In vivo bactericidal effect of colistin–linezolid combination in a murine model of MDR and XDR Acinetobacter baumannii pneumonia

Recently, paradoxical combinations of colistin with anti-Gram-positive bacterial agents were introduced as a treatment alternative for multidrug-resistant Acinetobacter baumannii (MDRAB) infection. We assessed the therapeutic efficacy of the colistin–linezolid combination regimen in vitro and in a murine model of Acinetobacter baumannii pneumonia. A multidrug-resistant clinical strain (MDRAB31) and an extensively drug-resistant clinical strain (XDRAB78) were used in this study. The survival rates of mice and bacterial counts in lung tissue were used to assess the effects of colistin–linezolid combination. The survival rates of colistin–linezolid combination groups significantly increased compared with colistin groups for MDRAB31 (72% versus 32%, P = 0.03) and for XDRAB78 (92% versus 68%, P = 0.031). The colistin–linezolid combination groups significantly reduced the bacterial counts in lung tissue compared with colistin groups for MDRAB31 and for XDRAB78 (P < 0.05). The colistin–linezolid combination had a bactericidal and synergistic effect compared with colistin alone in time-kill assay and in murine model of pneumonia. Our data demonstrated the synergistic effect of colistin–linezolid combination regimen as a treatment alternative for the severe pulmonary infection caused by MDRAB and XDRAB.

Time-kill assays. Figure 1 shows the time-kill curves of both A. baumannii strains. Although bactericidal action against MDRAB31 and XDRAB78 was detected using colistin alone at 0.5 μg/ml (1 × MIC), rapid regrowth was recorded after 8 h. The colistin-linezolid combination showed bactericidal and synergistic effects against MDRAB31 and XDRAB78, with minimal regrowth.
Survival rates. The survival curves of both strains are shown in the Fig. 2. For MRDAB31 strain, the survival rates within 4 days were 24% in control group, 20% in linezolid group, 32% in colistin group and 72% in colistin-linezolid combination group. The survival rate of colistin-linezolid combination group significantly increased compared with colistin group (72% versus 32%, P = 0.03). Significant differences in survival rates were observed between colistin-linezolid combination group and the other groups (P < 0.05). Comparing with MRDAB31 strain, a lower lethality rate of 48% was observed in control group for XDRAB78 strain. Survival rates of 52%, 52%, 68% and 92% were observed in the control group, linezolid group, colistin group, colistin-linezolid combination group, respectively. There were significant differences in survival rates between colistin-linezolid combination group and colistin group (92% versus 68%, P = 0.031), and between the colistin-linezolid combination group and the other two groups (P < 0.05). Figure 3 shows the evolution of bacterial loads of both strains in each group in lungs after intra-tracheal inoculation. The mean bacterial counts (log 10 CFU/g of lung) after inoculation 0 h and 4 h were respectively 5.89 ± 0.41 log 10 CFU/g and 8.08 ± 0.65 log 10 CFU/g for MDRAB31 strain, 5.84 ± 0.49 log 10 CFU/g and 8.22 ± 0.57 log 10 CFU/g for XDRAB78 strain. Colistin monotherapy did not show bacterial effects against both strains. The colistin-linezolid combination groups significantly reduced the bacterial counts in lung tissue compared with colistin groups for MDRAB31 and for XDRAB78 (P < 0.05). The colistin-linezolid combination demonstrated bactericidal effects and synergistic effects compared with colistin monotherapy on both strains. The bacterial counts continued to drop, and fell to 2.32 ± 0.41 log 10 CFU/g for MDRAB31 and 2.91 ± 1.29 log 10 CFU/g for XDRAB78 at the 76 h.

Discussion
Our study demonstrated the synergistic activity of colistin-linezolid combination in a murine model of MDR and XDR A. baumannii pneumonia for the first time.
Colistin, which was abandoned due to its neurotoxicity and nephrotoxicity, has been reused to fight MDR strains infection 16 . Like in other previous studies 17 , colistin exhibited good activity against A. baumannii at first and rapid regrowth was recorded subsequently in time-kill curves. In addition, in Dudhani et al. study 18 , similar phenomenon of colistin monotherapy appeared in murine model of A. baumannii infection. In 2006, Li et al. 19 defined colistin heteroresistance of A. baumannii as the emergence of resistant subpopulations from an otherwise susceptible population. Mutant prevention concentration (MPC) studies and pharmacokinetics (PKs)/ pharmacodynamics (PDs) studies revealed that monotherapy of colistin was unable to prevent the development of resistance, and may be substantially caused therapeutic failure 20,21 . Those views may explain the phenomenon of bacterial regrowth in vitro observed here when using colistin-monotherapy.
Linezolid is widely used against most Gram-positive bacteria by binding to rRNA on the 30S and 50S ribosomal subunits to prevent the synthesis of protein 22 . The intrapulmonary concentrations of linezolid in epithelial lining fluid were 64.3 ± 33.1 μg/ml at 4 h and 24.3 ± 13.3 μg/ml at 12 h in healthy volunteers for the recommended dosage regimen (600 mg every 12 h) 23 . And an important part of the application of linezolid was the treatment of pulmonary infection 24 . In this study, the MICs of linezolid in combination were significantly lower than the intrapulmonary concentrations in epithelial lining fluid for the recommended dosage regimen. So it provided the possibility of the linezolid-colistin combination to fight A. baumannii pneumonia.
Recently, linezolid was introduced as a colistin-combination option for A. baumannii infection basing on the mechanism that colistin exerted a subinhibitory permeabilizing effect allowing the second drug to enter cells 14 . Armengol et al. 12 from biophysical point of view, and Ritcher et al. 25     www.nature.com/scientificreports/ strains. Although all the A. baumannii strains were high resistance to linezolid, synergy between linezolid and colistin was observed in checkerboard assay and time-kill assay 15 . In addition, another study in checkerboard assay found when the sub-inhibitory concentrations of colistin were incorporated to colistin-linezolid combination, the MICs of linezolid in combination against all A. baumannii strains decreased dramatically, ranging from 4 to 16 μg/ml 13 . And in our time-kill assay and in vivo study, the colistin-linezolid combination also showed bactericidal and synergistic effects. More importantly, there were significant differences in survival rates between colistin-linezolid combination group and colistin group in a murine model of A. baumannii pneumonia. Further clinical trials are necessary to confirm this result. In this study, the result of colistin-linezolid combination for XDRAB78 strain was additive in checkerboard assay, while the result of combination was synergy in time-kill assay, when we used 1 × MIC colistin in combination. Sub-inhibitory concentrations of colistin could increase linezolid uptake in A. baumannii, and the accumulation of linezolid was colistin-concentration dependent 12 . Moreover, in the emergence of high concentrations of colistin (7 μg/ml), antimicrobials accumulation was obviously increased in E. coli 25 . This may explain above phenomenon and highly lights, even in combination therapies, the importance of optimizing the therapeutic regimen of colistin basing on PK/PD.
Although we did not detect the PKs of the bacterial agents used in this study, antibiotic doses were based on other published studies of same species murine model of pneumonia.

Conclusion
Colistin-linezolid combination therapy had a bactericidal and synergistic effect in vivo in a murine model of MDR and XDR A. baumannii pneumonia.

Materials and methods
Strains. Two clinical isolates of A. baumanni, which were isolated from two unrelated pulmonary infection patients with bacteremia, were studied. The first (MDRAB31) was tested to be a MDR A. baumanni strain (resistant to mezlocillin, piperacillin-tazobactam, cefepime, ceftazidime, ciprofloxacin, levofloxacin) and the second (XDRAB78) was tested to be a XDR A. baumanni strain (resistant to mezlocillin, piperacillin-tazobactam, cefepime, ceftazidime, ciprofloxacin, levofloxacin, gentamicin, amikacin, imipenem, meropenem) by the VITEK 2 testing system (bioMérieux, Craponne, France). The quality control stain used as internal standard for each batch of tests was Pseudomonas aeruginosa ATCC 27853. All strains were stored separately at − 70 °C in form of powder in airtight vials before being subcultured on containing 5% sheep blood Columbia plates (bioMérieux, Shanghai, China).
XuZhou Central Hospital Ethics Committee approved all experimental protocols, and the methods were carried out in strict accordance with the approved protocols. Informed consent was obtained from all subjects. Murine model of A. baumannii pneumonia. Healthy female, specific pathogen-free, immunocompetent, 6-week-old female C57BL/6 J mice (Animal Core Facility, Nanjing Medical University, Nanjing, Jiangsu, China) weighing 17-19 g were used for the A. baumannii pneumonia model. The mice were housed 5 per cage and had access to chow and drink ad libitum throughout the study. Cyclophosphamide (150 mg/kg of body weight in 0.15 ml) was intraperitoneally injected into the animals to render transiently neutropenic on days 4 and 1 before inoculation. All mice were anesthetized by a mixture of isoflurane and oxygen. Then 50 μl bacterial suspension containing 10 9 CFU/ml was inoculated by a needle through the nose. Antibiotics were initiated 4 h after inoculation and were administered intraperitoneally. www.nature.com/scientificreports/ Experimental Animal Welfare and Ethics Committee of the Nanjing Medical University approved all animal experiments, and the methods were carried out in strict accordance with the approved protocols.

Study groups.
In the first section of the experiment, the mice were randomized into four groups of 25 mice each group for each A. baumannii strain. The first group received colistin (125,000 UI/kg, every 6 h; 500,000 UI/ kg/day) 30 . The second group received linezolid (50 mg/kg, every 12 h) 31 . The third group received the combination of colistin (125,000 UI/kg, every 6 h) and linezolid (50 mg/kg, every 12 h). The control group received saline (every 12 h). The outcome was observed by survival rate.
In the second section of the experiment, the mice were also randomized into four groups (20 mice in each group): saline, colistin, linezolid, colistin-linezolid combination, for each A. baumannii strain. Three mice per group at 0 h, 4 h, 28 h, 52 h, 76 h were euthanized before next dosing and lungs were removed for quantitative bacteriological studies. Lungs were weighted and then homogenized in 1 ml of saline. 0.01 ml serial tenfold dilutions of homogenates were plated on containing 5% sheep blood Columbia plates at 37 °C for 24 h. The definition of bactericidal activity of agents was a ≥ 3 log 10 reduction compared with the bacterial concentration before first dosing. The definition of synergy was a ≥ 2 log 10 reduction in CFU/g for the combination compared with the most active single agent 32 .
Statistical analysis. The data were presented as the means ± SD (standard deviations). Log-rank test and Kruskal-Wallis test were used to compare survival and bacterial counts in lung tissue respectively between groups of each strain. In all experiments, statistical significance was accepted when the P value was < 0.05.