Resistance Mechanisms And Molecular Epidemiology In Carbapenem-Resistant Acinetobacter Spp. Clinical Isolates

Objectives:1. To investigated the role of Class B and D carbapenemase genes in the generation of drug resistance in carbapenem-resistant Acinetobacter spp. clinical isolates in Fuzhou.2. To explored the role of Class A carbapenemases, outer membrane proteins and efflux pumps in the carbapenem-resistant Acinetobacter spp. clinical isolates producing New Delhi metallo-β-lactamase-1(NDM-1).3. To studied the imipenem-hydrolyzing capability of NDM-1, NDM-6 and NDM-9.4. To explored the molecular epidemiological features of carbapenem-resistant Acinetobacter spp. clinical isolates in Fuzhou.Methods:1. A total of 391 strains of carbapenem-resistant Acinetobacter spp. were collected from September 2010 to August 2014 in Fuzhou. All strains were identified and antimicrobial susceptibility test(AST) of those strains were performed by VITEK-2compact system and then they were confirmed their imipenem resistance with Kirby-Bauer antibiotic testing. All Acinetobacter spp. isolates were classified by 42℃growth experiment, OXA-51-like gene detection and sequence analysis of rpo B gene.Class B and D carbapenemase genes were determined by PCR assay.2. Through PCR assay, Class A carbapenemase genes, efflux pump genes and outer membrane protein genes were screened in Acinetobacter spp. isolates that carried NDM-1 gene. The p CR 2.1 vectors that contained NDM-1, NDM-6 or NDM-9gene respectively was constructed and transformed into the TOP10 competent cells.The Minimal inhibitory concentration(MIC) values of different type of recombinant bacteria were determined with agar dilution method.3. The 58 clinical isolates of carbapenem-resistant Acinetobacter spp. were typed by Diversi Lab system, including 50 strains of carbapenem-resistant Acinetobacter baumannii(CRAB)(39 from Fujian Medical University Union Hospital, 9 strains from the First Affiliated Hospital of Fujian Medical University and 2 strains from Fuzhou General Hospital of Nanjing Military Command), 5 strains of carbapenem-resistant Acinetobacter pittii and 3 strains of carbapenem-resistant Acinetobacter nosocomialis(the latter two types were from Fujian Medical University Union Hospital).Results:1. ①Among the 391 clinical isolates, there were 382 strains of CRAB, 5 strains of carbapenem-resistant Acinetobacter pittii and 4 strains of carbapenem-resistant Acinetobacter nosocomialis. ②Among 382 strains of Acinetobacter baumannii, 93.46percent(357 strains) carried OXA-23-1ike gene, 1.05 percent(4 strains) carried OXA-24-1ike gene, 0.52 percent(2 strains) carried OXA-58-1ike gene; 2 strains carried NDM-1 gene. ③Among 5 strains of Acinetobacter pittii, 2 strains carried OXA-58-1ike gene, none strain carried OXA-23-like、OXA-24-like or OXA-143-like gene; All strains carried NDM-1 gene. ④Among 4 strains of Acinetobacter nosocomialis, none strain carried OXA-23-like gene、OXA-24-like、OXA-58-1ike and OXA-143-like gene, 3 strains carried NDM-1 gene. ⑤No other metallo-β-lactamase genes had been detected.2. ① Among 2 strains of Acinetobacter baumannii positive with NDM-1 gene, 1strain carried OXA-23-like gene and other carbapenemases were not detected; 2strains carried car O gene, 1 strain carried opr D gene; 2 strains carried ade B gene、ade I gene、ade J gene and ade K gene. ②Among the 5 strains of Acinetobacter pittii positive with NDM-1 gene, 2 strain carried OXA-58-like gene and other carbapenemases were not detected; 5 strain carried opr D gene, but car O gene was not detected; 5 strains carried ade I gene and ade K gene, 4 strains carried ade J gene, and 1 strain carried ade B gene. ③Among 3 strains of Acinetobacter nosocomialis positive with NDM-1 gene, 3 strains carried car O、opr D、ade B、ade I、ade J and ade K gene,but other carbapenemases were not detected. ④All the above 10 strains positive with NDM-1 gene did not carry ade S gene. ⑤ MICs of imipenem for Escherichia coli TOP10 expressing NDM-1, NDM-6 and NDM-9 were respectively 32, 128 and 64μg/ml.3. The 50 strains of Acinetobacter baumannii in Fuzhou were classified into 7 types from A to G. Type A, B, E and G each had 1 strain, type C had 4 strains, type D had 7strains, and type F had 35 strains. The 5 strains of Acinetobacter pittii and 3 strains of Acinetobacter nosocomialis were classified into type A and type B.Conclusions:1. The existence of OXA-23-like enzyme may be the main reason that CRAB isolates in Fuzhou were resistant to carbapenem.2. For the first time, NDM-1 gene and OXA-58-like gene were both detected in carbapenem-resistant Acinetobacter pittii.3. For the first time in Fujian province were found the CRAB carrying OXA-24-like gene, OXA-58-like gene or NDM-1 gene.4. In 2 strains of Acinetobacter baumannii, 5 strains of Acinetobacter pittii and 3strains of Acinetobacter nosocomiali, were all found NDM-1 gene. The carbapenem resistance of Acinetobacter pittii with NDM-1 gene was probably caused by the co-effect of efflux pump Ade IJK and NDM-1 enzyme and the lack of Car O, while that of Acinetobacter Nosocomialis and Acinetobacter baumannii might mainly be caused by co-effect of efflux pump Ade IJK and NDM-1 enzyme.5. As for the capability to hydrolyze imipenem, NDM-6 enzyme ranked top, and NDM-9 enzyme and NDM-1 enzyme followed.6. CRAB isolates of type F might be the major epidemic strain in Fuzhou. However,those in Burn ICU of Fujian Medical University Union Hospital was of type D, quite different from those F-type CRAB from General ICU.