Mechanism Of Drug Resistance Of Aminoglycosides In Multidrug Resistant Klebsiella Pneumoniae

Objective:To study the drug resistance characteristics of aminoglycoside-resistant Klebsiella pneumoniae（AGR-KP）in our hospital,to explore the drug resistance mechanism,molecular epidemic characteristics of AGR-KP and combined medication selection,which would provide laboratory data for the rational selection of antibiotics for clinical patients with AGR-KP infection,as well as the monitoring and control of drug resistant strains.Methods:Collected the aminoglycoside-resistant Klebsiella pneumoniae isolated from the clinical samples submitted by the First Hospital of Dalian Medical University from May 2020 to September 2021.The bacteria were identified by Clin-To F-ⅡTime-of-flight Mass Spectrometry,and the drug sensitivity analysis was derived from the VITEK 2 COMPACT system.Polymerase chain reaction（PCR）and sequencing were used to detect aminoglycoside resistance genes（16S rRNA methylase gene,aminoglycoside modifying enzyme gene）and carbapenemase gene(bla_KPC,bla_NDM,bla_IMP)of the strains.The PCR-based replication typing（PBRT）method was used to determine the plasmid carrying status of drug-resistant strains,and the plasmid conjugation experiment was used to clarify the transfer of aminoglycoside resistance genes between strains.The single nucleotide polymorphism（SNP）of high-level aminoglycoside-resistant Klebsiella pneumoniae（HLAGR-KP）was analyzed using whole-genome sequencing technology,and the molecular typing of HLAGR-KP strain was carried out using multilocus sequence typing（MLST）to explore the epidemiology of the strain at the molecular level.For CRKP,the synergistic,additive,irrelevant and antagonistic effects of amikacin+meropenem,amikacin+polymyxin,meropenem+polymyxin combinations were detected by chessboard method.Results:（1）All the 104 AGR-KP strains were multidrug-resistant,withβ-lactam antibiotics resistance,among which 52 strains were carbapenem-resistant（CRKP）,and the other 52 strains were Extended-Spectrumβ-Lactamases（ESBLs）producing strains.（2）75.0%of CRKP strains showed high level of aminoglycoside resistance,while only 11.5%of ESBLs-KP strains showed high level of aminoglycoside resistance.The 16S rRNA methylase gene rmtB was the main gene leading to high level of aminoglycoside resistance.In the ESBLs-KP group,the carrying rate of aminoglycoside modifying enzyme genes aac（3）-IIa and aph（3’）-Ia was significantly higher than that in the CRKP group.（3）In CRKP group,95.1%（39/41）of KPC-producing CRKP strains carried 16S rRNA methylase gene,which showed high level of aminoglycoside resistance;However,all 11 strains of CRKP that only produce NDM metalloproteinase have no high level of aminoglycoside resistance and are sensitive to amikacin.（4）Four types of plasmids were detected in 52 CRKP strains,and the carrier rate from high to low was IncFII（84.6%）,IncN（48.1%）,IncFIIK（42.3%）,and IncFIIS（26.9%）.Among 39 CRKP strains with high level of aminoglycoside resistance,IncFII+IncN（n=19）is the main combination type.Among the 13 CRKP strains sensitive to amikacin,IncFIIK+IncFIIS（n=8）is the main combination type.（5）Six plasmid types were detected in 52 strains of ESBLs-KP,and the carrier rate from high to low was IncFIIK（75.0%）,IncFIIS（51.9%）,IncFII（19.2%）,IncN（5.8%）,IncX（1.9%）,and IncI（1.9%）.Among the six strains of ESBLs-KP with high level of aminoglycoside resistance,IncFII（n=5）is the main plasmid type.Among 46 strains of ESBLs-KP sensitive to amikacin,IncFIIK+IncFIIS（n=22）was the main combination type,and 6 strains failed to amplify the plasmid type,considering that the resistance gene might be mediated by chromosome or other mobile elements.Through further comparison and analysis,it was found that the carrying rate of IncN and IncFII plasmids in CRKP group was significantly higher than that in ESBLs-KP group;However,the carrying rate of the plasmid types of IncFIIK and IncFIIS in ESBLs-KP group was significantly higher than that in CRKP group.（6）Plasmid conjugation experiment showed that rmtB gene could be transferred between different genera through plasmid.（7）Among the 45 HLAGR-KP strains,ST147 is the main epidemic strain,and ST11 is the secondary epidemic strain.The two groups of strains are mainly from the Emergency Intensive Care Unit（EICU）,and there are sporadic cases in other wards.ST147 strains have high homology,and the number and type of plasmids and drug resistance genes carried are high consistent.70.4%（19/27）of ST147 strains carry the combination of IncF II+IncN+rmtB+aac（6’）-Ib+aph（3’’）-Ib;The genetic distance between ST11 strains is relatively far,and the number and type of plasmids and drug resistance genes carried by ST11 strains vary.（8）For CRKP,the synergistic effect of amikacin combined with meropenem was 76.9%（10/13）.The synergistic effect of amikacin combined with polymyxin was 15.2%（2/13）.The synergistic effect of meropenem combined with polymyxin was 13.5%（7/52）.Conclusions:CRKP group was dominated by high level aminoglycoside resistance mediated by rmtB,and ESBLs-KP group was dominated by low level aminoglycoside resistance mediated by aminoglycoside modifying enzyme;High level of aminoglycoside resistance mainly occurs in KPC-enzyme-producing CRKP;The plasmids carried by aminoglycoside-resistant CRKP strain and ESBLs-KP strain were different;The rmtB gene can be transferred between genera by plasmid;High attention to the transmission and prevalence of ST147 and ST11 strains in EICU wards should be given,and effective prevention and control of nosocomial infection should be carried out;For CRKP,in vitro combined drug sensitivity test showed that amikacin+meropenem combined treatment had the highest synergistic effect rate.Therefore,in the control of CRKP infection,priority should be given to the selection of drug combinations with synergistic effect to enhance the efficacy,reduce toxic side effects,and prevent the further generation of multi-drug resistant strains.