Study On Clinical Characteristics And Transmission Mechanism Of Virulence Gene Of Highly Virulent Klebsiella Pneumoniae

Objective:1.Different detection methods of Highly virulent Klebsiella pneumoniae(Hv KP)were compared to provide laboratory basis for accurate identification of Hv KP strain.2.To analyze the specimen source,department distribution,Sequence Type(ST)and virulence gene distribution characteristics of Hv KP in our hospital from 2017 to 2021;Risk factors for death of Hv KP patients were analyzed.3.To investigate whether the resistance genes of carbapenem-resistant Klebsiella pneumonia(CRKP)and the virulence genes of Hv KP in our hospital have the ability of horizontal transmission.Methods:1.The strain Hv KP was identified by the drawing Test(String Test,ST)combined with the toxicology factor Rmp A and aerobactin,and the accuracy of the combined detection of the two methods was verified by the G.mellonella larva lethal Test.2.Clinical data of 50 strains of Hv KP in our hospital from 2017 to 2021 were collected,and the distribution characteristics of specimen sources and departments were analyzed.PCR amplification and agarose gel electrophoresis were used to detect the most common 18 virulence genotypes of 50 Hv KP strains.Seven housekeeping genes of 50 Hv KP strains were amplified by PCR.The target band was sent to Tianyi Huiyuan Biotechnology Co.,LTD for bidirectional DNA sequencing.DNAStar,Chromas,Primer5 and Alignx software were used to spline,confirm and correct the sequencing results.And the corrected spline results were uploaded to Pasteur website to confirm the specific sequence type.The phylogenetic tree was constructed by Mega 6.0 software.Clinical data,treatment and outcome of 50 Hv KP strains were collected,and the main risk factors of death caused by Hv KP infection were analyzed by statistical method(Chi-square test).3.The CRKP strain capable of horizontal transmission of drug resistance genes was screened through the plasmid conjugation test between CRKP strain and EScherichia coli J53;Then,the selected CRKP strain and Hv KP strain were subjected to plasmid conjugation test to screen the carbapenem-resistant hypervirulent Klebsiella pneumoniae(CR-HVKP).Results:1.50 strains showed positive in string test(string length>5mm);The virulence factor detection rate of aerobactin and Rmp A was 100%.The larval death test of G.mellonella showed that the larval death rate of G.mellonella was 100%within 72 hours after injection of Hv KP suspension at the concentration of 10~6cfu/ml.2.Hv KP strains were mainly from sputum and blood samples,accounting for 38%and 30%,respectively;Abscess and liver abscess puncture material accounted for 10%.The proportion of central venous catheterization and urine was 4%.The proportion of drainage fluid and cerebrospinal fluid was 2%.ICU and digestive ward were the main examination departments,accounting for 34%and 24%respectively.Respiratory wards accounted for 5%;Neurological wards accounted for 3%;Neurosurgery ward,urology ward,endocrine ward,cardiovascular ward,hepatobiliary ward accounted for 2%;Hematology ward,oncology ward and rheumatism ward accounted for 1%;ST23 was the main Hv KP strain with invasive infection.ST2861 was the main Hv KP strain with non-invasive infection.ST2861 was predominant in sputum,ST23 was predominant in blood and pus,and ST65 was predominant in liver abscess puncture.ST2861 was the main type in ICU,ST2861 and ST23 in respiratory ward,ST23,ST65 and ST828 in digestive ward.The distribution of the virulence factors of the 50 strains of Hv KP was as follows,and the virulence gene carrying rate of 100%belonged to the aerobactin,Rmp A,ycf M and ent B.The carrying rates of other virulence factors are as follows:Mag A 44%(22/50),wca G 80%(40/50),ure A 94%(47/50),ALLS 84%(42/50),mrk D 92%(46/50),fim H 98%(49/50),KPN 72%(36/50),Kfu 50%(25/50),IRP-1 80%(40/50),IRP-2 86%(43/50),YTBS 80%(40/50),fyu A 78%(39/50),UGE 92%46(/50),wab G 94%(47/50).ICU admission history,septic shock,acid-base imbalance,mechanical ventilation,deep vein catheterization,indwells catheterization,carbapenems and quinolones use were the main risk factors for death of Hv KP patients(P<0.05).3.It was found that one strain of CRKP was successfully conjugated with Hv KP strain to form CR-HVKP strain.The strain showed positive in string test,positive in drug resistance gene and virulence gene,and all G.mellonellalarvae died within 24h.Conclusion:1.The strain Klebsiella pneumoniae(Kp)that had the aerobactin and Rmp A toxicity factors positive at the same time could be identified as Hv KP strain.2.Hv KP strains in our hospital were mainly collected from sputum and blood samples and distributed in ICU and digestive ward.The main Hv KP strains were ST23 and ST2861,and the ST types were different due to the different sources of specimens and departments.The virulence factor carrying rate of Rmp A,aerobactin,ycf M and ent B was 100%.ICU admission history,septic shock,acid-base imbalance,mechanical ventilation,deep vein catheterization,indwells catheterization,using of carbapenems and quinolones were the main risk factors for death of Hv KP patients(P<0.05).3.CRKP and Hv KP strains in our hospital can achieve horizontal transmission of drug resistance genes and virulence genes through plasmid conjugation,suggesting that clinical and auxiliary departments should strengthen the prevention and control of nosocomiasis.