1 Public Health Research Institute, 225 Warren St., Newark, NJ 07103, USA; 2 Departments of Clinical Microbiology, St. Pauls Hospital (Grey Nuns) and Saskatoon and District Health; Department of Pathology, Royal University Hospital and the Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Received 11 November 2002; returned 18 January 2003; revised 4 March 2003; accepted 2 April 2003
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Abstract |
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Keywords: erythromycin, moxifloxacin, penicillin, tetracycline, chloramphenicol
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Introduction |
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In the present work, we examined the recovery of resistant mutants of Mycobacterium smegmatis and Staphylococcus aureus from agar plates containing compounds representing five types of antimicrobial agent. In each case, the reduction in mutant recovery due to increasing drug concentration became progressively steeper when large numbers of cells (1091010) were applied to drug-containing agar. This is the result expected as antimicrobial concentration approaches the MIC for the least susceptible single-step mutant, suggesting that MPC can be measured for antimicrobial agents of many types.
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Materials and methods |
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Wild-type M. smegmatis (strain mc2155), obtained from Dr S. Cole (Institute Pasteur, Paris, France), was grown at 37°C in 7H9 liquid medium and on 7H10 agar, both supplemented with 10% albumendextrose complex (ADC).14 S. aureus strain RN450, obtained from Dr B. Kreiswirth (Public Health Research Institute, Newark, NJ, USA), was grown at 37°C in CY broth (1% Casamino acids, 1% yeast extract, 0.1 M NaCl, 0.5% glucose and 0.05 M sodium glycerophosphate) and GL agar (0.3% Casamino acids, 0.3% yeast extract, 0.1 M NaCl, 0.2% sodium lactate, 0.1% glycerol and 1.5% agar, pH 7.8).15 Bacteria were stored at 80°C in growth medium plus 15% glycerol. Chloramphenicol, erythromycin, penicillin and tetracycline were purchased from SigmaAldrich Corp. (St Louis, MO, USA). Moxifloxacin was obtained from Bayer Corp. (West Haven, CT, USA). Stock solutions (10 mg/mL) were prepared by dissolving penicillin in distilled water, moxifloxacin in 0.1 M NaOH, tetracycline in 50% ethanol, chloramphenicol in 95% ethanol, and erythromycin in 100% ethanol.
Measurement of antimicrobial susceptibility
The minimum concentration that inhibited growth of 99% of the input cells [MIC(99)] was measured by applying serial dilutions of stationary phase cultures to agar plates containing various concentrations of antimicrobial agent. Colonies were counted after incubation (12 days for S. aureus; 34 days for M. smegmatis). Preliminary determinations using two-fold dilutions of drug provided an approximate value of MIC(99). This measurement was followed by a second determination, plus a replicate, that utilized linear drug concentration increments (about 20% per sequential increase). The fraction of colonies recovered was plotted against drug concentration to determine MIC(99) by interpolation.
MPC was defined as the concentration that blocked growth when at least 1010 cells were applied to agar plates.11 To measure MPC, cells were grown with vigorous shaking to reach a concentration of about 109 cfu/mL for M. smegmatis and 1010 for S. aureus. Cells were applied to drug-containing agar plates and, at the same time, the cell density of the culture was determined retrospectively by applying serial dilutions to drug-free agar. The maximal bacterial inoculum applied to each agar plate was 300 µL of 1010 cfu/mL for S. aureus and 1 mL at 1 to 5 x 109 cfu/mL for M. smegmatis. Multiple plates at a given drug concentration were used so that the total number of cells tested exceeded 1010. Agar plates were incubated at 37°C for various times depending on the species: 4 days with colony numbers recorded at 1-day intervals for S. aureus and 10 days with colony numbers recorded at 2-day intervals for M. smegmatis. Colonies were confirmed to contain mutant cells by regrowth on agar containing the selecting concentration of antimicrobial (control experiments in which mutant colonies were grown on drug-free agar before retesting on drug-containing agar showed that the mutants were stable).
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Results |
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It has been argued that resistant mutants are selectively enriched when antimicrobial concentrations fall between the minimal concentration that inhibits the growth of 99% of the cells [MIC(99)] and MPC, a range called the mutant selection window.10,13,16 [MIC(99) approximates the minimal concentration better than MIC because less selective pressure is present; however, for many antimicrobialpathogen combinations, little absolute difference is likely to exist between MIC(99) and MIC due to the steep dependence of colony recovery on drug concentration.] Values of MIC(99) and MPC were therefore calculated and are listed in Table 1. When the size of the selection window was expressed as the ratio of MPC to MIC(99), it varied considerably among bacterialantimicrobial combinations. For both bacterial species, the selection window was widest for erythromycin.
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Discussion |
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The second category of drug concentration dependence is illustrated by the combinations studied in the present work, by prior studies with fluoroquinolones,12,18,19 and by treatment of Candida albicans and C. glabrata with miconazole (J.-Y. Wang et al., unpublished observations). Increasing drug concentration causes colony recovery to drop sharply at the MIC, pass through an inflection point, and then drop sharply a second time. The second drop occurs at the MPC. Fluoroquinolone studies show that resistant mutants are selectively enriched at drug concentrations between MIC(99) (or MIC) and MPC.16,18 Whether monotherapy is appropriate for situations in this category depends on how long relevant tissue drug concentrations can be kept above MPC at each dosing interval. To address this issue, it is now necessary to measure MPC in vivo at the site of infection.
Confusion sometimes surrounds phenotypic or induced resistance. An example is the ß-lactam resistance that arises from the induction of ß-lactamases. When mutations are not responsible for this type of resistance, it is outside the scope of the present discussion. However, when ß-lactamases are expressed from plasmid-borne genes, they behave as category-one resistance and require combination therapy even if the plasmid-containing cells are members of rare subpopulations. Thus the dosing strategies that derive from consideration of the mutant selection window hypothesis may be broadly applicable.
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Acknowledgements |
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Footnotes |
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References |
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