Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada
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Abstract |
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Introduction |
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The apparent export of ß-lactams by MexAB-OprM is a unique feature of this efflux system which distinguishes it from the other multidrug efflux systems of P. aeruginosa. As such, inhibition of MexAB-OprM is likely to be a useful approach to enhance susceptibility of P. aeruginosa to ß-lactams. However, as resistance to ß-lactams arises in P. aeruginosa via means other than amplification of MexAB-OprM (e.g. mutations in penicillin-binding proteins (PBPs),6 derepression of chromosomally-encoded ß-lactamase,7 acquisition of ß-lactamase-encoding plasmids8), efflux pump inhibition may not adequately address the problem of ß-lactam resistance in this organism. We therefore examined the influence loss of MexAB-OprM had on ß-lactam resistance in strains displaying other mechanisms of ß-lactam resistance.
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Materials and methods |
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H736, a chromosomal ß-lactamase-derepressed mutant, and its parent H103, a PAO1 prototroph, were provided by R. E. W. Hancock (University of British Columbia, Canada). PAO4098,9 a non-derepressible ß-lactamase mutant, and its parent K767,10 a PAO1 prototroph, have been described previously. P2284,6 a penicillin-binding protein mutant of P. aeruginosa, and its parent P2076,6 a clinical isolate, have also been described previously. Both P. aeruginosa and Escherichia colistrains were routinely grown in LuriaBertani (LB) medium (1% (w/v) yeast extract, 0.05% (w/v) NaCl) at 37°C. Plasmid pQF202 (A. M. Kropinski, unpublished data) is a pBR322 derivative carrying a previously described broad-host range oriVwhich permitted its introduction into competent P. aeruginosa via transformation.2 Antibiotics (kanamycin (50 mg/L), HgCl2 (15 mg/L) and tetracycline (100 mg/L for PAO4098 carrying pQF202; 10 mg/L for K1232 carrying pQF202)) were included in the growth media as required. Construction of the mexAB-oprM deletion strains K1178, K1232 and K1177 from H736, PAO4098 and P2284, respectively, was carried out using pELCT0410 according to a previously described protocol.10 The presence of the deletion in these strains was confirmed by PCR using Taq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD, USA) and primers ABM-1 (5'-CAGCAGCTCTACCAGATCGAC-3'), which anneals 284 bp downstream of the mexA initiation codon, and ABM-2 (5'-GTGTCCTTGGTCAGCTGCAAC-3'), which anneals 844 bp upstream of the oprM stop codon. Loss of OprM in these strains was also confirmed by immunoblotting of isolated cell envelopes with an OprM-specific antiserum as described previously.10
PCR
Reaction mixtures (100 µmL), including 2.5 U Taq DNA polymerase, 0.3 µmM each primer, 0.2 mM each dNTP, 2 mM MgCl2, 10% (v/v) DMSO, 10 ng genomic DNA and 1 x 3 PCR buffer (Life Technologies) were heated for 1 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 56°C and 5 min at 72°C, before finishing with 10 min at 72°C.
Minimum inhibitory concentration
The susceptibility of P. aeruginosa strains to a number of antimicrobial agents (reported as MICs) was determined using the serial broth dilution protocol as described previously.3
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Results and discussion |
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All P. aeruginosa mexAB-oprMdeletion strains were shown to be more suceptible than their parents to several non-ß-lactam antimicrobial agents, as described previously.3,4 P. aeruginosa H736 is a chromosomal ß-lactamase-derepressed mutant of PAO1 strain H103 and, as expected, was markedly less susceptible to a variety of ß-lactams compared with its parent (Table I). Elimination of the MexAB-OprM efflux system via deletion (see strain K1178), however, increased the ß-lactam susceptibility of this strain such that the resultant deletion derivative was, for several antibiotics (cefepime, cefpirome, ceftazidime and cefoperazone), as susceptible as the original PAO1 strain. Thus, loss of the multidrug efflux system completely abrogated the effect of ß-lactamase derepression on susceptibility to these ß-lactams. It is interesting to note, however, that ß-lactamase derepression only afforded a modest two- to eight-fold increase in MICs of these antibiotics in the first place. In contrast, derepression of the chromosomal enzyme in H736 had a major impact (16- to 32-fold increase in MIC) on resistance to cefotaxime and ceftriaxone, and loss of MexAB-OprM (in K1178) had only a minor impact on susceptibility to these two antibiotics. K1178 remained eight- to 16-fold more resistant to cefotaxime and ceftriaxone than the wild-type strain. These latter ß-lactams are apparently good substrates for the chromosomal enzyme and are rapidly hydrolysed. As such, the increased ß-lactam accumulation expected to result from loss of MexAB-OprM in K1178 is, for the most part, readily accommodated by the derepressed enzyme as far as cefotaxime and ceftriaxone are concerned though apparently not for the other ß-lactams described above.
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P. aeruginosa P2284 is a PBP mutant derivative of a clinical isolate P2076 that showed reduced affinity of all of its PBPs for penicillin G.6 As expected, this strain was noticeably less susceptible to a number of ß-lactams (Table II). Elimination of mexAB-oprM in P2284 had, however, only a modest impact on ß-lactam resistance, reducing the MIC values only two- to four-fold in all cases and still two- to 32-fold higher than for the original parent strain P2076. Thus, the efflux system contributes only weakly to ß-lactam resistance attributable to PBP mutations, probably because the increased antibiotic accumulation expected in the deletion derivative is still insufficient to overcome the reduced affinity of the PBP for the ß-lactams.
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Finally, the results presented here suggest that for certain ß-lactams, pump inhibitors might be of therapeutic value in compromising both efflux-mediated ß-lactam resistance and resistance due to derepression of chromosomal ß-lactamase. The latter is of some significance, in light of several reports highlighting the appearance of ß-lactamase-derepressed mutants of P. aeruginosain cystic fibrosis patients undergoing ß-lactam therapy.7 Moreover, as evidence appears that resistance is multifactorial and that efflux plays a contributing role, the potential of such inhibitors is likely to increase.
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Acknowledgments |
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Notes |
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References |
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10
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Evans, K., Passador, L., Srikumar, R., Tsang, E.,
Nezezon, J. & Poole, K. (1998). Influence of the MexAB-OprM multidrug efflux
system on quorum sensing in Pseudomonas aeruginosa. Journal of
Bacteriology 180, 54437.
Received 3 February 1999; returned 26 March 1999; revised 21 April 1999; accepted 19 May 1999