The Study Of Multilicus Sequence Typing And The Carbapenem Resistance Mechanism Of Acinetobacter Baumannii From Psrtial Regions Of China

The carbapenem-resistant Acinetobacter baumannii (CRAB) has widely spread in China, which resulting in dilemma in clinical anti-infectious thrapy. Therefore, it is urgent to elucidate the distribution of CRAB in our country using molecular typing method. The results will be useful for prevention and control this pathogen.In Part Ⅰ of this study,226non-duplicated A. baumannii were collected from24tertiary hospitals of16cities in2005, including152CRAB and74carbapenem-susceptible A. baumannii. Multilocus sequence tying (MLST) was used as the method of molecular typing. The results of MLST were compared with those of the "golden standard" of bacterial molecular typing, pulsed-field gel electrophoresis (PFGE) to confirm the availability of MLST in molecular epidemiological study of A. baumannnii. eBURST was used to analyze the data of MLST and infer the evalutionary descent. The susceptibility testing of11antimicrobial agents were determined. PCR was used to detected the presence of carbapenemase genes.MLST revealed that the genetic background of CRAB was relatively simple, including8sequence types (STs). ST92and its single locus variants (SLVs), ST90and ST75were the dominant STs, followed by ST91. The genetic background of CSAB was more diverse, including36STs. ST92was also the major ST of CSAB.44pulsotypes were identified in226A. baumannii by PFGE. In generally, the results of MLST and PFGE were consistent. However, there were some disparities about the genotypes of ST92and its SLVs. MLST was more conservative in the interpreting of bacterial genetic relatedness, and could reveal the bacterial evolutionary descent in long term period.We compared our data with those in the MLST website database of A. baumannii (Pubmlst, http://pubmlst.org/abaumannii/). ST92and its SLVs were all grouped as clonal complex (CC)92(formerly named CC22), which is the largest CC in the database. The origins of CC92isolates were covered many countries of Asian, Europe, Oceania and Americas, confirming the worldwide spread of CC92. The CC92CRAB of China and other countries may evolve from a same progenitor.CC92and ST91were more resistant to carbapenems, third-and forth-generation cephalosporins, aminoglycosides, minocycline and fluoroquinolones than the other STs (P<0.001). There were no statistical difference between antibiotics resistance of ST92-CRAB and ST92-CSAB. The results implys multidrug resistance is an important feature of ST92.blaOXA-23was the main acquired carbapenemase gene of CRAB. ST91co-carried blaOXA-23and WaOXA-58.None of carbapenemase gene was detected in CSAB. The imipenem minimal inhibitory concentrations (MICs) of blaOXA-23-positive ST92-CRAB were32to128times to those of blaOXA-23-negative ST92-CSAB, implying blaOXA-23was an critical determinant of carbapenem resistance in ST92A. baumannii.In Part Ⅰ of this study, MLST was used for molecular epidemiological investigation of A. baumannii from multiple cities of China. CC92was the most prevalent CRAB clone of China, as well as the worldwide spread clone, and showed greater antibiotics resistance than the other STs. OXA-23-type carbapenemase was an resistance mechanism of CC92against carbapenems. The emergence and dissemination of CC92in China accounted for the increasing carbapenem resistance inA baumannii.The carbapenem hydrolyzing class D β-lactamases are the most important factors mediating carbapenem resistance in A. baumannii. blaOXA-58is a worldwide distributed CHDL gene. In Part Ⅰ of this study, we found the ST91CRAB co-carried blaOXA-23and blaOXA-58.On that basis, in Part Ⅱ of this study, we further studied the genetic background of blaOXA-58-harboring Acinetobacter spp., the feature of blaOXA-58-harboring plasmids, as well as the relationship between genetic contexts of blaOXA-58and susceptibility testing profiles13blaOXA-58-harboring non-duplicated Acinetobacter spp. were collected from seven tertiary hospitals of six cities, including two Acinetobacter pittii (formerly Acinetobacter genomic species3), three Acinetobacter nosocomialis (formerly Acinetobacter genomic species13TU) and eight A. baumannii (two A. baumannii isolates from Jinhua, Zhejiang Province were present in Part Ⅰ of this study). PFGE and MLST were used for the molecular epidemiological investigation. The MICs of13antimicrobial agents were determined. The genetic location of blaOXA-58was determined by Southern hybridization. The genetic contexts of blaOXA-58were acquired through restricted digestion and cloning experiments. The blaOXA-58-harboring plasmids were typed by A. baumannii PCR-based replicon typing method (AB-PBRT).PFGE identified the two A. pittii belonged to a same pulsotype (from Hangzhou and Taizhou respectively), and the three A. nosocomialis belonged to two pulsotypes (all from Wenzhou). Three pulsotypes were identified in eight A. baumannii, corresponding to three STs using MLST. ST91were detected in Jinhua, Zhejiang Province and Wuhan, Hubei Province, and recovered from Jinhua Center Hospital both in2005and2009, implying endemic prevalent in this hospital.The13Acinetobacter spp. displayed various carbapenem susceptibility testing profiles. All A. pittii and A. nosocomialis were carbapenem susceptible. However, the A. baumannii were almostly carbapenem resistance, except one showed carbapenem susceptible. Moreover, A. baumannii were more resistant to third-and forth-generation cephalosporins, β-lactams combination with β-lactamase inhibitor, aminoglycosides, minocycline and ciprofloxacin than A. pittii and A. nosocomialis.The plasmid location of blaOXA-58in13Acinetobacter spp. were confirmed by Southern hybridization, and the sizes of plasmid were ranged from ca.52kb to ca.305kb. All plasmids could transfer to carbapenem-susceptible recepient through electrotransformation except for the largest one. The plasmids did not belong to any known plasmid replicon typing. Aci10, a novel plasmid replicon typing was identified in the blaOXA-58-harboring plasmids of two A. pittii, three A. nosocomialis and two A. baumannii.An ISAba3-like element was identified upstream of blaOXA-58, and the element was disrupted by insertion sequence of IS6family in partial isolates, named IS6family-AISAba3-like-blaOXA-58structure. The special structure was present in seven carbapenem-resistant A. baumannii and one carbapenem-susceptible A. nosocomialis, and their transformants showed carbapenem resistant or mediated. In remaining carbapenem-susceptible isolates, the ISAba3-like element was intact. The special IS6family-△ISAba3-like-blaOXA-58structure provided strong promoter for transcription of blaOXA-58, which played an important role in the carbapenem resistance. Recombination sites around blaOXA-58were identified in partial isolates, implying the acquisition of blaOXA-58was associated with recombination events. An aminoglycoside O-phosphotransferases gene, aphA6, was identified downstream of blaOXA-58of ST91A. baumannii, and which located in a composite transposon named TnaphA6. The co-transfer of aphA6and blaOXA-58, resulted in the recepient developed carbapenem and aminoglycoside resistance at the same time.The part Ⅱ of this study demonstrated the plasmid-location of blaOXA-58in A.pittii, A. nosocomialis and A. baumannii, and the plamids could transfer to recepient by electrotransformation. A same plasmid replicon typing was identified in blaOXA-58-harboring plasmids of A. pittii, A. nosocomialis and partial A. baumannii, suggesting the transfer of plamids among Acientobacter spp. was involved in the rapid increasing of carbapenem resistant rate in this pathogen. The special IS6family-△ISAba3-like-blaOXA-58structure contributed to the carbapenem resistance through providing strong promoter for the transcription of blaOXA-58.