| Objective:To investigate the carbapenemase genotype of carhapenem-resistant Acinetobacter baumannii(CRAB) isolated from 301 Hospital, and in vitro, in vivo and intracellular activity of tigecycline in combination with cefoperazone/sulbactam against CRABMethods:(1)15 types of carbapenemase genes in the 87 CRAB isolates were amplified and sequenced. (2) In vitro antibiotic interactions were determined by chequerboard and time-kill assays. (3) The C57BL/6 mice were inoculated by intraperitoneal injection of minimal lethal dose of a CRAB isolate (AB-8, blood-borne, extensively drug-resistant). Bacterial clearance was compared in organs at 6h and 20h after inoculation, and organ tissue pathology was observed. (4) A 24-h model of RAW264.7 monocyte infection with a CRAB isolate (AB-7, blood-borne, sensitive to gentamicin) was developed. Phagocytosis was initiated at a bacterium-macrophage innocubation, and after removal of unphagocytosed and adherent CRAB with 400 μg/mL gentamicin, the cells were cultured for 24h. Morphological studies were performed by Wright’s staining, scanning electron microscope and transmission electron microscope. The model was used to quantitatively assess antibiotic activity against intracellular and extracellular bacteria by bacteriostatic concentrations (Cs) and maximal relative efficacies (Emax).Result:(1) Four carbapenemases of the 87 CRAB isolates were detected:blaoxA-51-like (97.7%), blaOXA-23-like (95.4%), blaTEM(85.1%) and blaOXA-58-like (2.3%). (2) A total of 12.6% isolates were susceptible to cefoperazone/sulbactam and 57.5% isolates’ MIC were< the Cmax of tigecycline in serum when used alone. However, a total of 44.8% isolates were susceptible to cefoperazone/sulbactamand 96.6% isolates’ MIC< the Cmax of tigecycline in serum when used in combination. Time-kill assays aslo showed synergistic activities of tigecycline and cefoperazone/sulbactam in AB-8, AB-14 isolates and AB-44, which were eradicated at 24h. However, the eradications were not observed in combinations of tigecycline and sulbactam (P<0.05). (3) In vivo tigecycline and the combination produced significant decreases in colony counts of blood and four organs compared to the untreated control group. Combination produced significant decreases in colony counts of lung and liver, compared to those in tigecycline (-0.63±0.11、-0.53±0.23) (P<0.05). The colony counts in blood and organs did not significantly different between combination therapy of tigecycline with cefoperazone/sulbactam and combination of tigecycline with sulbactam alone at 4h, but after 18 h of treatment, differences in liver between two combination therapies were statistically significant (4.40±0.17 VS 4.86±0.12 CFU/g, P<0.05) (4) A 24-h model of RAW264.7 monocyte infection with CRAB was developed in which bacteria are seen multiplying in vacuoles by electron microscopy. Cefoperazone/sulbactam required 24 h of incubation to prevent intracellularly bacterial growth while tigecycline reached a bacteriostatic effect after 6 h of incubation when used at extracellular concentrations lxMIC. Intracellular time-kill curves were found to be undistinguishable between combination and tigecycline alone. Intracellularly, bacteriostatic concentrations (Cs) values remained unchanged for cefoperazone/sulbactam, but were approximately only a tenth of MIC for tigecycline, whereas Emax values were markedly reduced, reaching-2.37log10 CFU/mg prot for tigecycline,-1.53log10 CFU/mg prot for cefoperazone/ sulbactam.Conclusions:(1)blaOXA-52-like, blaOXA-23-like, blaTEM and blaOX-58-like are main types of CRAB in the experiment. (2) In vitro and in vivo study suggest that the combination of tigecycline with cefoperazone/sulbactam has high synergistic activity against CRAB isolates, which was aslo superior than the combination of tigecycline with sulbactam alone. (3) A clear superiority of tigecycline over cefoperazone/sulbactam is showed when dealing with intracellular forms of CRAB. Combinations are not more effective than tigecycline against intracellular CRAB. |
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