Contribution of the MexAB-OprM multidrug efflux system to the ß-lactam resistance of penicillin-binding protein and ß-lactamase-derepressed mutants of Pseudomonas aeruginosa

Ramakrishnan Srikumar, Eric Tsang and Keith Poole*

Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Deletion of the mexAB-oprM multidrug efflux operon substantially compromised the ß-lactam resistance of ß-lactamase-derepressed mutants of Pseudomonas aeruginosa, although it had only a modest impact on resistance of a penicillin-binding protein mutant. This highlights the multifactorial nature of ß-lactam resistance in this organism. Moreover, the contribution of efflux to the net resistance seen in some ß-lactam-resistant mutants suggests that inhibition of MexAB-OprM-mediated drug efflux might be an effective approach to overcoming ß-lactam resistance attributed to efflux as well as to other mechanisms of ß-lactam resistance.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Pseudomonas aeruginosa is an opportunistic human pathogen which is intrinsically resistant to many antimicrobial agents, a property which results from the synergy between broadly specific drug efflux systems and low outer membrane permeability.1 One such efflux system, encoded by the mexAB-oprM operon,2 effluxes a range of antibiotics, including quinolones, ß-lactams, tetracycline, chloramphenicol, novobiocin and macrolides.3,4 Although expressed constitutively in wild-type cells, where it contributes to intrinsic drug resistance, the operon is also hyperexpressed in nalB5 mutants producing elevated levels of resistance to substrate antibiotics.3,4

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains and plasmids

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 Luria–Bertani (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


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
MexAB-OprM is expressed in wild-type P. aeruginosa growing under standard laboratory conditions, such that deletion of the efflux genes renders the organism highly susceptible to multiple antibiotics.3 It was anticipated, therefore, that antibiotic resistance that develops in the organism independent of MexAB-OprM-mediated efflux will still have efflux as a contributing factor. To assess the contribution that MexAB-OprM makes to net resistance of ß-lactam-resistant strains carrying PBP mutations, or hyperexpressing ß-lactamase as a result of stable derepression of the chromosomal enzyme or the presence of a ß-lactamase-encoding plasmid, the efflux genes were deleted from such mutants and the impact on ß-lactam resistance examined.

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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Table I. Influence of MexAB-OprM on the antibiotic resistance of a chromosomal ß-lactamase-derepressed mutant strain of P. aeruginosa
 
Introduction of the pBR322 plasmid-derived TEM-1 enzyme into the non-derepressible ß-lactamase mutant PAO40989 also had a marked impact >=16-fold increase in MIC) on resistance to several penicillin antibiotics (the preferred substrates of TEM-1) including carbenicillin, piperacillin and ticarcillin (data not shown). Again, however, resistance was little, if at all, affected by the deletion of mexAB-oprM (in K1232) (data not shown). Once again, it appears that for ß-lactams which are readily hydrolysed by ß-lactamases, the efflux status of the cells has less of an impact on net resistance, presumably because any increase in antibiotic accumulation in the efflux mutants is accommodated adequately by the enzyme.

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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Table II. Influence of MexAB-OprM on the antibiotic resistance of a penicillin-binding protein mutant strain of P. aeruginosa
 
In general, where enzyme-mediated resistance was high >=16-fold increase in MIC), the efflux status had little or no impact on ß-lactam susceptibility. Where enzyme-mediated increase in resistance was modest <=16-fold) the absence of the MexAB-OprM efflux system had a substantial impact on susceptibility and could abrogate the increased resistance afforded by the enzyme. Thus, for certain agents, efflux can operate somewhat co-operatively with other resistance determinants, playing a role in both intrinsic and acquired antibiotic resistance. Indeed, we recently observed that quinolone resistance provided by gyrase mutations could also be largely eliminated by deletion of mexAB-oprM (K. Poole, unpublished data). Thus, resistance generally attributed to gyrase mutations really represents co-operation between efflux and the gyrase alteration.

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.


    Acknowledgments
 
The authors thank Xian-Zhi Li and Nicole Barré for construction of P. aeruginosa strain K1232. The authors gratefully acknowledge the financial support of the Canadian Cystic Fibrosis Foundation (CCFF). R. S. is a Medical Research Council of Canada and CCFF Postdoctoral Fellow. E. T. was supported by a CCFF summer studentship. K. P. is a Natural Sciences and Engineering Research Council University Research Fellow.


    Notes
 
* Corresponding author. Tel: +1-613-533-6677; Fax: +1-613-533-6796; E-mail: poolek{at}post.queensu.ca Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Ma, D., Cook, D. N., Hearst, J. E. & Nikaido, H. (1994). Efflux pumps and drug resistance in Gram-negative bacteria. Trends in Microbiology 2, 489–93.[Medline]

2 . Poole, K., Heinrichs, D. E. & Neshat, S. (1993). Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine. Molecular Microbiology 10, 529–44.[ISI][Medline]

3 . Li, X.-Z., Nikaido, H. & Poole, K. (1995). Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1948–53.[Abstract]

4 . Poole, K., Krebes, K., McNally, C. & Neshat, S. (1993). Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. Journal of Bacteriology 175, 7363–72.[Abstract]

5 . Poole, K., Tetro, K., Zhao, Q., Neshat, S., Heinrichs, D. & Bianco, N. (1996). Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrobial Agents and Chemotherapy 40, 2021–8.[Abstract]

6 . Godfrey, A. J., Bryan, L. E. & Rabin, H. R. (1981). ß-Lactam-resistant Pseudomonas aeruginosa with modified penicillin-binding proteins emerging during cystic fibrosis treatment. Antimicrobial Agents and Chemotherapy 19, 705–11.[ISI][Medline]

7 . Giwercman, B., Meyer, C., Lambert, P. A., Reinert, C. & Hoiby, N. (1992). High-level ß-lactamase activity in sputum samples from cystic fibrosis patients during antipseudomonal treatment. Antimicrobial Agents and Chemotherapy 36, 71–6.[Abstract]

8 . Livermore, D. M. (1991). ß-Lactamases of Pseudomonas aeruginosa. Antibiotics and Chemotherapy 44, 215–20.[Medline]

9 . Li, X.-Z., Livermore, D. M. & Nikaido, H. (1994). Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin. Antimicrobial Agents and Chemotherapy 38, 1732–41.[Abstract]

10 . 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, 5443–7.[Abstract/Free Full Text]

Received 3 February 1999; returned 26 March 1999; revised 21 April 1999; accepted 19 May 1999