| Objective:1.To study the clinical characteristics,virulence and drug resistance of ceftazidime-avibactam resistance in carbapenem-resistant Klebsiella pneumoniae.2.To study the resistance mechanism of carbapenemase in ceftazidime/avibactam resistant CR-hv KP strains and assess the relationship with the virulence characteristics.3.To study the microbial characterization and genomics analysis of a non-enzymatic Producing ceftazidime/avibactam resistant CR-hv KP.Method:1.A total of 618 non-repeated Klebsiella pneumoniae strains of various samples from patients were collected during January 2014 to June 2020 from the First Affiliated Hospital of Nanchang University.ceftazidime/avibactam resistant CR-KP strains were screened by antibiotic susceptibility testing.Patient information were queried from the medical records.PCR and sequencing were used to detect the bacterial resistance genes,capsular serotype genes and virulence genes.S1-PFGE and southern blot hybridization was used to determine the location of plasmids.Multilocus sequence typing(MLST)and pulsed-field gel electrophoresis(PFGE)methods were used for molecular typing.2.By using string test and virulence genes detection,The ceftazidime/avibactam resistant CR-hv KP strains were screen out from the first part of the strains.We analyze the virulence and drug resistance of CR-KP strains.We used Multilocus sequence typing(MLST)and pulsed-field gel electrophoresis(PFGE)methods for molecular typing.S1-PFGE and southern blot hybridization was used to determine the location of plasmids.The virulence of CR-hv KP strains was analyzed by serum resistance assay and crystal violet assay.3.We found a ceftazidime/avibactam resistant CR-hv KP strain that did not produce carbapene Mase gene,sequenced the whole genome to explore the possible mechanism of drug resistance from the perspective of genomics.We analyzed its chromosome and virulence plasmid genomics,and compared it with the sequenced strain genome and analyzed its affinity.We determine their biofilm formation ability by crystal violet method.The results of serum resistance test reflected the virulence of the strain.Results:1.Most ceftazidime/avibactam resistant CR-KP strain in our hospital are from ICU and Burn department.Most patients with diabetes,hypertension,cerebrovascular accidents,hepatobiliary diseases and other serious basic diseases.89.5%of patients had invasive procedures.CR-KP strains mainly carry bla NDM-1and bla KPC-2gene,but bla KPC-3,bla NDM-5genes were also prevalent.The distribution of capsule serotyping was scattered,mainly K1?K57?K64 and untyped,but K2?K14?K20?K25 capsular serotypes were also found.ST23 and ST11 were the main epidemic clones of CR-KP strains.Most strains co-carried with ESBL and PMQR genes.In addition,CR-KP strains mostly carried virulence genes,such as sils?mrkd?sils?terw?rmp A?rmp A2,Among them sils carrying rate is highest,accounting for 71.1%.MLST showed that38 CR-KP strains belonged to 13 different ST such as ST11?ST23?ST4926?ST86?ST540?ST147?ST138?ST14?ST4065?ST345?ST2217?ST2673 and ST526,in which ST11 and ST23 were the main popular clones.PFGE results showed that 38ceftazidime/avibactam resistant CR-KP strains were divided into 14 different PFGE clusters.Clusters K and G were the main clusters,accounting for 26.3%(10/38)and23.7%(9/38)of the isolates,respectively.Some ST23 and ST11 CR-KP strains have high homology.2.Fifteen ceftazidime/avibactam resistant CR-hv KP strains isolated from the first part were mainly from ICU.All patients had invasive procedures such as tracheal intubation,catheter,central venous intubation,tracheotomy,ventilator,and5 of them underwent tracheotomy.Most carbapenem antibiotics,cephalosporins and other antibiotics are resistant,and the multidrug resistance rate is high.The drug resistance rate were consistent with CR-KP strains.The carbapenem gene was mainly bla NDM-1,followed by bla KPC-2gene type,3 strains carried bla NDM-5,and 1undetected.Most strains co-carried with ESBL and PMQR genes.Four hypervirulent serotypes K1,K2,K5 and K57 were found in capsule serotyping,of which K1 accounts for 66.7%.ST23 was the main type in the CR-hv KP strains,accounting for 60.0%,but hypervirulence ST typing such as ST86?ST11 were also existed.The carrying rates of rmp A?rmp A2?iuta?terw genes were 100%,significantly higher than those of CR-KP strains,there was a significant difference(P<0.001).Plasmid replicon typing was Inc F.The serum resistance was significantly higher than those of CR-KP strains(P<0.001),but there was no significant difference in biofilm formation.Plasmid replicon typing was Inc F.PFGE results showed that 15 ceftazidime/avibactam resistant CR-hv KP strains were divided into 5 different PFGE clusters.Clusters C was the main cluster,accounting for 60.0%(9/15)of the isolates.The 9 isolates(KP1?KP2?KP3?KP4?KP5?KP7?29?30?31)C cluster group all belong to ST23 type,which have high homology and indicate the existence of clonal transmission of CR-hv KP strains in our hospital.The mortality rate was as high as 60.0%,significantly higher than CR-KP group(34.2%),P<0.05.Southern blot showed the existion of rmp A2gene.Pick 6 representative CAZ/AVI resistant CR-hv KP strains and analyze the plasmid carrying rpm A2.SI-PFGE and Southern blotting confirmed the presence of approximately 216.9-244.4 Kb virulence plasmids in the six strains.3.The retrospective genomic study of the second part of the strains screened out the CR-hv KP S15.The strain caused pulmonary infection and liver abscess,and the capsule serotypes were K1 and K57,sequences were ST23,carrying rmp A?rmp A2?iuta?terw?mrkd 5 virulence genes.Serum resistance tests showed a high virulence phenotype.The whole genome sequencing revealed that S15 genome consists of a single chromosome and two plasmid.Six resistance genes and 13 virulence genes were found on chromosomes and plasmids.The comparative genome analysis showed that the S15 strain genomes had high homology with the other 9 highly virulent strains,and the plasmid 1 had high homology with the other 8 virulence plasmids.Virulence gene prediction showed carrying virulence genes such as clb B?fyu A?iro N?irp2?iuc C?iut A?mch F?ter C?tra T?mrkd?rmp A2?sils?terw.The prediction analysis of drug resistance genes showed that S15 mainly carried outer membrane pore protein gene ompk35?0mpk36??lactamase gene SHV-190 and CTX-M-14?quinolone gene,fosfomycin gene,etc,non carbapenem gene was found.Seventeen mobile genetic elements were predicted,7 on chromosomes and 10 on plasmids,including 1 integrative mobilizable element,13 insertion sequence,and 3composite transposon.Conclusion:1.Ceftazidime/avibactam resistant CR-KP strains mainly produced bla NDM-1and bla KPC-2carbapenemase in this area.Capsular serotypes were mainly K1?K57?K64and untyped,but K2?K14?K20?K25 were also found.ST23 and ST11 were the main epidemic clones of CR-KP strains.2.Ceftazidime/avibactam resistant CR-hv KP strains mainly carried bla NDM-1 gene,followed by bla KPC-2gene type.They also harbored several virulence genes and resistance gene.ST23 was the main type.Plasmid replicon typing was Inc F.The drug resistance mechanism was highly similar to CRKP strain.There was a local outbreak of CR-hv KP strains.3.S15 strain is a ST23 CAZ/AVI resistant CR-hv KP strain that does not produce carbapenemase,showing a high virulence phenotype,caz/avi the mechanism of drug resistance may be related to the SHV190. |
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