| Objectives To understand the changes of carbapenem-resistant Klebsiella pneumoniae dominant clones and to explore the mechanism of the changes of carbapenem-resistant Klebsiella pneumoniae dominant clones.Methods1.A total of 101 carbapenem-resistant Klebsiella pneumoniae strains collected from August,2014 to July,2015 were tested for carbapenemase gene and multiple sequence typing.The positive PCR products were sequenced and the results of the sequencing were analyzed.The analysis of the resistance gene carrying status and the multiple sequence analysis were compared with the previous data.2.The virulence phenotype and resistance to disinfectant gene of the dominant clones were detected,and the positive products were sequenced and the sequencing results were compared.The antimicrobial MICs of the two strains were determined by micro-broth dilution method.3.Analysis of the alternative mechanism of carbapenem-resistant Klebsiella pneumoniae.Survival characteristics of carbapenem-resistant Klebsiella pneumoniae.The growth curves of the two groups were determined by broth culture method,and the growth characteristics of the dominant clones in two different periods were studied.The two groups of strains were tested in vitro by broth co-culture method,and the growth of the two strains in the same environment Advantages and Disadvantages.Mutual inhibition experiments were conducted by broth culture.Results1.The detection rate of carbapenemase gene of carbapenem-resistant Klebsiella pneumoniae was the highest,the positive rate was 67.3%(68/101),followed by IMP-4 And 5.0%(5/101)respectively.The positive rate of NDM-1 was 1.0%(1/101).There were no significant differences in the number of Klebsiella pneumoniae strains producing KPC-2 from SME,GES,IMI,VIM,SIM,GIM,SPM and OXA-48.68 strains in 101 strains of carbapenem resistant Klebsiella pneumonia.The results of point sequence typing were ST11 and 5,respectively.Three strains of Klebsiella pneumoniae were identified as ST26 and 3 strains were ST334.2.The virulence factor and resistance to disinfectant gene showed that CR-KP-2014 strain had higher positive rate of virulence factor and resistance to disinfectant gene compared with CR-KP-2011 strain.The minimum inhibitory concentration(MIC)of four commonly used disinfectants showed that CR-KP-2014 had stronger resistance to disinfectants.3.The growth curve of ST11-KPC-2-KPN showed that the growth rate of ST11-KPC-2-KPN was faster than that of ST26-IMP-4-KPN.The growth curve of ST11-KPC-2-KPN showed a short lag phase and reached plateau after 12 hours.The growth retardation of ST11-KPC-2-KPN strain was about 3 hours,while the growth retardation of ST26-IMP-4-KPN strain was about 3.5-4 hours.In the in vitro competition experiment,the initial bacterial inoculation was consistent,but the colony forming units of ST11-KPC-2-KPN on the M-H plate was significantly higher than that of ST26-IMP-4-KPN in the four experimental groups after co-culture.There was no significant difference in the colony-forming units of the strains in the in vitro competition experiments.Cross-inhibition results showed that there was no significant cross-suppression between ST11-KPC-2-KPN and ST26-IMP-4-KPN.The slow growth of ST26-IMP-4-KPN strain in the culture supernatant of ST11-KPC-2-KPN strain 7h may be due to the rapid growth of previous ST11-KPC-2-KPN consuming most of the nutrients in the medium components.Conclusion1.From August 2011 to July 2015,the dominant clone strain of carbapenem-resistant Klebsiella pneumoniae was changed from ST26-IMP-4-KPN to ST11-KPC-2-KPN.2.CR-KP-2014 strain has stronger resistance to disinfectant in benzalkonium bromide and alcohol disinfectants compared to CR-KP-2011 strain.3.Compared with ST26-IMP-4-KPN strain,ST11-KPC-2-KPN had more growth ability,and the difference of survival characteristics could be one of the reasons for the transformation of the main epidemic clones. |
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