Skip to main content
NCBI home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 2014 Nov;58(11):6773–6781. doi: 10.1128/AAC.03505-14

Evaluation of Meropenem Regimens Suppressing Emergence of Resistance in Acinetobacter baumannii with Human Simulated Exposure in an In Vitro Intravenous-Infusion Hollow-Fiber Infection Model

Xin Li a,b, Lin Wang a, Xian-Jia Zhang a, Yang Yang b, Wei-Tao Gong a, Bin Xu a, Ying-Qun Zhu a, Wei Liu a,✉
PMCID: PMC4249358  PMID: 25182633

Abstract

The emergence of resistance to carbapenems in Pseudomonas aeruginosa can be suppressed by optimizing the administration of meropenem. However, whether the same is true for Acinetobacter baumannii is not fully understood. We assessed the bactericidal activity of meropenem and its potency to suppress the emergence of resistance in A. baumannii with human simulated exposure in an in vitro intravenous-infusion hollow-fiber infection model (HFIM). Two clinical strains of carbapenem-susceptible multidrug-resistant A. baumannii (CS-MDRAB), CSRA24 and CSRA91, were used, and their MICs and mutant prevention concentrations (MPCs) were determined. Six meropenem dosage regimens (0.5, 1.0, or 2.0 g given every 8 h [q8h] with a 0.5-h or 3-h infusion for seven consecutive days) were simulated and then evaluated in the HFIM. Both the total population and resistant subpopulations of the two strains were quantified. Drug concentrations were measured by high-performance liquid chromatography. All dosage regimens, except for the lowest dosage (0.5 g for both the 0.5-h and 3-h infusions), showed 3-log CFU/ml bacterial killing. Dosage regimens of 2.0 g with 0.5-h and 3-h infusions exhibited an obvious bactericidal effect and suppressed resistance. Selective amplification of subpopulations with reduced susceptibility to meropenem was suppressed with a percentage of the dosage interval in which meropenem concentrations exceeded the MPC (T>MPC) of ≥20% or with a ratio of T>MPC to the percentage of the dosage interval in which drug concentrations are within the mutant selection window of ≥0.25. Our in vitro data support the use of a high dosage of meropenem (2.0 g q8h) for the treatment of severe infection caused by CS-MDRAB.

INTRODUCTION

Acinetobacter baumannii is classified under the Acinetobacter genus and comprises strictly aerobic, Gram-negative, nonmotile, non-lactose-fermenting, oxidase-negative, catalase-positive coccobacilli (1). A. baumannii is an opportunistic pathogen associated with severe nosocomial infections and often exhibits multidrug resistance to most of the currently available antibiotic agents (1–3). Carbapenems, which are a class of β-lactam antibiotics including imipenem and meropenem, have been recommended as the last resort for the treatment of infection with multidrug-resistant A. baumannii (MDRAB) (4). However, a substantial increase in the prevalence of carbapenem-resistant A. baumannii strains has been documented during the last few years, which has become a serious problem that threatens our therapeutic armamentarium for A. baumannii infection (5–7). However, the development process for new drugs against A. baumannii infection is very slow, which indicates that overcoming multidrug resistance in A. baumannii cannot rely solely on the development and availability of new drugs; it is imperative that currently available antimicrobial agents be preserved as the mainstream treatment to fight against the emergence of resistance in A. baumannii by optimizing the use of these agents.

It has been shown that suboptimal dosing of antibiotics provides a selective pressure on bacteria and facilitates the emergence of resistance resulting from bacterial mutation under selective pressure of antibiotics (8, 9). In 1998, Nunez et al. reported a meningitis case caused by an Acinetobacter strain that developed resistance to meropenem during treatment with this agent (10). Several studies have identified the administration of carbapenems as an independent risk factor for the emergence of carbapenem-resistant MDRAB in patients (6, 11–15). It has also been confirmed that emergence of resistance in Pseudomonas aeruginosa can be suppressed by optimizing the administration of meropenem (16, 17). However, whether this phenomenon also applies to A. baumannii is not fully understood. The aim of the present study was to determine whether in vitro-simulated pharmacodynamic exposure to different meropenem dosage regimens is able to optimize bactericidal activity and suppress the emergence of resistance in carbapenem-susceptible MDRAB (CS-MDRAB) strains.

MATERIALS AND METHODS

Antimicrobial agents.

Meropenem reference substance, obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), was used for susceptibility testing and as the standard for measurement of meropenem by high-performance liquid chromatography (HPLC), as described below. The drug was dissolved and diluted to the desired concentrations with sterilized water for injection (Shijiazhuang No. 4 Pharmaceutical Company, Shijiazhuang, Hebei, China) prior to use. In addition, meropenem powder, kindly provided by SumitomoPharma Co., Ltd. (Suzhou, Jiangsu, China), was used for pharmacokinetic/pharmacodynamic (PK/PD) experiments in the hollow-fiber infection model (HFIM).

Microorganisms.

Two clinical strains of CS-MDRAB, CSRA24 and CSRA91, which we isolated from two unrelated patients with pulmonary infection, were used in this study. These two strains were tested to be multidrug resistant but carbapenem susceptible by routine antimicrobial susceptibility testing (Vitek 2 automated antimicrobial susceptibility testing system; bioMérieux). Briefly, CSRA24 and CSRA91 were both resistant to mezlocillin, ampicillin-sulbactam, piperacillin-tazobactam, ceftriaxone, ceftazidime, cefepime, cefotaxime, gentamicin, and tobramycin. However, CSRA91 was also resistant to ciprofloxacin and levofloxacin, whereas CSRA24 was susceptible to ciprofloxacin and levofloxacin. In addition, a P. aeruginosa strain, ATCC 27853, was used as a reference strain for quality control for susceptibility testing. In order to ensure the parallelism of experiments, all strains were dispensed into airtight vials in the form of a freeze-dried powder and stored at −70°C. Prior to each experiment, the strains were subcultured on Columbia plates with 5% sheep blood (bioMérieux, Shanghai, China) and incubated at 35°C for 24 h.

In vitro susceptibility testing.

Susceptibility testing was performed in triplicate by the agar dilution method according to the recommendations of the Clinical and Laboratory Standards Institute (18). Serial 2-fold dilutions of meropenem (0.125 to 64 μg/ml) were prepared and incorporated into designated Mueller-Hinton agar (MHA; Oxoid, Ltd., Hampshire, England) plates. The final CFU count of the bacterial inoculum on each plate was approximately 5 × 104 CFU. The MIC was defined as the lowest concentration of the drug that resulted in no visible growth on the agar after 24 h of incubation at 35°C in a biochemical incubator.

In addition, the mutant prevention concentration (MPC) was also determined, as described previously (19). Briefly, the tested strains were cultured in Ca–Mueller-Hinton broth (MHB; Oxoid, Ltd., Hampshire, England) and incubated at 35°C for 24 h. After centrifugation at 4,000 × g for 10 min, the strains were resuspended in MHB to yield a concentration of 3 × 1010 CFU/ml. Next, each of the MHA plates with one of the designated meropenem concentrations was inoculated with 1 × 1010 CFU of the strains. The inoculated plates were incubated for 72 h at 35°C in a biochemical incubator and screened visually for growth. MPC was defined as the lowest meropenem concentration that completely inhibited growth of the bacteria (20). Determination of the MPC was carried out in duplicate and was repeated two times on separate days.

Hollow-fiber infection model.

The HFIM, which was previously used and described in detail (21), was modified in the present study to simulate the PK/PD of meropenem. First, an intravenous-infusion model, instead of an injection model, was applied to simulate the rising phase of the concentration-time curve. Second, reverse external circulation of the extracapillary compartment with a low flow rate was added to assess the PD of meropenem accurately. Third, the peristaltic pumps were controlled by computer software to conveniently and precisely simulate the concentration-time curve under different meropenem regimens. An F4HPS capillary dialyzer (Fresenius Medical Care [Shanghai] Co., Ltd., Shanghai, China) was used as the hollow-fiber cartridge for the HFIM. The effective surface area of the hollow-fiber systems was 0.8 m2. A diagram of the HFIM is shown in Fig. 1.

FIG 1.

FIG 1

Open in a new tab

Diagram of the hollow-fiber infection model.

MHB containing the designated dose of meropenem was infused from the drug reservoir into the central reservoir by a peristaltic pump (Leadfluid BT-101L; Baoding Leadfluid Technology Co., Ltd., Baoding, China) from time zero to 3 h. Simultaneously, the medium in the central reservoir was pumped into the waste reservoir by another peristaltic pump. After 3 h, the pump controlling the drug reservoir was switched off, and the drug-free medium was infused from the diluent reservoir into the central reservoir by a third peristaltic pump. At 8 h, the peristaltic pump controlling the diluent reservoir was stopped, and the dosing process was repeated for another administration period of 7 days. The peristaltic pumps mentioned above were controlled by Labcomsoft 3.0 computer software (Baoding Leadfluid Technology Co., Ltd.) with the same designated flow rate. The 0.5-h infusion model for the designated dose of meropenem was carried out in the same fashion, except that the infusion time was 0.5 h instead of 3 h. MHB medium without meropenem was used as a control.

The bacterial suspension (approximately 70 ml) was injected and confined in the extracapillary compartment of the hollow-fiber cartridge, and nutrients and meropenem in the broth were able to be exchanged between the extracapillary and inner capillary compartments.

Simulated PK profiles.

Monoexponential profiles that mimicked three-times-daily administration (0.5-h or 3.0-h infusion) of meropenem with an elimination half-life (t1/2) of approximately 1.0 h were simulated as previously described (22, 23). Six meropenem dosage regimens (0.5, 1.0, or 2.0 g given every 8 h [q8h] with a 0.5-h or 3-h infusion for seven consecutive days) were simulated in the HFIM, as described above. The simulated single-dose peak concentrations (Cmax) were 22.76, 45.34, and 90.68 μg/ml for regimens of 0.5, 1.0, and 2.0 g with a 0.5-h infusion, respectively, and 9.20, 18.40, and 36.80 μg/ml for regimens of 0.5, 1.0, and 2.0 g with a 3-h infusion, respectively.

Experimental setup.

The freeze-dried powder of the strains was subcultured on Columbia plates with 5% sheep blood (bioMérieux, Shanghai, China) at 35°C for 24 h. The bacterial suspension was prepared by taking one freshly grown medium-sized colony, placing it into MHB, and incubating it further at 35°C until the bacteria reached the late log phase of growth (108 CFU/ml). In order to simulate the bacterial load of severe infection and detect the resistant mutant subpopulations at baseline, a bacterial suspension of 108 CFU/ml was then injected into the extracapillary compartment of the hollow-fiber cartridge and incubated continually at 37°C until the bacteria reached a concentration of approximately 109 CFU/ml (8, 24). The six meropenem regimens described above and a control (medium without meropenem) were investigated.

PK/PD validation and microbiological response.

For the regimens with a 0.5-h infusion, samples (1.0 ml) were collected from the central reservoir of the HFIM before each dosing and at 0.167 h (10 min), 0.33 h (20 min), 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, and 8 h after the beginning of the last dosing. For the regimens with a 3-h infusion, samples were collected before each dosing and 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, and 8 h after the beginning of the last dosing. The meropenem concentrations in these samples were assayed by HPLC, as described below. All drug samples were dispensed into 2.0-ml capped polypropylene tubes and stored at −70°C until analysis. The concentration-time profiles were modeled by fitting a one-compartment linear model with DAS version 3.0 (BioGuider Co., Shanghai, China). PK/PD parameters, including the ratio of the minimum concentration to MIC (Cmin/MIC ratio), the percentage of the dosage interval in which the drug concentrations exceeded the MIC (%T>MIC), the percentage of the dosage interval in which drug concentrations exceeded the MPC (%T>MPC), the percentage of the dosage interval in which drug concentrations were within the mutant selection window (%TMSW), and the ratio of T>MPC to TMSW (T>MPC/TMSW ratio) were calculated according to the steady-state concentration-time curves.

Serial bacterial samples were obtained from the extracapillary compartment of the hollow-fiber cartridge at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144, and 168 h. The samples were cultured to quantitatively assess the effects of the various drug regimens on the total bacterial population and the selection of bacterial subpopulations with reduced susceptibility, as described below. Total bacterial populations were quantified by inoculating, in triplicate, the 10-fold serially diluted samples (100 μl) onto drug-free MHA plates. Subpopulations with reduced susceptibility were quantified by inoculating, in triplicate, the 10-fold serially diluted samples (100 μl) onto MHA plates containing meropenem at 3× MIC. The plates were incubated at 35°C for 24 h (total population) or 72 h (subpopulations with reduced susceptibility) according to previous studies (16, 17). The bacterial colonies from each plate were counted by using an automatic colony counter (Hangzhou Shineso Co., Ltd., Zhejiang, China). Mutation was defined as occurring when any colony was present on the plates containing the drug at 3× MIC. Subsequently, the mutation frequency (MF) was calculated based on the following formula: MF = bacterial counts on plates containing the drug at 3× MIC/bacterial counts on drug-free plates. Enrichment of mutant strains was defined as occurring when the lower end of the 95% confidence interval (CI) of the bacterial colonies on the plates containing the drug at 3× MIC was at least the upper end of the 95% CI of the colonies in the control at the same point. Three colonies were randomly picked from the drug-containing plates and tested for MICs of meropenem to confirm the emergence of resistance. The limit of this detection method was 101 CFU/ml.

HPLC.

The drug concentrations of the collected samples were analyzed by HPLC (LC-20ATvp; Shimadzu, Japan) with an ODS (octadecyl silane) C18 column (250 mm by 4.6 mm, 5 μm) (Spursil; Dikma Technologies, Inc., Beijing, China). The mobile phase was a mixed solution of acetonitrile-methanol (10:3 [vol/vol] ratio of acetonitrile to methanol) and 0.05 mol/liter sodium dihydrogen phosphate at a ratio of 11:89 (vol/vol) (adjusted to pH 5.0 by phosphoric acid). The flow rate was 0.8 ml/min, and the detection wavelength was 298 nm. A total of 20 μl of sample was injected onto the HPLC instrument after being filtered by using a 0.22-μm polysulfone membrane. The calibration curve was linear over a range from 0.18 to 181.17 μg/ml (y = 6,498.23x + 5,581.00 [R2 = 0.9997], where y is the peak area of meropenem and x is the concentration of meropenem). The precision in intra- and interday assays was between 2.01% and 4.01%.

Statistical analysis.

Concentration-time curves for the six dosing regimens were simulated by DAS3.0 software (BioGuider Co., Shanghai, China). Numerical data are reported as means ± standard deviations (SD). Kaplan-Meier survival curves, with the Breslow-Gehan test, were performed with SPSS 13.0 for comparison of the bacterial killing effects (represented by the survival of bacteria at different time points) of 2.0 g q8h with a 0.5-h infusion and 2.0 g q8h with a 3-h infusion during the whole experiment. The significance level was set at a P value of <0.05.

RESULTS

MICs and MPCs.

The meropenem MICs for A. baumannii strains CSRA24 and CSRA91 were 2.0 μg/ml and 0.5 μg/ml, respectively. The MIC was 0.5 μg/ml for P. aeruginosa strain ATCC 27853. The meropenem MPC value was 28.8 μg/ml for both CSRA24 and CSRA91.

Simulated PK profiles.

The concentration-time curves of the six dosing regimens, simulated by DAS3.0 software, were close to nominal in all instances. The t1/2 value was 1.20 ± 0.36 h. For the 0.5-, 1.0-, and 2.0-g dose regimens with a 0.5-h infusion, the Cmax values for strains CSRA24 and CSRA91 were 24.07 and 24.10, 44.34 and 44.33, and 93.86 and 94.15 μg/ml, respectively. For the 0.5-, 1.0-, and 2.0-g dose regimens with a 3-h infusion, the Cmax values for strains CSRA24 and CSRA91 were 18.33 and 18.56, 9.91 and 9.87, and 37.13 and 36.10 μg/ml, respectively. The correlation coefficient between the measured and simulated values was 0.9878 ± 0.0147. Typical concentration-time curves are shown in Fig. 2.

FIG 2.

FIG 2

Open in a new tab

Mathematically simulated concentration-time curves of various meropenem regimens. (A) Regimens with 0.5- and 3-h infusions for strain CSRA24. (B) Regimens with 0.5- and 3-h infusions for strain CSRA91.

Microbiological response.

The MFs for both strains CSRA24 and CSRA91 were approximately 10−7. The effects of the six dosage-time regimens on the total-population burden for strains CSRA24 and CSRA91 are shown in Fig. 3. Initial bacterial counts at baseline were 1 × 109 to 4 × 109 CFU/ml. All dosage-time regimens, except for the lowest dosage (0.5 g), showed 3-log CFU/ml bacterial killing for both strains. For strain CSRA24, the regimens of 1.0 g with a 3-h infusion, 2.0 g with a 0.5-h infusion, and 2.0 g with a 3-h infusion showed a quick 3-log CFU/ml bacterial killing within 24 h, and the regimen of 1.0 g with a 0.5-h infusion attained a 3-log CFU/ml reduction at 72 h. For strain CSRA91, all six regimens showed a quick 3-log CFU/ml bacterial killing within 24 h.

FIG 3.

FIG 3

Open in a new tab

Effects of various regimens of meropenem on the total-population burden for strains CSRA24 (A) and CSRA91 (B).

Figure 4 illustrates the time courses of killing and enrichment of resistant mutant strains with the drug-free control (Fig. 4A) and six meropenem dosage regimens (Fig. 4B to G). For the two 0.5-g regimens, the resistant mutant strains enriched quickly after 48 h and rose to 107 to 108 CFU/ml at 168 h (Fig. 4B and C). Although the two 1.0-g regimens displayed 3-log CFU/ml bacterial killing for strains CSRA24 and CSRA91, enrichment of resistant mutant strains developed at 48 h and 72 h, respectively, and later reached 106 to 107 CFU/ml (Fig. 4D and E). From the baseline to 48 h, the MICs for the bacteria that grew on meropenem-containing plates were 8 μg/ml for CSRA24 and 4 to 8 μg/ml for CSRA91. From 72 h to 144 h, the MICs for all bacteria that grew on meropenem-containing plates were ≥16 μg/ml (16 to 64 μg/ml).

FIG 4.

FIG 4

Open in a new tab

Time courses of killing and enrichment of mutations with various regimens of meropenem. (A) Drug-free control; (B) 0.5 g with a 0.5-h infusion q8h; (C) 0.5 g with a 3-h infusion q8h; (D) 1.0 g with a 0.5-h infusion q8h; (E) 1.0 g with a 3-h infusion q8h; (F) 2.0 g with a 0.5-h infusion q8h; (G) 2.0 g with a 3-h infusion q8h.

The two 2.0-g regimens, including both the 0.5-h and 3-h infusions, demonstrated obvious bactericidal and resistance-suppressing effects on both strains. The total bacterial count fell to 101 to 102 CFU/ml for CSAR24 and fell to <101 CFU/ml (below the detection limit) for CSRA91 at the end of the experiment, and an enrichment of mutant strains was not observed (Fig. 4F and G). The Kaplan-Meier plot, with the Breslow-Gehan test, comparing the cumulated survival rates between regimens of 2.0 g with a 0.5-h infusion and 2.0 g with a 3.0-h infusion during the whole experiment demonstrated that there was no statistical difference between the two regimens (P = 0.597 and P = 1.000 for strains CSRA24 and CSRA91, respectively) (Fig. 5).

FIG 5.

FIG 5

Open in a new tab

Kaplan-Meier plot showing bacterial killing with meropenem dosage regimens of 2.0 g with a 0.5-h infusion and 2.0 g with a 3-h infusion for strains CSRA24 (A) and CSRA91 (B).

PK/PD indices for resistance suppression.

The PK/PD parameters for the six meropenem dosage regimens for both strains are shown in Table 1. The %T>MIC of all regimens was >60% for both strains CSRA24 and CSRA91. For strain CSRA24, the regimen of 2.0 g with a 3-h infusion had a %T>MIC of 100%, and the regimens of 1.0 g with a 3-h infusion and 2.0 g with a 0.5-h infusion almost reached a %T>MIC of 100%. For strain CSRA91, all 1.0- and 2.0-g regimens had a %T>MIC of 100%. However, resistance developed in both strains with the 1.0-g regimens along with the 0.5-g regimens (Table 1). Therefore, we did not consider %T>MIC to be a suitable parameter for resistance suppression. A similar phenomenon was observed for the Cmin/MIC ratio and %TMSW (Table 1). The two 2.0-g regimens (both 0.5-h and 3-h infusions) had a %T>MPC of ≥20% and a T>MPC/TMSW ratio of ≥0.25 for both strains and displayed rapid bacterial killing and resistance suppression effects, whereas other regimens had a %T>MPC of <20% or a T>MPC/TMSW ratio of <0.25 and failed to suppress enrichment of mutant strains, indicating that the %T>MPC or the T>MPC/TMSW ratio is a suitable PK/PD parameter.

TABLE 1.

PK/PD parameters of the six meropenem dosage regimensb

Strain Regimena %T>MIC Cmin/MIC ratio %T>MPC TMSW (%) T>MPC/TMSW ratio Resistance suppression
CSRA24 0.5 g with 0.5-h infusion 62 0.16 0 62 0 Failure
0.5 g with 3-h infusion 67 0.27 0 67 0 Failure
1.0 g with 0.5-h infusion 74 0.47 12 62 0.19 Failure
1.0 g with 3-h infusion 92 0.87 0 92 0 Failure
2.0 g with 0.5-h infusion 94 0.78 32 62 0.52 Success
2.0 g with 3-h infusion 100 1.01 22 78 0.28 Success
CSRA91 0.5 g with 0.5-h infusion 98 0.92 0 98 0 Failure
0.5 g with 3-h infusion 94 0.72 0 94 0 Failure
1.0 g with 0.5-h infusion 100 1.3 12 88 0.14 Failure
1.0 g with 3-h infusion 100 2.914 0 100 0 Failure
2.0 g with 0.5-h infusion 100 3.06 32 68 0.47 Success
2.0 g with 3-h infusion 100 3.784 20 80 0.25 Success
Open in a new tab
a

Meropenem was infused with the designated dose and infusion time at 8-h intervals for 7 days.

b

%T>MIC, percentage of the dosage interval in which drug concentrations are above the MIC; %T>MPC, percentage of the dosage interval in which drug concentrations are above the mutant prevention concentration; Cmin/MIC ratio, ratio of the minimum concentration to the MIC; TMSW, percentage of the dosage interval in which drug concentrations are within the mutant selection window.

DISCUSSION

In the present study, the two 2.0-g regimens, which achieved a %T>MPC of >20% or a T>MPC/TMSW ratio of >0.25, were observed to exhibit an obvious bactericidal effect and suppressed resistance. MPC is a “farsighted” concept focusing on the prevention of emergence of resistance by modifying antibiotic drug concentrations (25). According to the MPC theory, ideal regimens for antibiotics should involve not only the attainment of clinical efficacy but also the minimization of resistance emergence. However, studies on preventing carbapenem-resistant A. baumannii emergence based on MPC are scarce. Credito et al. tested MPCs of four carbapenems against 25 clinical strains of A. baumannii. Those researchers found that the MPC50 and MPC90 of meropenem were 8 and 128 μg/ml, respectively (26). In the present study, we assessed the PK/PD of meropenem for two clinical CS-MDRAB strains, CSRA24 and CSRA91, and further evaluated six routine clinical regimens for their effects on preventing resistance in the two strains. We observed that these two strains had similar MPCs but different MICs. More importantly, we revealed, for the first time, that the resistance of A. baumannii was suppressed by the optimized meropenem dosage regimens of 2 g q8h with a 0.5-h or 3-h infusion and that the %T>MPC or the T>MPC/TMSW ratio was an optimal PK/PD parameter for suppression of resistance of CS-MDRAB.

In the present study, we applied a modified HFIM to simulate human exposure and test the PK/PD of meropenem. In preliminary experiments, we observed that the degree and speed balance of drugs with a short half-life between the extracapillary and inner capillary compartments were seriously limited. This limitation occurred even when the flow rate of the extracapillary compartment reached the maximum, which could cause discordance in concentration-time curves between the extracapillary and inner capillary compartments. In order to solve this problem, reverse external circulation of the extracapillary compartment with a low flow rate was added. With this modification, the concentration-time curve between the extracapillary and central compartments can be kept consistent. Indeed, in our preliminary experiment, we validated the concordance of the PK profiles of 0.5-h and 3-h infusions of meropenem in the central and extracapillary compartments. The time to reach the Cmax in the extracapillary and central compartments was the same. The t1/2 values for the extracapillary and central compartments were 1.27 h and 1.25 h, respectively, for the regimen of 2 g with a 0.5-h infusion and 1.22 h and 1.18 h, respectively, for the regimen of 2 g with a 3-h infusion. The Cmax values for the extracapillary and central compartments were 85.33 μg/ml and 90.29 μg/ml, respectively, for the regimen of 2 g with a 0.5-h infusion and 37.13 μg/ml and 38.16 μg/ml, respectively, for the regimen of 2 g with a 3-h infusion. In addition, we observed that bacteria in the extracapillary compartment tended to concentrate close to the outlet and the bottom of the cartridge when there was only one high-rate flow passing through it. Consequently, the nonhomogeneous bacterial suspension could lead to errors in bacterial sample collection. The addition of reverse external circulation maintains homogeneity in the bacterial suspension in the extracapillary compartment.

The two strains used in the present study had different MICs but the same MPC. Whereas the MIC is the lowest drug concentration inhibiting the growth of 105 CFU/ml of the bacterium in in vitro measurements, the MPC is the lowest drug concentration preventing the growth of first-step resistant mutants and is measured based on 1010 CFU/ml. The factors that influence MPC are not fully understood. It has been reported that MIC correlates poorly with MPC and that MPC cannot be accurately predicted from MIC (27, 28). The two strains used in the present study had different drug resistance spectra and probably contained different susceptible subpopulations that determined the MPCs.

The MICs for the strains on plates containing antibiotic at 3× MIC at baseline were 4 μg/ml for CRSA91 and 8 μg/ml for CRSA24. These values are lower than the reported MICs of clinical isolates of meropenem-resistant A. baumannii (16 to 128 μg/ml) (29, 30) and showed only intermediate- and low-level resistance to meropenem. However, the MICs of the strains on antibiotic-containing plates increased gradually when enrichment occurred and reached 64 μg/ml at the end of the experiments. A similar tendency of variation in susceptibility was observed previously (31), which demonstrates that suboptimal exposure to antibiotics can lead to MIC creep of preexisting populations with reduced susceptibility and facilitate the emergence of resistance.

As a time-dependent antibiotic, a T>MIC of 40% is used as the best predictor of bacterial killing and the microbiological response of meropenem (32, 33). In our study, the %T>MIC of all regimens was >60%. However, the two 0.5-g regimens did not show an obvious bactericidal effect (3-log CFU/ml bacterial killing), which was consistent with findings from two previous studies showing that meropenem monotherapy did not achieve a 3-log CFU/ml bacterial killing effect in P. aeruginosa despite %T>MICs of 84% and 100% (16, 17). We believe that the phenomenon was caused by the enrichment of a resistance mutation that developed quickly and exerted an influence on the bactericidal effect.

In general, a prolonged infusion time can optimize %T>MIC by attaining a higher Cmin for a time-dependent antibiotic with a short t1/2. Theoretically, prolongation of the infusion time for the same dosage regimen should improve the %T>MIC. Compared to the 0.5-h infusion, prolonged infusions can optimize the efficacy of meropenem, especially for the treatment of infection caused by isolated pathogens with an MIC of 16 μg/ml (22, 32). However, when the MIC is 4 μg/ml or less, the regimen of 0.5 g with a 0.5-h infusion can also attain a T>MIC of 40%. Thus, a lower MIC and a higher dose are associated with a smaller difference in %T>MICs between continuous infusion and intermittent infusion. In the present study, the meropenem MICs for the two clinical strains CSRA24 and CSRA91 were 2.0 and 0.5 μg/ml, respectively, which corresponded to similar %T>MICs for the regimens of 2.0 g with a 0.5-h infusion and 2.0 g with a 3-h infusion. We compared the cumulative survival rates for the whole experiment period for these two 2.0-g regimens and found no statistical difference. Due to the fact that the infusion regimen of 2.0 g with a 3-h infusion did not display any advantage in the bacterial killing effect and resistance suppression compared with the infusion regimen of 2.0 g with a 0.5-h infusion, we assume that the regimen of 2.0 g q8h with a 3-h infusion may not be necessary when the MIC value of an isolated clinical strain of A. baumannii is 2 μg/ml or less. Therefore, we recommend that a regimen of 2.0 g q8h with a 0.5-h infusion be applied for target antimicrobial therapy when the MIC is known to be ≤2 μg/ml.

Traditionally, PK/PD parameters based on MIC are used to predict antibiotic efficacy, but there is now increasing interest in trying to use MPC-related PK/PD parameters to minimize the development of resistance. The correlation between MIC and MPC values is poor (6, 27, 34). Theoretically, MPC is more relevant to the development of resistance (24). Indeed, PK/PD parameters based on MPC are more predictive of bacterial resistance than MIC-related PK/PD parameters (8, 35, 36). In the present study, we also found that %T>MPC was a more preferred PK/PD parameter for suppressing mutant subpopulation proliferation than %T>MIC (Table 1). Previously, the Cmin/MIC ratio of meropenem was used as a PK/PD parameter to predict the development of P. aeruginosa resistance (16). Tam et al. demonstrated that selective amplification of P. aeruginosa subpopulations with reduced susceptibility to meropenem was suppressed with a Cmin/MIC ratio of ≥6.2 (16). However, Louie et al. reported conflicting results (17): the Cmin/MIC ratio associated with success in an isogenic MexAB pump-overexpressed mutant of P. aeruginosa PAO1 wild type was about the same as that associated with failure in the wild-type strains (1.88 versus 2.06, respectively). In the present study, we noted similar Cmin/MIC values between successful and failed regimens (Table 1), suggesting that the Cmin/MIC ratio is not a suitable index for predicting the development of resistance in A. baumannii with meropenem treatment.

According to the MPC theory, MSW is a “danger zone” for drug-selective amplification of resistant subpopulations to occur. TMSW is an important parameter in the enrichment of a mutant subpopulation (20, 24). However, we found that compared with the T>MPC/TMSW ratio, TMSW was not a suitable parameter relating to the enrichment of a mutant subpopulation, which was in agreement with findings reported previously by Kesteman et al. (37). The inability of TMSW to predict resistant mutant enrichment may be explained by the confounding influence of the actual antimicrobial concentrations at the edges of the selection window (38, 39). MSW is not a range of concentrations associated with a uniform probability of mutation selection; instead, it should be considered a range of concentrations within which multiple factors influence the likelihood of selecting for resistant microbial strains (40).

In the present study, the two 2.0-g q8h meropenem regimens (with 0.5-h or 3-h infusion) displayed resistance suppression for the two CS-MDRAB strains (CSRA24 and CSRA91). The two strains had different MICs but the same MPC, implying that MPC is critical in determining the resistance suppression of meropenem for A. baumannii. Louie et al. (17) demonstrated that meropenem monotherapy of 2.0 g q8h (1-h infusion time for PAO1 wild-type strains and 4-h infusion time for MexAB pump-overexpressing mutant strains) could not suppress resistance of P. aeruginosa. The MPCs of strains used in their study were unknown, and thus, we could not confirm whether or not a difference in MPC caused the discordant responses to similar meropenem regimens. It is noteworthy that Bulik et al. (41) reported that carbapenemase-producing Klebsiella pneumoniae and P. aeruginosa had discordant responses to the same meropenem regimen (2.0 g q8h with a 3-h infusion), although they had the same MIC. Those researchers considered that the reduced %T>MIC due to hydrolysis of carbapenemase-producing K. pneumoniae was the main reason for the discordant responses. However, different resistance mechanisms between P. aeruginosa and K. pneumoniae may also be responsible for their different responses to the same meropenem regimen, considering that one of the carbapenemase-producing K. pneumoniae strains with a %T>MIC of >40% in that study also displayed discordant responses (41). Accordingly, we presume that the different resistance mechanisms between P. aeruginosa and A. baumannii may also produce different responses to the same meropenem regimen.

In conclusion, administration of 2.0 g meropenem q8h with either a 0.5-h or 3-h infusion has an obvious bactericidal effect and suppresses resistance enrichment of CS-MDRAB. A T>MPC of ≥20% or a T>MPC/TMSW ratio of ≥0.25 can be used as a parameter for predicting suppression of meropenem resistance in CS-MDRAB. Our in vitro data support the use of a high dosage of meropenem (2.0 g q8h) for the treatment of severe infection caused by CS-MDRAB.

ACKNOWLEDGMENTS

This project was funded by the Changsha Science and Technology Fund (grant no. K1301012-31) and the Youth Foundation of The Third Hospital of Changsha (grant no. CSQJ2011-05).

Footnotes

Published ahead of print 2 September 2014

REFERENCES

  • 1.Bergogne-Berezin E, Towner KJ. 1996. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev. 9:148–165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Consales G, Gramigni E, Zamidei L, Bettocchi D, De Gaudio AR. 2011. A multidrug-resistant Acinetobacter baumannii outbreak in intensive care unit: antimicrobial and organizational strategies. J. Crit. Care 26:453–459. 10.1016/j.jcrc.2010.12.016. [DOI] [PubMed] [Google Scholar]
  • 3.Villegas MV, Hartstein AI. 2003. Acinetobacter outbreaks, 1977-2000. Infect. Control Hosp. Epidemiol. 24:284–295. 10.1086/502205. [DOI] [PubMed] [Google Scholar]
  • 4.Peleg AY, Hooper DC. 2010. Hospital-acquired infections due to gram-negative bacteria. N. Engl. J. Med. 362:1804–1813. 10.1056/NEJMra0904124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Brigante G, Migliavacca R, Bramati S, Motta E, Nucleo E, Manenti M, Migliorino G, Pagani L, Luzzaro F, Vigano FE. 2012. Emergence and spread of a multidrug-resistant Acinetobacter baumannii clone producing both the carbapenemase OXA-23 and the 16S rRNA methylase ArmA. J. Med. Microbiol. 61:653–661. 10.1099/jmm.0.040980-0. [DOI] [PubMed] [Google Scholar]
  • 6.Corbella X, Montero A, Pujol M, Dominguez MA, Ayats J, Argerich MJ, Garrigosa F, Ariza J, Gudiol F. 2000. Emergence and rapid spread of carbapenem resistance during a large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J. Clin. Microbiol. 38:4086–4095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Landman D, Babu E, Shah N, Kelly P, Olawole O, Backer M, Bratu S, Quale J. 2012. Transmission of carbapenem-resistant pathogens in New York City hospitals: progress and frustration. J. Antimicrob. Chemother. 67:1427–1431. 10.1093/jac/dks063. [DOI] [PubMed] [Google Scholar]
  • 8.Liang B, Bai N, Cai Y, Wang R, Drlica K, Zhao X. 2011. Mutant prevention concentration-based pharmacokinetic/pharmacodynamic indices as dosing targets for suppressing the enrichment of levofloxacin-resistant subpopulations of Staphylococcus aureus. Antimicrob. Agents Chemother. 55:2409–2412. 10.1128/AAC.00975-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Ba BB, Feghali H, Arpin C, Saux MC, Quentin C. 2004. Activities of ciprofloxacin and moxifloxacin against Stenotrophomonas maltophilia and emergence of resistant mutants in an in vitro pharmacokinetic-pharmacodynamic model. Antimicrob. Agents Chemother. 48:946–953. 10.1128/AAC.48.3.946-953.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Nunez ML, Martinez-Toldos MC, Bru M, Simarro E, Segovia M, Ruiz J. 1998. Appearance of resistance to meropenem during the treatment of a patient with meningitis by Acinetobacter. Scand. J. Infect. Dis. 30:421–423. 10.1080/00365549850160774. [DOI] [PubMed] [Google Scholar]
  • 11.Armand-Lefevre L, Angebault C, Barbier F, Hamelet E, Defrance G, Ruppe E, Bronchard R, Lepeule R, Lucet JC, El Mniai A, Wolff M, Montravers P, Plesiat P, Andremont A. 2013. Emergence of imipenem-resistant Gram-negative bacilli in intestinal flora of intensive care patients. Antimicrob. Agents Chemother. 57:1488–1495. 10.1128/AAC.01823-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Lee HY, Chang RC, Su LH, Liu SY, Wu SR, Chuang CH, Chen CL, Chiu CH. 2012. Wide spread of Tn2006 in an AbaR4-type resistance island among carbapenem-resistant Acinetobacter baumannii clinical isolates in Taiwan. Int. J. Antimicrob. Agents 40:163–167. 10.1016/j.ijantimicag.2012.04.018. [DOI] [PubMed] [Google Scholar]
  • 13.Lee HY, Chen CL, Wang SB, Su LH, Chen SH, Liu SY, Wu TL, Lin TY, Chiu CH. 2011. Imipenem heteroresistance induced by imipenem in multidrug-resistant Acinetobacter baumannii: mechanism and clinical implications. Int. J. Antimicrob. Agents 37:302–308. 10.1016/j.ijantimicag.2010.12.015. [DOI] [PubMed] [Google Scholar]
  • 14.Mentzelopoulos SD, Pratikaki M, Platsouka E, Kraniotaki H, Zervakis D, Koutsoukou A, Nanas S, Paniara O, Roussos C, Giamarellos-Bourboulis E, Routsi C, Zakynthinos SG. 2007. Prolonged use of carbapenems and colistin predisposes to ventilator-associated pneumonia by pandrug-resistant Pseudomonas aeruginosa. Intensive Care Med. 33:1524–1532. 10.1007/s00134-007-0683-2. [DOI] [PubMed] [Google Scholar]
  • 15.Ye JJ, Huang CT, Shie SS, Huang PY, Su LH, Chiu CH, Leu HS, Chiang PC. 2010. Multidrug resistant Acinetobacter baumannii: risk factors for appearance of imipenem resistant strains on patients formerly with susceptible strains. PLoS One 5:e9947. 10.1371/journal.pone.0009947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Tam VH, Schilling AN, Neshat S, Poole K, Melnick DA, Coyle EA. 2005. Optimization of meropenem minimum concentration/MIC ratio to suppress in vitro resistance of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 49:4920–4927. 10.1128/AAC.49.12.4920-4927.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Louie A, Grasso C, Bahniuk N, Van Scoy B, Brown DL, Kulawy R, Drusano GL. 2010. The combination of meropenem and levofloxacin is synergistic with respect to both Pseudomonas aeruginosa kill rate and resistance suppression. Antimicrob. Agents Chemother. 54:2646–2654. 10.1128/AAC.00065-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.CLSI. 2014. Performance standards for antimicrobial susceptibility testing: 22nd informational supplement. CLSI document M100-S24. CLSI, Wayne, PA. [Google Scholar]
  • 19.Zhao X, Drlica K. 2002. Restricting the selection of antibiotic-resistant mutant bacteria: measurement and potential use of the mutant selection window. J. Infect. Dis. 185:561–565. 10.1086/338571. [DOI] [PubMed] [Google Scholar]
  • 20.Firsov AA, Vostrov SN, Lubenko IY, Drlica K, Portnoy YA, Zinner SH. 2003. In vitro pharmacodynamic evaluation of the mutant selection window hypothesis using four fluoroquinolones against Staphylococcus aureus. Antimicrob. Agents Chemother. 47:1604–1613. 10.1128/AAC.47.5.1604-1613.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Louie A, VanScoy BD, Brown DL, Kulawy RW, Heine HS, Drusano GL. 2012. Impact of spores on the comparative efficacies of five antibiotics for treatment of Bacillus anthracis in an in vitro hollow fiber pharmacodynamic model. Antimicrob. Agents Chemother. 56:1229–1239. 10.1128/AAC.01109-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lee LS, Kinzig-Schippers M, Nafziger AN, Ma L, Sorgel F, Jones RN, Drusano GL, Bertino JS., Jr 2010. Comparison of 30-min and 3-h infusion regimens for imipenem/cilastatin and for meropenem evaluated by Monte Carlo simulation. Diagn. Microbiol. Infect. Dis. 68:251–258. 10.1016/j.diagmicrobio.2010.06.012. [DOI] [PubMed] [Google Scholar]
  • 23.Leelarasamee A, Rongrungruang Y, Trakulsomboon S, Pongpech P, Thanawattanawanich P, Jithavech P. 2008. Bioequivalence, antibacterial activity and therapeutic outcome of a generic meropenem (Mapenem). J. Med. Assoc. Thai. 91:980–988. [PubMed] [Google Scholar]
  • 24.Blondeau JM. 2009. New concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach. Vet. Dermatol. 20:383–396. 10.1111/j.1365-3164.2009.00856.x. [DOI] [PubMed] [Google Scholar]
  • 25.Canton R, Morosini MI. 2011. Emergence and spread of antibiotic resistance following exposure to antibiotics. FEMS Microbiol. Rev. 35:977–991. 10.1111/j.1574-6976.2011.00295.x. [DOI] [PubMed] [Google Scholar]
  • 26.Credito K, Kosowska-Shick K, Appelbaum PC. 2010. Mutant prevention concentrations of four carbapenems against Gram-negative rods. Antimicrob. Agents Chemother. 54:2692–2695. 10.1128/AAC.00033-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Drlica K, Zhao X, Blondeau JM, Hesje C. 2006. Low correlation between MIC and mutant prevention concentration. Antimicrob. Agents Chemother. 50:403–404. 10.1128/AAC.50.1.403-404.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Marcusson LL, Olofsson SK, Komp Lindgren P, Cars O, Hughes D. 2005. Mutant prevention concentrations of ciprofloxacin for urinary tract infection isolates of Escherichia coli. J. Antimicrob. Chemother. 55:938–943. 10.1093/jac/dki136. [DOI] [PubMed] [Google Scholar]
  • 29.Liang W, Liu XF, Huang J, Zhu DM, Li J, Zhang J. 2011. Activities of colistin- and minocycline-based combinations against extensive drug resistant Acinetobacter baumannii isolates from intensive care unit patients. BMC Infect. Dis. 11:109. 10.1186/1471-2334-11-109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wang H, Chen M, Ni Y, Liu Y, Sun H, Yu Y, Yu X, Mei Y, Liu M, Sun Z, Chu Y, Hu Z, Huang X. 2010. Antimicrobial resistance among clinical isolates from the Chinese Meropenem Surveillance Study (CMSS), 2003-2008. Int. J. Antimicrob. Agents 35:227–234. 10.1016/j.ijantimicag.2009.11.010. [DOI] [PubMed] [Google Scholar]
  • 31.Firsov AA, Smirnova MV, Lubenko IY, Vostrov SN, Portnoy YA, Zinner SH. 2006. Testing the mutant selection window hypothesis with Staphylococcus aureus exposed to daptomycin and vancomycin in an in vitro dynamic model. J. Antimicrob. Chemother. 58:1185–1192. 10.1093/jac/dkl387. [DOI] [PubMed] [Google Scholar]
  • 32.Jaruratanasirikul S, Sriwiriyajan S, Punyo J. 2005. Comparison of the pharmacodynamics of meropenem in patients with ventilator-associated pneumonia following administration by 3-hour infusion or bolus injection. Antimicrob. Agents Chemother. 49:1337–1339. 10.1128/AAC.49.4.1337-1339.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ong CT, Tessier PR, Li C, Nightingale CH, Nicolau DP. 2007. Comparative in vivo efficacy of meropenem, imipenem, and cefepime against Pseudomonas aeruginosa expressing MexA-MexB-OprM efflux pumps. Diagn. Microbiol. Infect. Dis. 57:153–161. 10.1016/j.diagmicrobio.2006.06.014. [DOI] [PubMed] [Google Scholar]
  • 34.Linde HJ, Lehn N. 2004. Mutant prevention concentration of nalidixic acid, ciprofloxacin, clinafloxacin, levofloxacin, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin for Escherichia coli under different growth conditions. J. Antimicrob. Chemother. 53:252–257. 10.1093/jac/dkh036. [DOI] [PubMed] [Google Scholar]
  • 35.Firsov AA, Strukova EN, Shlykova DS, Portnoy YA, Kozyreva VK, Edelstein MV, Dovzhenko SA, Kobrin MB, Zinner SH. 2013. Bacterial resistance studies using in vitro dynamic models: the predictive power of the mutant prevention and minimum inhibitory antibiotic concentrations. Antimicrob. Agents Chemother. 57:4956–4962. 10.1128/AAC.00578-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zhao X, Drlica K. 2008. A unified anti-mutant dosing strategy. J. Antimicrob. Chemother. 62:434–436. 10.1093/jac/dkn229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Kesteman AS, Ferran AA, Perrin-Guyomard A, Laurentie M, Sanders P, Toutain PL, Bousquet-Melou A. 2009. Influence of inoculum size and marbofloxacin plasma exposure on the amplification of resistant subpopulations of Klebsiella pneumoniae in a rat lung infection model. Antimicrob. Agents Chemother. 53:4740–4748. 10.1128/AAC.00608-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Cui J, Liu Y, Wang R, Tong W, Drlica K, Zhao X. 2006. The mutant selection window in rabbits infected with Staphylococcus aureus. J. Infect. Dis. 194:1601–1608. 10.1086/508752. [DOI] [PubMed] [Google Scholar]
  • 39.Firsov AA, Lubenko IY, Smirnova MV, Strukova EN, Zinner SH. 2008. Enrichment of fluoroquinolone-resistant Staphylococcus aureus: oscillating ciprofloxacin concentrations simulated at the upper and lower portions of the mutant selection window. Antimicrob. Agents Chemother. 52:1924–1928. 10.1128/AAC.01371-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Martinez MN, Papich MG, Drusano GL. 2012. Dosing regimen matters: the importance of early intervention and rapid attainment of the pharmacokinetic/pharmacodynamic target. Antimicrob. Agents Chemother. 56:2795–2805. 10.1128/AAC.05360-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Bulik CC, Christensen H, Li P, Sutherland CA, Nicolau DP, Kuti JL. 2010. Comparison of the activity of a human simulated, high-dose, prolonged infusion of meropenem against Klebsiella pneumoniae producing the KPC carbapenemase versus that against Pseudomonas aeruginosa in an in vitro pharmacodynamic model. Antimicrob. Agents Chemother. 54:804–810. 10.1128/AAC.01190-09. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES