a Northwestern Prevention Epicenter, Division of Microbiology, Department of Pathology and b Division of Infectious Diseases, Department of Medicine, Northwestern University Medical School and Northwestern Memorial Hospital, Chicago, IL, USA
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Abstract |
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Introduction |
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Thus far, PmrA is the only multidrug transport protein known to be involved in the fluoroquinolone resistance of S. pneumoniae.6 The genomes of other bacterial species encode several distinct transporters with overlapping substrate specificities that are involved in fluoroquinolone resistance.4,5,7 The objective of this study was to detect non-PmrA multidrug transporters in S. pneumoniae by inactivating pmrA and testing the knockout mutants for continued efflux of fluoroquinolones, using an archived strain of S. pneumoniae (CP1000) and a CP1000 mutant with an enhanced active efflux phenotype (EBR).
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Materials and methods |
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The wild-type pneumococcal strain, CP1000, was isolated before the introduction of fluoroquinolones into clinical practice. EBR is a laboratory derivative of CP1000 that overexpresses an active efflux mechanism,8 and probably carries a mutation in a regulatory locus pmrA situated outside the promoter region, since the pmrA promoter sequence in EBR was found to be unchanged (data not shown).
Antimicrobial agent susceptibility testing and growth inhibition studies
MICs of norfloxacin (Merck and Co., Inc., West Point, PA, USA), ciprofloxacin (Bayer Corporation, West Haven, CT, USA), levofloxacin (Ortho-McNeil Pharmaceuticals, Raritan, NJ, USA), moxifloxacin (Bayer Corporation), sparfloxacin (Rhone-Poulenc Rorer R-D, Vitry-sur-Seine, France) and ethidium bromide against CP1000, CP1000-pmr::cat (JJK01), EBR and EBR-pmr::cat (JJK02) were determined in duplicate by serial two-fold antibiotic dilution in ToddHewitt broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% yeast extract (THBY broth).
Growth inhibition studies using reserpine together with the same antimicrobial agents were conducted to detect operation of a reserpine-susceptible efflux system using our previously described method.9 S. pneumoniae cultures were started at a density of 1 x 106 cells/mL in THBY broth containing the respective fluoroquinolones at 0.25 x MIC, with or without the addition of reserpine 10 mg/L. Over a 12 h incubation period at 35°C, the OD550 reached by each culture was determined at various time points, and the extent of growth inhibition by each fluoroquinolone, with or without reserpine, was determined.
Disruption of pmrA in CP1000 and EBR strains
To create a disruption of pmrA, the gene (938 bp), excluding the promoter region, was amplified using primers PMRJ1 5'-TAATCTGCGCATTGCCTG-3' and PMRJ2 5'-AATCAAGGCACCGGTTCC-3', and cloned into pCR2.1 (Invitrogen, Carlsbad, CA, USA). An internal portion of the pmrA gene was replaced by a chloramphenicol resistance cassette (cat, 1047 bp) derived from pEVP3.10 In the resulting 5628 bp plasmid, cat is oriented in the opposite direction to that of pmrA. The recombinant plasmid was linearized and the DNA was used to transform both CP1000 and EBR to chloramphenicol resistance by replacement of bp 422688 of the chromosomal pmrA gene with the cat cassette. Transformants were selected with chloramphenicol 2 mg/L, and the presence of cat in pmrA was verified by PCR after overnight incubation of transformants in THBY broth containing chloramphenicol.
Selection of mutants resistant to ethidium bromide
Ethidium bromide-resistant mutants were obtained by exposing S. pneumoniae JJK01 to ethidium bromide 2 mg/L (2 x MIC). Between 107 and 108 cells from a S. pneumoniae JJK01 culture grown in THBY broth with chloramphenicol (2 mg/L) were plated on to a top layer of THBY agar, overlaid on THBY agar with ethidium bromide (4 mg/L). A total of 1010 cells on multiple plates were screened for mutant selection. Individual clones were taken after incubation for 48 h at 37°C, grown in THBY broth with chloramphenicol 2 mg/L and tested for susceptibility to ethidium bromide and various quinolones. Two of approximately 10 such clones, designated JJK01EB2 and JJK01EB5, were arbitrarily selected for further study.
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Results |
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To test whether the inactivation of PmrA influences the ability of the cell to produce drug-resistant mutants, mutant selection was undertaken by exposing strain JJK01 to ethidium bromide 2 mg/L. Resistant mutants were recovered at a frequency of 3 x 10-8 (28 colonies per 9 x 109 cells), which is similar to the rate of mutant selection observed with readily exported fluoroquinolones such as ciprofloxacin and levofloxacin,9 indicating that the loss of PmrA does not obviously affect the cell's ability to mutate to ethidium bromide resistance.
The MICs of ethidium bromide, norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin and sparfloxacin for the two mutant clones, JJK01EB2 and JJK01EB5, in the absence and presence of reserpine are shown in the Table. The data demonstrate a significant reserpine effect similar to the eight-fold reduction we have consistently observed for ethidium bromide against strain EBR (the diminishing effect of reserpine on ethidium bromide and fluoroquinolone resistance due to the inhibition of active efflux in CP1000 and EBR has been described3,8,9). The elevated levels of resistance to ethidium bromide and selected fluoroquinolones displayed by JJK01EB2 and JJK01EB5, with resistance profiles distinct from that of EBR, provide a strong indication for an additional resistance mechanism(s) in S. pneumoniae. The reductions in ethidium bromide, norfloxacin, levofloxacin and ciprofloxacin MICs effected by reserpine for JJK01EB2 and JJK01EB5 indicate operation of an alternative efflux mechanism(s) to PmrA in S. pneumoniae CP1000.
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Discussion |
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The operation of multiple exporters illustrates the complexity of some microbialantimicrobial interactions, and that a better understanding to optimize drug design and antimicrobial agent use is required. Our work provides additional support for the concept that agents that are not readily effluxed from the cell are, in principle, better chemotherapeutic agents than those that are, since agents that are not effluxed avoid the adverse consequences of export systems. As Tillotson and colleagues suggest,13 it is time to rethink our use of antimicrobial agents, with a clear focus on how best to eradicate invading pathogens that possess numerous pathways for survival.
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Acknowledgements |
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Notes |
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References |
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Received 10 May 2001; returned 22 August 2001; revised 14 November 2001; accepted 30 November 2001