Study On Carbapenem Resistance Mechanisms Of Acinetobacter Baumannii

Objective To know about the nosocomial infections, resistant characteristics and epidemics of Acinetobacter baumannii, we detect the resistance of the environmental isolates and clinical isolates in intensive care unit. Polymerase chain reaction(PCR)was used to detect the genes of β-lactamases and main structural genes and regulatory genes of ade ABC、ade IJK and ade FGH efflux pump system of the RND family, comparing the distribution and expression of these genes in the imipenem-resistant group and imipenem-sensitive group, the mechanism of outer membrane protein Omp34 mediated carbapenems resistance, and exploring carbapenems resistance mechanisms of Acinetobacter baumannii.Method 1、 We collected 78 Acinetobacter baumannii from 2012 July to October in our hospital. Enterbacteriat repetitive intergenic consensus(ERIC-PCR) and pulsed-field gel electrophoresis(PFGE) was used to type 78 clinical Acinetobacter baumannii isolates. 2、At the same time, we investigate the pollution of environmental in neurosurgical intensive care unit(ICU), isolated 28 Acinetobacter baumannii on the beds and desk of patients, surface of ventilator, door handles. The minimal inhibitory concentrations(MICs) of antimicrobial agents against 28 environmental isolates and 27 clinical isolates were determined by agar dilution method. Enterbacteriat repetitive intergenic consensus(ERIC-PCR) 、pulsed-field gel electrophoresis(PFGE) and Multi Locus Sequence Typing(MLST) was used to type 55 isolates. 3、 Polymerase chainreaction(PCR)was used to detect the genes of β-lactamases and main structural genes and regulatory genes of ade ABC、ade IJK and ade FGH efflux pump system of the RND family, comparing the distribution of these genes in the imipenem-resistant group and imipenem-sensitive group,further detecting its main efflux gene expression levels by using of the real-time fluorescence quantitative polymerase chain reaction(Realtime-PCR). 4、 Omp34 gene knockout mutations(ATCC 19606-△Omp34) was obtained by double homologous recombination technology, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis(SDS-PAGE) was used to analysis the expression of outer membrane proteins. Polymerase Chain Reaction(PCR) and gene sequencing further validated the gene knockout. Comparing of the resistance to commonly used antimicrobial agents, especially carbapenems, between the wild strain and knockout strain, in order to investigate the role of Omp34 in carbapenems-resistant Acinetobacter baumannii.Results 1、78 clinical isolates were commonly resistant to these antimicrobial agents. In the resisitant rates,minocyline had the lowest rate, was 30.9% and 53.9% for imipenem, others were all over 60%. The resistance rate to cefoxitin and co-trimoxazole were 97.5 % and 93.8 % respectively, all higher than other antimicrobial agents. 78 Acinetobacter baumannii strains were classified into 6 types based on ERIC-PCR profiles, including 62 genotyping A, 7 genotyping B and 6 genotyping C, D、E、F type was one isolate for each. There mainly were A genotypes, among which were 46 genotyping A1、16 genotyping A2. There mainly were 7 PFGE pulsotypes for 78 isolates,pulsotype A were 56 isolates(containing genotyping A1 A2 A3), pulsotype B were 7isolates, C were 4 isolates, D were 5 isolates, E were 3 isolates, F were 2 isolates, G were1 isolates. 2、27 clinical isolates of Acinetobacter baumannii are commenly resistant to antimicrobials except for tigecycline and colistin. In the resisitant rates,minocyline had the lowest rate, was 25.9% and 51.9% for cefoperazone/sulbactam, 59.3% for imipenem,others were all over 70%. The resistance rate to trimethoprim-sulfamethoxazole were100 % respectively. 28 environmental isolates of Acinetobacter baumannii are more sensitive compared with clinical isolates, to imipenem and meropenem resistance rate was only 7.1%、 3.6%, minocycline resistance rates was 10.7%. In the resisitant rates,cefoxitin had the highest rate, was 92.9%. 27 clinical isolates were classified into 3 types based on ERIC-PCR profiles, including 20 genotyping A, 6 genotyping B and 1genotyping C. 28 environmental isolates were divided into six main types, 13 genotyping A, 4 genotyping B,2 genotyping C, 4 genotyping D, 2 genotyping E, 3 genotyping F.There mainly were 5 MLST pulsotypes for 27 clinical isolates, including ST-208(1-3-3-2-2-97-3); ST-368(1-3-3-2-2-140-3); ST-191(1-3-3-2-2-94-3);ST-195(1-3-3-2-2-96-3); ST-540(1-3-3-2-2-160-3) and four new pulsotypes. MLST pulsotypes for 28 environment isolates mainly includ ST-208 and ST-229. 27 clinical isolates were classified into 3 types based on PFGE profiles, including 23 genotyping A,1 genotyping B, 2 genotyping D and 1 genotyping E. 28 Acinetobacter baumannii separated from neurosurgical ICU environment mainly divided into nine types, including15 of type A, B type were 2, C type was one, D type were 2, E type was 3, F type were 2,G, H, I type were 1 for each. 3、In this research, all of the strains carried OXA-51, and OXA-24、OXA-58、VIM-1and VIM-2 were all negative, the detection rate of Amp C、OXA-23、IMP-1 were84.6%、74.3%、56.4%. There were 66 isolates carrying Amp C,including 42 IRAB and 22 ISAB. 58 OXA-23-producing isolates included 42 IRAB and12 ISAB. 44 IMP-1-producing isolates included 23 IRAB and 19 ISAB. In Ade RS-ABC efflux pump, the detection of ade B、ade S、ade R was 77%, 77%, 73% respectively. 60 ade B-producing isolates included 39 IRAB and 19 ISAB. In Ade J and Ade FGH efflux pump, the detection of the ade J、ade F、ade G、ade H was 94%, 97%, 90%,92% respectively.72 ade J-producing isolates included 42 IRAB and 28 ISAB. 70 ade G-producing isolates included 42 IRAB and 26 ISAB. With the statistical analysis, the distribution rate differences of Amp C、OXA-23 and efflux pump genes ade B、ade G in imipenem-resistant A.baumannii(IRAB) group and imipenem-sensitive A.baumannii(ISAB)group were statistically significant. 4、ATCC 19606-△Omp34 strain was successfully obtained by double homologous recombination technology, and was verified by gene sequencing.SDS-PAGE showed that knockout strain had a lack of protein in the 34 k Da.Antimicrobial susceptibility test showed that knockout strain had significant change of carbapenems resistance compared with ATCC 19606.Conlusion 27 clinical Acinetobacter baumannii isolates from ICU was sensitive to tigecycline and colistin, but commonly resistant to these antimicrobial agents, only slightly lower to minocycline, followed by imipenem. Clinical could select the appropriate antimicrobial drugs according to susceptibility results. A clone Acinetobacter baumannii was the main type in our hospital. Clinical isolates and environmental isolates have high homology and distributed in many other departments, may be with cross-infection between departments. We should take effective measures to intervene in a timely manner. Producing β-lactamase、Amp-C OXA-23 and high expression of active efflux system Ade ABC 、 Ade FGH play an important role in mediating carbapenem resistance of Acinetobacter baumannii. By contrast, Omp34 deficiency seemed to only serve as a minor cooperative factor.