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Dkg268.fm

Journal of Antimicrobial Chemotherapy (2003) 52, 61–64
DOI: 10.1093/jac/dkg268
Advance Access publication 12 June 2003
Effect of chloramphenicol, erythromycin, moxifloxacin, penicillin and
tetracycline concentration on the recovery of resistant mutants of
Mycobacterium smegmatis and Staphylococcus aureus
Tao Lu1, Xilin Zhao1, Xinying Li1, Glen Hansen2, Joseph Blondeau2 and Karl Drlica1*
1Public Health Research Institute, 225 Warren St., Newark, NJ 07103, USA; 2Departments of Clinical Microbiology, St. Paul’s 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 The effect of antimicrobial concentration on colony-forming ability of resistant mutant subpopulations of
Mycobacterium smegmatis and Staphylococcus aureus was measured for chloramphenicol, erythromy-
cin, moxifloxacin, penicillin and tetracycline. The relationship between drug concentration and the recov-
ery of mutant colonies was distinct for each bacterium–antimicrobial combination; however, in each case
application of large numbers of cells to drug-containing agar plates revealed a progressive reduction in
mutant recovery as antimicrobial concentration increased. The minimal concentration that allowed no
mutant recovery from more than 1010 input cells was measured to estimate the minimum inhibitory concen-
tration (MIC) of the least susceptible, single-step mutant subpopulation, a parameter also called the mutant
prevention concentration (MPC). These data expand the number of antimicrobial–bacterial combinations
for which a mutant selection window can be measured.

Keywords: erythromycin, moxifloxacin, penicillin, tetracycline, chloramphenicol Introduction
always available for testing and since mutant susceptibility mightdiffer between pure cultures and small subpopulations, we have sug- Surveillance studies with a variety of pathogens show that antimicro- gested that MPC can be estimated as the concentration that allows bial resistance can develop rapidly.1–8 If this trend is allowed to recovery of no mutant when a large, susceptible population (1010 cells) continue without the introduction of new classes of agent, manymicrobial diseases are likely to become refractory to antimicrobial is applied to drug-containing agar plates.12 This microbiological treatment. It may be possible to slow the development of de novo, threshold can be readily measured in vitro for fluoroquinolones;12,13 step-wise resistance through more aggressive therapies that directly however, it is not clear how the concept applies to agents of other block the growth of the resistant mutant fraction of susceptible popu- classes. For example, single-step mutations could lower suscep- lations. In theory, antimicrobial therapy could be optimized such that tibility so much that MPC could not be measured, as is the case with failure due to the emergence of resistance during treatment occurs only rarely.9 Empirical determination of such a dose is likely to In the present work, we examined the recovery of resistant require large numbers of patients to ensure that a particular regimen mutants of Mycobacterium smegmatis and Staphylococcus aureus does not contribute to the rising prevalence of resistance or an from agar plates containing compounds representing five types of increase in the frequency of other adverse events. As an alternative, antimicrobial agent. In each case, the reduction in mutant recovery we have proposed a conceptual strategy based on in vitro data.10,11 In due to increasing drug concentration became progressively steeper principle, antimicrobial concentrations that require a cell to attaintwo concurrent resistance mutations for growth will rarely allow when large numbers of cells (109–1010) were applied to drug-contain- selective enrichment of mutant subpopulations. That concentration is ing agar. This is the result expected as antimicrobial concentration the minimum inhibitory concentration (MIC) of the least susceptible, approaches the MIC for the least susceptible single-step mutant, single-step mutant, which is termed the mutant prevention concentra- suggesting that MPC can be measured for antimicrobial agents of tion (MPC). Since the least susceptible, single-step mutant is not . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*Corresponding author. Tel: +1-973-854-3360; Fax: +1-973-854-3101; E-mail: drlica@phri.org . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2003 The British Society for Antimicrobial Chemotherapy T. Lu et al.
Materials and methods
Bacterial strains, growth conditions and antimicrobial agents Wild-type M. smegmatis (strain mc2155), obtained from Dr S. Cole(Institute Pasteur, Paris, France), was grown at 37°C in 7H9 liquidmedium and on 7H10 agar, both supplemented with 10% albumen–dextrose complex (ADC).14 S. aureus strain RN450, obtained from DrB. Kreiswirth (Public Health Research Institute, Newark, NJ, USA), wasgrown at 37°C in CY broth (1% Casamino acids, 1% yeast extract, 0.1 MNaCl, 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% sodiumlactate, 0.1% glycerol and 1.5% agar, pH 7.8).15 Bacteria were stored at–80°C in growth medium plus 15% glycerol. Chloramphenicol, erythro-mycin, penicillin and tetracycline were purchased from Sigma–AldrichCorp. (St Louis, MO, USA). Moxifloxacin was obtained from BayerCorp. (West Haven, CT, USA). Stock solutions (10 mg/mL) were pre-pared by dissolving penicillin in distilled water, moxifloxacin in 0.1 MNaOH, 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 inputcells [MIC ] was measured by applying serial dilutions of stationary phase cultures to agar plates containing various concentrations of anti-microbial agent. Colonies were counted after incubation (1–2 days forS. aureus; 3–4 days for M. smegmatis). Preliminary determinations usingtwo-fold dilutions of drug provided an approximate value of MIC This measurement was followed by a second determination, plus a rep-licate, that utilized linear drug concentration increments (about 20% persequential increase). The fraction of colonies recovered was plottedagainst 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 weregrown 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 culturewas determined retrospectively by applying serial dilutions to drug-freeagar. The maximal bacterial inoculum applied to each agar plate was300 µL of 1010 cfu/mL for S. aureus and 1 mL at 1 to 5 × 109 cfu/mL forM. smegmatis. Multiple plates at a given drug concentration were used sothat the total number of cells tested exceeded 1010. Agar plates were incu-bated at 37°C for various times depending on the species: 4 days withcolony numbers recorded at 1-day intervals for S. aureus and 10 dayswith colony numbers recorded at 2-day intervals for M. smegmatis. Colo-nies were confirmed to contain mutant cells by regrowth on agar contain-ing the selecting concentration of antimicrobial (control experiments inwhich mutant colonies were grown on drug-free agar before retesting ondrug-containing agar showed that the mutants were stable).
M. smegmatis or S. aureus were plated on agar containing variousantimicrobial concentrations. After incubation, colonies werecounted and the proportion of the initial inoculum recovered as Figure 1. Effect of antimicrobial concentration on recovery of resistant mutants.
colonies on the plates was calculated. As shown in Figure 1, raising M. smegmatis strain mc2155 (open symbols) and S. aureus strain (RN450) the antimicrobial concentration gave a sharp drop in colony recovery.
(filled symbols) were applied to agar plates containing the indicated concentra- For some bacterium–antimicrobial combinations, a distinct shoulder tions of (a) moxifloxacin, (b) erythromycin, (c) penicillin, (d) chloramphenicol or plateau was observed in the recovery curves at high drug concen- or (e) tetracycline. Triangles indicate concentrations at which no colony was tration (moxifloxacin, erythromycin and tetracycline with M. smeg- recovered when more than 1010 cells were applied to plates. Replicate experi- matis; erythromycin with S. aureus). For others only inflection points ments gave results similar to those shown.
Effect of antimicrobials on recovery of resistant mutants
Table 1. Mutant selection window for various antimicrobial agents with M. smegmatis and S. aureus
aSpecific activity 1600 U/µg.
were detected (penicillin with M. smegmatis; moxifloxacin, tetra- requires combination therapy even if the fraction of mutant cells is cycline, penicillin and chloramphenicol with S. aureus). In the case of chloramphenicol with M. smegmatis, no inflection point was The second category of drug concentration dependence is illus- trated by the combinations studied in the present work, by prior Although the shape of the mutant recovery–drug concentration studies with fluoroquinolones,12,18,19 and by treatment of Candida curves varied among the bacterium–antimicrobial combinations, in albicans and C. glabrata with miconazole (J.-Y. Wang et al., unpub- each case the drop in mutant recovery became progressively steeper lished observations). Increasing drug concentration causes colony as antimicrobial concentration increased and high cell numbers were recovery to drop sharply at the MIC, pass through an inflection point, tested [the drop is probably steeper than shown, since the drug and then drop sharply a second time. The second drop occurs at the concentrations at which no colony was recovered (triangles) over- MPC. Fluoroquinolone studies show that resistant mutants are selec- estimate the true drug concentrations required to block colony tively enriched at drug concentrations between MIC growth]. Increasing steepness is the result expected as the MIC for the MPC.16,18 Whether monotherapy is appropriate for situations in this least susceptible mutant (MPC) is approached.11 category depends on how long relevant tissue drug concentrations It has been argued that resistant mutants are selectively enriched 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.
when antimicrobial concentrations fall between the minimal concen- Confusion sometimes surrounds phenotypic or induced resist- tration that inhibits the growth of 99% of the cells [MIC ance. An example is the β-lactam resistance that arises from the a range called the mutant selection window.10,13,16 [MIC induction of β-lactamases. When mutations are not responsible for mates the minimal concentration better than MIC because less select- this type of resistance, it is outside the scope of the present discussion.
ive pressure is present; however, for many antimicrobial–pathogen However, when β-lactamases are expressed from plasmid-borne combinations, little absolute difference is likely to exist between genes, they behave as category-one resistance and require com- and MIC due to the steep dependence of colony recovery on bination therapy even if the plasmid-containing cells are members of rare subpopulations. Thus the dosing strategies that derive from con- calculated and are listed in Table 1. When the size of the selection sideration of the mutant selection window hypothesis may be broadly window was expressed as the ratio of MPC to MIC siderably among bacterial–antimicrobial combinations. For bothbacterial species, the selection window was widest for erythromycin.
Acknowledgements
Discussion
We thank Richard Burger and Marila Gennaro for critical commentson the manuscript prior to submission. The work was supported by Although the relationship between antimicrobial concentration and grants from the NIH (AI35257) and Bayer AG.
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