Article Figures & Data
Figures
- 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
- Table 1.
Bacterial strains and plasmids
Strain or plasmid Relevant characteristicsa Source or reference P. aeruginosa ML5087 ilv-220 thr-9001 leu-9001 met-9011 pur-67 aphA 29 K1111 ML5087 nfxB This study K1112 ML5087nalB This study E. coli DH5α supE44 ΔlacU169 (φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 acrAB+ Bethesda Research Laboratories KZM120b ΔacrAB::kan 21 LBB1201 KZM120btolC::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 K113 BL21(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 pKMJ002 pAK1900::nfxB mexCD-oprJ;mexCD-oprJ in orientation opposite to plac 31 pADD214 Mini-D replicon derived from phage D3112; Tcr 3 pPV1 pADD214 derivative carrying a P. aeruginosachromosomal fragment which includesmexAB-oprM 32 pPV20 pAK1900::mexAB-oprM mexAB-oprM in orientation opposite to plac 32 pT7-6 pBR322-based expression vector carrying MCS downstream of the strong gene 10 promoter of phage T7; Apr S. Tabor pRSK01 pT7-6::mexAB This study pPV6 pAK1900::mexAB; mexAB in orientation opposite to plac 32 pRSP01 pAK1900::mexAB-OprM This study pRK415 Low-copy-number, broad-host-range cloning vector carrying MCS downstream of plac; Tcr 13 pRSP06 pRK415::oprJ This study pRSP08 pRK415::oprM This study pRSP15 pRK415::nfxB mexCD-oprJ This study pRSP17 pRK415::mexAB-oprM This study pRSP19 pRK415::mexAB This study pRSP23 pAK1900::mexCD; mexCDin orientation opposite to plac This study pRSP25 pRK415::mexCD This study pRSP45 pAK1900::nfxB mexCD-oprJ This study pRSP46 pAK1900::mexCD This study pSL1180 Superlinker phagemid cloning vector Pharmacia pET21-d(+) pBR322-derived C-terminal His-Tag vector carrying a T7lac promoter upstream of a MCS; lacIApr Novagen pTK15 pET21-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
Strain Plasmid Efflux genes MIC (μg/ml)b,c ERY AZI CAM SDS CV CIP NOR SPAR PEF NOV KZM120 pRK415 —d 16 1 1 32 2 0.008 0.016 0.008 0.032 8 KZM120 pRSP17 mexAB-oprM 64 2 2 >256 8 0.008 0.016 0.016 0.064 32 KZM120 pRSP15 mexCD-oprJ >256 8 2 256 >16 0.016 0.064 0.016 0.064 8 KZM120 pRSP25 mexCD >256 8 2 256 >16 0.016 0.064 0.016 0.064 8 KZM120 pAK1900 —; TolC+ 4 0.5 NDe 128 0.25 ND 0.016 ND ND ND KZM120 pRSP45 mexCD-oprJ; TolC+ 64 4 ND >1,024 >16 ND 0.032 ND ND ND KZM120 pRSP46 mexCD; TolC+ 64 4 ND >1,024 >16 ND 0.032 ND ND ND LBB1201 pAK1900 —; TolC− 4 0.5 ND 16 0.25 ND 0.016 ND ND ND LBB1201 pRSP45 mexCD-oprJ; TolC− 256 16 ND >1,024 >16 ND 0.128 ND ND ND ↵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
Plasmid Efflux genes MIC (μg/ml)b,c AMP PEN TIC CPZ TAX pRK415 —d 16 32 4 0.128 0.064 pRSP17 mexAB-oprM 128 128 32 0.512 0.128 pRSP15 mexCD-oprJ 32 32 4 0.256 0.064 pRSP25 mexCD 32 32 4 0.256 0.064 ↵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.
