Study On Carbapenemase Gene Characteristics And Disinfectant Resistance Of Carbapenem-resistant Klebsiella Pneumoniae

Objectives:1.To investigate the clinical distribution and drug resistance of carbapenem-resistant Klebsiella pneumoniae（CRKP）in Sun Yat-sen Memorial Hospital from January 2015 to December 2019.2.To analyze the carbapenemase gene types carried by CRKP strains in our hospital,and to provide important clues and theoretical basis for the control of nosocomial infection and outbreak of CRKP strains,as well as effective clinical anti-infection therapy.3.To study the resistance of CRKP strains to7 kinds of disinfectants commonly used in clinic,so as to guide and assist the effective development of infection control work in hospital.4.To explore the mechanism of disinfectant resistance by detecting the main efflux pump genes carried by CRKP strains.Methods:1.WHONET 5.6 software was used to analyze the clinical data and resistance situation of 162 non-repetitive CRKP strains isolated from clinical infected patients in Sun Yat-sen Memorial Hospital from January 2015 to December 2019.2.Twelve carbapenemase genes(bla _KPC、bla_NDM-1、bla_VIM、bla_IMP、bla_SIM、bla_OXA-48、bla OXA-58、blaOXA-51、blaOXA-24、blaOXA-23、blaOXA-20、blaOXA-10)were detected by PCR technology in 64 non-repetitive CRKP strains which were preserved in our laboratory.3.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）experiments were selected to detect the resistance of CRKP strains to seven kinds of disinfectants（0.1%benzalkonium bromide,4%chlorhexidine,75%alcohol,entoiodine II,2%glutaraldehyde,2000mg/L chlorine-containing disinfectants,3%hydrogen peroxide）.Three efflux pump genes（oqxA、oqx B、and qac E△1-sul1）were detected by PCR technology.Results:1.A total of 162 strains of non-repetitive CRKP strains were isolated in Sun Yat-sen Memorial Hospital from 2015 to 2019,and the annual detection rates were 1.21%,2.26%,8.69%,8.29%and 9.89%,respectively,showing an increasing trend,especially in 2017.2.Most of the 162 non-repetitive CRKP strains were isolated from sputum（45.4%,74/162）,followed by urine（12.9%,21/162）,ascites/abdominal drainage fluid（11.0%,18/162）and blood（9.8%,16/162）.The top 3 departments were ICU,neurology/neurosurgery and rehabilitation,accounting for 41.1%（67/162）,10.4%（17/162）and 8.0%（13/162）,respectively.3.The antibiotic resistance rates of 162 non-repetitive CRKP strains to colistin and tigecycline were 0.00%and 10.00%,respectively,and the resistance rates of other antibiotics were more than 50%.The antibiotic resistance rate of tigecycline in our hospital（10%）was higher than the national average level in 2019（4.7%）and 2020（6.7%）.4.The positive rates of carbapenemase genes bla _KPC,bla _NDM-1,bla _VIM,bla _IMP,bla _SIM,bla _OXA-10,bla _OXA-51in 64 non-repetitive CRKP strains were 31.25%（20/64）,25.00%（16/64）,4.69%（3/64）,4.69%（3/64）,1.56%（1/64）,1.56%（1/64）and 1.56%（1/64）,respectively.However,bla OXA-58,bla OXA-48,bla OXA-24,bla OXA-23 and bla OXA-20 genes were not detected.5.Bla _KPC gene was mainly detected in ICU（60.00%）and sputum specimens（70.00%）,showing a highly centralized trend,while the concentration trend of bla _NDM-1gene was not found.6.64 non-repetitive CRKP strains preserved in our laboratory showed resistance to 6disinfectants,including 0.1%benzalkonium bromide,4%chlorhexidine,entoiodine II,2%glutaraldehyde,3%hydrogen peroxide and 2000mg/L chlorine-containing disinfectants,but no resistance was found in 75%alcohol.Attention should be paid to ensure that the effective chlorine concentration reached 1000mg/L when using chlorine-containing disinfectants and 0.1%benzalkonium bromide should be avoided dilution as far as possible.7.The mean MIC values of chlorhexidine in ICU ward and non-ICU wards were0.0034%and 0.0019%,respectively,and there was a statistical difference between them（P=0.02<0.05）,but the mean values were much lower than the current clinical routine concentration（2%or 4%）.8.The positive rates of 3 efflux pump genes oqxA,oqx B and qac E△1-sul1 of 64non-repetitive CRKP strains preserved in our laboratory were 68.75%（44/64）,15.63%（10/64）and 89.06%（57/64）,respectively.There were 5 positive detection patterns,in which the qac E△1-sul1+oqxA positive pattern was the main one（48.44%）,followed by the single qac E△1-sul1 gene positive pattern（28.13%）.9.There was a statistical difference in the MIC values of 0.1%benzalkonium bromide between the negative and positive oqxA gene groups（P=0.046<0.05）,while the MIC values of entoiodine II was significantly different between negative and positive qac E△1-sul1 gene groups（P=0.037<0.05）,and there was no statistical difference between the negative and positive efflux pump gene groups among the other disinfectants.Conclusions:1.Most of the CRKP strains isolated in our hospital between 2015 and 2019 were mainly from sputum specimens and ICU department.The CRKP strains showed high resistance rate to commonly used antibiotics.2.Bla _KPC was the main carbapenemase gene carried by CRKP strains isolated in our hospital,and it was mainly concentrated in ICU ward and sputum specimens.3.The CRKP strains had resistance to 0.1%benzalkonium bromide,4%chlorhexidine,entoiodine II,2%glutaraldehyde,3%hydrogen peroxide and 2000mg/L chlorine-containing disinfectants,but no resistance was found to 75%alcohol.All kinds of the disinfectants could kill CRKP strains according to the Regulation of disinfection technique in healthcare settings.4.The carrying rate of efflux pump genes in CRKP strains in our hospital was high,and the mechanism of efflux pump could be one of the mechanisms by which CRKP develops resistance to disinfectants.