| In recent years,carbapenem-resistant Klebsiella pneumoniae has evolved and spread around the world due to the extensive use of carbapenem-resistant drugs.In particular,carbapenemase-producing Klebsiella pneumoniae（CPKP）has become a serious clinical challenge due to its often untreatable infection.Yunnan is located in southwest China with limited economic and medical conditions and is adjacent to the Indian subcontinent where CPKP is prevalent.The epidemic situation of CPKP in Yunnan has a typical regional pattern,so it is necessary to monitor the change situation of drug-resistant bacteria in southwest China,especially In Kunming.In this study,multilocus sequence typing（MLST）and pulsed field gel electrophoresis（PFGE）were used to determine drug susceptibility and genetic relationship,and polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP）was used to detect gene polymorphism.The target gene structure was analyzed by genome walking and high-throughput sequencing,and the molecular genetic background of the target strain was analyzed by whole-genome sequencing.Finally,the target strains interact with nematodes to test their virulence,and make a preliminary study on their adaptability by observing and recording the growth of different strains in different concentrations of antibiotics.It can help to improve the understanding of the epidemic status and drug resistance factors of CPKP strains,and provide theoretical basis for the comprehensive prevention and treatment of CPKP and the development of targeted drugs in the future.The main results obtained are as follows:（1）Forty-one Klebsiella pneumoniae isolates were collected from the Second Affiliated Hospital of Kunming Medical University in 2011-2013,all isolates were resistant to the testedβ-lactam/β-lactamase inhibitor combination,cephems,Carbapenems and folate pathway inhibitors are resistant.The source of the strain is widely distributed in departments,which proves that this drug-resistant bacterium has been widely distributed in the hospital system.The bla KPC-2gene（resistance to carbapenemase-related antibiotics）was detected in 34（83%）isolates,and bla NDM-1（resistance toβ-lactamase-related antibiotics）was detected in 34 isolates（83%）.Gene)gene was detected in 3（7%）isolates KP67,KP72,KP82.（2）MLST analysis revealed five different Sequence types（Sequence types,a group of genotype combinations obtained by using bacterial 7 housekeeping genes as genetic markers）:ST11（34 strains,83%）,ST290（3 strains,7%）,ST340（1 strain）,ST395（2strains）and ST437（1 strain）,and all bla KPC-2positive isolates were ST11.Three bla NDM-1positive isolates were found in three different ST types,ST290,ST395 and ST437.（3）PCR-RFLP was used to detect gene polymorphisms in different strains,and the results showed that all bla KPC-2structures were highly similar except KP29.Bla KPC-2was not found in the other ST groups except ST11,indicating that horizontal transmission of bla KPC-2plasmid was limited.（4）The whole genome of KP21,KP29,KP65,KP67,KP72 and KP82 strains was sequenced.The genome length ranged from 1,049,428,229bp to 1,476,835,866bp,and each strain contained 3 to 7 plasmids.The plasmid sequence length ranged from2,469bp to 258,415bp,and GC content ranged from 44.62%to 55.62%.Comparative genomic analysis and epidemiological analysis were performed for antiplasmid.These results indicate that the transmission of enterobacter carbapenem resistant plasmid in southwest China may be from the southeast coast of China,suggesting that the origin of NDM in China may be from transnational communication and transmission in China..（5）It was found that the control group had the fastest death time and the strongest virulence when it did not contain the drug resistance genes.The group with the double resistance gene survived the longest and had the highest survival rate at the same time.In the KPC group,the NDM group died faster than the other resistant groups.ST11strains had the lowest virulence to nematodes and the highest survival rate of nematodes detected at the same time.The second was ST290.Compared with the control group,ST437 had similar death time for all nematodes,but its virulence to nematodes was lower at the same time.（6）The strains containing double genes obviously have the advantage of growing in the harsh environment with high concentration of antibiotics,and their resistance is relatively stronger.The KPC strain seems to have a stronger ability to adapt to the harsh environment than the NDM strain.ST11 strain can quickly reach the growth plateau,and its ability to adapt to the environment is relatively stronger than other types in this paper. |
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