Carbapenemase Genes Horizontal Transmission Mechanisms And Imipenem Synergistic Agents In Pseudomonas

The heavy use of antibiotics in clinical and breeding industries,and the increase in bacterial resistance have created a vicious cycle,leading to the emergence and prevalence of multi-drug resistant bacteria.Carbapenem drugs are most important antibiotics for the treatment of multi-drug resistant bacterial infections.It has been found to be resistant in a variety of Gram-negative bacteria,such as Pseudomonas.Pseudomonas aeruginosa is a common opportunistic pathogen in the environment,and it can cause many diseases,such as ventilator-associated pneumonia and cystic fibrosis,which seriously threatens human health.Pseudomonas hydrolyzes the corresponding drug by secreting carbapenemase.These resistance genes are often carried by mobile elements,which can be transmitted horizontally to increase the range and speed of transmission between and within species.And the slow development of new antibiotics has made it more difficult to treat infections with these bacteria.As a result,researchers have proposed a therapeutic strategy of antibiotic synergists that not only improves the effectiveness of antibiotics against drug-resistant bacterial infections,but also reduces the cost of their development.Natural compounds extracted from food have the advantages of being less toxic,less likely to induce bacterial resistance,and can be consumed for long periods of time.It is significance to develop it as antibiotic synergistic agent for the prevention and control of drug-resistant bacteria.In this study,40 carbapenem-resistant Pseudomonas（CRP）strains to 20 kinds of antibiotics was identified.And bacterial genetic sequence information was obtained by next-generation sequencing and single-molecule real-time sequencing,resolving the elaborate structure and evolutionary relationships within accessory genetic elements（AGEs）carrying carbapenemse genes and to explore their role in mediating the transmission of drug resistance genes.The novel carbapenems were extracted from 484 medicine food homology compounds,and the mechanism of action was explored.The main contents and results of this study are as follows:40 strains of CRP from Jilin Province were used as experimental objects.BD Phoenix^TM 100 automated identification and susceptibility testing system was used to identify bacterial species information and their susceptibility to 20 antibiotics.The bacterial draft genome was obtained by next-generation sequencing.Strains were identified by bioinformatics,screened for drug resistance genes,and analyzed phylogenetically for variation and genetic evolutionary relationships.The results showed that 39 strains of carbapenem-resistant Pseudomonas aeruginosa（CRPA）and one strain of carbapenem-resistant Pseudomonas juntendi（CRPJ）were identified.The resistance rate to 13 of the tested antibiotics exceeded 95%,with IP showing resistance to 3 strains of CRPA with minimum inhibitory concentration（MIC）for 4,096 μg/mL.A total of 27 acquired drug resistance genes were found from 40 CRP strains by ResFinder and CARD.And 6 strains carried 3 acquired carbapenemase genes(bla_IMP-1,bla_KPC-2 and bla_VIM-2),3 strains of which carried both bla_KPC-2 and bla_VIM-2.Virulence genes of 39 CRPA strains were screened by VFDB database,more than 90%of them with T3SS secreted protein genes exoY and exoT,and 85%of them with the exotoxin protein gene exoA.In this study,no exoA gene was screened in strains with acquired carbapenemase genes.A total of 9 serotypes and 17 STs were found in PAst and pbMLST database.There were no multi-drug resistance serotypes or pandemic clones,but a clinical CRPA isolate of new ST was identified.Phylogenetic tree showed that CRPJ 18091276 was genetically related to strains identified in the U.S.A.and Brazil,and CRPA carrying the same acquired carbapenemase genes were from the same ancestor.Complete chromosome and plasmid sequences of CRPJ 18091276 and CRPA 18083286 carrying bla_IMP-1 and CRPA 18102011 carrying both bla_KPC-2 and bla_VIM-2 were obtai ned by single-molecule real-time sequencing.Elaborate structures within AGEs carrying c arbapenemase genes were annotated and compared them with homologous elements to de termine genetic evolutionary relationships by bioinformatics and comparative genomics.C onjugation experiment was used to determine the horizontal transmission of acquired car bapenemase genes.The results showed that CRPJ 18091276 captured intI1-blIMP-1 region reconstituted downstream of res site IΔTn4662 via ICE1276,and then captured gene casse tte aacA’ form In1886.P.aeruginosa was further reconstituted by res site r1 of Tn402 transposition module to form substructure（intI1-blaIMP1-aacA4’-tniR-ISCfr1-aac（3）-IId-strB-strA-tniQ-tniB-tniA）of PA15W plasmid.P.aeruginosa 18083286 captured In992 with bl aIMP-1 to form carbapenem resistance by Tn7 transposon Tn6411,and Tn402-like transpos on module was missing,results in In992 unable to move.It was a stable presence in T n6411.bla_VIM-2 was captured as a gene cassette by In2057 and subsequently recombined into IncpRBL16 type mega plasmid pP2011-1,which carried by CRPA 18102011.By co mparing the structure of the same family plasmid,except existing 21 recombinant sites,and this plasmid formed 3 new recombinant sites（orf852,orf477 and orf432）.umuC wa s the stable recombinant site of this family plasmid.The plasmid captured the MBLs ge ne bla_VIM-2 by novel integron In2057,and then recombined into orf477 region,which be stowed bacteria with the ability to hydrolyze IP and resist enzyme inhibitors.bla_KPC-2 W as captured by the composite transposon consisting of ΔISKpn6 and ISKpn27,it subsequ ently recombined in IncP6 type plasmid pP2011-2 carried by CRPA 18102011.Of the AGEs carrying carbapenemase genes,only IncP6 and IncpRBL16 plasmids could deliver bl aKPC-2 and bla_VIM-2 horizontally into Escherichia coli DH5α,both with a conjugation effi ciency of 10-6.2"-O-galloylhyperin（HyG）with synergistic effect with carbapenem drug imipenem（IP）was selected from 484 medicine food homology compounds by checkboard method.Synergistic effects of compounds with IP were further determined by bacterial growth and time-kill curves,and the mechanism of action of food-derived synergist was determined by nitrocefin hydrolysis assay and transcriptome sequencing.The results showed that HyG（8 μg/mL）reduced the MIC value of IP against CRPA 18102011 and its transconjugant E.coli D2011 from 4,096 μg/mL to 1,024 μg/mL（FICI<0.5）,with synergistic effect.HyG（4 μg/mL）reduced the MIC value of IP against K.pneumonia 2445 with bla_KPC-2 from 128 μg/mL to 64 μg/mL.And the synergistic therapy showed no bactericidal activity against bacteria carrying other carbapenase types.The results of the bacterial growth and time-kill curves showed that HyG（8 μg/mL）had no effect on the growth of drug-resistant bacteria and significantly enhanced the bactericidal activity of IP.Transcriptional sequencing showed that the compound showed multiple locus regulation on the chromosomes and plasmids of tested strains.And gene ontology（GO）enrichment analysis showed the main reasons for the reduction of CRPA 18102011 resistance was the negative feedback regulation of some genes in the redox process to increase the level of radical oxygen species（ROS）in bacteria.This compound also significantly down-regulated the expression of IncP6 plasmid replicon gene repA.By observation of the color change in a nitrocefin hydrolysis experiment,this compound had an inhibitory effort on the expression of bla_KPC-2 downstream of repAIncP6,but inhibitory effort was not as significant as that of enzyme inhibitor avibactam.In summary,the results showed 40 CRP strains from Jilin Province carried three different types of acquired carbapenemase genes(bla_IMP-1,bla_VIM-2 and bla_KPC-2),mainly in the form of integron capture cassettes.There were cases where one strain carried multiple carbapenemase genes at the same time.One strain of CRPA carrying an IncpRBL16 type mega plasmid was identified,which has a highly diverse and complex mosaic molecular structure.The integration of large numbers of AGEs contributes to the accumulation and distribution of drug-resistant genes,improving the survival of Pseudomonas under drug selection pressure and increases the difficulty of treatment of infections caused by them.This plasmid can act as a mobile vector for carbapenemase genes and further dissemination between different species of bacteria.This study identifies the potential of HyG,a flavonoid from Pyrola,as an IP synergist through in vitro assays for the first time.It will provide a theoretical basis for subsequent studies related to drug resistance mechanisms and mitigation of resistance in CRPA,and offer a new food-derived natural compound as an adjunctive therapeutic strategy for CRPA.