Evaluation of the Synergistic Antibacterial Effects of Rifampicin-based Combination Therapies for the Management of Infections due to Carbapenem-Resistant Acinetobacter baumannii

Abstract

The increasing spread of carbapenem-resistant Acinetobacter baumanii (CRAB) is critical to public health due to the lack of treatment options and increased mortality rate. Herein, the synergistic and bactericidal effects of rifampicin in combination with conventional antibiotics were evaluated, and the molecular pathways of antibiotic resistance were predicted using bioinformatics and whole-genome sequencing. Phenotypic analysis including efflux pump detection and antibacterial activity of combination were evaluated against established biofilm cells using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and MTT assay, respectively. From the evaluations, about 89% of the 218 CRAB clinical isolates tested in the study showed resistance to rifampicin at zones of inhibition ≤16 mm, 9% were intermediate (17–19 mm), and 1% were susceptible (≥20 mm). The antibiotic-resistant profiles of the isolates were investigated in 31 representative clinical isolates. A total of (22/31) 71% and 94% (29/31) isolates demonstrated susceptibility to tigecycline and minocycline, respectively. The isolates showed multidrug resistance and exhibited a 100% resistance to gentamycin or tobramycin at Minimum inhibitory concentration (MIC) ≥ 1024 µg/mL, and chloramphenicol at ≥ 16µg/mL. Five isolates out of 20 were resistant to colistin at MIC = 4 µg/mL whereas 15 were intermediate at MIC=2 µg/mL. Combination therapy of rifampicin slightly improved antibiotic potency with synergism in 10/31, 7/31, 2/31, 4/31, 5/31, and 8/9 when combined with imipenem, meropenem doripenem, tigecycline, minocycline, and colistin respectively. In addition, time-kill kinetic revealed a bactericidal effect at higher concentrations and a bacteriostatic at lower concentrations. The combination of rifampicin plus imipenem and doripenem was bactericidal against TR123 at 1/4 MIC of rifampicin and 1/4MIC of doripenem and imipenem. Rifampicin combined with tigecycline or minocycline was bactericidal at 1/4MIC rifampicin plus 1/4 MIC of the antibiotics against TR131 out of three representative isolates. Rifampicin combination with tigecycline disclosed a 2-2.5 log reduction in CFU at all the combined concentrations including at 1/4 MIC rifampicin and 1/4 MIC of tigecycline. Rifampicin plus ciprofloxacin resulted in a 99% killing against TR023 out of three isolates indicating a bactericidal activity at MIC rifampicin plus 1/2 MIC ciprofloxacin, 1/2 MIC rifampicin plus 1/2 MIC ciprofloxacin and 1/2 MIC rifampicin plus 1/4 MIC ciprofloxacin. Rifampicin plus chloramphenicol or trimethoprim-sulfamethoxazole combinations were not effective at MIC and sub- Inhibitory concentrations among all the isolates used. The combination therapy of rifampicin and fosfomycin disclosed a bacteriostatic effect against two representative isolates. Notably, rifampicin with colistin exhibited bactericidal activity in three out of four representative isolates ( PT046, TR069, and TR082) at 1/4 MIC rifampicin plus 1/4 colistin. Antibacterial resistant mechanism assessment indicated a 4-fold reduction in the MIC of rifampicin in the presence of the efflux pump inhibitor CCCP in isolates SK056 and SK067 out of the 15 tested isolates. Biofilm viability test by MTT assay revealed a dose-dependent decrease of cell viability of established bacterial biofilm at 4 MIC rifampicin + 2 MIC carbapenems with a percentage reduction of 44–75%, compared with monotherapies at 16 MIC. The pan-genomic study of the isolates demonstrated a progressive evolution with 58% of accessory genes in the matrix. Seven of the ten sequenced isolates were of sequence type 2 (ST2), while one isolates each belongs to ST164, ST16, and ST25. Furthermore, 11 plasmids, 34 AMR genes, and 65 virulent genes were predicted to confer MDR. The blaOXA-23 blaADC-25, blaOXA-66, blaPER-7, aph(6)-Id, armA, and arr-3 were prevalent among the isolates. Sequence alignment of the bacteria genome to a reference strain revealed a deleterious mutation in the rpoB gene in 4 out of 29 isolates. Colistin-resistance-associated mutation on the PmrB and PmrC (two-component system), LPS biosynthetic protein lpxD, emrA, and emrB genes were detected among the five isolates that demonstrated resistance to colistin. This research emphasizes the specificity of isolates to antibiotics and suggests that the rifampicin combination with colistin, tigecycline, minocycline, imipenem, meropenem, and doripenem may be a potential treatment option for the management of CRAB isolates with low rifampicin minimum inhibitory concentration. It also demonstrates that the genotypic and phenotypic characterization of antimicrobial resistance (AMR) in CRAB clinical isolates may lessen the burden of AMR surveillance.