Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6
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Abstract |
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Introduction |
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Materials and methods |
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P. aeruginosa PAO1 was used as the wild-type strain.7 Strain OCR1, the MexAB-OprM-overproducing mutant and strain K1119, the MexAB-OprM-deficient mutant, have been described previously.7 LuriaBertani (LB) broth was the growth medium used throughout and bacteria were cultivated at 37°C.
Antibiotics and other agents
Carbenicillin, cefoperazone, ciprofloxacin, norfloxacin, erythromycin, tetracycline, chloramphenicol and novobiocin were purchased from SigmaAldrich Canada (Oakville, Ontario, Canada). Imipenem was from Merck Sharp Dohme Canada (Montreal, Canada). Nitrocefin was purchased from BectonDickinson & Company (Cockeysville, MD, USA). Disodium ethylenediaminetetraacetate (EDTA) and sodium hexametaphosphate (NaHMP) were obtained from BDH Inc. (Toronto, Canada) and Anachemia Science (Montreal, Canada), respectively.
Drug susceptibility testing
Susceptibility testing was carried out using the two-fold serial broth dilution method with an inoculum of 5 x 105 cells/mL. Data were reported as MICs, which reflected the lowest concentration of antibiotic inhibiting visible growth after 18 h incubation at 37°C. In some experiments OM permeabilizers (EDTA and NaHMP) were included to ascertain their effect on antibiotic MICs.
Permeabilization of the OM
The ability of two permeabilizers, EDTA and NaHMP, to permeabilize the OM of P. aeruginosa was assessed by examining the release of the chromosomally encoded ß-lactamase from P. aeruginosa PAO1 cells. Stationary-phase cells were diluted 1:59 into 30 mL of pre-warmed (37°C) LB broth and incubated with shaking for 2 h at 37°C. Following addition of imipenem (0.25 mg/L; to induce the ß-lactamase) the cultures were incubated with shaking for an additional 3 h and harvested by centrifugation at 5000g for 10 min at room temperature. Cell pellets were washed once with 50 mM sodium phosphate buffer (pH 7.2) and resuspended in a final volume of 10 mL of the same buffer. The permeabilizers were added to the cell suspensions at various concentrations as specified in the Results below. Aliquots (1.5 mL) were then withdrawn at various time points and centrifuged in an Eppendorf centrifuge at 15000g for 30 min at room temperature. The supernatants were saved and ß-lactamase activity was assayed as described previously using nitrocefin (100 µM) as the substrate.7
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Results and discussion |
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To assess the influence of OM permeabilization on the antibiotic susceptibility of P. aeruginosa strains of different MexAB-OprM status, it was necessary to ascertain the effective concentrations of known OM permeabilizers such as EDTA and NaHMP.8 All strains of P. aeruginosa tested (PAO1 wild-type, OCR1 and K1119) showed the same susceptibility to these permeabilizers [MIC values were EDTA (12.5 mM) and NaHMP (250 mM)]. Since ß-lactamases are located in the periplasm of Gram-negative bacteria, permeabilization of the OM can be assessed by measuring the release of ß-lactamases into cell-free supernatants. We treated cells of P. aeruginosa PAO1 with subinhibitory concentrations of EDTA or NaHMP following induction of ß-lactamase with a subinhibitory concentration of imipenem, and the release of ß-lactamase activity was assayed. As shown in the Figure, both EDTA and NaHMP increased the release of ß-lactamase from strain PAO1 at concentrations that were much lower than their MIC values, with maximal ß-lactamase release occurring at 2.5 mM EDTA or 10 mM NaHMP (Figure
). Treatment of cells with permeabilizers for various times (560 min) revealed that release of ß-lactamase occurred within 15 min (data not shown).
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Using isogenic mutants, the effects of OM permeabilizers and efflux pump status (MexAB-OprM pump overproduced or deficient) on antibiotic susceptibility were determined as described above. As shown in the Table, inactivation of the multidrug resistance (MDR) efflux pump (in strain K1119) or permeabilization of the OM strongly increased susceptibility to a variety of antibiotics. For example, pump inactivation decreased MIC values of carbenicillin 64-fold, from 512 mg/L for the pump-overproducing mutant (strain OCR1) to 8 mg/L for the pump-deficient mutant (K1119), while permeabilization of the OM with 1 mM (i.e. 1/12.5 MIC) EDTA produced a comparable decrease in the MICs of carbenicillin (16-fold) for strains producing high (OCR1) and moderate (PAO1) levels of MexAB-OprM. The combination of pump inactivation (K1119) and OM permeabilization, however, resulted in a c. 500-fold (with EDTA) to 5000-fold (with NaHMP) decrease in MIC values of carbenicillin. Similar trends were seen for all of the antibiotics tested (Table
). The permeabilization of the pump-deficient mutant K1119 rendered this strain extremely susceptible to erythromycin, an agent that is primarily used against Gram-positive bacteria. The MIC of erythromycin for the 1 mM EDTA-treated K1119 was, for example, 8 mg/L (a decrease from 512 mg/L for PAO1; Table
) and the reported MICs (MIC90) of erythromycin for Staphylococcus aureus and Streptococcus pneumoniae were 64 and 2 mg/L, respectively.9 Despite permeabilization, however, the MexAB-OprM-overproducing strain was more resistant to antibiotics than was wild-type PAO1, which was more resistant than the pump-deficient strain. Thus, MDR pumps were functional and still somewhat effective upon OM disruption or, more likely, the OM barrier remained somewhat intact despite EDTA and NaHMP treatment. Nevertheless, membrane disruption significantly increased the antibiotic susceptibility of the pump-overproducing strain, indicating that acquired MDR in P. aeruginosa can be reversed by OM disruption. Interestingly, pump inactivation and OM permeabilization appeared to affect antibiotic susceptibility selectively. For instance, MexAB-OprM inactivation did not substantially alter susceptibility of P. aeruginosa to the two fluoroquinolones tested (Table
; compare PAO1 and K1119). This may be due to the fact that P. aeruginosa possesses multiple efflux pumps capable of accommodating these compounds.2,5 On the other hand, OM permeabilization generally rendered the strains more susceptible to all antibiotics, and this effect increased with increasing permeabilizer concentration (Table
). Nevertheless, susceptibility to lipophilic compounds such as erythromycin seemed to be more affected by the permeabilizers than was susceptibility to hydrophilic compounds such as ciprofloxacin.
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Comments |
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Acknowledgments |
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Notes |
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References |
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2
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Nikaido, H. (1996). Multidrug efflux pumps in Gram-negative bacteria. Journal of Bacteriology 178, 58539.
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Mine, T, Morita, Y., Kataoka, A., Mizushima, T. & Tsuchiya, T. (1999). Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 43, 4157.
6 . Nikaido, H. (1998). The role of outer membrane and efflux pumps in the resistance of Gram-negative bacteria. Can we improve drug access? Drug Resistance Updates 1, 938.[ISI]
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Li, X.-Z., Zhang, L., Srikumar, R. & Poole, K. (1998). ß-Lactamase inhibitors are substrates of the multidrug efflux pumps of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 399403.
8 . Vaara, M. (1992). Agents that increase the permeability of the outer membrane. Microbiological Review 56, 395411.[ISI]
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Sefton, A. M., Maskell, J. P., Rafay, A. M., Whiley, A. & Williams, J. D. (1997). The in-vitro activity of trovafloxacin, a new fluoroquinolone, against Gram-positive bacteria. Journal of Antimicrobial Chemotherapy 39, Suppl. B, 5762.
Received 14 July 1999; returned 1 October 1999; revised 15 October 1999; accepted 1 November 1999