Study On The Plasmid-mediated Mechanism Of Bla_IMP-4 Horizontal Transmission And Virulence Genes In Klebsiella Pneumoniae

Objectives:1.To trace the causes of nosocomial infection outbreak caused by Carbapenem-resistant klebsiella pneumoniae（CRKP）in neonatal pediatrics of our hospital,so as to provide theoretical basis for its prevention and control;2.To explore the relation ship between multi-drug resistance and high virulence of CRKP,so as to lay a foundation for rational clinical antibiotic therapy;3.To analyze the molecular mechanism of plasmid-mediated horizontal transmission in antibiotic resistance gene s of CRKP,and to provide experimental basis for delaying the spread of antibiotic resistance.Methods:1.Haitai electronic case system was used to retrospectively analyze the clinical background data of CRKP strains in neonatal pediatrics in our hospital during 2019;2.The species and antibiotic sensitivity tests of the experimental strains were re-identified by VITEK2 Compact microbial identification system;3.The phenotype of carbapenem was screened by disk suppression synergy test and colloidal gold immunochromatography;4.PCR was used to amplify the common antibiotic resistance genes and virulence genes of CRKP,and the MLST of experimental strains was confirmed;5.Plasmid conjugation transfer experiment was used to screen the target strains which can be transmitted horizontally by plasmid-mediated antibiotic resistance genes;6.The whole genome sequencing of the target strain was carried out to analyze the full length sequence of the plasmid and the environmental characteristics of antibiotic resistance genes,so as to understand the molecular transmission mechanism of the antibiotic resistance gene.Results:1.Clinical background data showed that there were 4 cases of CRKP infection in neonatal pediatrics in our hospital from May to September 2019,of which 2 strains were isolated from the end of endotracheal intubation tube,1 strain was isolated from urine and 1 strain was isolated from sputum.Most of the infected infants were low weight premature infants with malnutrition,pulmonary infection,（invasive/non-invasive）ventilator-assisted ventilation treatment,long-term use of multiple antibiotics and so on;2.The results of antibiotic sensitivity tests showed that the 4 strains of CRKP from neonatal pediatrics were completely resistant to penicillins（piperacillin/tazobactam）、cephalosporins（ceftazidime、cefoperazone/sulbactam、cefepime）、carbapenem（imipenem、meropenem）、tetracyclines（doxycycline、minocycline）and partially resistant to aztreonam、ciprofloxacin and sulfamethoxazole,with a resistance rate of 50%.And they were completely sensitive to amikacin、tobramycin、levofloxacin、tigecycline and polymyxin;3.The results of disk suppression synergy test showed that all the 4 strains produced metallo-β-lactamases.Colloidal gold immunochromatography indicated that all strains produced bla _IMP.The results of phenotype screening of carbapenemase were consistent with that of bla _IMP-4gene amplified by PCR.MLST was confirmed as ST76.And the detection of 18 common virulence genes showed that all of them contained wca G+ure A+mrk D+fim H+Kpn+irp-1+uge;4.The results of plasmid conjugation transfer test showed that the 4 strains of ST76 CRKP producing bla _IMP-4from neonatal pediatrics were conjugated and transferred successfully.The conjugate was carbapenem-resistant Escherichia coli J53 carrying bla _IMP-4;5.The results of whole genome sequencing showed that the genome of strain 110 was composed of chromosome and two plasmid.And the antibiotic resistance gene bla _IMP-4 was located on the Inc N with the length of 52656bp.The upstream of bla _IMP-4 was IS26-IS26-Int I1,and the downstream was IS6100.Conclusions:1.A small-scale epidemic of ST76 CRKP carrying bla _IMP-4broke out in the neonatal pediatrics of our hospital from May to September 2019.Attention should be paid to the prevention and control of nosocomial infection;2.Carbapenem-resistant Klebsiella pneumoniae in the neonatal pediatrics of our hospital detected 7 virulence genes at the same time.It is necessary to guard against the further fusion of the antibiotic resistance genes and virulence genes to form CRKP with high virulence;3.bla _IMP-4 could be transmitted horizontally by plasmid,suggesting that it is a severe challenge to prevent and control the spread of antibiotic resistance in CRKP.