| Purpose:We report a pandrug resistant?PDR?Klebsiella pneumoniae?KP13223?isolated from a urine sample,which was resistant to carbapenems,tigecycline,polymyxin andceftazidime/avibactam.The purpose of this study is to clarify the drug-resistancemechanism of the PDR strain and provide the basis for controlling the spread of such superbugs.Methods:Broth dilution method and agar dilution method were used to determine the resistance characterization.The carbapenemase genes of bla KPC,bla NDM,bla IMP,bla VIM,bla OXA-48 and colistin resistant gene mcr-1 were amplified by PCR amplification and sequenced.Localization of resistant genes and the sizes of their harboring plasmids were analyzed by S1-PFGE and Southern blot hybridization.The strain conjugation experiment was performed to prove the transferability of plasmids that carring bla KPC,bla NDM and mcr-1.Complete genome sequence of the KP13223 were carried out to analyze the ST type and the surrounding environment of related resistance genes.The role of RND-type efflux pumps and its regulators in tigecycline resistance were evaluated by the efflux pump inhibition experiments and Quantitative PCR?q RT-PCR?.Results:KP13223 showed resistance to all nine antibiotics we tested,including amikacin,fosfomycin,aztreonam,carbapenems,tigecycline,polymyxin and ceftazidime/avibactam.And the MIC of imipenem was 128mg/L,that of tigecycline was 8mg/L,polymyxomycin was 4mg/L,and ceftazidine/avibactan was more than 128mg/L.PCR and sequence revealed KP13223 was positive of bla KPC-2,bla NDM-5and mcr-1.S1 PFGE and Southern blot showed the resisitant genes were located on different plasmids,the presence of the bla KPC-2 in a 104.5-138.9 kb plasmid,bla NDM-5in a 33.3-54.7kb plasmid and mcr-1 in a 33.3-40kb plasmid.Conjugation experiment confirmed the transfer capacity of the three related resistant plasmids.Whole genome sequencing showed the KP13223 belongs to Sequence Type 11.The bla KPC-2 gene was located on the Inc FII plasmid,which also contained aminoglycoside resistance gene rmt B,beta-lactam resistance genes bla SHV-12,bla TEM-1B.Around the bla KPC-2,it had a structure of ISkpn6-bla KPC-2-hp-IS481-hp-IS6-Tni A.The bla NDM-5and the mcr-1 gene were located on the Inc X3 and Inc X4 plasmid,respectively.The surrounding structure of bla NDM-5gene and mcr-1 gene were bla NDM-5-IS5-ISAba125-IS3000-hp-Tn3 and IS5-mcr-1-hp respectively.Furthermore,our research investigated the tigecycline resistant mechanism of the isolate KP13223.The efflux pump inhibition experiment confirmed that the three efflux pump inhibitors NMP,PA?N,and CCCP all had obvious inhibitory effects,among which NMP was the most significant which could restore the tigacycline sensitivity of the KP13223.Gene analysis and quantitative PCR detection found that the resistance of KP13223 to tigecycline was due to acr A,acr B and mar R mutation,which caused high expression of Acr AB efflux pump.Conclusions:KP13223 was resistanant to all the antibiotics tested,which was rare reported in China.The bla KPC-2,bla NDM-5and mcr-1 gene were located on three different plasmids respectively,and these plasmids were transferable.Mutations in acr A,acr B and mar R in this strain caused activation of the Acr AB efflux pump,leading to the tigecycline resistance.The spread of such pandrug-resistant pathogen poses a huge threat to global public health.Therefore,it's imperative to establish a comprehensive monitoring system to avoid the horizontal spread of drug-resistant genes. |
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