Study on resistance genes and virulence factors of carbapenem resistant Klebsiella pneumoniae isolates from hospital acquired pneumonia patients
Objective To analyze the drug resistance genes and virulence factors of carbapenem-resistant Klebsiella pneumoniae isolates from patients with hospital-acquired pneumonia.Methods Ninety-six hospital acquired pneumonia patients admitted to Anyang People's Hospital from January 2021 to December 2022 were selected as the research subjects,and 100 carbapenem-resistant Klebsiella pneumoniae isolates were isolated.Microbial identification instrument and drug sensitivity test were used to identify the strains,and clinical data of the patients were statistically analyzed(including gender,age,department,underlying disease,specimen type,ICU admission,invasive operation,antibiotic exposure and prognosis,etc.).The strain phenotype was detected by Hodge method,carbapenase inactivation method and wire drawing test.PCR was used to detect 19 β-lactam(carbapenemase,AmpC enzyme and ESBL)and quinolone resistance genes and virulence genes,and the positive products were selected and their genotypes were identified by bidirectional sequencing.The epidemiology of carbapenem-resistant Klebsiella pneumoniae was analyzed by multi-site sequence analysis,and the cloning of carbapenem-resistant Klebsiella pneumoniae was analyzed by pulsed field gel electrophoresis.Results In this study,100 strains of Klebsiella pneumoniae were tested in vitro,and the drug resistance rates of cephalosporins,enzyme inhibitors,monocyclic antibiotics and carbapenems were the highest,all higher than 95.0％,while the drug resistance rates of aminoglycoside antibiotics and tigacycline were lower,all about 60.0％.The most widespread department was the ICU.The main source of bacteria was sputum,followed by blood and urine.Eighty-four positive strains were detected by Hodge test and 93 positive strains were detected by mCIM method.The results showed that NDM,KPC,SHV,IMP,TEM,CTX-M and qnrS could be amplified with positive bands of corresponding location size.However,no corresponding bands were amplified for 12 resistance genes:SME,VIM,GIM,GES,OXA-23,OXA-48,VEB,PER,DHA,AAC,qnrA and qnrB.Twenty KPC-positive DNA products were randomly selected for sequencing,and all of them were found to be KPC-2 genotypes by sequence comparison,while four NDM-positive DNA products were found to be NDM-1 genotypes and two NDM-5 genotypes by sequence comparison.Multiple sequence alignment showed that there were two mutations in NDM-5 and NDM-1 genes.The strains carrying 4 different drug resistance genes accounted for the most(53.0％),followed by the strains carrying 5(24.0％)and 3(16.0％)different drug resistance genes.Eleven strains were found to be positive by wire drawing test.The capsular virulence genes were detected and 11 virulence genes were found,among which ureA,wabG,fimH,entB and ybtA had higher detection rates(＞90.0％).The detection rate of rmpA gene was 20％,and wcaG gene was detected in only 1 strain.The detection of the above 11 virulence genes showed that there were more strains carrying 6(36.0％)and 7(32.0％)virulence genes,and 3 strains of Klebsiella pneumoniae had 10 virulence genes.Conclusions The antimicrobial resistance of carbapenem-resistant Klebsiella pneumoniae is serious,and invasive operation and antibiotic exposure are the main reasons leading to the epidemic of drug-resistant bacteria.The mechanism of drug resistance was that the strain expressed IMP-4,NDM and KPC carbapenemase,ST11 was the main ST type of CRKP in our hospital,ICU was one of the main disease areas of the strain cloning,and many virulence genes were detected.
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