Molecular Characteristics Of Klebsiella Pneumoniae With High Virulence And Drug Resistance Analysis Of Sputum Cultured Bacteria In Respiratory Department Of A Hospital

Hypervirulent Klebsiella pneumoniae(hvKp)poses a serious threat to people’s health.With the growing problem of bacterial resistance,K.pneumoniae with hypervirulence and drug resistance poses great challenges for clinical treatment.In this study,we analyzed the clinical and microbiological characteristics of hvKp in a hospital from Hebei Province,and examined the genetic structure basis of a multidrug-resistant the hvKp strains by whole genome sequencing,hence to provide insights for the development of effective therapeutic strategies for controlling hvKp infections in clinic.The respiratory department is the main department where infections occur,and the sputum specimen is the main source of bacteria isolations.We analyzed the distribution and drug resistance profile of sputum cultured bacteria in the respiratory department of the First Affiliated Hospital of Hebei North University,and provide reference for clinical rational use of antibiotics.In the first part,we analyzed the clinical distribution,molecular characteristics and the genome characterization of hvKp.Firstly,hvKp was identified by PCR amplification of hypervirulence-related genes,the hypermucoviscous phenotype was determined by the"string test".Logistic regression was used to identify risk factors for hvKp infection.The drug susceptibility analysis was performed using the VITEK(?)2 Compact Bacterial Identification and Monitoring System.The capsular serotype of hvKp strain was detected by PCR,and the molecular epidemiological characteristics of the strains were analyzed by pulsed-field gel electrophoresis(PFGE).The result showed that a total of 113 non-repetitive K.pneumoniae was isolated in 2015,of which,59(52%)isolates were identified to be hvKp.Preliminary analysis of clinical characteristics showed that compared with cKp patients,hvKp-infected patients have a higher average age,higher polymorphonuclear(PMN)white blood cell count,and urinary catheter was more frequently to be the source of hvKp infection.hvKp was more susceptible to antibacterial drugs than cKp,and one ESBLs-producing hvKp strain was detected.The main capsular serotype of hvKp were K2,K57 and K1.PFGE indicated that the 59 strains of hvKp could be classified into 51 PFGE band types,forming 6 PFGE clusters.At the same time,we found a multidrug-resistant hvKp Kpn1693.Drug susceptibility testing of the strain was performed by broth microdilution method,and the hypervirulent phenotype of the strain was analyzed by string test and mice systemic infection model.The whole genome of Kpn1693 was sequenced by PacBio Sequel platform,and annotated by related software,the phylogenetic relationship of pKpn1693-CTXM with other IncFⅡ plasmids carrying variants of blaCTXD-M was analyzed by MEGA X software.The results showed that Kpn1693 was resistant to multiple antibiotics and exhibited a multidrug-resistant phenotype.The virulence assay showed that Kpnl 693 was positive for the string test,and showed a hypervirulent phenotype in the mouse systemic infection model.Whole genome sequencing showed that the Kpn1693 genome contained one circular chromosome and two circular plasmids.The IncHI1B/IncFIB plasmid(pKpn1693-Vir)had a high similarity with the hypervirulence plasmid pK2044.The IncFⅡ plasmid(pKpn1693-CTXM)carried a gene integrated in the ISEcp1-like element,suggesting possible formation from an IncFⅡ plasmid recruiting a blacTX-M-24 gene via mobile genetic elements.As far as we know,this is the first report of blacTx-M-24-bearing IncFⅡ plasmid carried by a multidrug-resistant hvKp.In the second part,we analyzed the distribution and drug resistance of sputum culture bacteria from January 1,2014 to December 31,2018 in the respiratory department of the First Affiliated Hospital of Hebei North University.The drug susceptibility was detected by BD PhoenixTM-100 Automated Microbiology System,and the data were analyzed using WHONET 5.6 software.A total of 1268 strains were isolated,of which 91.5%were gram-negative strains,and 8.5%were gram-positive strains.The top five bacteria were Klebsiella pneumoniae(15.9%),Pseudomonas aeruginosa(10.4%),Klebsiella oxytoca(10.4%),Acinetobacter baumannii(9.8%)and Enterobacter cloacae(8.9%).Gram-negative bacteria have high resistance rates to ampicillin,cefazolin,ampicillin/sulbactam,amoxicillin/clavulanic acid,being 72.2%(255/353),55.6%(293/527),47.8%(367/767)and 46.8%(252/538),respectively.And the resistance rates to colistin,amikacin,meropenem,and imipenem were lower,being 3.5%(39/1101),5.6%(61/1082),6.4%(68/1065)and 7.7%(83/1081),respectively.The detection rates of gram-positive bacteria were low,with staphylococcus of the highest proportion(75%,81/108),but the detection rate of methicillin-resistant staphylococcus(MRS)was increasing.In summary,the detection rate of hvKp in this hospital was high,and it is speculated that people with older ages or a higher PMN cell count are more likely to gain hvKp infection.ESBLs-producing hvKp was emerging,indicating the importance of epidemiologic surveillance and clinical awareness of this pathogen in this region.The strain Kpn1693 was a multidrug-resistant hvKp strain with a blaCTX-M-24 carrying IncFⅡ-type plasmid and a pK2044-like virulent plasmid,and this is the first report of blaCTX-M-24-bearing IncFⅡ plasmid carried by a multidrug-resistant hvKp.From 2014 to 2018,the sputum culture bacteria in the respiratory department of the First Affiliated Hospital of Hebei North University were mainly Gram-negative bacteria.The resistance rates of Gram-negative bacteria to antibiotics didn’t show trends of increase year by year,but decreased first and then increased,indicating that the control of antibiotic use in clinic needs to be strictly and long-term implemented.