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• W2144783616 abstract "Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics. Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics. Pseudomonas aeruginosa is a Gram-negative bacterium thriving in environments polluted with organic matter. It is also an opportunistic pathogen frequently encountered in the hospital, causing morbidity and mortality in immunocompromised and cystic fibrosis patients (1Rosenfeld M. Ramsey B.W. Gibson R.L. Curr. Opin. Pulm. Med. 2003; 9: 492-497Crossref PubMed Scopus (107) Google Scholar). P. aeruginosa is characterized by an intrinsically high level of resistance to xenobiotics including antimicrobial agents, solvents, and heavy metals (2Wang C.L. Michels P.C. Dawson S.C. Kitisakkul S. Baross J.A. Keasling J.D. Clark D.S. Appl. Environ. Microbiol. 1997; 63: 4075-4078Crossref PubMed Google Scholar), which can be accounted for by a combination of its low outer membrane permeability and the presence of multiple efflux pumps (3Nikaido H. Science. 1994; 264: 382-388Crossref PubMed Scopus (1271) Google Scholar). These pumps belong to the resistance, nodulation, cell division (RND) 1The abbreviations used are: RND, resistance, nodulation, cell division; TYG, tryptone-yeast extract-glucose; MTC, maximal tolerable concentration; qRT-PCR, quantitative real time PCR; LUC, latex urinary catheter. transporter family, present in many Gram-negative bacteria (4Saier M.H. Tam R. Reizer A. Reizer J. Mol. Microbiol. 1994; 11: 841-847Crossref PubMed Scopus (275) Google Scholar). To extrude substrates from the cytoplasm across the two membranes, these systems are composed of a proton antiporter located in the cytoplasmic membrane, a membrane fusion protein spanning the periplasmic space, and an outer membrane protein (5Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar). Members of the RND family, namely the Mex pumps, have recently gained increasing interest. In particular, the constitutively expressed MexAB-OprM (6Poole K. Krebes K. McNally C. Neshat S. J. Bacteriol. 1993; 175: 7363-7372Crossref PubMed Scopus (559) Google Scholar, 7Li X.-Z. Nikaido H. Poole K. Antimicrob. Agents Chemother. 1995; 39: 1948-1953Crossref PubMed Scopus (513) Google Scholar) and the inducible MexXY (8Ramos-Aires J. Köhler T. Nikaido H. Plésiat P. Antimicrob. Agents Chemother. 1999; 43: 2624-2628Crossref PubMed Google Scholar) efflux pumps endow the PAO1 reference strain and other clinical isolates (9Ziha-Zarifi I. Llanes C. Köhler T. Pechere J.C. Plésiat P. Antimicrob. Agents Chemother. 1998; 43: 287-291Crossref Google Scholar) with a natural resistance to a wide range of antimicrobial agents. Proton-driven RND type efflux pumps conferring heavy metal resistance have been described in Ralstonia metallidurans (for a recent review, see Ref. 10Mergeay M. Monchy S. Vallaeys T. Auquier V. Benotmane A. Bertin P. Taghavi S. Dunn J. van der Lelie D. Wattiez R. FEMS Microbiol. Rev. 2003; 27: 385-410Crossref PubMed Scopus (355) Google Scholar) and include the Cnr system (nickel/cobalt) (11Liesegang H. Lemke K. Siddiqui R.A. Schlegel H.G. J. Bacteriol. 1993; 175: 767-778Crossref PubMed Google Scholar), the Ncc system (nickel/cobalt/cadmium) (12Schmidt T. Schlegel H.G. J. Bacteriol. 1994; 176: 7045-7054Crossref PubMed Google Scholar), and the Czc system (cobalt/zinc/cadmium) (13Mergeay M. Nies D. Schlegel H.G. Gerits J. Charles P. Van Gijsegem F. J. Bacteriol. 1985; 162: 328-334Crossref PubMed Google Scholar, 14Nies D. Mergeay M. Friedrich B. Schlegel H.G. J. Bacteriol. 1987; 169: 4865-4868Crossref PubMed Google Scholar). In P. aeruginosa, an RND type efflux pump called CzrCBA was recently described in an environmental isolate where it contributes to the intrinsic resistance to zinc and cadmium (15Hassan M.T. van der Lelie D. Springael D. Romling U. Ahmed N. Mergeay M. Gene (Amst.). 1999; 238: 417-425Crossref PubMed Scopus (126) Google Scholar). Cross-resistance between heavy metal and antibiotic pumps has not been reported so far. In the few cases where associations have been observed they were either plasmid-mediated (16Marques A.M. Congregado F. Simon-Pujol D.M. J. Appl. Bacteriol. 1979; 47: 347-350Crossref PubMed Scopus (44) Google Scholar) or resulted from uncharacterized multiple resistance mechanisms (17Filali B.K. Taoufik J. Zeroual Y. Dzairi F.Z. Talbi M. Blaghen M. Curr. Microbiol. 2000; 41: 151-156Crossref PubMed Scopus (113) Google Scholar, 18de Vicente A. Aviles M. Codina J.C. Borrego J.J. Romero P. J. Appl. Bacteriol. 1990; 68: 625-632Crossref PubMed Scopus (81) Google Scholar). In P. aeruginosa, the question of heavy metal and antibiotic resistance is of particular concern since this organism is a possible candidate for bioremediation processes where selection of antibiotic resistance upon heavy metal exposure is undesirable. On the other hand, zinc was found to be released from urinary catheters resulting in antibiotic resistance (19de Haan K.E. Woroniecka U.D. Boxma H. de Groot C.J. van den Hamer C.J. Burns. 1990; 16: 393-395Crossref PubMed Scopus (4) Google Scholar, 20Conejo M.C. Garcia I. Martinez-Martinez L. Picabea L. Pascual A. Antimicrob. Agents Chemother. 2003; 47: 2313-2315Crossref PubMed Scopus (60) Google Scholar). Therefore the possibility of cross-resistance selection by either heavy metals or antibiotics is of concern for both environmental and clinical issues. In the present study, we addressed this question by exposing the P. aeruginosa reference strain PAO1 to either zinc or to the antibiotic imipenem. Surprisingly exposure to zinc selected strains that were resistant to both heavy metals (zinc, cadmium, and cobalt) and to the carbapenem antibiotic imipenem. Analysis of the underlying mechanism revealed a co-regulation between carbapenem influx and heavy metal efflux. A single amino acid change located in the two-component sensor protein CzcS, regulating heavy metal efflux pump expression, was found to be responsible for the observed cross-resistance in P. aeruginosa. Bacterial Strains and Growth Conditions—Bacterial strains used in this study are listed in Table I. Strain BdB4 was isolated from soil of a heavy metal-contaminated site (zinc and lead) near Geneva (Bois de Bay) by successive subculturing in tryptone-yeast extract-glucose (TYG) medium (21Mergeay M. Gerits J. Houba C. C. R. Seances Soc. Biol. Fil. 1978; 172: 575-579PubMed Google Scholar) provided with increasing ZnCl2 concentrations (10–35 mm; dose increase, 5 mm). Strain BdB4 was confirmed as P. aeruginosa using an API20NE gallery (Biomérieux, Marcy l’Etoile, France). Luria-Bertani (LB) (22Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar) and TYG medium were used as rich media. Some experiments were performed in liquid mineral medium (13Mergeay M. Nies D. Schlegel H.G. Gerits J. Charles P. Van Gijsegem F. J. Bacteriol. 1985; 162: 328-334Crossref PubMed Google Scholar) supplemented with 0.4% glucose and 50 mm sodium Hepes, pH 7.0, instead of Tris buffer. The phosphate content (0.64 mm) of this Hepes-buffered minimal medium minimized the interference of phosphate with heavy metals. Cultures were grown at 37 °C on a rotary shaker in 200-ml Erlenmeyer flasks containing 25 ml of minimal medium.Table IBacterial strains and plasmids used in this studyStrainsRelevant characteristicsaZnR, CdR, and CoR, resistance to zinc, cadmium, and cobalt, respectively; IPMR, resistance to imipenem; WT, wild type.Reference or sourcePT5PAO1 wild typeLaboratory collectionPT364PT5 oprD::ΩTc57Epp S.F. Köhler T. Plésiat P. Michéa-Hamzehpour M. Frey J. Pechère J.C. Antimicrob. Agents Chemother. 2001; 45: 1780-1787Crossref PubMed Scopus (61) Google ScholarPT1173PT5ΔczcAThis studyPT1102PT5, IPMR, selected on imipenemThis studyPT1105PT5, IPMR, ZnR, CdR, CoR, selected on imipenemThis studyPT1108PT5, IPMR, ZnR, CdR, CoR, selected on zincThis studyPT1152PT5 (pMMB66EH)This studyPT1151PT5 (pRWT), overexpression of CzcR-WTThis studyPT1153PT5 (pSWT), overexpression of CzcS-WTThis studyPT1154PT5 (pSV194L), overexpression of CzcS(V194L)This studyBdB4P. aeruginosa environmental isolateThis studyBdB4-3BdB4, IPMR, ZnR, CdR, CoR, selected on zincThis studya ZnR, CdR, and CoR, resistance to zinc, cadmium, and cobalt, respectively; IPMR, resistance to imipenem; WT, wild type. Open table in a new tab Isolation of Spontaneous Mutants Resistant to Heavy Metals or Imipenem—For selection of zinc-resistant mutants, PT5 was inoculated at a final concentration of about 2 × 107 cells/ml in 5 ml of liquid TYG medium in test tubes that were incubated for 5 days at 30 °C. The medium was supplemented with 15 (maximal tolerable concentration (MTC)), 20, 25, or 30 mm ZnCl2. From each condition, clones were isolated on TYG medium by serial dilutions. 50 clones were tested for zinc resistance after growth for 48 h at 30 °C on TYG plates containing 20 mm ZnCl2. 16 independent clones were then tested for antibiotic resistance and tolerance to other heavy metals. PT5 was also inoculated on Mueller-Hinton agar plates and exposed to imipenem-impregnated disks (10 μg, Biomérieux). After overnight incubation at 37 °C, 42 imipenem-resistant clones appearing inside the inhibition zone were picked, streaked on TYG medium, and tested for zinc resistance. Resistant mutants were then tested for tolerance to other metals (cadmium, cobalt, copper, and nickel) and antibiotics (imipenem, ticarcillin, carbenicillin, nalidixic acid, ciprofloxacin, norfloxacin, tetracycline, amikacin, chloramphenicol, and polymyxin B). Determination of Heavy Metal and Antibiotic Resistance—The MTCs of heavy metals were determined on solidified TYG medium containing different concentrations of heavy metal salts. The MTCs were scored in 10 × 10 × 2-cm Sterilin plates (Bibby Sterilin Ltd., Stone, Staffs, UK) provided with 25 compartments. 2-ml of medium were poured into each compartment, plates were air-dried, and subdivisions were inoculated with 10 μl of an overnight culture. The MTC is defined as the highest metal concentration at which growth was still observed after 48 h of incubation at 30 °C. Resistance to antibiotics was determined by the Kirby-Bauer method on Mueller-Hinton agar plates using antibiotic-impregnated disks (Biomérieux). An overnight culture grown at 37 °C in TYG medium was diluted 1:500 in 0.9% NaCl, and 2 ml of the suspension were spread on Mueller-Hinton agar plates. The plate was air-dried, and antibiotic disks were applied. After 1 h of diffusion at room temperature, plates were incubated overnight at 37 °C. Inhibition zones were measured and compared with those of the reference strain PT5. Killing Curves in Presence of Zinc—PT5 was grown for 48 h at 37 °C in phosphate-deficient (0.1 mm) minimal medium to increase bioavailability of heavy metals. Cultures were performed in the absence (uninduced control cells) or in the presence of 1 mm ZnCl2 (metal-induced cells). Treated cultures were found to exhibit a lag phase of about 24 h followed by a period of active growth. Both cultures were then diluted 1:200 in fresh medium containing either no zinc or a lethal zinc concentration of 5 mm. Cultures were incubated at 37 °C with shaking (150 rpm) for 5 h. Kinetics of killing of uninduced versus metal-induced cells was then followed. At defined intervals, samples were removed, diluted in TYG medium, and plated on the same medium for viable counts. DNA Manipulations—Standard techniques were used for DNA manipulation (22Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar). The gene encoding the CzcR protein was amplified by PCR from PT5 genomic DNA (Pfu DNA polymerase, Promega) with primers czcR-F and czcR-R (see below). The 900-bp product was cloned into the SmaI site of vector pMMB66EH (23Furste J.P. Pansegrau W. Frank R. Blocker H. Scholz P. Bagdasarian M. Lanka E. Gene (Amst.). 1986; 48: 119-131Crossref PubMed Scopus (813) Google Scholar) under the tac promoter, yielding plasmid pRWT. The czcS gene from PT5 and PT1105 was amplified by PCR using primers S58 (5′-cggaattcgcggcgtcggctacgtcc) and S59 (5′-cgggatcctgcggcgagtaccggctgtggc) containing an EcoRI and a HindIII site, respectively. The 1,600-bp product was EcoRI/HindIII-digested and cloned into the EcoRI/HindIII sites of pMMB66EH under the tac promoter, yielding plasmids pSWT and pSV194L, respectively. Correct orientation of the genes was verified by sequencing. Plasmids were electroporated into P. aeruginosa strain PT5 (24Smith A.W. Iglewski B.H. Nucleic Acids Res. 1989; 1710509Crossref PubMed Scopus (230) Google Scholar). To delete the czcA gene from strain PT5, a 600-bp PCR fragment corresponding to the 5′-end of czcA was generated with primers 48 (5′-cccaagcttcgaacgcatcatccaattcg) and 49 (5′-gcttcttcggatccggggcg) and ligated to an 800-bp PCR fragment containing the 3′-end of czcA generated with primers 50 (5′-cgggatcctgttcgagggcgaccgcc) and 51 (5′-gctctagatccagcgatagagcaccggc). The gentamicin resistance cassette from plasmid pPS858 (25Hoang T.T. Karkhoff-Schweizer R.R. Kutchma A.J. Schweizer H.P. Gene (Amst.). 1998; 212: 77-86Crossref PubMed Scopus (1510) Google Scholar) was inserted between the two parts in the BamHI site. This construct was cloned as a PCR fragment into the HindIII-cleaved plasmid pEX18Ap (25Hoang T.T. Karkhoff-Schweizer R.R. Kutchma A.J. Schweizer H.P. Gene (Amst.). 1998; 212: 77-86Crossref PubMed Scopus (1510) Google Scholar). After transfer and homologous recombination into strain PT5, excision of the gentamicin cassette was performed as described previously (25Hoang T.T. Karkhoff-Schweizer R.R. Kutchma A.J. Schweizer H.P. Gene (Amst.). 1998; 212: 77-86Crossref PubMed Scopus (1510) Google Scholar). The resulting strain PT1173 carries a 1,700-bp deletion inside the czcA gene as verified by PCR. Sequencing of czcRS—The DNA region from position 2,843,235 to 2,845,974 on the PAO1 chromosome (26Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3446) Google Scholar) including the promoter region of czcR-czcC and the czcR-czcS operon were amplified using four different sets of primers: proczcC-F (5′-ccaggcagagtcccatcagtagc) and proczcC-R (5′-tggtgcaggtagtcggcagtctt), czcR-F (5′-aggcaacgcccgaaatgtaactt) and czcR-R (5′-ccagcttcaattgcaggttttcc), czcS1-F (5′-tctcgctgatctgggacatgaa) and czcS1-R (5′-gggatgcggtaggagagatcctg), and czcS2-F (5′-gcctgctcgacggtttcct) and czcS2-R (5′-ctgttcctcgccggtttctg). DNA sequencing was performed on double-stranded DNA templates obtained from genomic DNA by PCR amplification. Sequencing reactions were performed by the core facility of the Medical School of the University of Geneva using an Applied Biosystems (Foster City, CA) capillary sequencing machine (model 3100). Western Blot Analysis—An overnight preculture of P. aeruginosa grown in minimal medium was diluted 100-fold in the same medium and grown at 37 °C to an A600 of 1. Total protein was solubilized by resuspending a bacterial pellet in 1× SDS gel loading buffer. Samples were boiled 5 min and centrifuged 10 min in a microcentrifuge to remove bacterial debris. A duplicate sample was used for protein quantification with the Lowry method (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) on NaOH-solubilized extracts (28Hanson R.S. Philipps J.A. Gerhardt P. Murray R.G.E. Costilow R.N. Nester E.W. Wood W.A. Krieg N.R. Briggs Philipps G. Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, D. C.1981: 328-364Google Scholar). 10 μg of total protein were separated on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. Blots were incubated with anti-OprD, anti-OprF, or anti-Hsp70 antibodies and revealed by chemiluminescence. All antibody incubations and washes were performed in TBS-T (20 mm Tris, 137 mm NaCl, 0.1% Tween 20, pH 7.6) supplemented with 5% powdered milk. Real Time PCR Analysis—For RNA isolation, strains were cultured in 5 ml of minimal glucose medium described above or in LB medium and grown at 37 °C to midexponential growth phase. 0.25 ml of this culture, corresponding to 5 × 108 cells, was added to 0.5 ml of RNeasy Protect bacteria solution (Qiagen, Hildesheim, Germany), and total RNA was isolated with RNeasy columns according to the instructions of the supplier. Residual DNA was eliminated by DNase treatment using 20 units of RQ1 RNase-free DNase (Promega). After removal of DNase by phenol/chloroform extraction, RNA was precipitated, and the pellet resuspended in 30 μl of RNase-free H2O. For cDNA synthesis, 1 μg of RNA was reverse-transcribed using random hexamer primers (Promega) and Improm-II reverse transcriptase (Promega) according to the supplier’s instructions. Reverse transcriptase was inactivated by incubation at 70 °C for 15 min, and the obtained cDNAs were stored at –20 °C until use. The following primer sequences for the PCR amplification of cDNA were designed using the Primer3 program 2See www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.: czcR-1 (5′-gtcatcacccggacgcagatcat) and czcR-2 (5′-gtagccgacgccgcgaatggtat), czcS-1 (5′-tacgcagctctcgcagttctcc) and czcS-2 (5′-tgtccacctgcaccaggaacagc), czcC-1 (5′-ggtcagcatcggcagcaagtacg) and czcC-2 (5′-ggtcgtaggcctgtaccgcttcg), and rpsL-3 (5′-gcaactatcaaccagctggtg) and rpsL-5 (5′-gctgtgctcttgcaggttgtg). A RotorGene real time PCR machine (model RG3000, software version 4.6.67) was used for the quantification of cDNA. PCRs were performed using a Sybr Green Quantitect kit (Qiagen, Hilden, Germany) according to the specifications of the supplier. To check for residual contaminating genomic DNA, control reactions without reverse transcriptase were analyzed in the real time PCR apparatus using the rpsL primer set. No amplification signal above the non-template control was detected indicating that the RNA samples were free of contaminating DNA. The cDNA samples were diluted 10-fold, and 3 μl of this dilution served as the template in the PCRs that were performed in duplicate for each gene and sample. To correct for differences in the amount of starting material, the ribosomal rpsL gene was chosen as a reference gene. Results are presented as ratios of gene expression between the target gene (target) and the reference gene (rpsL), which were obtained according to the following equation: ratio = (Etarget gene)ΔCttarget(PT5 – test strain)/(ErpsL)ΔCt rpsL(PT5 – test strain) (29Pfaffl M.W. Horgan G.W. Dempfle L. Nucleic Acids Res. 2002; 30: e36Crossref PubMed Google Scholar) where E is the real time PCR efficiency for a given gene and Ct the crossing point of the amplification curve with the threshold. An effect on gene transcription was considered significant when the corresponding ratios were ≥2.0 or ≤0.5. Growth in Latex Urinary Catheters—Filter-sterilized urine (0.45-μm Nalgene 150-ml filters) obtained from healthy volunteers was inoculated with PT5 at a concentration of about 5 × 106 bacteria/ml and introduced into latex urinary catheters (Silkolatex Rüsch Gold, Rüsch, Kamunting, Malaysia). Catheters were incubated for 48 h at 37 °C in 13-cm Petri dishes. Cell densities reached 1.5 × 109 bacteria/ml. Control PT5 bacteria unexposed to latex catheters were grown in urine in test tubes at 37 °C. To assay for the MTC of zinc, PT5 cells grown in urine were centrifuged and resuspended in the same volume of TYG medium before inoculation. Zinc concentrations in urine incubated in latex urinary catheters were measured by atomic absorption spectroscopy (Philips-Pye-Unicam SP9). Urine was diluted 1:400 in Milli-Q water before measurements. Characterization of the CzcCBA Heavy Metal Efflux Pump from PAO1—An efflux pump of the RND transporter family, called CzrCBA, was recently shown to produce tolerance to cadmium and zinc in the P. aeruginosa strain CMG103 isolated from a metal-polluted river in Pakistan (15Hassan M.T. van der Lelie D. Springael D. Romling U. Ahmed N. Mergeay M. Gene (Amst.). 1999; 238: 417-425Crossref PubMed Scopus (126) Google Scholar). A highly similar efflux pump, annotated as CzcCBA, 3See www.pseudomonas.com. is present on the completely sequenced genome of the P. aeruginosa reference strain PAO1 (26Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3446) Google Scholar). Sequence alignment between the czrCBA genes from strain CMG103 (15Hassan M.T. van der Lelie D. Springael D. Romling U. Ahmed N. Mergeay M. Gene (Amst.). 1999; 238: 417-425Crossref PubMed Scopus (126) Google Scholar) and the czcCBA genes in the PAO1 genome (26Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3446) Google Scholar) revealed a >99% amino acid identity between the CzrA and CzcA efflux pump proteins. However, sequence variations were present in the N termini of the outer membrane proteins CzrC (CMG103) and CzcC (PAO1). Furthermore the membrane fusion protein CzrB from strain CMG103 lacks a stretch of 31 amino acids present in CzcB from PAO1. This 31-amino acid stretch is present in the CzcB protein from R. metallidurans and is 70% identical to that of PAO1 (data not shown). To analyze the contribution of the CzcCBA system to heavy metal resistance, we inactivated by homologous recombination the efflux pump gene, annotated as czcA (PA2520) in our PAO1 strain called PT5 (Table I). The resulting mutant, termed PT1173, was indeed more susceptible than its parent to zinc and cadmium as well as to cobalt (Table II). A similar increase in susceptibility to these three heavy metals was observed in strain CMG103-13 containing a transposon insertion in the czrCBA region of strain CMG103 (15Hassan M.T. van der Lelie D. Springael D. Romling U. Ahmed N. Mergeay M. Gene (Amst.). 1999; 238: 417-425Crossref PubMed Scopus (126) Google Scholar). With respect to the hypersusceptibility of the czcA deletion mutant PT1173 to zinc, cadmium, and cobalt, we decided to comply with the current annotation of the PAO1 genome 3See www.pseudomonas.com. and adopted the designation CzcCBA for this heavy metal efflux pump.Table IIHeavy metal and antibiotic susceptibilities of P. aeruginosa wild type PT5 and derivatives and environmental isolate BdB4StrainsSelectionMTCsZones of inhibitionZincCadmiumCobaltCopperNickelIPMCIPmmmmPT5NA1562-3632532PT1173 (ΔczcA)NA521-2NDND2730PT5 + zincaInduced overnight at 37 °C (2 mm zinc or 0.4 mm cadmium).NA30836314bSelective pressure maintained in the Mueller-Hinton agar test plate (0.5 mm zinc, 0.1 mm cadmium).31bSelective pressure maintained in the Mueller-Hinton agar test plate (0.5 mm zinc, 0.1 mm cadmium).PT5 + cadmiumaInduced overnight at 37 °C (2 mm zinc or 0.4 mm cadmium).NA30836313bSelective pressure maintained in the Mueller-Hinton agar test plate (0.5 mm zinc, 0.1 mm cadmium).35bSelective pressure maintained in the Mueller-Hinton agar test plate (0.5 mm zinc, 0.1 mm cadmium).PT1102IPM1562-3631332PT1105IPM2583631434PT1108Zinc2583631434PT364NA1562-3631033BdB4NA156-82422528BdB4-3Zinc30104421032a Induced overnight at 37 °C (2 mm zinc or 0.4 mm cadmium).b Selective pressure maintained in the Mueller-Hinton agar test plate (0.5 mm zinc, 0.1 mm cadmium). Open table in a new tab To further characterize the CzcCBA pump, we tested its inducibility by heavy metals. Without induction, the PT5 wild type strain reached a MTC of 15, 6, and 2–3 mm for zinc, cadmium, and cobalt, respectively (Table II). After preculturing PT5 in the presence of zinc (1 mm) or cadmium (0.4 mm) the MTCs increased to 30, 8, and 3 mm for zinc, cadmium, and cobalt, respectively (Table II). MTCs of copper and nickel were not affected. A killing rate experiment showed that uninduced PT5 cells were killed after 3 h in the presence of 5 mm ZnCl2, while about 50% of cells, preincubated in the presence of 1 mm ZnCl2, stayed viable after 5 h of incubation (Fig. 1). Therefore, the induced cells are strongly protected by the CzcCBA efflux pump against a challenge with lethal heavy metal concentrations. Selection of Mutants Resistant to Either Zinc or Imipenem—We attempted to select stable, heavy metal-resistant mutants from our wild type strain. We therefore exposed PT5 to elevated zinc concentrations (20–25 mm). Sixteen independent clones displayed resistance to zinc, cadmium, and cobalt with MTCs comparable to those of the induced PT5 strain (data not shown). This resistance phenotype was stable even after several passages in the absence of zinc. Since the CzcCBA pump belongs to the RND transporter family that also includes all the Mex multidrug efflux pumps, we tested the susceptibilities of the 16 zinc-selected mutants to various antibiotics (see “Experimental Procedures”). Surprisingly all the tested mutants displayed increased imipenem resistance, while susceptibility to the other antibiotics was unaffected (data not shown). One zinc- and imipenem-resistant mutant, called PT1108, was selected for further experiments (Table II). To investigate a possible link between heavy metal and carbapenem resistance, the reciprocal experiment was performed by exposing strain PT5 to imipenem. Spontaneous mutants appearing after 24 h in the inhibition zone around the imipenem disk were picked, and their resistance profiles were analyzed. Two groups of mutants were identified. Members of the first group were resistant only to imipenem (24 of 30 mutants), while those of the second group were resistant to both heavy metals (zinc, cadmium, and c" @default.
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• W2144783616 date "2004-03-01" @default.
• W2144783616 modified "2023-10-12" @default.
• W2144783616 title "CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa" @default.
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• W2144783616 doi "https://doi.org/10.1074/jbc.m312080200" @default.
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