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Mechanisms of Resistance

Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

Ramakrishnan Srikumar, Tatiana Kon, Naomasa Gotoh, Keith Poole
Ramakrishnan Srikumar
Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario K7L 3N6, Canada, and
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Tatiana Kon
Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario K7L 3N6, Canada, and
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Naomasa Gotoh
Department of Microbiology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan
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Keith Poole
Department of Microbiology and Immunology, Queen’s University, Kingston, Ontario K7L 3N6, Canada, and
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DOI: 10.1128/AAC.42.1.65
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    Fig. 1.

    Western immunoblots of bacterial cell envelopes (10 μg of protein) developed with antibodies raised against MexB (A), OprM (B), and OprJ (C). Lanes 1, K1111 (nfxB); lanes 2, K1112 (nalB); lanes 3, KZM120 carrying pRK415; lanes 4, KZM120 carrying pRSP06 (oprJ); lanes 5, KZM120 carrying pRSP15 (nfxB mexCD-oprJ); lanes 6, KZM120 carrying pRSP25 (mexCD); lanes 7, KZM120 carrying pRSP08 (oprM); lanes 8, KZM120 carrying pRSP17 (mexAB-oprM); lanes 9, KZM120 carrying pRSP19 (mexAB). (The relevant efflux phenotype or genes expressed are indicated in parentheses.)

Tables

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  • Table 1.

    Bacterial strains and plasmids

    Strain or plasmidRelevant characteristicsaSource or reference
    P. aeruginosa
     ML5087 ilv-220 thr-9001 leu-9001 met-9011 pur-67 aphA 29
     K1111ML5087 nfxB This study
     K1112ML5087nalB This study
    E. coli
     DH5α supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 acrAB+ Bethesda Research Laboratories
     KZM120b ΔacrAB::kan 21
     LBB1201KZM120btolC::Tn10 5
     BL21(DE3)F− ompT r B − m B −; DE3 is a lambda derivative carrying lacI and T7 RNA polymerase genes under placUV5 control 40
     K113BL21(DE3) carrying the phage T7 lysozyme vector pLysS; Cmr 39
    Plasmids
     pAK1900 E. coli-P. aeruginosa shuttle cloning vector carrying plac upstream of MCS; AprCbr R. Sharp, Queen’s University
     pKMJ002pAK1900::nfxB mexCD-oprJ;mexCD-oprJ in orientation opposite to plac 31
     pADD214Mini-D replicon derived from phage D3112; Tcr 3
     pPV1pADD214 derivative carrying a P. aeruginosachromosomal fragment which includesmexAB-oprM 32
     pPV20pAK1900::mexAB-oprM mexAB-oprM in orientation opposite to plac 32
     pT7-6pBR322-based expression vector carrying MCS downstream of the strong gene 10 promoter of phage T7; Apr S. Tabor
     pRSK01pT7-6::mexAB This study
     pPV6pAK1900::mexAB; mexAB in orientation opposite to plac 32
     pRSP01pAK1900::mexAB-OprM This study
     pRK415Low-copy-number, broad-host-range cloning vector carrying MCS downstream of plac; Tcr 13
     pRSP06pRK415::oprJ This study
     pRSP08pRK415::oprM This study
     pRSP15pRK415::nfxB mexCD-oprJ This study
     pRSP17pRK415::mexAB-oprM This study
     pRSP19pRK415::mexAB This study
     pRSP23pAK1900::mexCD; mexCDin orientation opposite to plac This study
     pRSP25pRK415::mexCD This study
     pRSP45pAK1900::nfxB mexCD-oprJ This study
     pRSP46pAK1900::mexCD This study
     pSL1180Superlinker phagemid cloning vectorPharmacia
     pET21-d(+)pBR322-derived C-terminal His-Tag vector carrying a T7lac promoter upstream of a MCS; lacIApr Novagen
     pTK15pET21-d(+)::mexB This study
    • ↵a MCS, multiple cloning site; Apr, ampicillin resistant; Cbr, carbenicillin resistant; Tcr, tetracycline resistant; T7lac, phage T7 promoter followed by a lac operator. Unless otherwise indicated, all genes were cloned in the same orientation as plac of pAK1900 or pRK415.

    • ↵b KZM120 is the same strain as HN818.

  • Table 2.

    Antimicrobial susceptibility of E. coliexpressing components of the P. aeruginosa multidrug efflux pumpsa

    StrainPlasmidEfflux genesMIC (μg/ml)b,c
    ERYAZICAMSDSCVCIPNORSPARPEFNOV
    KZM120pRK415—d 16113220.0080.0160.0080.0328
    KZM120pRSP17 mexAB-oprM 6422>25680.0080.0160.0160.06432
    KZM120pRSP15 mexCD-oprJ >25682256>160.0160.0640.0160.0648
    KZM120pRSP25 mexCD >25682256>160.0160.0640.0160.0648
    KZM120pAK1900—; TolC+ 40.5NDe 1280.25ND0.016NDNDND
    KZM120pRSP45 mexCD-oprJ; TolC+ 644ND >1,024>16ND0.032NDNDND
    KZM120pRSP46 mexCD; TolC+ 644ND >1,024>16ND0.032NDNDND
    LBB1201pAK1900—; TolC− 40.5ND 160.25ND0.016NDNDND
    LBB1201pRSP45 mexCD-oprJ; TolC− 25616ND >1,024>16ND0.128NDNDND
    • ↵a The susceptibility of E. coli KZM120 (ΔacrAB::kan) and LBB1201 (KZM120 tolC::Tn10) carrying P. aeruginosa multidrug efflux genes on the indicated plasmids to various antimicrobial agents was assayed as described in Materials and Methods.

    • ↵b ERY, erythromycin; AZI, azithromycin; CAM, chloramphenicol; SDS, sodium dodecyl sulfate; CV, crystal violet; CIP, ciprofloxacin; NOR, norfloxacin; SPAR, sparfloxacin; PEF, pefloxacin; NOV, novobiocin.

    • ↵c E. coli KZM120 carrying plasmids pRSP08 (oprM), pRSP19 (mexAB), or pRSP06 (oprJ) showed the same pattern of resistance as exhibited by KZM120 carrying the pRK415 vector control. E. coli LBB1201 carrying pRSP46 (mexCD) showed the same pattern of resistance as LBB1201 carrying the pAK1900 vector control.

    • ↵d —, no plasmid-borne efflux genes.

    • ↵e ND, not determined.

  • Table 3.

    β-lactam susceptibility of E. coli strains expressing components of the P. aeruginosa multidrug efflux pumpsa

    PlasmidEfflux genesMIC (μg/ml)b,c
    AMPPENTICCPZTAX
    pRK415—d 163240.1280.064
    pRSP17 mexAB-oprM 128128320.5120.128
    pRSP15 mexCD-oprJ 323240.2560.064
    pRSP25 mexCD 323240.2560.064
    • ↵a The susceptibility of E. coli KZM120 (ΔacrAB::kan) carryingP. aeruginosa multidrug efflux genes on the indicated plasmids to various antimicrobial agents was assayed as described in Materials and Methods.

    • ↵b AMP, ampicillin; PEN, penicillin G; TIC, ticarcillin; CPZ, cefoperazone; TAX, cefotaxime.

    • ↵c E. coli KZM120 carrying pRSP08 (oprM), pRSP19 (mexAB) and pRSP06 (oprJ) showed the same resistance pattern as that exhibited by KZM120 carrying pRK415. β-lactams for which no change in susceptibility was observed for KZM120 with or without plasmids containing efflux genes included cephaloridine, cefsulodin, cefepime, cefpirome, imipenem, ceftriaxone, and ceftazidime.

    • ↵d —, no plasmid-borne efflux genes.

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Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain
Ramakrishnan Srikumar, Tatiana Kon, Naomasa Gotoh, Keith Poole
Antimicrobial Agents and Chemotherapy Jan 1998, 42 (1) 65-71; DOI: 10.1128/AAC.42.1.65

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Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain
Ramakrishnan Srikumar, Tatiana Kon, Naomasa Gotoh, Keith Poole
Antimicrobial Agents and Chemotherapy Jan 1998, 42 (1) 65-71; DOI: 10.1128/AAC.42.1.65
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KEYWORDS

Bacterial Proteins
Drug Resistance, Multiple
Escherichia coli
Pseudomonas aeruginosa

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