JAC Advance Access originally published online on September 28, 2007
Journal of Antimicrobial Chemotherapy 2007 60(6):1380-1383; doi:10.1093/jac/dkm375
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Daptomycin inoculum effects and mutant prevention concentration with Staphylococcus aureus
Public Health Research Institute and Department of Microbiology and Molecular Genetics, UMDNJ—New Jersey Medical School, 225 Warren Street, Newark, NJ 07103, USA
* Corresponding author. Tel: +1-973-854-3364; Fax: +1-973-854-3101; E-mail: zhaox5{at}umdnj.edu
Received 31 May 2007; returned 3 July 2007; revised 4 September 2007; accepted 5 September 2007
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Abstract |
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Background: Antimutant activity of antimicrobials can be estimated by comparing drug pharmacokinetics with mutant prevention concentration (MPC). Large bacterial inocula known to reduce susceptibility have not been studied for effects on MPC determination.
Methods: Staphylococcus aureus inoculum size was varied with solid and liquid media containing daptomycin and Ca2+, a cation expected to lower inoculum effects, to assess effects on MIC and MPC.
Results: With drug-containing agar, individual colonies were obtained over a narrow range of inoculum size that shifted to higher inoculum size as daptomycin concentration increased. Increasing Ca2+ supplementation from 1 to 50 mM lowered MIC by 2-fold and MPC from 20 to 3 mg/L, the latter determined by extrapolation of population analysis profiles to an inoculum size of 1010 cfu. Cells of colonies recovered from daptomycin-containing agar had wild-type MIC. With liquid medium, supplemented with 1 mM Ca2+and containing 1010 cfu, MPC was between 2.5 and 5 mg/L at an inoculum density of 107 cfu/mL. Bacteria recovered from liquid assays exhibited a 4- to 8-fold increase in MIC and contained point mutations in mprF.
Conclusions: Inoculum effects on MPC can be reduced by measurement with low-density (large volume) liquid bacterial cultures. Retesting putative mutants for susceptibility can be important: stable mutants having genetic variations in the mprF gene were recovered from liquid medium, but not from agar. Daptomycin MPC with S. aureus was below minimal plasma drug concentration with approved doses, which is consistent with resistance to daptomycin arising rarely.
Keywords: mutant selection window , liquid medium , daptomycin-resistant mutants , resistant allele
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Introduction |
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Controlling antimicrobial resistance is difficult, in part because subpopulations of resistant pathogens are selectively enriched by antimicrobials. Selective enrichment and amplification are proposed to occur when antimicrobial concentrations fall in a specific range called the mutant selection window.1 The upper boundary of the selection window is the minimal concentration that blocks growth of the least susceptible, single-step resistant mutant subpopulation, a parameter termed the mutant prevention concentration (MPC). In principle, growth of resistant subpopulations is blocked by drug concentrations above MPC, just as susceptible growth is restricted by concentrations above MIC. Although agar-plate determinations of MPC can be used to define conditions that restrict mutant amplification when fluoroquinolone concentrations fluctuate, both in vitro and in vivo,1–3 the selection window hypothesis has not been tested for many antimicrobial–pathogen combinations.
Treatment of Staphylococcus aureus with daptomycin illustrates a potential problem for MPC determination. Daptomycin is a bactericidal4 cyclic lipopeptide to which cells rarely exhibit resistance.5,6 However, ‘false mutants’ are readily recovered from daptomycin-containing agar.6 ‘False mutants’, which exhibit wild-type MIC, probably grow on daptomycin-containing agar because large bacterial inocula protect them from drug action.6 Such protective inoculum effects make agar-based MPC determination difficult, as large numbers of bacterial cells (109–1010) must be applied to drug-containing plates.
The present work examined the effect of inoculum size on measurement of daptomycin MIC and MPC with S. aureus. Mueller–Hinton agar (MHA) was supplemented with Ca2+ to reduce recovery of ‘false mutants’,6 but colony recovery remained a complex function of both inoculum size and drug concentration. Most of the inoculum effect was eliminated by measuring MPC using liquid medium.
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Materials and methods |
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Strains, drugs and culture conditions
S. aureus strain RN450, provided by Barry Kreiswirth (Public Health Research Institute), was stored at –80°C in CY medium,7 supplemented with 15% glycerol. Daptomycin (Cubist Pharmaceuticals, Lexington, MA, USA) was dissolved in 0.9% NaCl to make 10 mg/mL stock solutions, which were diluted with molten MHA or Mueller–Hinton broth (MHB).
MIC was measured for 104–105 cfu by broth dilution in MHB or by agar dilution on MHA, both supplemented with various concentrations of Ca2+. To measure apparent MPC with agar, S. aureus was grown overnight in CY medium7 at 37°C, washed three times with fresh CY, diluted 50-fold in fresh CY and grown to 
109 cfu/mL. Cells were concentrated 10-fold by centrifugation and spread on daptomycin-containing MHA (supplemented with 0, 1 or 50 mM Ca2+) at inoculum sizes ranging from 104 to 1010 cfu. All plates were incubated at 37°C for 48 h with assessment of colony number every 24 h.
MPC was measured in liquid medium by growth of cells to 109 cfu/mL in CY, dilution of 1010 total cfu at various cell densities into MHB and treatment with various concentrations of daptomycin. The minimal daptomycin concentration that blocked growth of 1010 total cells after 24 h of incubation was taken as MPC.
Characterization of daptomycin-resistant mutants
Putative mutants were passaged five times in drug-free MHB or on MHA before MIC was determined as above. Portions of mprF and yycG containing alleles implicated in daptomycin resistance8 were amplified by PCR from chromosomal DNA using primers mprF-F1 (5'-CGGTGGCTTTATTGGTGCAGGCGT), mprF-R1 (5'-CGGTGGCTTTATTGGTGCAGGCGT), mprF-F2 (5'-GTGGTTCTTGGAGATCCGTTAGGTGATGA), mprF-R2 (5'-CCAAGCGCATCAGGCATAACTGTATACC), yycG-F (5'-GGCGATGTACGTACGGTCGATGTAACGA) and yycG-R(5'-ACGTCCACGGCGGTCTGTTGCA). Nucleotide sequences were determined using primers mprF-S1 (5'-GCTTTATTCCTGGTGGTTTCGGCGCT), mprF-S2 (5'-CGACTTGCAGGCCGTGTCTTTGAAC) and yycG-S (5'-TGGCGGTGGTAAGGACCGTGTCTG).
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Effect of Ca2+ supplementation on daptomycin MIC, inoculum effects and MPC
Daptomycin MIC with S. aureus, measured using agar dilution, was 0.64, 0.16 and 0.08 mg/L when agar was supplemented with 0, 1 or 50 mM CaCl2, respectively. Thus increasing Ca2+ concentration lowers MIC.6
When S. aureus was applied to agar, supplemented with either 1 or 50 mM CaCl2 plus various concentrations of daptomycin, increasing inoculum size increased colony number more than expected (non-proportional recovery, e.g. a 10-fold increase in bacterial load caused colony number to increase 50-fold or more). Such an inoculum effect could not be eliminated by increasing CaCl2 supplementation to 50 mM. Proportional recovery of individual colonies did occur within a narrow (10-fold) inoculum range that was shifted upward as daptomycin concentration increased (shown by vertical lines connecting symbols in Figure 1a and b). The number of individual colonies observed under proportional recovery conditions provided apparent population analysis profiles for daptomycin with 1 and 50 mM Ca2+ supplementation (Figure 1c and d). The profiles were roughly parallel, with inhibition of colony formation occurring at a lower daptomycin concentration for 50 mM Ca2+ than for 1 mM Ca2+. MPC (no colony formation with 1010 cells tested1) could only be estimated by extrapolation. Two independent estimates of MPC at 1 mM Ca2+ and three estimates at 50 mM Ca2+ averaged 20 and 3 mg/L, respectively.
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Determination of MPC with liquid medium
Uncertainties associated with extrapolation prompted us to develop an alternate measure of MPC. S. aureus cultures were grown in liquid medium to 109 cfu/mL, and they were diluted in MHB at various cell densities so total cell number was 1010 cfu (5–12 x 109). Daptomycin concentrations that blocked subsequent growth, as judged by the lack of increase in culture turbidity following a 24 h incubation, were defined as MPC. MPC, determined with 107 cfu/mL in 1 L of MHB supplemented with 1 mM Ca2+, was between 2.5 (three determinations) and 5 mg/L (two determinations). When 1010 cells were tested at 5 x 107 and 2.5 x 108 cfu/mL (200 and 40 mL, respectively), MPC was higher (10 and 20 mg/L, respectively). Thus cell density also affected MPC determination using liquid medium.
Characterization of putative mutants
Ten independent colonies, obtained from daptomycin-containing agar supplemented with either 1 or 50 mM CaCl2, were passaged five times on drug-free agar before testing for MIC at each Ca2+ concentration. MIC was the same as with the parental strain, i.e. the colonies comprised ‘false mutants’.6 With 1 L liquid cultures (107 cfu/mL), cells recovered from the highest drug concentration allowing growth were passaged five times as bulk subcultures in drug-free MHB (passages preceded by 1:1000 dilutions). After passage, these subcultures exhibited an 8-fold increase in MIC99 (minimal concentration reducing colony formation on MHA by 99%). With batch subcultures, increasing daptomycin concentration in agar from 2x to 8x MIC99 decreased the number of colonies gradually; thus, these cultures contained multiple mutant types having various levels of susceptibility. With homogeneous cultures prepared after purifying batch subcultures to single colonies on agar, daptomycin MIC and MIC99 increased by 4- and 8-fold, respectively. When three independent mutants were analysed for variations in mprF and yycG, genes previously implicated in daptomycin resistance with serially passaged and clinical samples,8 one mutant contained a previously reported8 serine to leucine change at position 295 of MprF and two mutants contained a new mprF daptomycin-resistant allele (Ser-337
Leu). No change was found in YccG. Thus, the primary determinant for daptomycin resistance maps to mprF.
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Discussion |
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A complex relationship exists between bacterial inoculum size, daptomycin concentration and formation of individual colonies on agar. Agar-plate determination of MPC, which required extrapolation of population analysis curves, produced values of 10–20 mg/L at 1 mM Ca2+. These values fit with those obtained previously using several other strains of S. aureus.9 Inoculum effects, plus the inability to recover bona fide mutants, indicate that agar-plate determinations overestimated MPC. Liquid assays allowed the use of lower inoculum densities, which explains liquid-determined MPC (2.5–5 mg/L at 1 mM Ca2+) being lower than agar-determined values. Inoculum effects also caused liquid assays to overestimate MPC. However, the overestimate was 2-fold or less when determined with 107 cfu/mL cultures, as mutants recovered had MIC values of 1.28–2.56 mg/L, about half the measured MPC (MPC can be defined as MIC for the least susceptible single-step mutant).
With humans, daptomycin pharmacokinetics determined with 4 mg/kg doses administered once daily indicates that minimal serum concentrations of daptomycin are 6 mg/L,4 which is higher than MPC (2.5–5 mg/L). With the higher approved dose (6 mg/kg), peak serum concentration is 100 mg/L and half-life is 9 h.10 In this situation, even after taking into account 90% protein binding,4 plasma daptomycin concentration should be above MPC for most of the 24 h dosing interval. These considerations are consistent with resistance arising rarely during daptomycin treatment.5,6
Although we did not recover bona fide resistant mutants from agar-plate assays, they can be obtained in liquid by serial passage.8 Such mutants explain the increase in MIC observed by Firsov et al.9 when S. aureus is challenged with moderate concentrations of daptomycin using a dynamic model. No MIC increase was observed when concentrations were kept above MPC, as expected when selective amplification of mutants is prevented.9 We obtained daptomycin-resistant mutants containing variations in MprF by a single challenge using a large volume of low-density liquid cultures. To the best of our knowledge, this is the first report of single-challenge recovery of daptomycin-resistant mutants.
We conclude that MPC can be determined using liquid assays even when inoculum effects occur. The present study, plus work by Firsov et al.,9 show that the mutant selection window hypothesis applies to antimicrobials other than fluoroquinolones.
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The work was supported by grants from Cubist Pharmaceutical Co. and the National Institutes of Health (AI35257).
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X. Z. has stock investment in Cubist Pharmaceuticals. Other authors have no declaration.
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Acknowledgements |
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We thank the following for critical comments on the manuscript: Marila Gennaro, Jared Silverman and Judith Steenbergen.
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References |
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1 Drlica K, Zhao X. Mutant selection window hypothesis updated. Clin Infect Dis (2007) 44:681–8.[CrossRef][Web of Science][Medline]
2
Firsov A, Vostrov S, Lubenko I, et al. In vitro pharmacodynamic evaluation of the mutant selection window hypothesis: four fluoroquinolones against Staphylococcus aureus. Antimicrob Agents Chemother (2003) 47:1604–13.
3 Cui J, Liu Y, Wang R, et al. The mutant selection window demonstrated in rabbits infected with Staphylococcus aureus. J Infect Dis (2006) 194:1601–8.[CrossRef][Web of Science][Medline]
4
DeRyke CA, Sutherland C, Zhang B, et al. Serum bactericidal activities of high-dose daptomycin with and without coadministration of gentamicin against isolates of Staphylococcus aureus and Enterococcus species. Antimicrob Agents Chemother (2006) 50:3529–34.
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Lewis JS, Owens A, Cadena J, et al. Emergence of daptomycin resistance in Enterococcus faecium during daptomycin therapy. Antimicrob Agents Chemother (2005) 49:1664–5.
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Silverman J, Oliver N, Andrew T, et al. Resistance studies with daptomycin. Antimicrob Agents Chemother (2001) 45:1799–802.
7 Novick RP, Brodsky R. Studies on plasmid replication. I. Plasmid incompatibility and establishment in Staphylococcus aureus. J Mol Biol (1972) 68:285–302.[CrossRef][Web of Science][Medline]
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Friedman L, Adler JD, Silverman JA. Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob Agents Chemother (2006) 50:2137–45.
9
Firsov A, Smirnova M, Lubenko I, et al. Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model. J Antimicrob Chemother (2006) 58:1185–92.
10
Dvorchik B, Brazier D, DeBruin M, et al. Daptomycin pharmacokinetics and safety following administration of escalating doses once daily to healthy subjects. Antimicrob Agents Chemother (2003) 47:1318–23.
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