Vol. 175, No. 22JOURNAL OF BACrERIOLOGY, Nov. 1993, p. 7363-73720021-9193/93/227363-10$02.00/0Copyright C) 1993, American Society for Microbiology

Multiple Antibiotic Resistance in Pseudomonas aeruginosa:Evidence for Involvement of an Efflux Operon

KEITH POOLE,* KATHLEEN KREBES, CATHERINE McNALLY, AND SHADI NESHATDepartment of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received 7 June 1993/Accepted 2 September 1993

An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant ofPseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a

number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, andstreptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, forexample, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previouslyidentified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretionof the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibilityto tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for theORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis ofORFC (oprK) revealedthat its product is homologous to a class of outer membrane proteins involved in export. Similarly, the productsof ORFA and ORFB exhibit homology to previously described bacterial export proteins located in thecytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug eftlux contributesto multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multipleefflux) for ORFAB.

Pseudomonas aeruginosa is a clinically significant pathogencharacterized by intrinsic resistance to a number of antimicro-bial agents. Moreover, problems with the development ofresistance to agents generally exhibiting potent antibacterialactivity against this organism (e.g., carbepenems and fluoro-quinolones) are encountered with increasing frequency (12, 14,19, 41, 43, 47, 48, 54, 55, 65). In addition, cross-resistance tochemically unrelated antibiotics can be associated with fluoro-quinolone resistance (33, 43, 48, 54, 55, 62, 65). In vitro studiesof fluoroquinolone-resistant strains exhibiting cross-resistancehave indicated that resistance is attributable to decreased drugaccumulation resulting from alterations in outer membranepermeability (12, 14, 19, 24, 41, 43, 47, 65). In some instances,this conclusion stems from the identification of novel outermembrane proteins in these mutants (24, 33, 43, 47).

Fluoroquinolone resistance has also been reported for Esch-erichia coli, for which an energy-dependent efflux mechanismhas been implicated (10, 11, 38). Interestingly, cross-resistanceto unrelated antibiotics is also a property of some of thesemutants (32, 37, 38), and multiple-antibiotic-resistant E. colialso shows cross-resistance to fluoroquinolones (11).

Recently, we identified an operon (ORFABC) in P. aerugi-nosa apparently involved in pyoverdine secretion (57). Wereport here the characterization of ORFC, which encodes anouter membrane protein whose overproduction is associatedwith multiple antibiotic resistance. Moreover, we demonstratethat the products of ORFABC show substantial homology tobacterial efflux proteins.

MATERIALS AND METHODS

Strains and plasmids. The bacterial strains and plasmidsused in this study are described in Table 1. Strain K385 is aK372 derivative isolated as a spontaneous mutant growing on

* Corresponding author.

iron-deficient succinate minimal medium (see below) contain-ing 0.5 mM 2,2'-dipyridyl.Growth media. Iron-deficient succinate minimal medium

has been described elsewhere (60) and was made iron sufficientby the addition of FeSO4 (100 ,uM). For culturing of E. colicells, glucose (0.4% [wt/vol]) replaced succinate in the above-described medium. In some experiments, a phosphate-suffi-cient N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid(HEPES)-buffered minimal medium (28) was used as theiron-deficient medium and was similarly made iron sufficient bythe addition of 100 ,uM FeSO4. Amino acids (1 mM) andthiamine-HCl (30 ,uM) were added to growth media as re-quired. L broth (16) was used as the rich medium throughout.Ampicillin (100 ,ug/ml), carbenicillin (200 ,ug/ml), tetracycline(P. aeruginosa, 100 ,ug/ml; E. coli, 10 ,ug/ml), kanamycin (50,ug/ml), and HgCl2 (15 ,ug/ml) were included in growth mediaas necessary. Solid media were obtained by the addition ofBacto Agar (Difco; 1.5% [wt/vol]).Membrane isolation and SDS-polyacrylamide gel electro-

phoresis. Outer membranes were prepared by differentialTriton X-100 solubilization of isolated cell envelopes (66) or bysucrose gradient density centrifugation (29). Inner membraneswere prepared on sucrose gradients (29). Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis was carriedout as described previously (46) with 9 or 14% (wt/vol)acrylamide in the running gel.

Purification of OprK. Outer membranes prepared from 8liters of P. aeruginosa K385 grown overnight in iron-sufficientsuccinate minimal medium were resuspended in 80 ml of 2%(vol/vol) Triton X-100-20 mM Tris-HCl (pH 8.0)-i M NaCland centrifuged (180,000 x g; 60 min). The pellets obtainedwere subsequently reextracted with 80 ml of the same solutionand then resuspended in 40 ml of 1% (wt/vol) Zwittergent 3-14(Calbiochem)-20 mM Tris-HCl (pH 8.0). Following centrifu-gation (180,000 x g; 60 min), the resultant supernatantcontaining Zwittergent 3-14-soluble material was recoveredand loaded onto a 5-ml DEAE-Sepharose CL-4B column (1.5

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TABLE 1. Bacterial strains and plasmids

Source orStrain or plasmid Description' reference

StrainsP. aeruginosaPA06609K372

K385

K590K613ML5087

K635

K636

E. coli5KK38S17-1

Plasmids

met-9011 amiE200 rpsL pvd-9Derivative of PA06609 deficient

in production of pyochelin andthe ferripyochelin receptor

Dipyridyl-resistant derivative ofK372 overproducing an outermembrane protein of 50 kDaand inner membrane proteinsof 40 and ca. 110 kDa

K372 ORFA::tetK372 ORFC::QlHgilv-220 thr-9001 leu-9001 met-

9011 pur-67 aphAPyoverdine-deficient derivative ofML5087

K635 (ORFB::mini-Tn]O-kan)

thr lacZ rpsL thi ser hsdR hsdMX+ (HfrC) relA1thi pro hsdR recA Tra+

pADD214 Mini-D replicon derived fromphage D3112; used for in vivocloning in P. aeruginosa; Tcr

pPV1 pADD214 derivative carrying P.aeruginosa chromosomal DNAwhich restores growth of P.aeruginosa K437 on dipyridyl-containing iron-deficientmedium

pAK1900 E. coli-P. aeruginosa shuttlecloning vector; Apr Cbr

pGP1-2 pACYC177 derivative carryingthe phage T7 RNA polymerasegene under X P,. control andthe c1857 repressor gene; Kmr

pT7-5 pBR322 derivative carrying a

multicloning site downstreamof the strong gene 10 promoterof phage T7; Apr

pT7-6 Same as pT7-5 but with themulticloning site in theopposite orientation; Apr

pSUP301 pACYC177 derivative carryingthe Mob (mobilization) site ofplasmid RP4; Apr Kmr

" Apr, ampicillin resistance; Cb', carbenicillin resistance; Tcr, tetracyclineresistance; Kmr, kanamycin resistance.

by 3.0 cm; Pharmacia-LKB) equilibrated with 1% Zwittergent3-14-20 mM Tris-HCl (pH 8.0) (column buf-er A). The columnwas subsequently washed with 10 ml of column buffer A andthen with 5 ml of column buffer A containing 0.1 M NaCl.Bound protein was eluted from the column with a 40-ml lineargradient of NaCl (0.1 to 0.4 M) in column buffer A andcollected in 1-ml fractions. Fractions enriched for OprK (asdetermined by SDS-polyacrylamide gel electrophoresis) were

pooled and dialyzed overnight against 1% Zwittergent 3-14-10mM Na2HPO4-NaH2PO4 buffer (pH 7.0)-0.1 M NaCl (columnbuffer B). The dialyzed sample was then applied to a 3-ml

hydroxyl apatite column (1 by 4 cm; Bio-Rad Laboratories,Mississauga, Ontario, Canada) equilibrated with column bufferB. After the column was washed with 10 ml of column buffer B,bound protein was eluted with a 40-ml linear gradient ofNa2HPO4-NaH2PO4 buffer (pH 7.0) (10 to 350 mM) in 1%Zwittergent 3-14-0.1 M NaCl. One-milliliter fractions werecollected, and those containing pure OprK (as determined bySDS-polyacrylamide gel electrophoresis) were pooled andstored at - 20°C.CNBr digestion of OprK and isolation of digestion products.

CNBr digestion of OprK was based on the methodologydescribed by Schultz and Oroszlan (67). In brief, purified OprK(500 pLg) was incubated with CNBr (4 mg/ml in 70% formicacid) at 37°C for 24 h in a final volume of 500 pL.. Followingdilution with I ml of 3-mercaptoethanol (0.2%), the mixturewas lyophilized and resuspended in 100 pl. of water. Lyophili-zation was repeated twice more, and OprK was finally resus-pended in 20 .1l of SDS-polyacrylamide gel electrophoresissample buffer (46) and electrophoresed on 14% (wt/vol)polyacrylamide gels. Electrophoresed proteins were subse-quently transferred electrophoretically to ProBlott membranes(Applied Biosciences Inc., Mississauga, Ontario, Canada) in3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer asdetailed by the manufacturer. Proteins were visualized withCoomassie blue according to a procedure described by themanufacturer, and bands corresponding to digestion productsof OprK were excised with a razor blade. An N-terminal aminoacid sequence determination was carried out on electroblottedmaterial at the Centres of Excellence Core Facility for Proteinand DNA Chemistry at Queen's University.

Antimicrobial susceptibility testing. The susceptibilities ofP. aeruginosa strains to a number of antimicrobial agents weretested by inoculating 1-ml cultures of iron-deficient BM2succinate minimal medium containing serial twofold dilutionsof each antimicrobial agent with 5 x 106 organisms. Growthwas assessed visually after 18 h of incubation at 37°C. The MICwas defined as the lowest concentration of antimicrobial agentthat inhibited visible growth.DNA methodology. Plasmid DNA was routinely prepared by

the alkaline lysis procedure (64). Restriction endonucleasesand T4 DNA ligase were obtained from Gibco-BRL or Phar-macia-LKB and used according to manufacturer's instructionsor as described by Sambrook et al. (64). Transformation of E.coli (64) and P. aeruginosa (4) with plasmid DNA has beendescribed elsewhere. Restriction fragments were isolated, asrequired, from agarose gels (0.8 to 1.5% [wt/vol]) with Gene-clean (Bio 101, Inc., La Jolla, Calif.) or Prep-a-gene (Bio-Rad)glass matrices as detailed by the manufacturer. Subcloning ofDNA was performed initially with E. coli 5K prior to itsintroduction into P. aeruginosa.

Cloning and sequencing of ORFC. ORFC was cloned to-gether with ORFA and ORFB (57) by use of the in vivocloning system described by Darzins and Casadaban (15).Plasmid DNA carrying ORFC was prepared on CsCl2 gradi-ents (64) and sequenced at the Centres of Excellence CoreFacility for Protein and DNA Chemistry at Queen's University.An overlapping sequence from both strands was obtained byuse of a series of custom-synthesized primers. Nucleotide anddeduced amino acid sequences were analyzed with the PCGene software package (Intelligenetics Inc., Mountain View,Calif.).

Expression of cloned genes in pT7 vectors. To identify theproducts of cloned genes, the phage T7-based expressionsystem of Tabor and Richardson (70) was used. In brief, E. coliK38 harboring T7 RNA polymerase plasmid pGPI-2 and arecombinant pT7 plasmid carrying cloned genes of interest was

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grown to the log phase in L broth containing the appropriateantibiotics. Cells (200 pll) were harvested by centrifugation andwashed twice in an iron-replete glucose minimal mediumcontaining thiamine before being resuspended in 1 ml of thismedium supplemented with 1 mM concentrations of all aminoacids, except for methionine and cysteine. Following incuba-tion at 37°C for 30 to 45 min, the cultures were shifted to 42°Cfor 15 min, at which time rifampin (400 p.g/ml) was added.After a further 15 min of incubation at 42°C, the cells werepulse-labeled with Tran35Slabel (20 VLCi; ICN BiomedicalsCanada Ltd., Mississauga, Ontario) for 5 min at 37°C. The cellswere then harvested by centrifugation, resuspended in 100 [lIof gel loading buffer (46), and heated at 95°C for 5 min. Theresulting whole-cell extracts were resolved on SDS-polyacryl-amide gels, which were subsequently stained and destainedbriefly, dried, and exposed to Cronex-4 X-ray film (DupontCanada, Mississauga, Ontario) for 24 h.

In vitro mutagenesis and gene replacement. To construct P.aeruginosa strains mutated in ORFC (see Fig. 6), ORFC wasfirst isolated on a 4-kb PstI fragment derived from pPV20 andthen cloned into the unique PstI site on pSUP301 (68) to yieldpPV21. Plasmid pPV21 was partially digested with BamHI,which cuts once in pSUP301 and once in ORFC, and full-length DNA was recovered from agarose gels. Full-lengthDNA was identified by simultaneously running an EcoRIdigest of the same DNA. The mercury resistance determinantof plasmid pPH45fQHg (22) was isolated as a BamHI fragmentwhich was subsequently ligated to BamHI-digested (partially)pPV21 and transformed into E. coli 5K. Plasmids carrying themercury resistance determinant within ORFC were identifiedvia restriction analysis. One such plasmid (pPV22) was trans-formed into mobilizing E. coli S17-1 (68), which was subse-quently conjugated with P. aeruginosa strains by pelleting equalvolumes (100 [I1) of overnight cultures of donor (grown at37°C) and recipient (grown at 42°C) cells in microcentrifugetubes, resuspending the cells in 25 [L1 of L broth, and spottingthe cells onto the center of L broth plates. After overnightincubation at 37°C, bacteria on the plates were suspended in 1ml of L broth and subsequently plated on HgCl2-containing Lbroth plates. P. aeruginosa recipients carrying chromosomalORFC::flHg were HgCl2 resistant and carbenicillin sensitive.

RESULTS

Identification of a 50-kDa outer membrane protein inmutants of P. aeruginosa growing in the presence of 2,2'-dipyridyl. In an attempt to identify outer membrane compo-nents involved in iron acquisition in P. aeruginosa, spontaneousmutants of strain K372 able to grow in the presence ofgrowth-inhibitory concentrations of the nonmetabolizable ironchelator 2,2'-dipyridyl (0.5 mM) were isolated, and outermembranes were screened for the presence of novel proteins.It was reasoned that the ability to grow under such iron-restricted conditions would necessitate an improvement in theiron-acquiring ability of the mutants and that their character-ization might identify outer membrane proteins involved iniron uptake. A number of mutants capable of growth in thepresence of 0.5 mM 2,2'-dipyridyl were recovered, and many ofthese (e.g., strain K385) expressed high levels of a 50-kDaouter membrane protein (Fig. IA, compare lanes 1 and 2)which we designated OprK by using the nomenclature sug-gested by Hancock et al. (30). OprK was not repressible by ironin K385 (Fig. IA, lane 4), even at 200 p.M FeSO4 (data notshown), and was not inducible in the parent strain duringgrowth in phosphate-buffered (BM2) iron-deficient succinateminimal medium (Fig. IA, compare lanes I and 3). Induction

Av E

B

-4

12 34FIG. 1. Cell envelope proteins of P. aeruginosa K372 and its

OprK-overproducing derivative K385. (A) Outer membrane proteinsof K372 (lanes I and 3) and K385 (lanes 2 and 4) grown iniron-deficient (lanes 1 and 2) and iron-sufficient (lanes 3 and 4) BM2succinate minimal medium. OprK is indicated by an arrowhead. (B)Cytoplasmic membrane proteins of K385 (lane 1) and K372 (lane 2)grown in iron-deficient succinate minimal medium. Proteins overpro-duced in K385 are indicated by arrowheads. Molecular mass standards(phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbu-min, 42.7 kDa; carbonic anhydrase, 31 kDa; and soybean trypsininhibitor, 21.5 kDa) are indicated by horizontal lines (from top tobottom).

of the protein was, however, achieved by growth in HEPES-based iron-deficient minimal medium (Fig. 2, lane 5). Inaddition, extended (24 to 36 h) incubation of K372 in thepresence of 0.5 mM 2,2'-dipyridyl resulted in the growth ofK372 and the concomitant induction of OprK (Fig. 2, lane 3).Interestingly, maximal production of the protein was achievedby growth in the presence of ZnSO4 (Fig. 2, lane 7).

Susceptibility of K385 to antimicrobial agents. The produc-tion of a 50-kDa outer membrane protein was previouslyobserved for mutants of P. aeruginosa resistant to streptonigrin(56), and K385 exhibited enhanced resistance to this com-pound (Table 2). This association between the production of a50-kDa outer membrane protein and resistance to an antimi-crobial agent was intriguing, since a number of previouslyreported quinolone-resistant P. aeruginosa strains also pro-duced novel outer membrane proteins with a ca. 50-kDamolecular mass (24, 33, 43). Furthermore, these mutantsshowed cross-resistance to additional nonquinolone antibiot-ics. Examination of the antibiotic susceptibility of K385 re-vealed that it too exhibited resistance to quinolones (cipro-floxacin and nalidixic acid) as well as to chloramphenicol andtetracycline (Table 2).

Overproduction of cytoplasmic membrane proteins in K385.Quinolone-resistant E. coli and multiple-antibiotic-resistant E.coli have been described (11, 32, 37, 38), and resistance has, inpart, been attributable to an efflux component located in thecytoplasmic membrane (10). To determine whether the resis-tance phenotype of K385 was characterized by alterations inthe cytoplasmic membrane, in addition to overproduction ofouter membrane protein OprK, cytoplasmic membrane frac-tions were prepared from K385 and its parent and examined

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1 2 3 4 5 6 7 8FIG. 2. Induction of OprK in P. aeruginosa K372. Outer mem-

branes were prepared from P. aeruginosa K372 cultured in iron-deficient BM2 succinate minimal medium (lane 2), iron-deficient BM2succinate minimal medium with dipyridyl (0.5 mM) (lane 3), iron-sufficient HEPES-buffered minimal medium (lane 4), iron-deficientHEPES-buffered minimal medium (lane 5), iron-deficient BM2 succi-nate minimal medium (lane 6), and iron-deficient BM2 succinateminimal medium with ZnSO4 (0.1 mM) (lane 7). Lanes 1 and 8 showouter membranes of K385 grown in iron-deficient BM2 succinateminimal medium. OprK is indicated by arrowheads.

on SDS-polyacrylamide gels. A protein of ca. 40 kDa, absent inK372, was readily observed in the cytoplasmic membranepreparations of K385 (Fig. 1B, lane 1). In addition, thereappeared to be increased staining in the region of a diffuseband at ca. 108 kDa in the cytoplasmic membrane preparationsof K385 (Fig. IB, lane 1), consistent with increased productionof this protein or production of an additional protein of a

similar size. Interestingly, we recently identified an operon ofat least three genes (ORFABC) (57) (Fig. 3), the first two ofwhich encode products of 40 and ca. 108 kDa and predicted tooccur in the cytoplasmic membrane (57). Moreover, mutantsdefective in these genes show a decrease ability to grow in thepresence of 2,2'-dipyridyl (57). Given that the ORFAB genesare important for growth on dipyridyl and encode products ofthe same molecular masses as the cytoplasmic membraneproteins identified in a mutant (K385) growing on elevatedlevels of dipyridyl suggest that these cytoplasmic membraneproteins are the products of ORFAB. Moreover, they suggestthat OprK is the product of the third gene of this operon,ORFC.

Nucleotide sequence of ORFC. A portion of ORFC waspreviously sequenced (57); to determine whether OprK was

the product of ORFC, its sequence was completed (Fig. 4).ORFC, which begins 2 bp downstream from the stop codon ofORFB, encompasses 1,430 bp and is predicted to encode aproduct of 476 amino acids (excluding the initiation methio-nine) and with a molecular mass of 51,481 Da. The deducedORFC product possesses a sequence at its N terminus char-acteristic of signal sequences as well as a putative lipoproteinsignal peptidase cleavage site (75). The predicted mature

polypeptide comprises 460 amino acids and has a molecularmass of 50,010 Da. The expression of ORFC on a 6.5-kbSmaI-HindIll fragment (Fig. 3B) in E. coli by use of the phageT7-based expression system (70) yielded a major product of 49kDa and a minor product of 51 kDa (Fig. 5). This result was inexcellent agreement with the predicted molecular masses ofthe mature and precursor forms, respectively, of the ORFCproduct and with the estimated molecular mass of OprK. Aweakly detected band running above the 97-kDa molecularmass marker is apparently derived from ORFB, most of whichis present on the 6.5-kb Smal-HindlIl fragment. N-terminalamino acid sequencing of OprK was unsuccessful, as might beexpected for a possible lipoprotein. Problems were also en-countered with CNBr digestion of the protein, owing to theapparent acid lability of the protein, although a 43-kDadigestion product was recovered in sufficient quantity forN-terminal amino acid sequencing. The N-terminal sequenceobtained for this degradation product (ALXNNRXL) matchesthe predicted ORFC amino acid sequence beginning at residue73 (bp 228). Moreover, an ORFC-derived product beginninghere would have a molecular mass of 44,110 Da, in goodagreement with the estimated size of the OprK-derived CNBrdigestion product which was sequenced. Thus, OprK is, indeed,the product of ORFC, which should now be referred to asoprK. Furthermore, it is likely that the cytoplasmic membraneproteins identified in K385 are the products of ORFAB.

Characterization of mutants defective in ORFABC. As themutation in strain K385 has yet to be mapped or fullycharacterized, it is possible that additional alterations whichmay be responsible for the multiple antibiotic resistance phe-notype of strain K385 occur. To demonstrate a direct involve-ment of ORFABC in multiple antibiotic resistance, then,mutants specifically deficient in the ORFABC products wereconstructed and examined for antibiotic susceptibility. AnORFA mutant had been constructed previously by insertion ofthe tet gene of plasmid pBR322 into the cloned gene, whichwas then introduced into P. aeruginosa by gene replacement(57). Similarly, a stable ORFB mutant had been obtainedearlier by insertion of a mini-Tn]O-kan element into the clonedgene, which was also introduced into P. aeruginosa by genereplacement (57). Finally, an ORFC mutant was constructed(Fig. 6) by insertion of the HgCl2 resistance determinant ofpHP45flHg (22) into the cloned gene, which was then intro-duced into P. aeruginosa by gene replacement. Interestingly,attempts at introducing these mutations into wild-type P.aeruginosa PAO1 or mutant strain K385 were unsuccessful,despite repeated attempts. This was unfortunate, since itprecluded direct confirmation that the ORFAB genes wereresponsible for the production of the cytoplasmic membraneproteins overproduced in K385. Moreover, while mutatedORFA and ORFB were readily introduced into strain K372,the recovery of K372 carrying the ORFB::mini-TnlO-kan mu-

TABLE 2. Susceptibilities of P. aeruginosa strains to selectedantimicrobial agents

MIC' of:Strain

DIP SN CIP NAL CAM TET

K372 0.5 10 0.125 62.5 12.5 2.5K385 1.0 40 1.5 250 100 25

a Values are reported in micrograms per milliliter, except that values fordipyridyl are reported as micromolar concentrations. DIP, 2,2'-dipyridyl; SN,streptonigrin; CIP, ciprofloxacin; NAL, nalidixic acid; CAM, chloramphenicol;TET, tetracycline.

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MULTIPLE ANTIBIOTIC RESISTANCE IN P. AERUGINOSA 7367

1 kb

PstIEcoRI

PstISmaIBamHI

PstI1+ jP" PstI

SphI BamHIPstI SphI XhoI

I II

A-

ORFAF

ORFB

A

ORFC

SmaIpT7-5 >>

FIG. 3. Restriction map of the ORFABC operon as derived from restriction and sequence analyses. (A) An 8.5-kb Hindlll subclone ofphagemid pPV1 (57) carrying the entire ORFABC operon and the attR region (thick line) of phage D3112. (B) A 6.5-kb SmaI-HindIII fragment(derived from the subclone shown in panel A) carrying ORFC and subcloned into expression vectors pT7-5 and pT7-6. The orientation of ORFCwith respect to the strong T7 promoters on pT7-5 and pT7-6 is indicated.

tation was complicated by the high frequency with which K372(and most P. aeruginosa PAO strains) mutates to kanamycinresistance. This is attributable, at least in part, to the presence

in P. aeruginosa PAO of a gene, aphA4, which encodes an

aminoglycoside 3'-phosphotransferase (53). Thus, theORFB::mini-TnlO-kan mutation was introduced into strainK635, a pyoverdine-deficient derivative of aphA mutant strainML5084 (53). Strains carrying mutated ORFA (K590), ORFB(K636), and ORFC (K613) all showed increased susceptibility(relative to that of their parent strains) to a number ofantibiotics, including tetracycline, chloramphenicol, cipro-floxacin, and the iron-binding compounds streptonigrin and2,2'-dipyridyl (Table 3). These data confirm a direct role forthe ORFABC operon in antibiotic resistance in P. aeruginosa.Homology of the ORFABC products to bacterial proteins

involved in resistance to antimicrobial agents. The products ofORFAB were previously demonstrated to be highly homolo-gous (40 to 60% identity) to the envCD gene products (57)(Table 4), reported to be involved in septum formation in E.coli (42). Mutants defective in ORFAB were not, however,defective in septum formation, and it was suggested that theenvCD gene products were likely not involved directly inseptum formation (57). A more recent scan of the GenBankdata library identified an additional pair of proteins, AcrA andAcrB (76) (accession number M94248), exhibiting substantialhomology to the ORFAB products (Table 4). These proteins

are the products of an operon responsible for resistance, in E.coli, to acriflavine as well as a number of hydrophobic antibi-otics, dyes, and detergents (5). The ORFA product shares57.7% identity and 8.9% similarity with AcrA while, remark-ably, the ORFB product exhibits 69.8% identity and 10.9%similarity with AcrB. The homologous protein pairs are ofcomparable sizes (Table 4) and, like the ORFA product, AcrApossesses the consensus lipoprotein box (data not shown).FusA, the product of a gene required for fusaric acid resistancein Pseudomonas cepacia (71), was identified as homologous tothe oprK (ORFC) product. Although the level of homologybetween the two proteins over their entire sequences was low(Table 4), alignment of residues 118 to 380 of OprK andresidues 19 to 277 of FusA revealed that 29.4% of the residueswere identical and 12.3% were conservative substitutions (datanot shown). No homology was observed, however, between theORFA-ORFB-oprK (ORFC) operon or its products and therecently sequenced E. coli mar (multiple antibiotic resistance)locus (9) (accession number M96235) or its deduced products.Homology between the ORFABC products and bacterial

export proteins. The multiple antibiotic resistance phenotypein E. coli has been attributed, at least in part, to an effluxcomponent present in the cytoplasmic membrane (10). More-over, we previously showed (57) that the ORFB product ishomologous to the cation efflux protein CzcA from Alcaligeneseutrophus (51). A scan of the GenBank data library revealed

HindIIISstI

il

(A)HindIII

(B)

att

HindIII

HindIII<< pT7-6

v v w

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G Q - M K R S F L S L A V A A V V L S GIC S L I P D Y Q R P E A P1 GGGGCAATGATATGAAACGGTCCTTCCTTTCCCTGGCGGTAGCCGCTGTCGTTCTGTCCGGCTGCTCGCTGATCCCCGACTACCAGCGCCCCGAGGCGCC

V A A A Y P Q G Q A Y G Q N T G A A A V P A A D I G W R E F F R D101 GGTAGCCGCGGCCTACCCGCMGGGCAGGCCTACGGGCAGMCACCGGCGCGGCGGCCGTTCCGGCCGCCGACATCGGCTGGCGCGAGTTCTTCCGCGAC

P Q L Q Q L I G V A L E N N R D L R V A A L N V E A F R A Q Y R I201 CCGCAGTTGCAGCMCTGATCGGCGTGGCGCTGGAAAACAACCGCGACCTGCGGGTCGCCGCGCTGMCGTCGAGGCCTTCCGGGCGCAGTACCGCATCC

Q R A D L F P R I G V D G S G T R Q R L P G D L S T T G S P A I S S301 AGCGGGCCGACCTGTTCCCGCGGATCGGCGTGGACGGTAGCGGCACCCGCCAGCGTTTGCCGGGCGACCTGTCGACCACCGGCAGTCCGGCGATTTCCAG

Q Y G V T L G T T A W E L D L F G R L R S L R D Q A L E Q Y L A T401 CCAGTACGGGGTGACCCTGGGCACTACCGCCTGGGAACTCGATCTCTTCGGCCGCCTGCGCAGCCTGCGCGACCAGGCCCTGGAGCAGTACCTGGCGACC

E Q A Q R S A Q T T L V A S V A T A Y L T L K A D Q A Q L Q L T K501 GAACAGGCGCAGCGCAGCGCGCAGACCACCCTGGTGGCCAGCGTGGCGACCGCCTACCTGACGCTGMGGCCGACCAGGCGCAGTTGCAGCTGACCMGG

D T L G T Y Q K S F D L T Q R S Y D V G V A S A L D L R Q A Q T A V601 ACACCCTGGGCACCTACCAGAAGAGTTTCGACCTGACCCAGCGCAGCTACGACGTCGGCGTCGCCTCCGCGCTCGACCTGCGCCAGGCGCAGACCGCCGT

E G A R A T L A Q Y T R L V A Q D Q N A L V L L L G S G I P A N L701 GGAAGGCGCCCGCGCGACCCTGGCGCAGTACACCCGCCTGGTAGCCCAGGACCAGAATGCGCTGGTCCTGCTGCTGGGCTCCGGGATCCCGGCGMCCTG

BamHIP Q G L G L D Q T L L T E V P A G L P S D L L Q R R P D I L E A E

801 CCGCAAGGCCTGGGCCTGGACCAGACCCTGCTGACCGAAGTGCCGGCGGGTCTGCCGTCGGACCTGCTGCAACGGCGCCCGGACATCCTCGAGGCCGAGCXhoI

H Q L M A A N A S I G A A R A A F F P S I S L T A N A G T M S R Q L901 ACCAGCTCATGGCTGCCAACGCCAGCATCGGCGCCGCGCGCGCGGCGTTCTTCCCGAGCATCAGCCTGACCGCCAACGCCGGCACCATGAGCCGCCAACT

S G L F D A G S G S W L F Q P S I N L P I F T A G S L R A S L D Y1001 GTCCGGCCTGTTCGACGCCGGTTCGGGTTCCTGGTTGTTCCAGCCGTCGATCAACCTGCCGATCTTCACCGCCGGCAGCCTGCGTGCCAGCCTGGACTAC

A K I Q K D I N V A Q Y E K A I Q T A F Q E V A D G L A A R G T F1101 GCGMGATCCAGAAGGACATCAACGTCGCGCAGTACGAGAAGGCGATCCAGACGGCGTTCCAGGAAGTCGCCGACGGCCTGGCCGCGCGCGGTACCTTCA

T E Q L Q A Q R D L V K A S D E Y Y Q L A D K R Y R T G V D N Y L T1201 CCGAGCAGTTGCAGGCGCAGCGCGATCTGGTCAAGGCCAGCGACGAGTACTACCAGCTCGCCGACAAGCGCTATCGCACGGGGGTGGACAACTACCTGAC

L L D A Q R S L F T A Q Q Q L. I T D R L N Q L T S E V N L Y K A L1301 CCTGCTCGACGCGCAACGCTCGCTGTTCACCGCGCAGCAGCAACTGATCACCGACCGCCTCAATCAGCTGACCAGCGAGGTCMCCTGTACAAGGCCCTG

W G G D C F D T C Q K R A G -1401 TGGGGAGGTGACTGTTTTGACACATGCCAAAAGAGGGCGGGATAGGCTAGAGCCCCTATAGCACTAGG

FIG. 4. Nucleotide sequence of ORFC. The deduced amino acid sequence of the ORFC product is also presented, initiating 2 bp downstreamfrom the stop codon of ORFB (Fig. 3). A lipoprotein box and selected restriction sites are highlighted. The sequence of ORFC up to the XhoIsite was published previously (57). The sequence of ORFC has been deposited in the GenBank data base.

additional efflux proteins with homology to the ORFABCproducts (Table 4). Proteins exhibiting homology to ORFAincluded CzcB (51), a cytoplasmic membrane protein involvedin cation (heavy metal) efflux in A. eutrophus; HlyD (31), acytoplasmic membrane protein involved in hemolysin export inE. coli; and LktD (26), a leukocidin export protein ofActinoba-cillus actinomycetemcomitans. Similarly, the ORFB productwas homologous to a second cation efflux protein, CnrA (45),also from A. eutrophus. Finally, the oprK (ORFC) productshowed homology to a number of outer membrane proteinswith an export function, including CyaE (cyclolysin export inBordetella pertussis) (25) and PrtF (protease export in Erwiniachrysanthemi) (44). It also showed homology to NodT, apredicted outer membrane lipoprotein reportedly involved innodulation in Rhizobium leguminosarum (69). The nodT geneis clustered with a number of genes involved in the export ofnodulation factors (72), and the NodT product is presumed tofunction in nodulation factor export (18). Interestingly, ORFBis also homologous to two nodulation proteins from Rhizobiummeliloti, NodH and NodI, which may also be involved innodulation factor secretion (3). Both Nod proteins are mark-

edly smaller than the ORFB product, with NodH exhibitinghomology solely to the central region of ORFB, while NodI ishomologous to the C terminus of this protein (Table 4).

DISCUSSION

The production of a 50-kDa outer membrane protein(OprK) in P. aeruginosa K385 was associated with decreasedsusceptibility to 2,2'-dipyridyl as well as to a number ofantimicrobial agents. The previously described norfloxacin-resistant P. aeruginosa nfxB (33) and nfxC (24) strains alsoshowed decreased susceptibility to several antimicrobial agentsconcomitant with the production of 54- and 50-kDa outermembrane proteins, respectively. Unlike the nfxC mutants,however, K385 and the nfxB mutants did not show a decreasein the level of OprD. A ciprofloxacin-resistant mutant of P.aeruginosa exhibiting cross-resistance to nonquinolone antibi-otics also expressed a novel outer membrane protein of 54 kDa(43). Finally, an outer membrane protein estimated at 49 kDa(OprM) was identified in multiple antibiotic resistance mutantsof P. aeruginosa selected by meropenem or a combination of

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1 2FIG. 5. Identification of the ORFC product. E. coli K38 cells

harboring ORFC-containing pT7-5 (lane 1) or pT7-6 (lane 2) werepulse-labeled with a mixture of [35S]methionine and [35S]cysteine(Tran35S label) following induction of the cloned genes (see Materialsand Methods) and were subjected to SDS-polyacrylamide gels electro-phoresis and autoradiography. Molecular mass standards (lines atright, from top to bottom): phosphorylase b, 97.4 kDa; bovine serumalbumin, 66.2 kDa; ovalbumin, 42.7 kDa; carbonic anhydrase, 31 kDa.

ofloxacin and cefsulodin (47). Despite some subtle differencesin the resistance phenotypes of the aforementioned mutants, itis tempting to speculate that the outer membrane proteinidentified in all cases is the same. If so, it is likely thatdecreased drug accumulation leading to resistance in the nfxB,

nfxC, OprM-producing, and ciprofloxacin-resistant mutantswas due not to altered outer membrane permeability, asoriginally suggested, but to antibiotic efflux, given the homol-ogy between OprK (i.e., the ORFC product) and severalbacterial outer membrane efflux proteins. Interestingly, quin-olone resistance and multiple antibiotic resistance in E. colihave also been attributed, in part, to an efflux mechanism (10).The similarities in resistance phenotypes exhibited by the

aforementioned mutants are also intriguing, in that the cfxB(nalB) mutation (61, 62) and the mutation resulting in theoverproduction of OprM (47) have been mapped to the 20-minregion of the P. aeruginosa PAO chromosome, while theORFA-ORFB-oprK (ORFC) operon has been physicallymapped to the 16- to 20-min region (21). In contrast, the nfxCmutation has been mapped near the catA1 gene at min 46 onthe P. aeruginosa PAO chromosome (24), the same location atwhich the gene(s) required for pyoverdine synthesis has beenmapped (2). This is interesting because, as discussed below, theORFA-ORFB-oprK (ORFC) operon is proposed to play a rolein pyoverdine secretion.

In addition to OprK, the ORFA-ORFB-oprK (ORFC)operon encodes two proteins, of 40 and 108 kDa, predicted tooccur in the cytoplasmic membrane. Strikingly, OprK-produc-ing K385 overproduces two cytoplasmic proteins of thesemolecular masses, suggesting that antibiotic resistance in K385is due to overexpression of the ORFA-ORFB-oprK (ORFC)operon. In light of the homology between the ORFAB prod-ucts and cytoplasmic membrane efflux proteins, it seems likelythat the ORFAB products function, in antibiotic resistance, inthe efflux of antimicrobial agents across the cytoplasmic mem-

1 kb

HindIII PstIPstI PstI PstI

HindIII.PstI*PstI

-A

ORFA

A

ORFB ORFC

attry

I I

PstI BamHI PstI

BamHI omega-Hg BamHIFIG. 6. Insertion mutagenesis of cloned ORFC. ORFC was recovered on a 4-kb Pstl fragment from pPV20 (top) and cloned into the unique

Pstl site of plasmid pSUP301. The HgCl2 resistance determinant of pHP45-Hg (recovered on a BamHI fragment) was subsequently inserted intothe unique BamHI site in ORFC. The mutated ORFC was then inserted into the chromosome of P. aeruginosa by gene replacement. PstI* indicatesa restriction site present in the pAK1900 cloning vector into which the ORFABC-carrying HindIll fragment was originally cloned.

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TABLE 3. Susceptibilities of P. aeruginosa ORFABC mutants toantimicrobial agents

MIC" of:Strain

DIP SN CIP CAM TET

K372 0.5 10 0.25 12.5 2.5K590 (ORFA-) 0.25 2.5 0.125 3.125 NDK613 (ORFC-) 0.25 2.5 0.125 3.125 '0.15K635 2 10 0.125 25 5K636 (ORFB-) 0.5 1.25 0.0625 6.25 0.312

a See Table 2, footnote a. ND, not determined.

brane. For this reason, we propose to rename ORFAB mexAB(multiple efflux).The ORFABC (mexA-mexB-oprK) operon is regulated by

iron (57) and, indeed, OprK was inducible under certain condi-tions of iron limitation. The failure to observe induction of theprotein during growth in iron-deficient BM2 minimal medium isprobably attributable to substantial iron contamination of thephosphate component of BM2, rendering the medium less irondeficient than other minimal media. This suggests that substan-tial OprK (and therefore ORFABC [mexA-mexB-oprK]) expres-sion requires more severe iron limitation. OprK was, for exam-ple, readily induced during growth in an iron-deficient HEPES-buffered minimal medium, which may be contaminated to alesser extent with iron. Certainly, siderophore yields are three-to fourfold higher in this medium than in iron-deficient BM2

TABLE 4. Identification of proteins exhibiting homology to theORFABC products"

Protein Size Alignment(no. of residues) score F

ORFA 383AcrA 397 50.174 Acriflavine resistanceEnvC 384 32.514 ?LktD 477 5.656 Leukocidin exportCzcB 520 4.625 Heavy metal effluxHlyD 478 2.789 Hemolysin export

ORFB 1,046AcrB 1,049 162.56 Acriflavine resistanceEnvD 964 74.827 ?CzcA 1,063 18.893 Heavy metal effluxCnrA 1,075 14.135 Heavy metal effluxNodH 215 48.151c Nodulation factor exportNodI 454 23.47d Nodulation factor export

ORFC 477NodT 467 32.048 Nodulation factor exportCyaE 474 14.376 Cyclolysin exportPrtF 462 4.505 Protease exportFusA 433 0.272 Fusaric acid resistance

C The BLAST (1) network service offered by the National Center for Biotech-nology Information (Bethesda, Md.) was used to identify proteins exhibitinghomology to the ORFABC products.

h Protein sequences exhibiting homology to the products of ORFA (firstgrouping), ORFB (second grouping), or ORFC (oprK) (third grouping) were in-dividually aligned to the sequence of the appropriate ORFABC product by useof the PCOMPARE program available with the PC Gene software package. Thealignment score was obtained by the method of Needleman and Wunsch (50) asimplemented by Feng et al. (23) by use of the structure-genetic matrix with a gappenalty of 6 and a bias parameter of 0. A value of >3.0 suggests significantsimilarity.

Score obtained for alignment of NodH to residues 357 to 569 of the ORFBproduct.

d Score obtained for alignment of NodI to residues 734 to 1024 of the ORFBproduct.

minimal medium (56), consistent with a lower iron content.Similarly, growth in the presence of the iron chelator 2,2'-dipyridyl, which would be expected to reduce available iron,resulted in the induction of OprK. The observed induction ofOprK by Zn2+ is also consistent with iron regulation of thisprotein, since Zn2+ is known to enhance the expression ofiron-regulated constituents in P. aeruginosa, including sid-erophores (34, 35) and their receptors (56). A similar effect ofZn + on siderophore production has been noted for Pseudomo-nas fluorescens (8) and Azotobacter vinelandii (39). In the latterinstance, Zn2+-enhanced siderophore production was attribut-able to a reduction in cytoplasmic ferrous iron levels resultingfrom the inhibition of ferric reductase activity by Zn2+ (40).

In addition to the iron regulation of ORFABC (mexA-mexB-oprK), this operon is also coregulated with components ofpyoverdine production and uptake (57), and we have suggestedthat it functions in pyoverdine secretion (57). The observedhomology between the ORFABC (mexA-mexB-oprK) productsand a number of bacterial export proteins is certainly consis-tent with such a conclusion. Moreover, while antibiotics pur-ported to be substrates of an ORFABC (mexA-mexB-oprK)efflux system are structurally quite distinct, they do retain somecommon features (an aromatic ring), and most exhibit anability to bind cations, including iron (6, 20, 49, 56, 73, 77). Inthis regard, they resemble the catechol-containing chromophoreof pyoverdine (17). It is possible, then, that ORFABC (mexA-mexB-oprK)-dependent drug resistance results from similaritiesbetween certain antimicrobial agents and pyoverdine, whichmay be the true substrate for the ORFABC (mexA-mexB-oprK)efflux system. Obviously, the ORFABC (mexA-mexB-oprK)products exhibit a very broad substrate specificity, and it may bethat not only pyoverdine but also metabolites thereof are thenatural substrates for this efflux system. Thus, ORFABC (mexA-mexB-oprK) may function not only in de novo pyoverdinesecretion but also in the secretion of recycled pyoverdine andmetabolites resulting from that process.

Precedence for broad substrate specificity in components ofiron transport can be found in studies of the E. coli iron-regulated outer membrane proteins Fiu (7) and Cir (7, 50),reportedly involved in the uptake of hydrolytic products of thesiderophore enterobactin (52). These proteins also facilitatethe uptake of antibiotics containing iron-chelating moieties,including catechol-substituted 3-lactams (13, 52).The strikingly high degree of homology between the OR-

FAB (mexAB) products and proteins AcrA and AcrB is astrong indication of a common function. While AcrA and AcrBare putative efflux proteins involved in resistance to acriflavineand other antimicrobial agents (5), it is unlikely that acriflavineis the normal cellular substrate for these proteins. It is inter-esting to note, however, that like pyoverdine, the E. colisiderophore enterobactin is a catechol-containing molecule(74). It is tempting to speculate, therefore, that AcrA and AcrBfunction in the secretion of enterobactin and/or its metabolites.The previously identified homology between the ORFAB(mexAB) products and EnvCD (57) (which is also highlyhomologous to AcrAB) suggests that E. coli may possessmultiple systems for enterobactin export.

ACKNOWLEDGMENTS

We thank R. E. W. Hancock for initially suggesting a possibleconnection between K385 and the previously described nfx mutants.

This work was supported by operating grants from the MedicalResearch Council of Canada and the Canadian Cystic Fibrosis Foun-dation. K.P. is a Natural Sciences and Engineering Research CouncilUniversity Research Fellow. C.M. was supported by a CCFF summerstudentship.

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