IMP-4 Carbapenemase Plasmid Dissemination Mechanism And Drug Combination Strategy In Enterobacteriaceae

Object To understand the drug resistance,molecular characteristics and virulence factors of Enterobacteriaceae carrying bla _IMP-4,to explore the optimal treatment of Enterobacteriaceae carrying bla _IMP-4 with different antibiotics,and the mechanism of horizontal transfer of plasmids in Enterobacteriaceae,aiming at it can provide ideas for the development of a new generation of preventive measures for plasmid-mediated drug resistance transmission,and provide laboratory evidence for the treatment selection of carbapenem-resistant bacteria.Method A total of 259 strains of Carbapenem-resistant Enterobacteriaceae（CRE）isolated from two tertiary grade A teaching hospitals were collected,of which 4 strains of IMP-4carbapenemase positive strains were screened.1.Use polymerase chain reaction（PCR）to detect related drug resistance genes:β-lactam gene,Amp C enzyme coding gene,plasmid-mediated quinolone resistance gene,polymyxin resistance gene.2.Minimum inhibitory concentration（MIC）was detected by broth dilution method.3.Checkerboard titration and time-killing experiments were used to explore the optimal combination of different antibiotics for CRE carrying bla _IMP-4 gene.4.The virulence of carrying bla _IMP-4 was detected by biofilm formation test and serum killing test.5.Conjugation test,natural passage method and SDS-high temperature alternation method were used to verify the transfer and stability of plasmids.6.Whole genome sequencing technology was used to detect the detailed information of drug resistance genes,virulence genes,drug resistance plasmids,multi-locus sequence typing（MLST）and the composition of type IV secretion system（T4SS）.7.Quantitative Real-time PCR（RT-q PCR）was used to detect the expression of the conjugation gene tra D.Result 1.Detected bla _IMP-4（3 strains）and bla _IMP-26（1 strain）carbapenem resistance genes,and also detected mcr-9,bla _SHV,bla _TEM,bla _DHA,acc（6’）-lb and bla _OXA-1 resistance genes,no bla _KPC,bla _NDM,bla _VIM,bla _ACC and bla _CTX-M resistance genes were detected.2.All isolates were resistant to meropenem,polymyxin,imipenem,ceftazidime-avibactam,and 25%of the isolates were resistant to tigecycline,amikacin and levofloxacin.3.The FICI value of MEM+LEV on CRECL42 was 0.25,which showed synergistic effect.The FICI values of MEM+AK,AK+LEV and PB+IMP on CRECL60 were all 0.5,showing a synergistic effect.No synergy was seen for CRECL42 in any combination in the time-killing experiments.The combination of TIG（0.5MIC and 1MIC）+MEM/IMP（1MIC）and TIG（2MIC）+IMP（0.5MIC and 1MIC）showed a synergistic effect on CRECL60.The combination of TIG（0.5MIC and 1MIC）+IMP（0.5MIC and 1MIC）showed synergistic effect on CRKP294.The combination of TIG（0.5MIC,1MIC and 2MIC）+MEM/IMP（0.5MIC）exhibited an antagonistic effect on CRECL352.1MIC IMP showed stronger bactericidal and bacteriostatic ability to all strains than all combinations at 4-12h,TIG+1MIC IMP showed stronger synergistic effect on CRECL60(carrying bla _IMP-26)than other combinations at 24h.4.One strain of CRKP showed strong biofilm formation,and three strains of CRECL showed weak biofilm formation.One strain of CRKP was resistant to serum killing,two CRECL strains were mediators of serum killing,and one CRECL was extremely sensitive to serum killing.5.Among the 4 strains carrying IMP metalloenzyme,only the CRECL42 strain was successfully conjugated,and the Inc P plasmid was the most stable in the plasmid stability test.After 50 passages,the plasmid was not lost.The second is Inc C and Inc HI2,the most unstable is Inc U,the plasmid of the zygote is more stable than that of the original strain.6.Whole genome sequencing showed that the MLST types of 3CRECL strains were ST520,ST528 and ST25,and 1 strain of CRKP was ST37.The plasmid types of the four CRE strains were Inc C,Inc HI2,Inc U,and Inc P,respectively.Plasmids carrying bla _IMP from different strains carry multiple different drug resistance genes and virulence factors at the same time.Both bla _IMP-4 and bla _IMP-26 are on class 1 integron,and the surroundings of bla _IMP-4 are acc（6’）-lb-qac G2-ltr A-bla _IMP-4-intl1 and acc（6’）-lb-qac G2-bla _IMP-4-intl1,the surroundings of bla _IMP-26 are IS6100-sul1-qac E-ltr A-bla _IMP-26-intl1-drf A19.Three strains detected"F"type T4SS,and the constituent proteins included tra C,tra D,tra I,tra G,tra A,etc.,and one strain detected"P"type T4SS,and the constituent proteins included trb G,trb B,trb H,trb F,etc.7.The relative expression level of tra D gene of zygote J42 is 1.08 times that of CRECL42,while the relative expression level of tra D of CRECL60 and CRKP294 is 0.31times that of CRECL42.Conclusion 1.According to the checkerboard titration method and the time-kill test to evaluate the antibacterial activity in vitro,the antibacterial activity of imipenem with bla _IMP-4alone was the best at 8-12h,and the bactericidal effect of TIG+1MIC IMP with bla _IMP-26 was the best at 24h.It is recommended that the clinical treatment of Enterobacteriaceae carrying bla _IMP-4and the minimum inhibitory concentration of imipenem≤4μg/m L can use imipenem（short-acting）for the clinical treatment of Enterobacteriaceae carrying bla _IMP-26 can be combined with TIG+1MIC IMP（long-acting）.2.Carrying IMP-4 and IMP-26 carbapenem resistance gene has little effect on virulence.3.In this study,bla _IMP-4 and bla _IMP-26 are located on the class 1 integron of different plasmids.The surrounding environment of the two Enterobacter cloacae bla _IMP-4is the same,both acc（6’）-lb-qac G2-bla _IMP-4-intl1,and one of the Inc C-type plasmids was successfully conjugated,indicating that the Inc C plasmid could lead to the horizontal spread of drug-resistant plasmids and played an important role in the spread of bla _IMP-4.4.Different plasmids carrying bla _IMP-4 and bla _IMP-26 have different stability.Inc P plasmid is the most stable,followed by Inc C and Inc HI2,and the most unstable is Inc U.The plasmid of the zygote is more stable than the plasmid of the original bacteria.Stability is related to successful engagement.5.In the plasmid carrying bla _IMP-4,whether the expression of vir D encoding T4SS and the level of the tra D gene expression may affect the ability of bacteria to conjugate,thereby promoting the spread of drug-resistant plasmids.