interspecies interactions during the course ofinfection and therapy is currently not understood. Thus, studies ofinteraction patterns between species and the evolution withinpolymicrobial infections are a critical first step toward developingnovel interference strategies against such infections.

Chronic cystic fibrosis (CF) airway infections caused by thebacterium Pseudomonas aeruginosa offer optimal opportunities tostudy evolutionary dynamics within a natural environment be-cause of systematic routine sampling of the ecosystem over ex-tended time periods (years) and because of the well-characterizedecological properties of the system (5). We have recently deter-mined the genetic basis of adaptation in a highly successful P.aeruginosa lineage (DK2) that disseminated across multiple pa-tients and evolved in the CF airways for more than 38 years (58).During evolution in the host environment, the DK2 lineage un-derwent significant genetic and phenotypic changes and thus pro-vides a unique opportunity for us to explore how the adaptationalevents may have affected the ability of this lineage to interact withother pathogenic bacterial species, such as Staphylococcus aureus.Importantly, in vitro coculture experiments have demonstrated

complex interactions between P. aeruginosa and S. aureus thatresult in phenotypes with potential relevance for disease develop-ment and therapy. For example, peptidoglycan shed by S. aureuswas recently found to stimulate production of virulence factors inP. aeruginosa and thereby increase its lytic activity and virulence ina Drosophila model of infection (1). In addition, P. aeruginosaexoproducts were found to inhibit S. aureus respiration, induceresistance toward antibiotic killing (9), and stimulate pigment(e.g., staphyloxanthin) production, virulence, and biofilm forma-tion by S. aureus (1012). The virulence mechanisms of P. aerugi-nosa and S. aureus and their regulation have been widely studied(1315). In P. aeruginosa, the majority of the extracellular viru-lence factors are controlled by the Pseudomonas quorum sensing(QS) system, which consists of two hierarchical systems, las andrhl, induced by cell density-dependent acyl-homoserine lactone(AHL) signaling molecules. In addition, the 4-hydroxy-2-al-kylquinoline (HAQ) molecule 3,4-dihydroxy-2-heptylquinoline(PQS) and its biosynthetic precursor 4-hydroxy-2-heptylquino-line (HHQ) act as signalingmolecules regulating thePseudomonasQS system (1517). The transcriptional regulator LasR controlsthe conversion of HHQ to PQS, thereby providing a link betweenthe AHL and HAQ regulatory systems (17). Mutations in the lasR

Received 19 June 2014 Accepted 25 August 2014

Published ahead of print 2 September 2014

Address correspondence to Lars Jelsbak, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.02006-14.

Copyright 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.02006-14

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gene are frequently observed in P. aeruginosa strains isolated fromthe airways of CF patients (5, 7, 18) and can be identified by adistinctive colony morphology described as metallic iridescentsheen coverage due to an accumulation of the iridescent mole-cule, HHQ (19). In addition, this phenotype has been linked toautolysis in P. aeruginosa (20).

In S. aureus, although not required for virulence itself, the agrtwo-component system is a major regulator of most extracellularproteins essential for staphylococcal virulence and is activated bycell density-dependent autoinducer peptides (AIPs) (13, 14). Inaddition, the sarA global regulatory gene controls S. aureus viru-lence (21). Furthermore, besides a direct importance for survivalunder stress conditions, the ClpP proteolytic complexes have alsoshown to be essential for S. aureus virulence by regulating theexpression of major virulence factors (22).

Staphylococcus aureus is frequently found together with P.aeruginosa in polymicrobial infections, including CF airway infec-tions (23, 24). Therefore, the aim of this study was to explore acollection of longitudinally sampled P. aeruginosa DK2 strains,which represent different evolutionary stages of host adaptation ofthe DK2 lineage, in order to characterize the colony developmentand in vitro interaction patterns with S. aureus.

MATERIALS AND METHODSBacterial strains and growth conditions. The Pseudomonas aeruginosaand Staphylococcus aureus strains used in this study are listed in Table 1and Table S1 in the supplemental material. The P. aeruginosa strains (Ta-ble 1 and Fig. 1A) were derived from a collection of clinical P. aeruginosastrains of the DK2 clone lineage isolated from chronically infected DanishCF patients (68). The Pseudomonas strains were cultured in liquid or onsolid Luria Bertani (LB) medium (2% agar) at 37C unless otherwisespecified.

The S. aureus strains and the S. aureus transposon mutant subcollec-tion (from the Nebraska Transposon Mutant Library, the Nebraska Cen-ter for Staphylococcal Research) (25) weremaintained on solid tryptic soyagar (TSA) medium (2% agar) at 37C.

Gfp tagging P. aeruginosa CF224-2003 by triparental mating. P.aeruginosa DK2-P24M2-2003 was tagged with a mini-Tn7 transposon,mini-Tn7(Gm)PrrnB P1 gfpAGA, containing an unstable version of theGfpprotein (gfpAGA) expressed by a growth rate-dependent Escherichia coliribosomal promoter (rrnB P1) (26) by conjugative transfer using thehelper plasmids pUX-BF13 (carrying the transposase genes) and the mo-bilizing plasmid pRK600 as previously described (27). Transconjugantswere selected on selective Pseudomonas isolation plates (PIA) containing60 g/ml of gentamicin and checked for Gfp expression using a fluores-cence microscope (Axioplan 2; Zeiss, USA).

Cross-streak assay and spot inoculation. Stationary-phase culturesof S. aureus (optical density at 600 nm [OD600] of2) and P. aeruginosa(OD600 1.5) cultured in LB were streaked or spotted onto dry LB agar(2%) plates. In the cross-streak assay, one loopful of P. aeruginosa culturewas first streaked in one direction on the plate and left to dry before theplate was cross-streaked with one loopful of the S. aureus culture. Duringspot inoculation, 5 l of the P. aeruginosa and S. aureus cultures werespotted next to each other onto dry LB agar plates. The experiments werecarried out in triplicate, and the plates were incubated at 37C. After twoto four days of incubation, the plateswere inspected visually aswell aswiththe fluorescence microscope when required (Axioplan 2; Zeiss, USA).

Screening of the S. aureus JE2 mutant library. The S. aureus JE2(USA300) wild type (WT) and the transposon mutant collection (seeTable S1 in the supplemental material) were screened to detect mutantsthat no longer decreased metallic sheen coverage of P. aeruginosa DK2-P24M2-2003. The S. aureus JE2 WT and the mutant collection were cul-

tured in 96-well microtiter plates (Nunc) in LB at 37C (150 rpm) over-night.

A layer of stationary-phase P. aeruginosaDK2-P24M2-2003 (1ml cul-ture plus 6.5 ml LB soft agar, 0.5%) was spread on top of an OmniTray(Nunc) containing LB agar (2%) and left to dry under a lid for 1 h. Cul-tures of S. aureus JE2 WT and mutant strains were applied at one timeonto the P. aeruginosa lawn by using a sterile replicator. The plates wereincubated at 37C for 24 h before they were inspected visually for zoneswith suppressed metallic sheen/autolysis. The experiment was repeatedthree times.

Preparation of S. aureus culture supernatants and fractionation ofsupernatants. For preparing S. aureus culture supernatants, the strainswere cultured for 3, 6, 17, or 24 h in tryptic soy broth (TSB) mediumwithshaking (200 rpm) at 37C. Supernatants were collected by centrifugationof cultures (10,000 g, 10 min, 4C) and sterile filtration (0.2-m poresize) to remove all cells. Vivaspin 6-ml columns with 5- and 10-kDa mo-lecular-mass cutoff filters (Sartorius Stedim Biotech GmbH) were used tofractionate the S. aureus JE2 WT supernatant as described by the manu-facturer. The fractions and supernatants were kept at20C until use.

The culture supernatants and fractions were screened against a lawn ofP. aeruginosa cells as described below.

Antibiotic plate screening with P. aeruginosa culture and S. aureusculture supernatants. A total of 100l of P. aeruginosa overnight culture(diluted to OD600 0.2) was spread on top of LB agar plates, containingdifferent concentrations of tobramycin (i.e., 0, 5, 10, 12.5, 14, 15, 16, 17.5,and 20g/ml), gentamicin (i.e., 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, and 40g/ml), and ciprofloxacin (i.e., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, and 10g/ml)and left to dry under a lid for 30 min. Wells were prepared in the P.aeruginosa-covered agar plates by removing 6-mm agar plugs. Fortymicroliters of S. aureus supernatant from stationary-phase cultures(OD600 2.5) prepared as described above was applied to each well, andthe plates were incubated at 37C after the supernatants had dried. Theplates were made in duplicate, and the experiment was repeated threetimes. The plates were visually inspected after 2 to 3 days of incubation.

Liquid culture assay. An overnight culture of P. aeruginosa DK2-P24M2-2003 was diluted 1:100 in fresh LB followed by supplementationwith either 10% unused TSB (control) or S. aureus JE2 culture superna-tant prepared as described above. Cultures were incubated in shakingflasks at 37C (200 rpm), and growth wasmonitored bymeasuring OD600at the indicated time points. Each condition was assayed in triplicate, andthe experiment was repeated three times using different S. aureus JE2culture supernatant preparations. Doubling time was calculated usingregression up to 5.5 h, and the P value was calculated by Tukeys honestlysignificant difference (HSD) test in conjunction with analysis of variance(ANOVA) using R version 3.0.3 (28).

Live/dead staining. The fraction of dead (or damaged) P. aeruginosaDK2-P24M2-2003 cells in cultures treated with either 10% unused TSB(control) or S. aureus JE2 culture supernatant was assessed by flow cytom-etry using thiazole orange (TO; Sigma-Aldrich) and propidium iodide(PI; Sigma-Aldrich) as live and dead stains, respectively. Cultured cellswere diluted in 0.9% NaCl (sterile filtered) containing 1 mM EDTA tofacilitate TO uptake and stained for 15 min with TO (420 nM, final con-centration) and PI (7.2M,final concentration) followed by sample anal-ysis (acquisition of 20,000 events) using a BD Accuri C6 flow cytometer.Controls included unstained cells and cells killed by treatment with 70%isopropanol. Samples from triplicate cultures were analyzed, and sampledata were gated appropriately according to forward scatter (FSC)/sidescatter (SSC) distribution and TO staining to exclude unstained cells andunspecific noncell events. Gatings were kept constant between samplesand treatments for each experiment.

Antibiotic resistance assay using Etest strips. Stationary-phase cul-tures of the P. aeruginosa DK2-P24M2-2003 parent strain and DK2-P24M2-TM1werewashed twicewith LB, diluted to anOD600 of0.5, andspread on top of LB agar plates. The tobramycin (Tm), gentamicin (Gm),and ciprofloxacin (Ci) Etests (bioMrieux, Sweden) were performed ac-

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cording to the provided protocol. The experiment was repeated threetimes.

RESULTSP. aeruginosaDK2 isolates showdifferent colonymorphologiesand interaction patternswith S. aureus.Colonymorphologies ofa longitudinal collection of genetically different P. aeruginosaDK2isolates shown to cover an infection history of 35 years (i.e., from1973 to 2008) (6, 7) were characterized by culturing on LB agarmedium (Fig. 1A; see Table 2). Among these P. aeruginosa DK2strains, visual variations in their colony morphotypes were ob-

served. P. aeruginosa DK2 strain CF114-1973 showed a smoothcolony morphology similar to the P. aeruginosa PAO1 referencestrain (Fig. 1A and data not shown), whereas the strains CF43-1973 and CF66-1973 and the rest of the P. aeruginosaDK2 collec-tion showed centric zones of autolysis and an iridescent metallicsheen coverage of the colony streaks (Fig. 1A and Table 2). Loss-of-function mutations in lasR have been shown to result in thischaracteristic colony phenotype (19), and we have previouslyshown that all P. aeruginosa DK2 strains sampled between 1979and 2008 harbored identical lasR loss-of-function mutations intheir genomes (Fig. 1A) (7, 8). However, CF43-1973 and CF66-1973 containednomutations in lasR, and othermutations in theseisolates therefore may result in this characteristic colony mor-phology. In addition, introduction of a loss-of-functionmutationin lasR inP. aeruginosaPAO1 reference strain (i.e., SD2) producedcolonies with centric zones of autolysis and metallic sheen cover-age (Table 2).

The collection of P. aeruginosa DK2 isolates was subsequentlytested for their interplay with a panel of S. aureus strains (Table 1)

TABLE 1 Bacterial strains and plasmids used in this study

Strain or plasmidRelevant genotype, phenotype,and/or description

Referenceor source

StrainsP. aeruginosa

PAO1 WT 33SD2 PAO1 lasR 8CF114-1973 DK2 isolate from 1973 7CF43-1973 DK2 isolate from 1973 7CF66-1973 DK2 isolate from 1973 7CF30-1979 DK2 isolate from 1979 7CF173-1984 DK2 isolate from 1984 7CF333-1991 DK2 isolate from 1991 7CF66-1992 DK2 isolate from 1992 7CF333-1997 DK2 isolate from 1997 7CF333-2003 DK2 isolate from 2003 7CF173-2005 DK2 isolate from 2005 7CF333-2007 DK2 isolate from 2007 7CF66-2008 DK2 isolate from 2008 7DK2-P24M2-2003 DK2 isolate from 2003 This studyDK2-P24M2-2003/gfpAGA DK2-P24M2-2003 tagged with

PrrnB P1-gfpAGAThis study

DK2-P24M2-TM1 SCV of DK2-P24M2-2003 This studyS. aureus

JE2 Plasmid-cured derivative ofFPR3757

25

FPR3757 USA300 community-acquiredmethicillin-resistantS. aureus isolate

34

Newman Clinical isolate (NCTC 8178) 358325-4 Laboratory strain 3685959 CF isolate This study

E. coli HB101 recA thi pro leu hsdRM; Smr 37

PlasmidsprK600 Cmr; ori ColE1 RK2-mob

RK2-tra helper plasmidfor conjugation

37

pUX-BF13 Apr; mob RK6 replicon-based helper plasmid forconjugation

38

miniTn7PrrnB P1-gfpAGA Gmr; PrrnB P1 gfpAGA 26

FIG 1 Overview of P. aeruginosa DK2 strains used in this study. (A) Treeshowing the genetic relationship based on accumulations of single-nucleotidepolymorphisms (SNPs) identified from genome sequencing (6, 7). The num-bers in italics indicate the number of SNPs between the isolates. Symbolsrepresent DK2 isolates sampled at different time points (indicated on the timeline) fromdifferent patients with CF (indicated by symbol shape). On the rightare colony morphologies of selected P. aeruginosa DK2 strains. (B) Cross-streak assay between selected P. aeruginosaDK2 stains and S. aureus strain JE2cocultured on LB agar medium. White or black arrowheads indicate zones ofbacterial inhibition or altered colony morphology (increased cell density),respectively. (C) Experimental setup of the cross-streak analysis between P.aeruginosa and S. aureus. The dashed square indicates the zone of interaction.(D) Zoom of interaction zone between P. aeruginosaDK2-P24M2-2003 and S.aureus JE2.

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using an in vitro cross-streak assay (Fig. 1C). This analysis revealeddifferential interaction patterns among the collection of P. aerugi-nosa DK2 strains. An antagonistic interaction, with clear inhibi-tion of S. aureus growth, was observed at the interface between P.aeruginosa DK2 strain CF114-1973 and S. aureus strain JE2, a de-rivative of USA300 (25) (Fig. 1B and Table 2). This antagonisticinteraction was the same at the interface between strain CF114-1973 and all the S. aureus strains listed in Table 1, including the S.aureus CF isolate (strain 85959), and corresponded to the inhibi-tion of S. aureus observed by the P. aeruginosa reference strain,PAO1 (Table 2). Inhibition of S. aureus strain JE2 was also ob-served by P. aeruginosa DK2 strain CF43-1973 in the zone of in-teraction (Fig. 1B). However, in the same interaction zone, analtered colony morphology of the Pseudomonas strain also ap-peared in the presence of S. aureus, which could reflect an area ofhigher cell density (Fig. 1B and D and Table 2). On the contrary,for the remaining P. aeruginosaDK2 collection (i.e., strains CF66-1973 to CF66-2008), we observed a commensal-like interactionpattern with S. aureus, with both an altered Pseudomonas colonymorphology in the interaction zone with S. aureus as well as noinhibition of S. aureus growth by P. aeruginosa (Fig. 1B and Table2). Similar commensal interaction patterns were seen with theentire panel of S. aureus strains fromTable 1. To gainmore knowl-edge about the altered Pseudomonas colonymorphology in cocul-ture with S. aureus, the interaction between one representative ofthe P. aeruginosaDK2 strains, DK2-P24M2-2003 (Fig. 1B and D),and S. aureus strain JE2 was subjected to further analysis.

S. aureus extracellular factors increase growth activity andsuppress autolysis ofP. aeruginosaDK2.The effects onP. aerugi-nosa DK2-P24M2-2003 colony morphology in the presence of S.aureus JE2were examined in vitroby spot inoculating S. aureus JE2next to P. aeruginosaDK2-P24M2-2003, which is chromosomallytagged with a gene encoding an unstable Gfp protein (gfpAGA)and expressed by the growth rate-dependentE. coli ribosomal pro-moter, rrnB P1 (i.e., strain DK2-P24M2-2003/gfpAGA) (Fig. 2).After 3 days of incubation, a centric zone of autolysis andmetallic

sheen coverage was observed with the DK2-P24M2-2003/gfpAGAcolony, whereas an area of altered colony morphology appearedon the section of the Pseudomonas colony situated next to the S.aureus JE2 colony (Fig. 2B). In addition, the same area of thePseudomonas colony gave a stronger Gfp signal in fluorescencemicroscopy analysis (Fig. 2C) than the rest of the colony (data notshown), suggesting an increased expression of Gfp in this area.This increased Gfp expression may be due to a higher growth rateof P. aeruginosa or an increase in P. aeruginosa cell density or both.To examine whether the S. aureus JE2 active factors, which con-tributed to the observed alteration in P. aeruginosa growth activ-ity, were secreted or associatedwith the S. aureus cells, supernatantfrom an overnight culture (i.e., 17 h) was filtered through a 0.2-m-pore-size filter and tested against a lawn of P. aeruginosaDK2-P24M2-2003 cells by applying the supernatant to a well cutin the P. aeruginosa-covered agar plate (Fig. 3A). After 2 days ofincubation, a lawn of DK2-P24M2-2003 cells covered the entireLB agar plate, and metallic sheen coverage of the cells was ob-served (Fig. 3A). Surrounding the applied S. aureus JE2 superna-tant, a darker zone appeared on the metallic sheen coverage (Fig.3A). This zonewas not inhibition of bacterial growth but rather anarea of suppressed metallic sheen of the DK2-P24M2-2003 lawn.The bioactivity of the S. aureus JE2 supernatant was not observed

TABLE 2 Phenotypes by monocultures of the P. aeruginosa DK2 strains,strain PAO1, the PAO1 lasR mutant, and SD2 and in interactions withS. aureus

Pseudomonasstrain

Metallic sheen/autolysisb

Increased cell densityof P. aeruginosaa

Inhibition ofS. aureusb

CF114-1973 CF43-1973 CF66-1973 CF30-1979 CF173-1984 CF333-1991 CF66-1992 CF333-1997 CF333-2003 DK2-P24M2-2003 CF173-2005 CF333-2007 CF66-2008 PAO1 SD2 a, none;, low;, medium;, high cell density observed by P. aeruginosa inthe zone of interaction with S. aureus.b, yes;, no.

FIG 2 Monocultures of S. aureus JE2 WT (A) and the agrC mutant (D) orcoculture with P. aeruginosa DK2-P24M2-2003/gfpAGA (B and E, respec-tively) by spot inoculation onto LB agar medium. The black arrowhead indi-cates altered P. aeruginosa colony morphology. The Gfp fluorescence signal ofP. aeruginosa DK2-P24M2-2003/gfpAGA after 3 days of incubation is visu-alized in the zone of interaction with S. aureus JE2 WT (C) or the agrCmutant (F). The white arrowhead indicates increased Gfp expression byDK2-P24M2-2003/gfpAGA.

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in samples from mid-exponential cultures (i.e., after 3 h of incu-bation) but was observed in samples from late-exponential cul-tures (i.e., after 6 h of incubation) and from stationary cultures(i.e., after 17 and 24 h of incubation) (see Fig. S1B in the supple-mental material) and could be removed by heat treatment ortreatment with proteinase K (data not shown). In addition, frac-tionation of the S. aureus JE2 supernatant using Vivaspin 6-mlcolumns (Sartorius Stedim Biotech GmbH) with 5- and 10-kDamolecular-mass cutoff filters was carried out in order to deter-mine if the S. aureus JE2 active factors were low-molecular-massmolecules. The different fractions were tested against a lawn of P.aeruginosa DK2-P24M2-2003 cells, and suppression of metallicsheen was observed with the fractions containing molecules withmolecularmasses over 5 or 10 kDa (see Fig. S1A in the supplemen-tal material). All together, these results point to the S. aureus JE2bioactive extracellular factors as proteins with molecular massesgreater than 10 kDa.

To further assess the growth stimulatory activity provided bythe S. aureus JE2 extracellular factors, the growth of P. aeruginosaDK2-P24M2-2003 was monitored in liquid cultures in LB me-dium supplemented with JE2 supernatant and compared to

growth in control cultures supplemented with unused S. aureusgrowthmedium (TSB) (Fig. 4A). Amoderate yet significant stim-ulation of P. aeruginosa growth in the exponential phase was ob-served in cultures supplemented with the S. aureus JE2 superna-tant, as visualized by a doubling time of 0.99 h compared to adoubling time of 1.19 h (P 0.01) in the control cultures, corre-sponding to an over 15% increase in growth rate. The increase ingrowth rate by S. aureus JE2 supernatant compared to the growthrate of the control was not due to an inhibitory effect of TSBcomponents, since treatment with unused LB medium gave agrowth rate similar to that of the control culture. In addition, nochange in pH was observed in the different treatments (data notshown). In order to test whether the increased cell density ob-served in the treatment with JE2 supernatant compared to that ofthe control was due to differences in Pseudomonas cell damage/death, the viability of P. aeruginosa DK2-P24M2-2003 cells wasevaluated in late-exponential-phase cultures by employing a live/dead stain flow cytometric analysis of the respective cultures. Nodifferences in cell damage/death between the cultures treated withS. aureus JE2 supernatant and the TSB-treated control culturesduring this growth phase were observed (Fig. 4B and C). In con-trast, a striking observation in the stationary-phase liquid cultureswas a considerable amount of cell debris (or cell aggregation) gen-erated inP. aeruginosaDK2-P24M2-2003 treatedwith TSB, whichwas absent from cultures treated with S. aureus JE2 supernatant(Fig. 4D and E, respectively). Intriguingly, a very large populationof the cells appeared dead or damaged after 22 h of growth in thecontrol cultures, and notably this distinct population was absentin cultures grown with S. aureus JE2 supernatant (Fig. 4F and G,respectively). Hence, S. aureus JE2 extracellular factors seem tosuppress or delay an intrinsic tendency of DK2-P24M2-2003 toundergo autolysis during the stationary growth phase.

S. aureus bioactive extracellular proteins are controlled bythe S. aureus quorum sensing system and major regulatorygenes.A subcollection (see Table S1 in the supplementalmaterial)of S. aureus JE2 mutant strains derived from the Nebraska Trans-poson Mutant Library (25) was screened in vitro to test for S.aureusmutants that no longerwere able to suppressmetallic sheencoverage of P. aeruginosaDK2-P24M2-2003. From this screening,we found quorum sensing (QS)-deficient mutant strains, i.e., S.aureus JE2 strains NE95 and NE873, mutated in agrB and agrC,respectively, as well as the NE912 and NE1193 mutant strains,mutated in the clpP and sarA regulatory genes, respectively, whichall failed to suppress metallic sheen coverage of DK2-P24M2-2003, as observed for the S. aureus JE2 wild-type (WT) strain (Fig.3A; see also Table S1 in the supplemental material). For example,the culture supernatant of the S. aureus agrC mutant revealed nosuppression of metallic sheen coverage of the P. aeruginosa DK2-P24M2-2003 lawn compared to that of the S. aureus JE2 WT cul-ture supernatant (Fig. 3A). Similarly, the interaction between theS. aureusmutant agrC andDK2-P24M2-2003 showed no effect onDK2-P24M2-2003 colony morphology in contrast to the interac-tion with the S. aureus JE2 WT strain (Fig. 2). In addition, P.aeruginosa DK2-P24M2-2003 was found strongly to induce pig-ment production of S. aureus agrC during coculture (Fig. 2E),corroborating previous observations (10).

The stationary-phase culture of P. aeruginosa DK2-P24M2-2003 treated with supernatant from S. aureus agrC showed anamount of cell debris similar to that observed with the controlculture. Interestingly, however, the growth rate and cell damage/

FIG3 P. aeruginosaDK2-P24M2-2003 plated on top of LB agar plates withoutantibiotics (A) or with inhibitory levels of the antibiotics tobramycin (i.e., 15g/ml) (B), gentamicin (i.e., 38 g/ml) (C), or ciprofloxacin (i.e., 3.5 g/ml)(D). (A) Suppression of metallic sheen coverage of the P. aeruginosa DK2-P24M2-2003 lawn is observed in a zone surrounding the S. aureus JE2 WTculture supernatant (indicated by the arrowhead and scale bar) but not theagrC mutant supernatant. (B, C, and D) A halo of small colonies of P. aerugi-nosa DK2-P24M2-2003 is observed around the S. aureus JE2 WT culture su-pernatant (indicated by arrowheads) on antibiotic plates but not around theagrC mutant supernatant.




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death profile of the cultures treated with the agrC supernatantwere not significantly different from those of cultures treated withthe S. aureus JE2 WT supernatant (data not shown). Hence, theeffects on P. aeruginosa DK2-P24M2-2003 growth activity by S.aureus JE2 may be multifactorial.

S. aureus extracellular proteins induce selection for P.aeruginosa small-colony variants (SCVs) under antibiotic pres-sure. The effects of S. aureus JE2-derived extracellular proteins onP. aeruginosa DK2-P24M2-2003 were further examined in thepresence of various antibiotics. Culture supernatants from the S.aureus JE2 WT and the agrCmutant strains were tested against P.aeruginosaDK2-P24M2-2003 cells spread on top of LB plates con-taining different antibiotic concentrations. Notably, P. aeruginosagrew as small colonies in a zone surrounding the applied S. aureusJE2WT supernatant (Fig. 3B) despite inhibitory concentrations oftobramycin (15 g/ml) in the growth medium. In contrast, theculture supernatant from the S. aureus agrC mutant did not pro-tect P. aeruginosaDK2-P24M2-2003 from this inhibitory effect oftobramycin (Fig. 3B). The same increased antibiotic tolerance ofDK2-P24M2-2003 induced by the S. aureus JE2 WT culture su-pernatant was observed when cultured on LB plates containinggrowth inhibitory concentrations of another aminoglycoside an-tibiotic, gentamicin (Fig. 3C), as well as the fluoroquinolone an-tibiotic, ciprofloxacin (Fig. 3D), although the zone here was lesspronounced than that with the treatments with aminoglycosideantibiotics.

The small colonies of P. aeruginosa DK2-P24M2-2003 wererecovered from the zones of cells surrounding the S. aureus JE2WT culture supernatant (Fig. 3B, C, and D) and restreaked oncorresponding antibiotic plates. One of the collected strains, DK2-P24M2-TM1, isolated from the tobramycin plate, was analyzedfor its MIC profile compared to that of the DK2-P24M2-2003parent strain using tobramycin, gentamicin, and ciprofloxacinEtest strips (bioMrieux, Sweden). Indeed, DK2-P24M2-TM1had increased MIC values (up to 2.5-fold) to all the antibioticstested compared to those of DK2-P24M2-2003 (Fig. 5B). In addi-tion, the DK2-P24M2-TM1 strain was slow growing (OD600 [18h] 0.75) compared to DK2-P24M2-2003 (OD600 [18 h] 1.45)and displayed a smaller colony size (Fig. 5A), which suggests thatDK2-P24M2-TM1 is an SCV of the DK2-P24M2-2003 parentstrain. These phenotypes were stable when cultured in both theabsence and presence of tobramycin. Thus, exposure of P. aerugi-nosa DK2-P24M2-2003 to the S. aureus JE2 supernatant suggestsinduced selection of Pseudomonas antibiotic-tolerant SCVs underantibiotic pressure.

DISCUSSION

Interspecies interactions betweenpathogenicmicroorganisms cancomplicate the treatment of polymicrobial infections (1, 9). Insome cases, these interactions are antagonistic, where one popu-lation is inhibited by the presence of the other (9, 12). In othercases, synergistic interactions occur, in which the consequences

FIG 4 (A) Growth experiment with liquid cultures of P. aeruginosa DK2-P24M2-2003 treated with 10% unused TSB medium or control or S. aureus JE2 WTsupernatant by measuring OD600 over time. (B, C, F, and G) The PI intensity histograms represent live/dead staining data from late-exponential-phase andstationary-phase DK2-P24M2-2003 control cultures (B and F, respectively) or from late-exponential-phase and stationary-phase cultures treated with S. aureusJE2 WT supernatant (C and G, respectively) as generated by flow cytometry. A distinct population of damaged/dead cells characterized by high PI uptake isevident only among cells from stationary-phase cultures treated with TSB (F). Miniature inserts display the TO-PI distribution of events from identical samples.Graphs represent sample data from a single culture representative of several independent experiments. (D and E) Control cultures of P. aeruginosaDK2-P24M2-2003 (D) but not cultures treated with S. aureus JE2 WT supernatant (E) show cell debris (indicated by the arrowhead) after 22 h of incubation.

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manifested by the interaction are not achieved by the individualmicrobe alone and can result in, e.g., increased persistence, viru-lence, or antibiotic resistance of the pathogens involved (1, 911).These latter findings point toward an unexplored area for novelinterference treatment strategies in connection to polymicrobialinfections.

However, bacterial pathogens evolve during long-term infec-tion of their human host, and our understanding of how suchevolutionary pathways influence microbial interaction processesis limited. In this study, we investigate a specific P. aeruginosalineage (DK2) with a well-characterized evolutionary history thatrepresents 30 years of adaptation to human hosts (68). Thisstrain collection enabled us to study the relationship between dif-ferent steps in the evolution of the DK2 lineage and the in vitrointeraction with another clinically relevant species, S. aureus. Ourresults suggest that the evolution of the DK2 lineage has affectedthe interaction patterns with S. aureus from an antagonistic tocommensal-like interplay.

We have previously shown that a loss-of-function mutation inthe lasRQS regulator gene is among the key adaptivemutations inthe evolution of P. aeruginosaDK2 (7, 8). A loss-of-function mu-tation in lasR results in an inactivation of the last step in the PQSbiosynthetic pathway, which leads to an accumulation of the iri-descent intercellular signal molecule, HHQ, and gives the bacteriacharacteristic colony morphologies, i.e., zones of autolysis and aniridescentmetallic sheen coverage (19, 20). In addition,mutationsin lasR have been reported to decrease production of acute viru-lence factors, such as siderophores, exotoxins, pyocins, and pro-teases (18, 19), which not only are important for host adaptationbut also could benefit the interplay with other pathogens residingat the infection site. In this study, themajority of the P. aeruginosaDK2 strain collection that showed a commensal-like interactionwith S. aureus harbored a loss-of-functionmutation in lasR. How-ever, P. aeruginosaDK2 strain CF66-1973 was notmutated in lasRbut still displayed notable autolysis and metallic sheen coverageand a commensal interaction with S. aureus. The observed autol-

ysis andmetallic sheen coverage of CF66-1973 and of strain CF43-1973 colonies, despite no lasR mutation, may result from muta-tions of additional mechanisms involved in PQS biosynthesis(20). Furthermore, P. aeruginosa strain SD2, in which a loss-of-function mutation of lasR was genetically engineered, still inhib-ited the growth of S. aureus. Thus, our data suggest that the in vitrocommensal interaction pattern observed with the majority of theP. aeruginosaDK2 collection is not only restricted to the effect of aloss-of-functionmutation in lasR but ismore likely a combinationof several mutational events.

The in vitro coculturing of S. aureus JE2 with P. aeruginosaDK2-P24M2-2003, chromosomally tagged with a gfp gene encod-ing an unstable Gfp protein and expressed by a growth-dependentribosomal promoter, which previously has been used to discrim-inate between fast- and slow-growing cells (29, 30), resulted inincreased Gfp expression in the zone of interaction with S. aureus.This implies either an increased growth rate or increased cell den-sity of P. aeruginosa in this region. In addition, we observed amoderate increase in growth rate and cell density but also a strongeffect on cell damage/death in liquid P. aeruginosa cultures treatedwith S. aureus JE2 WT supernatant compared to that of the con-trol cultures. An explanation could be that bioactive factors in theJE2 WT supernatant could suppress autolysis of P. aeruginosaDK2-P24M2-2003 and thereby result in increased cell density.Indeed, screening with the S. aureus JE2WT supernatant against alawn of P. aeruginosa cells resulted in suppressed metallic sheencoverage of P. aeruginosa cells in a zone surrounding the applied S.aureus JE2WT supernatant, which previously has been correlatedwith suppressed autolysis (19). Furthermore, a considerable re-duction/delay of autolysis (cell damage/death) was observed instationary-phase P. aeruginosa cultures treated with JE2 WT su-pernatant compared to the control treatment.However, no differ-ences were observed in cell damage/death during the late expo-nential growth phase, which therefore suggests that the effect ofthe JE2 WT supernatant on P. aeruginosa growth activity is mul-tifactorial. The fractionation of the culture supernatant, togetherwith the loss in bioactivity by the S. aureus JE2 culture supernatantduring proteinase K or heat treatment, indicates that the presenceof one ormore extracellular proteinswith amolecularmass higherthan 10 kDa is responsible for the altered P. aeruginosa growthactivities. In addition, S. aureus JE2 transposon mutant strains(25) deficient in the agr quorum sensing system or the sarA andclpP regulatory genes were no longer able to suppress metallicsheen coverage/autolysis or alter growth activity of P. aeruginosaDK2-P24M2-2003 during in vitro coculturing on agar plates. ClpPis, besides its role in stress survival, a central regulatory protein inS. aureus that among other major response pathways also is re-quired for induction of the agr response (22). SarA belongs to afamily of transcriptional regulators that control virulence factorexpression in S. aureus in part through agr (21). Thus, our datafurther suggest that S. aureus agr-controlled extracellular proteinsare likely responsible for promoting the interactionwith P. aerugi-nosa during in vitro coculturing on agar plates. The suppression ofmetallic sheen coverage/autolysis of P. aeruginosa has previouslybeen shown to be mediated by compounds known to inhibit thePQSbiosynthetic pathway inPseudomonas (19). S. aureus JE2 pro-duces awide range of extracellular proteins in vitro,many ofwhichhave been associated with the stress tolerance and virulence of thebacterium (22, 31), and further analysis will reveal the (multifac-

FIG 5 (A) Colonymorphologies of P. aeruginosaDK2-P24M2-2003 (left) andDK2-P24M2-TM1 (right) spotted (2 l OD600 1) on top of LB agar me-dium. (B) Antibiotic resistance of P. aeruginosa DK2-P24M2-2003 (DK2-2003) andDK2-P24M2-TM1 (DK2-TM1) by determining theMICusing Eteststrips of tobramycin (Tm), gentamicin (Gm), or ciprofloxacin (Ci).




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torial) mechanisms involved in the commensal interaction withthe P. aeruginosa DK2 lineage described in this study.

Besides the suppressed autolysis and altered growth activity ofP. aeruginosaDK2-P24M2-2003 during interactionwith S. aureus,our results also suggest a protection of P. aeruginosa against inhi-bition by different classes of antibiotics, i.e., aminoglycosides (to-bramycin and gentamicin) and the fluoroquinolone antibiotic,ciprofloxacin, in the presence S. aureus JE2 extracellular proteins.

The presence of small P. aeruginosa colonies was observed in azone surrounding the S. aureus JE2WT supernatant on agar platescontaining inhibitory levels of antibiotics, and further analysis ofone of the recovered strains (i.e., DK2-P24M2-TM1) revealedcharacteristics of P. aeruginosa small-colony variants (SCVs) (32),such as increased MIC values toward various antibiotics, a de-crease in growth rate, as well as a smaller colony size. Increasedantibiotic resistance by inducing a selection for SCV has beendescribed for S. aureus in the presence of the P. aeruginosa exo-product 4-hydroxy-2-heptylquinoline N-oxide (HQNO); we be-lieve this is the first report to show that the opposite also can occur.The resistance to aminoglycosides by P. aeruginosa SCVs has pre-viously been correlated with the overexpression of efflux mecha-nisms, lipopolysaccharide (LPS) modification, and downregula-tion of genes involved in PQS biosynthesis (32) and could explainthe increased antibiotic resistance by Pseudomonas observed inthis study.

Using a unique collection of strains from the long-term-per-sistent P. aeruginosaDK2 lineage, we show that evolutionary hostadaptation may affect the interspecies interaction potential of thelineage from an initial antagonistic relationship toward commen-sal-like interactions with S. aureus. Although S. aureus is a clini-cally relevant organism often found in the polymicrobial CF air-way infections, the main focus of this study was to investigate thealtered interspecies interaction patterns by P. aeruginosa DK2throughout the evolution of this lineage. Therefore, the S. aureusstrains chosen for this study were not coisolated together with theDK2 strains analyzed here, and further studies are required toaddress the occurrence and clinical significance of the commensalP. aeruginosa-S. aureus relationship in coinfected CF patients.

ACKNOWLEDGMENT

This study was supported by Villum Foundation project no. VKR023113.

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Staphylococcus aureus Alters Growth Activity, Autolysis, and Antibiotic Tolerance in a Human Host-Adapted Pseudomonas aeruginosa LineageMATERIALS AND METHODSGfp tagging P. aeruginosa CF224-2003 by triparental mating.Cross-streak assay and spot inoculation.Screening of the S. aureus JE2 mutant library.Preparation of S. aureus culture supernatants and fractionation of supernatants.Antibiotic plate screening with P. aeruginosa culture and S. aureus culture supernatants.Liquid culture assay.Live/dead staining.Antibiotic resistance assay using Etest strips.RESULTSP. aeruginosa DK2 isolates show different colony morphologies and interaction patterns with S. aureus.S. aureus extracellular factors increase growth activity and suppress autolysis of P. aeruginosa DK2.S. aureus bioactive extracellular proteins are controlled by the S. aureus quorum sensing system and major regulatory genes.S. aureus extracellular proteins induce selection for P. aeruginosa small-colony variants (SCVs) under antibiotic pressure.

DISCUSSIONACKNOWLEDGMENTREFERENCES

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Staphylococcus Aureus Alters Growth Activity, Autolysis, And Antibiotic Tolerance