| Objective:1.To study the clinical distribution,antibiotic resistance status,epidemiological characteristics and infection-related risk factors of carbapenemase resistant hypervirulent Klebsiella pneumoniae(CR-hvKP);2.To analyze the drug resistance mechanism and virulence characteristics of CR-hvKP strain,carbapenemase resistant Klebsiella pneumoniae(CRKP)and hypervirulent Klebsiella pneumoniae(hvKP),and possible evolutionary mechanisms;3.To analyze the difference between CR-hvKP strain and CRKP strain against neutrophil phagocytosis,and explore the pathogenic mechanism of CR-hvKP strain.Methods:1.Klebsiella pneumoniae strains isolated for the first time clinically were collected from the affiliated hospital of Xuzhou Medical University from January 01,2018 to December 31,2018,and were analyzed relevant clinical data and risk factors.All Klebsiella pneumoniae were identified and confirmed using a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF MS),and the microbial mass spectrometer was used for identification and confirmation,and the hypervirulent Klebsiella pneumoniae(hvKP)was screened by the wire drawing test and virulence gene detection.Agar dilution method was used to detect the minimum inhibitory concentration(MIC)of hvKP against commonly used antibacterial drugs.WHONET5.6 soft was analyzed the MIC data and screened out CR-hvKP.2.PCR method was used to amplify related drug resistance Genes:carbapenem resistance genes(blaKPC,blaSME,blaGES,blaNDM,blaVIM,blaIMP,blaoXA-48),extended-spectrum ?-lactamases(blaSHV,blaTEM,blaCTX-M-1 group,blacTXM-2 group,blaCTX-M-8 group,blaCTX-M-9 group).PFGE and MLST were used to analyze the homology of the strains to predict the prevalence of CR-hvKP strains in the hospital.The plasmid conjugation test was used to confirm the ability of the carbapenemase plasmid to spread;3.PCR method was used to detect common capsular serotypes(K1,K2,K5,K20,K54,K57)and virulence genes(rmpA,kfu,aerobactin,iroN,entB,ybtS,fimH,mrkD,allS,rmpA2,iucA),S1-PFGE combined with Southern blot hybridization technology was used to analyze the virulence plasmid characteristics of CR-hvKP strain.4.The difference between CR-hvKP strain and CRKP strain was detected by anti-neutrophil phagocytosis test,and the pathogenic mechanism of CR-hvKP strain was preliminarily discussed.Results:1.A total of 1053 unreplicated Klebsiella pneumoniae were collected in this study,including 409 CRKP strains(38.8%,409/1053),189 hvKP strains(17.9%,189/1053),and 26 CR-hvKP Strain(2.5%,26/1053),429 other Klebsiella pneumoniae strains(40.7%,429/1053).The 26 CR-hvKP strains were all multi-drug resistant bacteria.They were 100%resistant to lactams,quinolones,and aminoglycosides.The resistance rate to tetracycline was 69.2%.No strains were found to be resistant to tigecycline and polymyxin.2.25 CR-hvKP strains(96.2%,25/26)isolated from adults carried blaKPC-2,but one strain(3.8%,1/26)isolated from newborns was detected to carry blaNDM-5.All 26 CR-hvKP strains were blaTEM-1 positive,23 strains were blaSHV-11 positive,and 3 strains were blaSHV-31 positive.14 isolates carried blaCTX-M-65,and 2 isolates carried blaCTX-M-14.A total of 5 ST genotypes were identified in 26 CR-hvKP strains.Among strains ST11 type was the most important ST type(87.5%,21/26),followed by ST 37(7.7%,2/26),ST337(3.9%,1/26),ST23(3.9%,1/26)And ST218(3.9%,1/26).PFGE results showed that 26 CR-hvKP strains were divided into 7 different PFGE clusters.Clusters A and C were the main clusters,accounting for 19.2%(5/26)and 53.8%(14/26)of the isolates,respectively.A detailed epidemiological investigation found that 7 ST11 isolates belonging to cluster C(KP 15,KP 16,KP 16,KP 17,KP 18,KP 19 and KP 20 strains)were isolated from 5 patients in an intensive care unit ward,which indicated that these strains were highly homologous.Further investigation found that during the same period,7 CR-hvKP strains were isolated from 5 patients in the intensive care unit,they had the same resistance phenotype,carbapenem resistance gene,MLST typing and PFGE map,which indicated that the CR-hvKP infections had locally occurred in this ward.We conducted an epidemiological survey of this outbreak.All five patients had serious underlying diseases and all underwent brain surgery,followed by antibacterial treatment and mechanical ventilation.All patients developed severe ventilator-associated pneumonia.CR-hvKP strains were isolated from sputum or blood samples of 5 patients.Although all five received targeted antimicrobial treatment and strict infection control measures,they unfortunately died.3.Among the 26 CR-hvKP strains,all strains had hypermucoviscousity(HM)phenotype,3 strains of capsular serotype K1 accounted for 11.5%,4 strains of K2 type accounted for 15.4%,and the remaining 21 strains were untyped(73.1%).The carrying rate of most common virulence genes exceeded 50%,of which entB(100%,26/26),aerobactin(92.3%,24/26),mrkD(92.3%,24/26),and fimH(88.5%,23/26),rmpA(84.5%,22/26),iroN(80.7%,21/26)and ybtS(65.4%,17/26),The virulence genes usually located on the virulence plasmid have a high carrying rate,such as iucA(100%,26/26),rmpA(73.1%,19/26),rmpA2(84.6%,22/26),iroN(80.7%,21/26).Pick 5 representative CR-hvKP strains and analyze the plasmid carrying rpmA.S1-PFGE and Southern blotting confirmed the presence of approximately 219 Kb virulence plasmids in the four strains.In addition to the?219 kb plasmid,the KP 15 isolate contained at least three plasmids with plasmid sizes of?88 Kb,?80 Kb,and?50 Kb,respectively.The KP18 isolate had a virulence plasmid of about 130 Kb.4.CR-hvKP group and CRKP group showed different degrees of anti-phagocytic ability against neutrophils,and the phagocytic rate of CR-hvKP group was significantly lower than CRKP group(p<0.01).7Conclusion:1.The clinical distribution of CRKP strains had the absolute advantage and remained the challenge of clinical anti-infection treatment;CR-hvKP strains are similar to CRKP strains in resistance rate,and are highly resistant to commonly used antibacterial drugs.This is quite different from hvKP strains,which are highly sensitive to most antibacterial drugs.Similar to the classic CRKP strain,advanced age,severe underlying diseases,various invasive procedures,and history of anti-drug use were high-risk factors for CR-hvKP strain infection.2.The CR-hvKP strains popular in this area were mostly ST11 type plasmid-mediated CRKP strain producing KPC-2 enzyme,and the drug resistance mechanism was highly similar to CRKP strain.There was a local outbreak of CRhvKP strains,and an effective prevention and control method should be implemented to reduce the prevalence of such bacteria.3.The CR-hvKP strains in this area were most likely due to the evolution of the classic CRKP strain producing ST 11 KPC-2,which has obtained a toxic plasmid(such as pLVPK)and got hypervirulent characteristics.4.Most of the CR-hvKP strains isolated in this area were rich in capsular polysaccharides,with a large number of virulence genes and strong anti-neutrophil phagocytosis ability,which resulted in CR-hvKP strains having a higher Klebsiella pneumonia stronger pathogenic ability.Significance:In this study,the "super bacteria" CR-hvKP was taken as the research object,the clinical distribution,drug resistance characteristics,risk factors and other epidemiological characteristics of Klebsiella pneumoniae were investigated.The molecular biology method was used to reveal the resistance mechanism and virulence factors of CR-hvKP,and provide laboratory data support for clinical and hospital infection control departments to treat and prevent the spread of such bacteria in the hospital. |
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