Beruflich Dokumente
Kultur Dokumente
Quantitative modeling of combination therapy can describe the effects of each antibiotic against multiple bacterial populations.
Our aim was to develop an efficient experimental and modeling strategy that evaluates different synergy mechanisms using a
rapidly killing peptide antibiotic (nisin) combined with amikacin or linezolid as probe drugs. Serial viable counts over 48 h were
obtained in time-kill experiments with all three antibiotics in monotherapy against a methicillin-resistant Staphylococcus aureus
USA300 strain (inoculum, 108 CFU/ml). A sequential design (initial dosing of 8 or 32 mg/liter nisin, switched to amikacin or lin-
ezolid at 1.5 h) assessed the rate of killing by amikacin and linezolid against nisin-intermediate and nisin-resistant populations.
May 2013 Volume 57 Number 5 Antimicrobial Agents and Chemotherapy p. 2343–2351 aac.asm.org 2343
Landersdorfer et al.
FIG 2 Mechanistic synergy with drug B enhancing the rate of killing by drug
FIG 1 Subpopulation synergy concept. Bacterial cells inside the solid line are A for the population susceptible to drug A, the population resistant to drug A,
susceptible to drug A; bacterial cells inside the long dashed lines are susceptible or both populations.
to drug B. Bacteria within the dotted lines are resistant to either drug A or drug
B. The combination of drug A and drug B will eradiate these bacterial popu-
lations due to the lack of a population resistant to both drugs.
ic(s). Viable counts representing the initial inoculum were obtained
within less than 10 min prior to dosing.
Monotherapy. Static time-kill studies assessed nisin (4, 8, 32, and 128
counted for the time-dependent upregulation and minimized the mg/liter), amikacin (1, 4, 16, and 64 mg/liter), and linezolid (2, 8, and 32
FIG 3 Life cycle growth model for the population susceptible to both nisin
and amikacin. This two-state life cycle growth model was applied to each of the
six populations in Fig. 4. k12, first-order growth rate constant; k21, first-order
replication rate constant (representing doubling); InhRep, inhibition of the
probability of successful replication; Inhk12, inhibition of the first-order
growth rate; other symbols are explained in Table 1 and Materials and
Methods. FIG 4 Subpopulation synergy model for nisin and amikacin comprising six
populations with different susceptibility to each antibiotic. Niss, nisin suscep-
tible; Nisi, nisin intermediate; Nisr, nisin resistant; Amis, amikacin susceptible;
Amir, amikacin resistant. Thick arrows represent fast growth or fast killing.
rate constant, k12 (Fig. 3), and replication (k21, or doubling) was assumed Solid downward arrows represent killing by nisin, and dashed arrows represent
冊
Population model. Models with one, two, or three preexisting popu-
KmaxS · CAmiHill
lations with different susceptibilities to the respective antibiotic were con- ⫹ · CFUSS,2 (4)
sidered similarly to previously described models (8, 14, 19, 22, 25–30). CAmiHill ⫹ KC50Hill
The susceptibility of each population was estimated via a specific rate of The differential equations for the five populations that were less sus-
bacterial killing by the respective antibiotic (Fig. 4). ceptible to nisin, amikacin, or both are shown in the supplemental mate-
Nisin-plus-amikacin combination. The mathematical model for ni- rial. These differential equations accounted for the decreased susceptibil-
sin plus amikacin contained six populations, including all combina- ity to nisin (using k2I or k2R instead of k2S; Table 1) or to amikacin (using
tions of a nisin-susceptible (Niss), -intermediate (Nisi), and -resistant KmaxR instead of KmaxS). Some less-susceptible populations were esti-
(Nisr) population and of an amikacin-susceptible (Amis) and -resis- mated to have a decreased biofitness described by a longer mean genera-
tant (Amir) population. With the two states (i ⫽ 1 and i ⫽ 2 in equa- tion time (denoted by the rate constants k12_RS_SR, k12_IR, and k12_RR
tion 1) for each population from the life cycle growth model, the total [Table 1]).
population (CFUALL) contained 12 compartments (i.e., two compart- Survival fraction. The survival fraction after pretreatment with nisin
ments for each of the six populations). for 1.5 h was calculated for each population based on the second-order
2 killing rate constants (i.e., k2 was either k2S, k2I, or k2R) as FrSurvive ⫽
CFUALL ⫽ 冱 CFUSS,i ⫹ CFUIS,i ⫹ CFURS,i ⫹ CFUSR,i exp(⫺k2 · CNis · 1.5 h).
i⫽1
Initial conditions. The total inoculum and the mutation frequency of
⫹ CFUIR,i ⫹ CFURR,i (1)
each less-susceptible population were estimated model parameters. The
The differential equation for the concentration of bacteria from the product of the total inoculum and the mutation frequency defined the size
Niss/Amis population in state 1 (CFUSS,1) comprised the second-order of each of the less-susceptible populations at 0 h (i.e., initial condition).
killing by nisin and a saturable killing by amikacin (initial condition de- The initial condition of the population susceptible to both antibiotics was
scribed below): calculated by subtracting the size of all less-susceptible populations from
dCFUSS,1
dt 冉
⫽ REP · k21 · CFUSS,2 ⫺ k12 ⫹ k2S · CNis
that of the total initial inoculum. All bacteria of a population were initial-
ized into state 1 of the respective population (i.e., for i ⫽ 1 in equation 1).
冊
Initial conditions for the compartments representing state 2 (i ⫽ 2 in
KmaxS · CAmiHill equation 1) were zero, since k21 was rapid.
⫹ · CFUSS,1 (2)
CAmiHill ⫹ KC50Hill Nisin-plus-linezolid combination. The model for nisin plus linezolid
where CNis is the nisin and CAmi the amikacin concentration in broth contained three populations, including nisin-susceptible and linezolid-
medium. CFUSS,2 is the concentration of bacteria in state 2 of the Niss/ susceptible (Niss/Lins), nisin-intermediate and linezolid-susceptible
Amis population. The rate constants k21, k12, and k2S and the parameters (Nisi/Lins), and nisin-resistant and linezolid-intermediate (Nisr/Lini)
for the Hill-type killing function for amikacin (KmaxS, KC50, and Hill) are populations. The differential equation for the Niss/Lins population in
defined in Table 1. The replication factor REP ensures that the total con- state 1 (CFUSS,1) contained second-order killing by nisin and two inhib-
centration of bacteria in all populations (CFUALL) does not exceed the itory effects of linezolid (Fig. 3; initial condition calculated as for the
maximum population size (CFUmax): nisin-plus-amikacin model):
冉
REP ⫽ 2 · 1 ⫺
CFUALL
CFUmax ⫹ CFUALL 冊 (3)
dCFUSS,1
dt
⫽ REP · (1 ⫺ InhRep) · k21 · CFUSS,2 ⫺ (k12 · Inhk12
At low viable counts, REP approaches 2, representing a 100% proba- ⫹ k2S · CNis) · CFUSS,1 (5)
bility of successful replication (i.e., doubling). As CFUALL approaches where REP was defined as above and CFUALL was the sum of both states
Killing by amikacin
Maximum killing rate constants
Susceptible population KmaxS (h⫺1) 10.1 (19) 9.10 (21)
Resistant population KmaxR (h⫺1) 0.771 (18) 0.635 (10)
Amikacin concn causing 50% of Kmax KC50 (mg/liter) 14.7 (7.7) 16.5 (7.2)
Hill coefficient Hill 2.45 (12)b 2.83 (14)
for the three populations of the nisin-plus-linezolid model. Parameters in the linezolid concentration (CLin) required for half-maximal inhibition of
equations 5 to 9 are explained in Table 1. Linezolid inhibited protein protein synthesis. In the absence of linezolid, the protein pool is at its
synthesis, and the protein constituent pool (P), with an initial condition of hypothetical baseline of 100%. Depletion of the protein pool by linezolid
1.0, was defined as: increases the probability of unsuccessful replication (InhRep):
dP
dt
⫽ kprot · 冋冉 1⫺
CLin
CLin ⫹ IC50,Prot 冊 册
⫺P (6)
InhRep ⫽ ImaxRep · (1 ⫺ P)
As described previously (22), an InhRep (equations 5 and 7) of 0.50
(7)
kprot is the turnover rate constant for the protein pool, and IC50,Prot is results in net stasis of the respective bacterial population and an InhRep of
⬎0.5 results in bacterial killing, since bacteria that replicate unsuccessfully and Fig. 6C). The modeled survival fraction (FrSurvive; based on
are eliminated (i.e., lost from the system) (Fig. 3). Linezolid additionally estimated k2S in Table 1) of the Niss populations after 1.5 h pre-
inhibited the growth rate (Inhk12): treatment was ⬍10⫺18, indicating a complete killing of Niss bac-
Imax,k12 · CLinHillk12 teria (calculated from k2S in Table 1). For the Nisi populations,
Inhk12 ⫽ 1 ⫺ (8) FrSurvive was 0.056 (equivalent to 1.25 log10 killing) for 32 mg/liter
CLinHillk12 ⫹ IC50,k12Hillk12
nisin and 0.49 (equivalent to 0.3 log10 killing) for 8 mg/liter nisin
The differential equation for bacteria in state 2 (CFUSS2) was:
pretreatment. Thus, 32 mg/liter nisin pretreatment killed the Nisi
dCFUSS,2 populations to levels below those of the Nisr populations. For the
⫽ k12 · Inhk12 · CFUSS,1 ⫺ (k21 ⫹ k2S · CNis) · CFUSS,2
dt Nisr populations, the FrSurvive values were 0.79 for 32 mg/liter and
(9) 0.94 for 8 mg/liter nisin pretreatment, suggesting that these pop-
The differential equations for state 1 (CFUIS,1) and state 2 (CFUIS,2) of ulations were killed by ⱕ0.1 log10 by either pretreatment. These
the Nisi/Lins population were: FrSurvive results were in good agreement with results from the pilot
dCFUIS,1 experiments (results not shown) that optimized the nisin concen-
⫽ REP · (1 ⫺ InhRep) · k21 · CFUIS,2 ⫺ (k12 · Inhk12 trations and duration of pretreatment.
dt
⫹ k2S · CNis) · CFUIS,1 (10)
Nisin switched to amikacin. Control arms that switched from
nisin pretreatment to no antibiotic showed growth and thus con-
therapy, sequential and simultaneous combinations (Fig. 5) were the high inoculum, 32 mg/liter linezolid achieved slow killing by 1
successfully comodeled with unbiased and precise fits (r, 0.99; log10 from 8 to 56 h (Fig. 6B), whereas 2 mg/liter linezolid essen-
slope, 0.996; intercept, 0.05 log10 CFU/ml for observed versus in- tially paralleled the growth control. After nisin pretreatment, lin-
dividually fitted log10 viable counts). For the 1- and 4-mg/liter ezolid yielded mostly static profiles, suggesting that linezolid dis-
amikacin curves, the observation at 4 h was overpredicted played some activity against the Nisi and Nisr populations
(Fig. 5B); however, this did not have a large influence, since the (Fig. 6C). The simultaneous combinations of linezolid with 16
overall extent of killing by 1 and 4 mg/liter amikacin was very or 32 mg/liter nisin achieved 3 to 6 log10 killing at 48 h (Fig. 6D
modest and the 1-h and 8-h samples were reasonably fitted. The and E).
observed versus population fitted log10 viable counts (i.e., in the Modeling nisin and linezolid. For nisin and linezolid, all 20
absence of random between curve variability) were also unbiased profiles were adequately fit simultaneously (Fig. 6) with an overall
and reasonably precise (r ⫽ 0.94). All parameters for the nisin and r of 0.99, a slope of 1.001, and an intercept of 0.002 log10 CFU/ml
amikacin model were estimated with good precision (relative for the observed versus individual fitted log10 viable counts and an
standard errors, ⱕ23% except for fk12_IR and fk12_RR [Table 1]). r of 0.96 for the population fits. Only the 8- and 32-mg/liter curves
Inclusion of an enhanced rate of killing for the simultaneous com- for the nisin monotherapy and the 2-mg/liter linezolid mono-
bination improved neither the objective function nor the curve therapy showed slight misfits (Fig. 6A and B) which did not affect
fits, suggesting a lack of mechanistic synergy. This result on the the characterization of the type of synergy. In contrast to nisin and
types of synergy was based on thorough modeling analyses in amikacin, linezolid did not exhibit fast bacterial killing (Fig. 6B).
S-ADAPT and NONMEM which yielded comparable parameter In agreement with its effect on inhibiting protein synthesis, lin-
estimates (Table 1) and led to the same conclusion regarding a lack ezolid prolonged the mean generation time (Inhk12 in Fig. 3) of all
of mechanistic synergy. populations. For the Niss/Lins and Nisi/Lins populations, linezolid
Linezolid and nisin. The MIC was 2 mg/liter for linezolid. At additionally inhibited the probability of successful replication
(InhRep), most likely by affecting the synthesis of proteins that are was defined here as a higher rate of killing of a bacterial population
necessary for replication. Therefore, linezolid (slowly) killed these under the simultaneous presence of two antibiotics (Fig. 2) com-
populations at linezolid concentrations above the IC50,Prot of 3.92 pared to the rate of killing of this bacterial population predicted by
mg/liter (Table 1) at the high inoculum studied. Linezolid the independent effects of both antibiotics in monotherapy.
achieved a static response only against the Nisr/Lini population The proposed approach accounted for the limited time avail-
which was modeled by linezolid prolonging the mean generation able to bacteria to elaborate their tolerance and resistance mech-
time (Inhk12). Nisin killed this population slowly. anisms to survive standard, simultaneously dosed antibiotic com-
The Nisr/Lini population was estimated to have a lower growth binations. Short-term (1.5-h) pretreatment with a rapidly killing
rate than the Lins populations. The estimated log10 mutation fre- first antibiotic (nisin) was utilized to select less-susceptible popu-
quencies suggested that the initial inoculum comprised a rela- lations. This approach allows one to test second antibiotics against
tively high fraction (10⫺4.15) of the Nisr/Lini population (Table 1). the entire surviving bacterial population and does not require
This result indicated that high nisin and linezolid concentrations isolating presumably more stably resistant colonies after
in combination would be required to achieve more than ⬃4.15 growth over 24 h or even longer on agar plates containing the
log10 killing against this high inoculum. first antibiotics.
Nisin very rapidly killed MRSA at a high inoculum which al-
DISCUSSION lowed an efficient selection of the Nisi and Nisr populations. Ami-
The experimental and modeling strategy developed in the present kacin achieved substantial killing against the Nisi and Nisr popu-
study was designed to efficiently identify and characterize synergy lations (Fig. 5B), whereas linezolid yielded only stasis or slow
mechanisms for antibiotic combinations. We proposed to distin- killing (Fig. 6B) against these populations. The Nisr/Amir popula-
guish between two types of synergy. Subpopulation synergy can be tion had a log10 mutation frequency of ⫺7.42 (Table 1), suggest-
greatly beneficial, if one antibiotic kills the resistant population of ing that nisin in combination with amikacin can achieve more
a second antibiotic and vice versa (Fig. 1). Mechanistic synergy than 7 log10 killing at high drug concentrations. This was success-
fully confirmed for both sequential and simultaneously dosed models for aminoglycosides against Pseudomonas aeruginosa and
combinations (Fig. 5). Staphylococcus aureus utilized Hill-type killing functions in agree-
The nisin-plus-amikacin combination leverages subpopula- ment with the present combination model (39, 40). Consistent
tion synergy via nisin killing the Amir populations and amikacin with previously developed models for linezolid against Enterococ-
killing the Nisi and Nisr populations (except for the small Nisr/ cus faecalis (41) and MRSA (18), the present model included an
Amir population that was only [slowly] killed by 16 mg/liter ami- effect of linezolid prolonging the mean generation time and inhib-
kacin [Fig. 5E]). This sequential dosing strategy was critical, since iting the probability of successful replication due to linezolid in-
it provided experimental insights into why eradication was hibiting protein synthesis. The population analysis in S-ADAPT,
achieved by the simultaneously dosed combination. In immuno- in addition to the analysis in NONMEM, demonstrated that the
competent patients the small Nisr/Amir population might be erad- model structure is sufficiently complex to capture all viable count
icated by the immune system (12, 13), as the vast majority of profiles well, as shown by the r ⱖ 0.99 for the individual fitted
bacteria are rapidly killed by the antibiotic combination. versus observed log10 CFU/ml, and it suggested there is a small
In contrast, the Nisr/Lini population had a high mutation fre- between-curve variability that is reflected by variability in the pa-
quency of ⫺4.15 log10 (Table 1). This suggested limited promise rameter estimates.
for the nisin-plus-linezolid combination against this MRSA strain In summary, nisin achieved subpopulation synergy with ami-
at a high inoculum. However, future studies are required to assess kacin but not with linezolid against a high inoculum of MRSA.
policy recommendations to save lives. Clin. Infect. Dis. 52(Suppl 5):S397– modeling of anti-cancer activity of tetraiodothyroacetic acid in a perfused
S4428. cell culture system. PLoS Comput. Biol. 7:e1001073. doi:10.1371/journal
7. Bulitta JB, Li J, Poudyal A, Yu HH, Owen RJ, Tsuji BT, Nation RL, .pcbi.1001073.
Forrest A. 2009. Quantifying synergy of colistin combinations against 24. Maidhof H, Johannsen L, Labischinski H, Giesbrecht P. 1989. Onset of
MDR gram negatives by mechanism-based models, abstr A1-573. Intersci. penicillin-induced bacteriolysis in staphylococci is cell cycle dependent. J.
Conf. Antimicrob. Agents Chemother., San Francisco, CA, 12 to 15 Sep- Bacteriol. 171:2252–2257.
tember 2009. 25. Meagher AK, Forrest A, Dalhoff A, Stass H, Schentag JJ. 2004. Novel
8. Bulitta JB, Li J, Poudyal A, Yu HH, Owen RJ, Tsuji BT, Nation RL, pharmacokinetic-pharmacodynamic model for prediction of outcomes
Forrest A. 2010. Mechanism-based modelling of the synergy of colistin with an extended-release formulation of ciprofloxacin. Antimicrob.
combinations against multidrug-resistant gram negative bacteria, abstr Agents Chemother. 48:2061–2068.
1918. Abstr. Annu. Meet. Population Approach Group in Europe (PAGE). 26. Campion JJ, Chung P, McNamara PJ, Titlow WB, Evans ME. 2005.
www.page-meeting.org/?abstract⫽1918. Pharmacodynamic modeling of the evolution of levofloxacin resistance in
9. Bulitta JB, Landersdorfer CB, Forrest A, Brown SV, Neely MN, Tsuji Staphylococcus aureus. Antimicrob. Agents Chemother. 49:2189 –2199.
BT, Louie A. 2011. Relevance of pharmacokinetic and pharmacodynamic 27. Mouton JW, Vinks AA, Punt NC. 1997. Pharmacokinetic-
modeling to clinical care of critically ill patients. Curr. Pharm. Biotechnol. pharmacodynamic modeling of activity of ceftazidime during continuous
12:2044 –2061. and intermittent infusion. Antimicrob. Agents Chemother. 41:733–738.
10. Greco WR, Bravo G, Parsons JC. 1995. The search for synergy: a critical 28. Czock D, Keller F. 2007. Mechanism-based pharmacokinetic-
review from a response surface perspective. Pharmacol. Rev. 47:331–385. pharmacodynamic modeling of antimicrobial drug effects. J. Pharmaco-
11. White DB, Slocum HK, Brun Y, Wrzosek C, Greco WR. 2003. A new kinet. Pharmacodyn. 34:727–751.
nonlinear mixture response surface paradigm for the study of synergism: a 29. Nielsen EI, Viberg A, Lowdin E, Cars O, Karlsson MO, Sandstrom M.