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JAC Advance Access originally published online on February 1, 2008
Journal of Antimicrobial Chemotherapy 2008 61(3):629-635; doi:10.1093/jac/dkm542
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Selection of linezolid-resistant Enterococcus faecium in an in vitro dynamic model: protective effect of doxycycline

Stephen H. Zinner1, Deborah Gilbert2, Irene Yu. Lubenko3, Kenneth Greer2 and Alexander A. Firsov3,*

1 Mount Auburn Hospital, Harvard Medical School, Cambridge, MA, USA 2 Roger Williams Medical Center, Providence, RI, USA 3 Department of Pharmacokinetics and Pharmacodynamics, Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11 Bolshaya Pirogovskaya Street, Moscow 119021, Russia

* Corresponding author. Tel: +7-495-708-3341; Fax: +7-495-245-0295; E-mail: firsov{at}dol.ru

Received 11 October 2007; returned 17 December 2007; revised 16 November 2007; accepted 18 December 2007


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Objectives: To relate the enrichment of linezolid-resistant Enterococcus faecium with linezolid pharmacokinetics, the pharmacodynamics of linezolid and its ability to prevent the selection of resistant mutants were studied in an in vitro model that simulates antibiotic concentrations in and out of the mutant selection window (MSW), i.e. the concentration range from the MIC to the mutant prevention concentration (MPC).

Methods: A clinical isolate of E. faecium (MIC 1.8 mg/L and MPC 7 mg/L) at a starting inoculum of 8 log cfu/mL was exposed to twice-daily linezolid, alone and in combination with once-daily doxycycline (MIC 0.2 mg/L and MPC 3.4 mg/L), for 3 consecutive days in a hollow-fibre two-compartment model.

Results: The ratios of 24 h area under the curve (AUC24) to MIC of linezolid were estimated at 70, 100 and 230 h and those of doxycycline were estimated at 230 and 720 h. At the two lower AUC24/MIC ratios of linezolid given alone, E. faecium resistant to 2 x MIC–16 x MIC and 2 x MIC–8 x MIC of linezolid, respectively, were selectively enriched with a concomitant slight loss in susceptibility. Neither growth on linezolid-containing media nor changes in susceptibility occurred at the high AUC24/MIC ratio. A similar protective effect was observed with the minimal AUC24/MIC ratio of linezolid (70 h) combined with doxycycline at an AUC24/MIC of 230 h.

Conclusions: This study suggests that selection of linezolid-resistant enterococci can be predicted from the MSW concept and can be prevented by linezolid given in combination with doxycycline, each at suboptimal AUC24/MIC ratios.

Keywords: pharmacodynamics , resistant E. faecium , oxazolidinones , enterococci


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Linezolid is a new oxazolidinone antibiotic active against Gram-positive cocci including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. However, despite its relatively recent clinical introduction, linezolid-resistant S. aureus1 and E. faecium2–4 have been isolated from patients who received prolonged courses of the drug. Recent attempts in our laboratory to enrich resistant S. aureus exposed in vitro to fluctuating concentrations of linezolid in a dynamic model that simulates antibiotic pharmacokinetics were unsuccessful,5,6 possibly because of small numbers of resistant cells in the starting inoculum. The aim of this study was to use the same model to detect linezolid-resistant E. faecium at antibiotic concentrations in and out of the mutant selection window (MSW)7 and to evaluate the ability of doxycycline to prevent the enrichment of resistant enterococci.


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Antimicrobial agents, bacterial strain and susceptibility testing

Linezolid and doxycycline powders were provided by Pfizer, Inc. (Groton, CT, USA). A vancomycin-resistant clinical isolate of E. faecium, strain 392, was used for this study; vancomycin MIC was 80 mg/L, linezolid MIC was 1.8 mg/L, and doxycycline MIC was 0.20 mg/L. Susceptibility testing was performed by broth macrodilution techniques with the organism grown in brain–heart infusion (BHI) broth at an inoculum size of 5 x 105 cfu/mL.

The mutant prevention concentrations (MPCs) were determined as described elsewhere.7 Briefly, the tested microorganisms were cultured in fresh BHI broth and incubated overnight at 36°C. Then, two 50 mL aliquots were centrifuged at 2000 rpm for 30 min. The supernatant was discarded and the remaining pellets resuspended in 3 mL of fresh BHI. The 3 mL aliquots were thoroughly vortexed and combined in one tube to yield a concentration of 1010 cfu/mL. From the combined 6 mL suspension, one to eight dilution steps were made in sterile saline and 200 µL from each dilution step was plated on antibiotic-containing Trypticase soy agar (TSA) supplemented with 4% sheep blood (SB) plates that were prepared in our laboratory. Concentrations for the linezolid plates ranged from 1.80 to 46.15 mg/L, and from 0.20 to 5.12 mg/L for doxycycline. The inoculated plates were incubated for 48 h at 36°C and screened visually for growth. To estimate the MPC, logarithms of bacterial numbers were plotted against antibiotic concentrations. MPC was taken as the point where the plot intersected the x-axis, i.e. the lowest antibiotic concentration that completely inhibited growth when ~1010 cells were tested. The MPCs of linezolid and doxycycline were estimated at 7 and 3.4 mg/L, respectively.

In vitro dynamic model and simulated pharmacokinetic profiles

A previously described dynamic model8 was used in the study. Briefly, this two-compartment model consists of a central compartment and three bioreactors, artificial chambers (Fibercell Systems Inc., Frederick, MD, USA) connected in series, which represent the peripheral compartments. For all experiments, the bacterial inoculum was prepared from previously frozen inocula by thawing, diluting with an equal part of BHI and incubated for 90 min at 36°C to bring the organisms into growth phase.

This mixture was then inoculated into each peripheral compartment, which also contained BHI, via an entry port and incubated until a density of ~108 cfu/mL was achieved, at which time the antibiotic was introduced into the central compartment (time zero). Given a 20 mL volume of the peripheral compartment, the total number of organisms in the starting inoculum reached ~2 x 109 cfu. Control experiments without antimicrobial agent were performed to characterize growth kinetics. All dynamic model experiments were performed in triplicate.

A series of monoexponential profiles that mimic twice-daily administration (60 min infusion) of linezolid (half-life 6 h)9 and once-daily doxycycline (half-life 15 h)10 alone and in combination (1:3 ratio based on 24 h AUC/MICs) were simulated for 3 consecutive days. Overall, six dosing regimens were simulated: linezolid AUC24/MIC 60 h; linezolid AUC24/MIC 120 h; linezolid AUC24/MIC 240 h; doxycycline AUC24/MIC 240 h; doxycycline AUC24/MIC 720 h; linezolid AUC24/MIC 60 h+doxycycline AUC24/MIC 240 h.

To provide simultaneous monoexponential elimination of linezolid and doxycycline, a previously described dynamic model was modified according to Zinner et al.11

As the antimicrobial effect depends on antibiotic concentration in the peripheral compartments (where the organisms come into contact with antibiotic), peripheral compartments were sampled to determine linezolid concentrations by bioassay using well plates of TSA seeded with a clinical strain of coagulase-negative Staphylococcus and doxycycline with bioassay well plates of Antibiotic Medium #8 seeded with Bacillus cereus ATCC 11778.

Quantification of the antimicrobial effect and susceptibility changes

In each experiment, the peripheral compartments were sampled to determine bacterial concentrations. The numbers of surviving organisms were determined by serial dilution of samples in cold sterile saline and inoculating 20 µL in triplicate onto commercially available Mueller–Hinton agar supplemented with 5% SB. After overnight incubation at 36°C, the resulting bacterial colonies were counted and the numbers of cfu/mL were calculated. The detection limit was 10 cfu/mL. The duration of the experiments was defined as the time (after the last dose) until antibiotic-exposed bacteria reached the same maximum numbers as observed in the absence of antibiotic (≥109 cfu/mL).

On the basis of the time–kill data, the intensity of the antimicrobial effect (IE—the area between control growth and time–kill curves)12,13 was determined from time zero to the time after the last antibiotic dose at which the number of antibiotic-exposed bacteria reached 108 cfu/mL.

Changes in susceptibility of E. faecium 392 were examined by repeated MIC determinations and by plating a standard loopful of each 24 h specimen on TSA-SB plates containing no antibiotic, 2xMIC, 4xMIC, 8xMIC and 16xMIC of linezolid or doxycycline. The stability of observed resistance was determined daily by consecutive passaging E. faecium on antibiotic-free agar plates for 5 consecutive days.


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Pharmacodynamics and resistance in single-drug treatments with linezolid

Simulated pharmacokinetics and killing kinetics of linezolid-exposed E. faecium are presented in Figure 1. As seen in the top panel of the figure, the estimated AUC24/MIC ratios were close to the target values: 70 h versus 60 h, 100 h versus 120 h, and 230 h versus 240 h. At the lowest AUC24/MIC, linezolid concentrations fell in the MSW for most of the dosing interval: after the first dose, the time within the MSW (TMSW) was 100% of the interval. At the intermediate AUC24/MIC, linezolid concentrations were within the MSW for a shorter period: TMSW of 70%, whereas at the highest AUC24/MIC, the simulated concentrations were above the MPC throughout the dosing interval (out of the MSW): TMSW approached zero. The bottom panel of Figure 1 shows similar patterns of the time–kill curves: a gradual decrease in viable counts over 80 h (AUC24/MIC 70 h), 90 h (AUC24/MIC 100 h) and 100 h (AUC24/MIC 230 h) was followed by bacterial regrowth. The respective IEs were AUC24/MIC-dependent: the higher the AUC24/MIC, the greater the IE (see diagram in the right corner of the figure). The AUC24/MIC-response relationship was log-linear (data not shown).


Figure 1
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  		Figure 1. In vitro simulated pharmacokinetics of linezolid (upper panel) and time–kill curves of E. faecium exposed to linezolid (bottom panel). Arrows indicate linezolid administration. The respective AUC24/MIC ratio (h) is indicated for each curve. Diagram in the right corner of the bottom panel shows AUC/MIC-dependent antimicrobial effects of linezolid (see text for a description of the determination of IE).

 
Population analysis of specimens sampled from peripheral units of the dynamic model demonstrates a clear relationship between the enrichment of resistant mutants and simulated pharmacokinetics of linezolid (Figure 2). Treatments of E. faecium at AUC24/MIC of 70 and 100 h that provide antibiotic concentrations within the MSW over at least a part of the first dosing interval (TMSW 100% and 70%, respectively) were accompanied by bacterial growth on the antibiotic-containing plates. This growth in the presence of 2x, 4x and 16xMIC, but not 8xMIC of linezolid, began earlier at AUC24/MIC of 70 rather than 100 h. The concomitant loss in susceptibility observed by 72–96 h after the start of treatment was more pronounced at AUC24/MIC = 70 h (post-exposure MIC 2.2-fold higher than the pre-exposure MIC) than at AUC24/MIC = 100 h (1.5-fold difference between pre- and post-exposure MICs). This loss at both simulated AUC24/MICs was unstable: the differences between pre- and post-exposure MICs disappeared after six passages (data not shown). Unlike the lower AUC24/MICs, at the highest AUC24/MIC ratio (230 h) when antibiotic concentrations were out of the MSW, the starting population was not enriched by resistant mutants and the susceptibility of E. faecium was not changed.


Figure 2
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  		Figure 2. Effect of AUC24/MIC on the selection of linezolid-resistant E. faecium after exposure to linezolid: population analysis. Simulated AUC24/MIC ratios (in hours) are indicated for the curves.

 
Pharmacodynamics and resistance in combined treatments with linezolid and doxycycline

Comparative killing kinetics of E. faecium in treatments with linezolid and doxycycline given alone and in combination are shown in Figure 3. As seen in the figure, despite the rapid initial killing of E. faecium exposed to doxycycline, its effect was transient: bacterial regrowth was observed after the first dose. Much more stable effects were seen with linezolid (regrowth only after the end of treatment), although the simulated AUC24/MIC of linezolid was 3.4 times lower than that of doxycycline (70 h versus 230 h). Both in terms of the minimal number of surviving organisms and the time to regrowth, even more pronounced killing was observed in the combined treatment of E. faecium with linezolid and doxycycline. On the basis of the comparison of the respective IEs (see the diagram in the right corner of Figure 3), the combination of linezolid with doxycycline was synergistic: the IE that reflects the effect of combined treatment was 4.5 times greater than the sum of linezolid and doxycycline IEs in the single-drug treatments.


Figure 3
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  		Figure 3. Pharmacodynamics of linezolid (LZD) and doxycycline (DOX) alone and in combination with E. faecium. Simulated AUC24/MIC ratios (in hours) are indicated in parentheses.

 
The studied combination also appeared to be efficient in preventing the selection of linezolid-resistant E. faecium. As seen in Figure 4 (left-hand panel), organisms resistant to 2xMIC were enriched much later in the combined treatment than with linezolid monotherapy at the same AUC24/MIC. Moreover, enterococci resistant to the higher linezolid concentrations (4x, 8x and 16xMIC) were not enriched at all in the presence of doxycycline, with no loss in susceptibility of antibiotic-exposed E. faecium. In contrast, the selection of doxycycline-resistant organisms also began later in combinations with linezolid, but it occurred at all doxycycline concentrations (from 2xMIC to 16xMIC) (Figure 4, right-hand panel).


Figure 4
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  		Figure 4. Selection of linezolid (LZD)- and doxycycline (DOX)-resistant E. faecium after exposure to LZD alone and in combination with DOX: population analysis.

 
The described effects of linezolid on doxycycline pharmacodynamics and the emergence of enterococcal resistance to doxycycline could not be achieved by using a greater AUC24/MIC ratio of doxycycline given alone. As seen in Figure 5, an almost 4-fold increase in the simulated AUC24/MIC (from 230 to 720 h) with TMSWs of 100% and 35% of the first dosing interval, respectively (upper panel), provided only a 1 day delay in regrowth of the doxycycline-susceptible subpopulation with similar minimal numbers of surviving organisms (upper middle panel). In contrast to these minor differences between the time–kill curves, the combination of doxycycline (AUC24/MIC 230 h) with linezolid (AUC24/MIC 70 h) delayed regrowth for 4 days and yielded a much smaller minimal number of survivors. Similarly, at the increased AUC24/MIC (720 h), the amplification of E. faecium resistant to 2x–16xMIC of doxycycline occurred at the same time as at the lower AUC24/MIC (230 h)—both much earlier than with the combined treatment—see the lower middle panel in Figure 5 showing the respective data on plates with 4xMIC of doxycycline. Similar data were obtained on antibiotic-containing plates at 2x, 8x and 16xMIC (data not shown). Minimal differences in the amplification of resistant E. faecium exposed to the smaller and larger AUC24/MICs of doxycycline are in concordance with similar concomitant changes in susceptibility of doxycycline- and doxycycline plus linezolid-exposed organisms (Figure 5, bottom panel). At both AUC24/MICs, the MICs of doxycycline increased 3 days earlier with doxycycline monotherapy than with the doxycycline/linezolid combination, although a similar loss in doxycycline susceptibility (30- to 35-fold difference between pre-exposure and post-exposure MICs) occurred in all treatments.


Figure 5
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  		Figure 5. In vitro simulated pharmacokinetics of doxycycline (DOX)—upper panel; time–kill curves of E. faecium exposed to DOX alone and in combination with linezolid (LZD)—upper middle panel; bacterial survival on plates with 4xMIC of DOX—lower middle panel; and time courses of changes in susceptibility of E. faecium to DOX—bottom panel. Arrows indicate DOX administration. Simulated AUC24/MIC ratios (in hours) are indicated in parentheses.

 

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This in vitro study demonstrates concentration-dependent pharmacodynamics of linezolid and selection of linezolid-resistant mutants, in this case with E. faecium. A log-linear relationship was established between the intensity of the antimicrobial effect and simulated AUC24/MIC ratios in 3 day treatments with linezolid. In concordance with the MSW hypothesis,7 E. faecium mutants resistant to 2–16xMIC of linezolid were enriched when the antibiotic concentrations were within the MSW (AUC24/MIC 70 and 100 h), but not when concentrations were out of the MSW (AUC24/MIC of 230 h). The estimated ‘anti-mutant AUC24/MIC ratio’ is comparable to those reported in our studies with fluoroquinolone- and glycopeptide-exposed S. aureus (201–244 h14 and 200 h15) and with moxifloxacin-exposed Streptococcus pneumoniae (>100 h),16 as well as in a similar in vitro study with garenoxacin-exposed S. aureus (190 h).17 Given these consistent findings to other antibiotic–pathogen pair organisms, it is unclear why linezolid-resistant S. aureus did not emerge resistance in a similar experimental setting.5,6

In the present study with a specific strain of E. faecium, an AUC24/MIC ratio >200 h was estimated to protect against the selection of linezolid-resistant enterococci. This estimated value is two times larger than the AUC24/MIC of 100 h (186 mgxh/L9/1.8 mg/L) that can be provided by a 600 mg clinical dose of twice-daily linezolid. One approach for restricting resistance is the use of linezolid in combination with other antibiotics. The combination of linezolid plus doxycycline (both at AUC24/MICs at which amplification of resistant E. faecium occurred in monotherapy) appears promising. Doxycycline was shown to strengthen the effects of linezolid on both susceptible and resistant E. faecium subpopulations. Unlike monotherapy, organisms resistant to 2xMIC of linezolid were enriched only after the end of the combined treatment, and those resistant to 4–16xMIC were not enriched at all. In contrast to linezolid-resistant staphylococci, the combined use of linezolid with doxycycline was able to delay the selection of enterococcal mutants resistant to 4–16xMIC of doxycycline, but did not prevent it. This can be explained by the fact that the simulated concentrations of linezolid (AUC24/MIC = 70 h) were within its MSW over only part of the dosing interval, whereas concomitant concentrations of doxycycline (AUC24/MIC = 240 h) were within its MSW over the entire dosing interval. Although linezolid influenced the pharmacodynamics of doxycycline to a lesser extent than doxycycline did with linezolid, the linezolid-induced delay in the selection of doxycycline-resistant enterococci was much more pronounced than what could be provided by a 4-fold greater doxycycline AUC24/MIC ratio given alone. Earlier, a similar combination of suboptimal AUC24/MICs of moxifloxacin and doxycycline, i.e. AUC24/MICs smaller than the anti-mutant values, was shown to prevent the enrichment of resistant staphylococci.18

On the basis of the comparison of IEs determined in simulations of the pharmacokinetics of linezolid and doxycycline given alone and in combination, the combination's effect on the susceptible subpopulation of E. faecium was synergistic. Recently, similar synergism was reported in our pharmacodynamic studies with the same combination against S. aureus.5,6 These findings are consistent with a clinical report on bacteriologic cure of a neutropenic patient with fever caused by VRE with the addition of doxycycline to linezolid after unsuccessful monotherapy with daptomycin and linezolid.2 These data are not consistent with an in vitro study with E. faecium19 that demonstrated only minimal if any advantages of the linezolid/doxycycline combination in a relatively short 48 h simulation.

Overall, these data suggest that linezolid-resistant enterococci are enriched when drug concentrations fall into the MSW and that the amplification of resistant mutants may be restricted by using linezolid in combination with doxycycline, even if both agents are given at suboptimal individual doses.


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This study was supported by a grant from Pfizer, Inc.


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None to declare.


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1 Tsiodras S, Gold HS, Sakoulas G, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet (2001) 358:207–8.[CrossRef][Web of Science][Medline]

2 Lewis JS 2nd, Owens A, Cadena J, et al. Emergence of daptomycin resistance in Enterococcus faecium during daptomycin therapy. Antimicrob Agents Chemother (2005) 49:1664–5.[Free Full Text]

3 Auckland C, Teare L, Cooke F, et al. Linezolid-resistant enterococci: report of the first isolates in the United Kingdom. J Antimicrob Chemother (2002) 50:743–6.[Abstract/Free Full Text]

4 Swoboda S, Fritz S, Martignoni ME, et al. Varying linezolid susceptibility of vancomycin-resistant Enterococcus faecium isolates during therapy: a case report. J Antimicrob Chemother (2005) 56:787–9.[Abstract/Free Full Text]

5 Zinner S, Smirnova M, Lubenko I, et al. Comparative pharmacodynamics of linezolid, vancomycin and doxycycline with susceptible and resistant subpopulations of Staphylococcus aureus. In: Abstracts of the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, 2006. Washington, DC, USA: American Society for Microbiology. Abstract A-641, p. 15.

6 Smirnova M, Alferova I, Lubenko I, et al. Bacterial strain-independent pharmacodynamics of linezolid/doxycycline combinations with Staphylococcus aureus: 5-day simulations using an in vitro dynamic model. Clin Microbiol Infect (2006) 12(Suppl_4). Abstract P1519.

7 Zhao X, Drlica K. Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies. Clin Infect Dis (2001) 33:147–56.[CrossRef]

8 Blaser J, Stone BB, Zinner SH. Two compartment kinetic model with multiple artificial capillary units. J Antimicrob Chemother (1985) 15(Suppl A):131–7.[Free Full Text]

9 Stalker DJ, Jungbluth GL. Clinical pharmacokinetics of linezolid, a novel oxazolidinone antibacterial. Clin Pharmacokinet (2003) 42:1129–40.[CrossRef][Web of Science][Medline]

10 Deppermann KM, Lode H, Hoffken G, et al. Influence of ranitidine, pirenzepine, and aluminum magnesium hydroxide on the bioavailability of various antibiotics, including amoxicillin, cephalexin, doxycycline, and amoxicillin–clavulanic acid. Antimicrob Agents Chemother (1989) 33:1901–7.[Abstract/Free Full Text]

11 Zinner SH, Blaser J, Stone BB, et al. Use of an in-vitro kinetic model to study antibiotic combinations. J Antimicrob Chemother (1985) 15(Suppl A):221–6.[Abstract/Free Full Text]

12 Firsov AA, Chernykh VM, Navashin SM. Quantitative analysis of antimicrobial effect kinetics in an in vitro dynamic model. Antimicrob Agents Chemother (1990) 34:1312–7.[Abstract/Free Full Text]

13 Firsov AA, Vostrov SN, Shevchenko AA, et al. Parameters of bacterial killing and regrowth kinetics and antimicrobial effect examined in terms of area under the concentration–time curve relationships: action of ciprofloxacin against Escherichia coli in an in vitro dynamic model. Antimicrob Agents Chemother (1997) 41:1281–7.[Abstract]

14 Firsov AA, Vostrov SN, Lubenko IY, et al. In vitro pharmacodynamic evaluation of the mutant selection window hypothesis using four fluoroquinolones against Staphylococcus aureus. Antimicrob Agents Chemother (2003) 47:1604–13.[Abstract/Free Full Text]

15 Firsov AA, Smirnova MV, Lubenko IY, 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.[Abstract/Free Full Text]

16 Zinner SH, Lubenko IY, Gilbert D, et al. Emergence of resistant Streptococcus pneumoniae in an in vitro dynamic model that simulates moxifloxacin concentrations inside and outside the mutant selection window: related changes in susceptibility, resistance frequency and bacterial killing. J Antimicrob Chemother (2003) 52:616–22.[Abstract/Free Full Text]

17 Tam VH, Louie A, Deziel MR, et al. Bacterial-population responses to drug-selective pressure: examination of garenoxacin's effect on Pseudomonas aeruginosa. J Infect Dis (2005) 192:420–8.[CrossRef][Web of Science][Medline]

18 Firsov AA, Vostrov SN, Lubenko IY, et al. Prevention of the selection of resistant Staphylococcus aureus by moxifloxacin plus doxycycline in an in vitro dynamic model: an additive effect of the combination. Int J Antimicrob Agents (2004) 23:451–6.[CrossRef][Web of Science][Medline]

19 Allen GP, Cha R, Rybak MJ. In vitro activities of quinupristin–dalfopristin and cefepime, alone and in combination with various antimicrobials, against multidrug- resistant staphylococci and enterococci in an in vitro pharmacodynamic model. Antimicrob Agents Chemother (2002) 46:2606–12.[Abstract/Free Full Text]


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