In Vitro Susceptibility And Resistance Characteristic Of Carbapenem-resistant Enterobacteriaceae To Ceftazidime-Avibactam

ObjectivesIn this study,we focused on monitoring the epidemic status of carbapenem-resistant Enterobacteriaceae(CRE),detecting the in vitro susceptibility of KPC-producing CRE with no prior treatment with ceftazidime-avibactam(CAZ-AVI)to this combination and studying on the resistance characteristic of KPC-producing Klebsiella pneumoniae(KPC-KP).MethodsCRE isolates:The unique clinical CRE were collected from October 2016 to June 2018 from 6 tertiary hospitals in six different cities in China.The clinical information of the isolates was collected using EPIINFO software based on the medical records.We re-identified the isolates by MALDI-TOF MS,meanwhile we confirmed the isolates by 16S rRNA sequencing.Sequence types(STs)were determined according to Multi locus sequence typing(MLST).The carbopenemase genes were selected by polymerase chain reaction(PCR),and were identified by gene sequencing.The CAZ-AVI minimal inhibitory concentrations(MICs)of KPC-CRE were determined using the broth microdilution method.KPC-CRE with reduced CAZ-AVI susceptibility:In vitro susceptibility against the commonly used clinical antimicrobial was evaluated by Phoenix 100 Automated Microbiology System.Multiplex PCR analyses were applied to investigate the four virulence genes(rmpA,rmpA2,iroN,iutA)and the capsular types(K locus type,KL).The clonal relatedness between these was investigated by pulsed-field gel electrophoresis(PFGE)and the results were evaluated using GelJ v.2.0 analyzing software.The extended-spectrum β-lactamase(ESBL)genes,outer membrane porin genes,blaKPC-2 and the promoter regions were detected by PCR,and were identified by gene sequencing.Quantitative real-time PCR(qRT-PCR)was applied to assess the blaKPC-2 copy numbers and expressions relative to an internal K.pneumoniae housekeeping gene,rpoB.The experimental isolates were continuously cultured in a gradient concentration of CAZ-AVI liquid medium,and the changes of the CAZ-AVI MICs were detected and recorded.Selected resistance KPC-CRE:The outer membrane porin genes,blaKPC-2,the blaKPC-2 copy numbers and expressions were analyzed.The experimental isolates were successively passaged for 200 generations,and the CAZ-AVI MICs were detected and recorded.The results of the blaKPC-2 copy numbers,blaKPC-2 expressions and changes of the CAZ-AVI MICs were analyzed using Graph Pad Prism5.Results1.A total of 902 CRE were collected including 616(63.8%)K.pneumoniae,the most common of which was ST11,140(15.5%)E.coli belonging to 11 STs.2.There were 741 carbapenemase-producing Enterobacteriaceae(CPE)among which 410(55.3%)isolates carried blaKPC,320(43.2%)isolates carried blaNDM,31 isolates carried blaIMP and 9 isolates carried blaVIM and no blaoxA-48 were detected.With the exception of amikacin,tobramycin and trimethoprim-sulfamethoxazole,the commonly used clinical antimicrobials resistance rates of CRE were>70%.3.The susceptibility rate of the 361 CPE containing blaKPC without coexistence of metallo-β-lactamases genes was 99.7%,among which 12 isolates reduced susceptibility to CAZ-AVI(MIC≥4/4 g/ml).The 12 isolates are resistant to almost all antibiotics tested.All of the 12 isolates belonged to ST11 while 8 of which were belonged to capsular type KL64 and the other 4 isolates were from KL47.6 of the 12 isolates carried the four virulence genes and shared the same PFGE pattern.The 12 isolates carried ESBLs genes and contained a mutant OmpK35 as well as a mutant OmpK36.The relative copy number and transcription expression of blaKPC in the reduced susceptibility group were 2.6 and 3.9-fold up to the susceptibility group,respectively.4.The CAZ-AVI MICs of the isolates with reduced susceptibility were≥32/4μg/ml when the selection concentration reached 4/4 μg/ml,while the MICs of the isolates in the susceptibility group were≤8/4 μg/ml.5.The KPC-2 D179Y mutation was detected in one isolate among the selected CAZ-AVI-resistant isolates.The relative copy number and transcription expression of blaKPC in the other isolates of the resistance group were 2.5 and 3.4-fold up to the susceptibility group isolates,respectively.6.Among the 200 generations of the selected CAZ-AVI-resistant isolates,only 1 isolate lost the resistance(MIC 8/4 μg/ml),and the other 6 isolates remained resistant to CAZ-AVI.Two of the MICs were≥128/4 μg/ml,and the others were 16/4 μg/ml.Conclusions1.KPC-CRE is the most common CRE in six tertiary hospitals.The clinical treatment status is severe.2.CAZ-AVI has potent in vitro activities against KPC-CRE without previous CAZ-AVI treatment history.3.The mechanisms of reduced susceptibility to CAZ-AVI in KPC-KP are possibly related to the increased copy number and/or transcription expression of blaKPC.4.The KPC-KP with reduced susceptibility to CAZ-AVI were more prone to develop CAZ-AVI resistance under the pressure of CAZ-AVI exposing and performed stable resistant phenotype.