In Vitro And In Vivo Antimicrobial Combinations For Extensively Drug Resistant Acinetobacter Baumanni

Objective To investigate the distribution and antimicrobial resistance of Acinetobacter baumannii infection in Anhui province, providing references of clinical medication. To detect carbapenemase gene in carbapenem-resistant A. baumannii(CRAB), supplying further basis to study the mechanisms of drug resistance. To evaluate the in vitro activity of colistin(COL) alone and in combination with fosfomycin(FOS), sulbactam(SUP), imipenem(IMP), rifampicin(RIF) and levofloxacin(LVX) against extensively drug resistant A. baumannii(XDRAB). To performe synergy studies using the microtitre plate chequerboard assay, time–kill methodology and the mutant prevention concentrations(MPCs) in vitro. Use a simple invertebrate model(Galleria mellonella) to assess the in vivo activity of antimicrobial therapies.Materials and Methods Isolates A total of 120 A. baumannii clinical strains were isolated from patients hospitalized at 21 different hospitals located in Anhui, China from 2012 to 2013. Among these strains, 95 were from sputum, 12 from urine, 9 from secretion, 4 from blood. Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as quality control strains for antimicrobial susceptibility testing. A. baumannii ATCC19606 was also used as control in time-kill study and MPC assay. All the control strains were stored in Anhui Center of Surveillance of Bacterial Resistance.Methods 1. Minimum inhibitory concentration(MIC) values were determined with agar dilution method according to Clinical and Laboratory Standards Institute guideline(CLSI, 2014). 2. Carbapenem-resistant A. baumannii(CRAB) isolates were selected by the MICs. The carbapenemase genes(blaOXA-23-like gene, blaOXA-24-like gene, blaOXA-51-like gene, blaOXA-58-like gene, blaVIM, blaNDM, blaIMP and blaKPC gene) were detected by polymerase chain reaction(PCR). 3. We selected XDRAB through methods of genotypic test and phenotypic test. Antimicrobial combination testing was used to study the in vitro activity of colistin-based combinations including COL+RIF, COL+LVX, COL+IMP, COL+SUP and COL+FOS against XDRAB. 4. An A. baumannii type strain ATCC19606, a colistin-resistant strain AB19606 R and two clinical isolates of XDRAB isolated in Anhui, China were uesd to investigate the in vitro and in vivo efficacy of levofloxacin-colistin combination. Synergy studies were performed using the microtitre plate chequerboard assay and time–kill methodology. The MPCs were also studied in vitro. G. mellonella infection model was used to text the efficacy of COL alone and in combination with LVX in A. baumannii infection in vivo.Results 1. Our results indicated that all the 13 antibiotics we tested didn’t show good susceptibility against the 120 A. baumannii isolates. Among the 13 antibiotics, AZM(14.3%) and CRO(14.7%) showed the lowest sensitivity. Susceptibility rates of IMP, MEM, TZP, AMK and CIP were 38.9%, 31.5%, 37.1%, 40.4%, and 33.6%, respectivily. 70 out of 120 isolates showed resistance to IMP.2. All the 70 CRAB strains were found to carry blaOXA-23 and blaOXA-51 gene. blaOXA-24, blaOXA-58, blaVIM, blaIMP, blaNDM and blaKPC gene were not found in all the tested strains. 3. Comparing to the single-drug strategy, all the antibiotic combinations showed enhanced activity. COL plus FOS/ RIF/ IMP/ SUP/ LVX showed synergistic effect with the ratios of 50%, 72%, 88%, 92% and 64%, respectively. 4. The LVX-COL combination was bactericidal against the COL-susceptible clinical isolate. In checkerboard assays, synergy was observed between COL and LVX. The MPCs of COL were very high in the two tested strains(MPCs, >128 mg/L) with CST monotherapy. When 2mg/L LVX was used in combination with COL, a decrease in MPCs of COL could be found(MPCs, 16mg/L and 16 mg/L, respectively). COL mutation prevention index(MPIs) decreased >8-fold and >16-fold for the strains of ATCC19606 and COL-susceptible clinical isolate, respectively. Treatment of G. mellonella larvae infected with lethal doses of A. baumannii resulted in significantly enhanced survival rates when LVX was given with COL in vivo.Conclusions 1. A. baumannii is an important pathogen that causes hospital-acquired infections in Anhui province, China. The increasing rate of A. baumannii infection and the emergence of XDRAB further limits the treatment options. Our research can provide a new way of thinking for treatment options. 2. In our study, the blaOXA-23 and blaOXA-51 genes are more prevalent than the other genes in tested A. baumannii strains. 3. COL is the last option in the treatment of infections caused by XDR A. baumannii. To date, most A. baumannii strains are still susceptible to COL. However, in recent years COL-resistant A. baumannii clinical isolates and heteroresistance to COL have been reported. Considering the limitations of single-drug therapy, combination therapy should be a good alternative choice of treatment.