The synergistic activity of antibiotic combinations against colistin-resistant Acinetobacter baumannii clinical isolates and its mechanisms related to colistin resistance

The increasing development of colistin resistance in Acinetobacter baumannii clinical isolates is a global concern. Due to the limitation of effective antibiotics, the antibiotic combination therapies are the alternative choices. Here, we investigated the prevalence of colistin resistant A. baumannii (CoR-AB) clinical isolates and their resistance mechanisms, and examined the in vitro synergistic activities of the antibiotic combinations against CoR-AB. Among 341 carbapenem-resistant Acinetobacter spp., 317 (92.0%) were identified as A. baumannii by gyrB multiplex PCR. The rate of colistin resistance was 15.1% in A. baumannii isolates with MIC range of 0.125 – 64 mg/L. A total of 30 representative CoR-AB isolates were selected for the studies of colistin resistance mechanisms and synergistic activity of antibiotic combinations. The phosphoethanolamine (pEtN) addition to lipid A was found in 27 of 30 (90%) CoR-AB clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). In PmrC, The N248D mutation was found in 30 CoR-AB isolates. The 22 of 30 (73.3%) CoR-AB isolates harbored identical substitution in 48 amino acid positions and the other 8 isolates harbored different amino acid substitutions. For PmrA, the 4 amino acid stitutions were found in 22 (73.3%) CoR-AB isolates. Twenty-five amino acid substitutions were found in PmrB. Twenty-nine (96.7%) isolates contained the A227V mutation in PmrB that was previously described as the cause of colistin resistance. Alterations of the lipopolysaccharide production enzyme (LpxD) were identified at V3A and E117K (6.7% and 93.3%, respectively). Other mechanisms including overexpression of efflux pumps or mutations in genes associated with lipopolysaccharide production might be associated with colistin resistance in our isolates. The synergistic activities of colistin/sulbactam, colistin/fosfomycin, and sulbactam/fosfomycin combinations were identified in 86.7%, 33.3%, and 70% of CoR-AB isolates, respectively, by checkerboard assay. The time-kill study confirmed the synergistic effect of colistin/sulbactam with various concentration combinations against 6 representative CoR-AB isolates. In conclusion, this study indicated the increasing prevalence of colistin resistance in A. baumannii. The pEtN modification is the major mechanism of colistin resistance, which is contributed to the mutations in PmrC, PmrB, and PmrA proteins. The colistin/sulbactam combination could be used as alternative therapy for CoR-AB infections.