Enzyme Type Analysis And Clinical Significance Of Carbapenem Producing Klebsiella Pneumoniae In Northern Henan

BackgroundThe widespread use or even abuse of carbapenems in the clinic has caused the detection rate of carbapenem resistant Enterobacteriaceae in the detection of clinical pathogenic organisms to climb year by year.Among them,carbapenem resistant Klebsiella pneumoniae（CRKP）has been detected in several hospitals in several provinces of China,and the proportion is the highest,which has become one of the main causes of clinical infection increasing year by year.Resistance mechanisms of CRKP are diverse,among which carbapenemase producing enzymes are the main cause of Klebsiella pneumoniae carbapenemase（KPC）type resistance.There are various mutants of KPC type,and the common one in China is KPC-2,but there are differences in different regions.This discrepancy leads to differences in molecular characteristics and resistance mechanisms,which in turn produce resistance to carbapenems such as ceftazidime avibactam.PurposeTo understand the current resistance status of Klebsiella pneumoniae in Northern Henan,to explore the enzymatic genotyping and molecular characteristics of enzyme producing Klebsiella pneumoniae in Northern Henan,and to analyze the resistance rates of different enzyme types to provide guidance for clinical drug use.Method1.To collect 135 strains of CRKP strain identified from January 2018 to December2021 in the First Affiliated Hospital of Xinxiang Medical College,the enzymatic type of135 strains was detected using carbapenemase detection kit and counted;2.Extract DNA of carbapenemase producing Klebsiella pneumoniae,design primers,and PCR amplify KPC,NDM,IMP genes;3.Separate the amplified products by agarose gel electrophoresis,contrasting with the band of interest;4.DNA is recovered for sequencing,and the resulting genes of interest are subjected to blast alignment of sequencing results through the NCBI online functional network;5.Statistic the subtypes of different enzyme type genes,the same enzyme subtypes were subjected to multiple sequence alignment by mega software to construct evolutionary trees,statistic the difference of different branch sequences;6.Statistics of drug susceptibility of strains of different enzyme subtypes by drug susceptibility test.Result1.Of the 135 CRKP strains collected,133 produced carbapenemases.Among them,124 carried KPC resistance genes,9 carried NDM resistance genes,and 1 carried both IMP and NDM resistance genes;2.124 strains carried KPC resistance gene agarose gel electrophoresis test all obtained the bands of interest,consistent with the results obtained from carbapenemase test kit.KPC-2,which all shared 99%-100% identity by blast alignment,Nine NDM type strains were NDM-1-like by blast alignment,and One IMP type strain was IMP-4-like by blast alignment.3.The constructed evolutionary tree divided the 124 KPC-2-type resistance genes into three major clades and three minor clades,and the strains with less than 100% homology in the multiple sequence gene alignment had point mutations at some base positions.4.Drug susceptibility tests showed that 133 enzyme producing pairs of β-Lactam antibiotics were all resistant,and among 124 KPC producing isolates,83.1% were resistant to amikacin（aminoglycoside）,49.4% to tetracycline（tetracycline）,44.4% and 58.9% to chloramphenicol and CO trimoxazole,and 2.42% to ceftazidime avibactam;Eight of the nine NDM producing isolates were resistant to ceftazidime avibactam,and one was susceptible;Strains producing both NDM and IMP in nine were also resistant to ceftazidime avibactam.Conclusion1.The mechanism of Klebsiella pneumoniae unsensitive to carbapenems in Northern Henan province is produced carbapenem,and KPC type is the most common among many genotypes encoding carbapenem gene,and its subtype is KPC-2;2.Some KPC-2 carbapenemase gene mutation,such as deletions,insertions,and substitutions do not affect the expression of KPC-2 carbapenemase,therefore sensitive toβ-Lactam resistance is not affected;3.Ceftazidime avibactam is effective in treating clinical infections due to KPC type carbapenemase producing Klebsiella pneumoniae.