Molecular Epidemiology And Resistance Mechanisms Of Multi-drug Resistant Acinetobacter Baumannii

Part 1Molecular Epidemiology of Multi-drug Resistant Acinetobacter baumanniiObjective:The purpose of this study was to investigate the molecular epidemiology multidrug-resistant Acinetobacter baumannii (MDRAB). Methods:96 A.baumannii were collected from Huadong Hospital Affiliated to Fudan University from 2011-2013,including 55 MDRAB. Multilocus sequence tying (MLST) were used as the method of molecular typing for MDRAB, eBURST was applied to analyze the data of MLST and infer the evolutionary. The homology of A. baumannii isolates was detected by Pulsed-field gel electrophoresis (PFGE). Results:According to the MLST, the 55 A.baumannii isolates could be grouped into five STs, including three existing STs(ST208,1-3-3-2-2-97-3; ST191, 1-3-3-2-2-94-3;ST195,1-3-3-2-2-96-3) and four new STs (STnl,1-3-G1-2-2-94-3; STn2,1-12-3-71-2-145). Seven pulsotype were identified in 55 A.baumannii by PFGE.52 related (≥80%similartity) or indistinguishable (100% homology) MDRAB clones were grouped into five clusters(A to E) and three MDRAB was genetically unrelated to the others. Cluster A and Cluster B revealed a similarity coefficient value of≥89%, which makes them epidemiologically very closely related. ST208, ST191,ST195,STnl were grouped as clonal comples(CC) 92. ST208,ST191,ST195 were SLVs of ST92, which were different with each other at gpi locus.Compared with the founder ST92, STn2 (1-12-3-71-2-145-3) had different alleles at gyrB,recA and gpi of the 7 loci and was not grouped as clonal comples 92. STn2 had a unique PFGE profile and was not homologous with other pulsetype, which was consistant with eBURST analysis. Conclusions:ST208 and ST191 were the dominant STs of MDRAB and CC92 was the main MDRAB clone complex in our hospital. CC92 spread across regions with high antibiotic resistance.The strains of CC92 can survive under clinical antibiotic pressure and posses the risk of nosocomial prevalence. PFGE has higher resolution than MLST. However, MLST is more precise in the interpreting of bacterial genetic relatedness.Part 2Resistance Profiles and Molecular Mechanisms of Multi-drug Resistant Acinetobacter baumanniiObjective:The purpose of this study was to investigate the drug resistance of multidrug-resistant Acinetobacter baumannii (MDRAB). Methods:PCR and sequencing was used to identify the main resistance genes in 55 MDRAB isolates. Minimum inhibitory concentrations were determined for key antibiotics. The genetic environment of these genes and the gyrA and parC QRDRs was also analysed by PCR amplification and sequencing.Results:CRAB was more resistant to cephalosporin, cefotaxime, cefepime, cefoperazone/sulbactam and piperacillin/tazobactam, levofloxacin, gentamycin, amikacin, polymyxin than CSAB(P<0.01). The drug resistance rate of A.baumannii derived from ICU to piperacillin/tazobactam,ceftazidime, cefotaxime, levofloxacin, gentamycin, cefepime, polymycin B were higher than that of the isolates derived from non-ICU(P<0.05). The antibiotic resistance of A.baumannii derived from ICU to common antibiotics other than cefoperazone/sulbactam and polymycin B were comparatively higher, (P< 0.05).96 A.baumannii naturally carry blaOXA-66, blaOXA-23 gene was found in 81.8% of the isolates from CRAB, and 7.9% of CSAB(P<0.01). Transposon Tn2008 was found in 90.9% of the isolates from CRAB and 17.5% of the isolates from CSAB(P<0.01). The drug resistance rate of A.baumannii carrying Tn2008 was higher than that of isolates carrying blaOXA-23 without Tn2008(P< 0.01).Other CHDLs(blaOXA-24-like, blaOXA-58-like), Metallo-β-lactamases (blaIMP-type,blaVIM-type, blaSIM-1)and Tn2006(ISAbal-blaOXA-23-ISAba1) were not identified in 96 A.baumannii. aac(6’)-Ib was found in 45.5% A.baumannii,which was not detected in 70.9% A.baumannii. Other aminoglycosides resistance genes such as aac(6’)-Ih and aph(3’)-VI gene were negative. Sequencing of the QRDRs of the gyrB and parC showed that all the MDRAB clinical isolates had a point mutation on the gyrA gene that converted the serine at position 83(Ser83) to leucine(Leu) in GyrA. Sequencing of the parCgenes indicated that all the MDRAB clinical isolates had a point mutation in the parCgene that caused an amino acid change in Ser-80(TCG) to leucine(TTA). The Ser83 mutation in GyrA and Ser80 in ParC is consistent with a fluoroquinolone-resistant phenotype. In addition, the efflux pump gene adeA, adeB, adeC were positive in 100% of the MDRAB clinical isolates. Conclusions:blaOXA-23 and blaOXA-66 were the main acquired carbapenemase gene of CRAB.Tn2008 was the vehicle mobilizing blaOXA-23 for local isolates of ST208,ST191. AAC(6’)-Ib,APH(3’)-I,AAC(3)-II are the main gene that contribute the aminoglycosides resistance mechanism. Concurrent GyrA and ParC aminoacid substitutions in the "hot spots" of their respective QRDRs were primarily responsible for these MDRAB isolates.