Mechanisms Of Resistance To Ceftazidime-avibactam In KPC-90-Producing Carbapenem-Resistant Pseudomonas Aeruginosa

Objective:Multidrug-resistant Pseudomonas aeruginosa has currently become a serious threat in the clinic.As a new antibiotic,ceftazidime-avibactam has unique advantages in the treatment of Multidrug-resistant P.aeruginosa.However,P.aeruginosa resistant to ceftazidime avibactam has appeared clinically,and the detailed mechanism of its resistance is still unknown.In this study,a KPC-90-producing P.aeruginosa strain was screened from the clinic,and its resistance mechanism to ceftazidime avibactam was explored to provide a theoretical basis for its further clinical prevention and treatment.Methods:762 strains of carbapenem-resistant P.aeruginosa was isolated from the clinic,and the minimum inhibitory concentrations（MICs）of ceftazidime-avibactam were performed by the broth microdilution method recommended by Clinical Laboratory Standards Institute（CLSI）;Resistance genes were screened by using polymerase chain reaction（PCR）in ceftazidime-avibactam resistant isolates,including bla _KPC、bla _VIM、、bla _NDM、bla _IMP、bla _OXA;Multilocus sequence typing（MLST）was performed;Enzyme digestion cloning combined with drug susceptibility testing were performed to verify the resistance gene-mediated ceftazidime-avibactam resistance phenotype;Plasmid conjugation experiment was performed to verify whether the drug-resistant gene can be transferred horizontally;The genomic characteristics of the strain were clarified by sequencing the whole genome of the strain;The relevance of efflux pump genes in P.aeruginosa in mediating the development of ceftazidime-avibactam resistance was verified by using real-time polymerase chain reaction（RT-q PCR）and efflux pumps inhibition experiments.Results:Among 14 ceftazidime-avibactam resistant CRPAs,one P.aeruginosa strain PA2207 carrying a bla _KPC-2mutant gene was screened,and we named this mutant gene bla _KPC-90according to the gene naming rules.The whole genome sequencing results showed that PA2207 was ST463 type,and its plasmid carried bla _KPC-90gene.The deletion mutation of OPRD membrane porin resulted in carbapenem resistance.Compared with bla _KPC-2,bla _KPC-90inserted six nucleotides between 538-539 pairs of bases,resulting in the insertion of two amino acids（tyrosine and threonine）between 180th and 181th amino acids.The whole genome sequence analysis showed that the plasmid was highly similar to the plasmid p P23carrying bla _KPC-2on ST463 carbapenem resistant P.aeruginosa p23.In addition,the study of the expression level of the efflux pump gene found that the mex Y gene was increased by 7times.After adding the efflux pump inhibitor PaβN,the MIC value of ceftazidime-avibactam decreased from 512mg/L to 16mg/L.The results confirmed that the high expression of the mex Y gene of the efflux pump system of PA2207 was also an important reason for the resistance of the strain to ceftazidime-avibactam.Conclusion:This study is the first to report the mechanism of a new variant KPC-90generated by insertion of tyrosine and threonine outside theΩ-loop region of KPC protein to mediate the development of ST463 P.aeruginosa resistance to ceftazidime-avibactam.In addition,the high expression of efflux pump gene in P.aeruginosa can also lead to ceftazidime-avibactam resistance.PA2207 was isolated from the patient with hematological diseases who used antibiotics for a long time.Clinicians should pay attention to the possibility of multidrug-resistant bacteria infection in this population and take measures to prevent the spread of multidrug-resistant strains.