| BackgroundGram-negative bacilli are important hospital opportunistic pathogens,including Enterobacteriaceae and non-fermenting bacteria.Generally,these bacteria are primarily colonized in the gastrointestinal tract,respiratory tract,and urethra.They can cause multiple types of clinical infections,such as primary or ductal bacteremia,peritonitis,endocarditis,surgical and wound infections,mediastinitis,and urinary tract infections,closely related to increased clinical fatality rate.Furthermore,cross-transmission between hospitalized patients can cause outbreaks and epidemics of nosocomial infections.Carbapenems are effective and broad-spectrum β-lactam antibiotics that effectively treat severe conditions caused by multidrug-resistant gram-negative bacilli.However,the emergence and spread of carbapenem-resistant gram-negative bacilli(CRGNB)poses tremendous challenges for clinical anti-infective treatment.Most CPGNBs exhibit extensive drug-resistant phenotypes,resistance to almost all β-lactam drugs and less sensitivity to multiple inhibitors,which can cause serious infections.Studies have shown that the wide range of resistance spectrum,potent carbapenemase activity,and resistance to inhibitors made VIM has become a predominant carbapenemase of clinical resistance in Gram-negative bacilli.In most instances,the blaVIMs co-exist with one or more aminoglycoside resistance genes,such as aacA4,aacA7,aadA1,aadA2,aadB and aacC1,embedded in the class Ⅰintegron,integrated into the transposon,and transferred along with the mobile elements between bacteria.Moreover,blaVIM also co-exists with other β-lactamase genes,such as blaoxA and blaPSE-1 causing a global public health crisis for the emergence of multidrug-resistant strains.Currently,the emergence of VIM-type carbapenemase has been reported in different regions of China.However,there is still a lack of large-scale screening about the distribution,expression,and genetic environment of VIM-producing gram-negative bacilli.In this study,1587 specimens collected from the clinical laboratory of the First Affiliated Hospital of Zhengzhou University were screened for VIM-type carbapenemase.A variety of experimental methods are used,such as PCR sequencing,antimicrobial susceptibility testing,S1-PFGE,Southern blot hybridization,conjugation assays,construction and expression of recombinant plasmids,as well as whole-genome sequencing and bioinformatics.Finally,we investigate the distribution characteristics of VIM,clarify the drug-resistant phenotype of the strains,elucidated the genetic environment and transfer mechanism of blaVIM,explore the expression of recombinant proteins.All the above results provide an essential basis for clinical control of the spread of drug-resistant genes and other inhibitors’ development.Method1.We first purified the collected samples of 1587 non-repetitive gram-negative bacilli.Then screen common carbapenemase genes(including blaNDM,blaKPC,blaVIM,blaIMP,and blaOXA-48)using PCR sequencing,identified the species of bacteria via MALDI-TOF.We finally identified ten strains of VIM-producing gram-negative bacilli in this study.2.Homology analysis of ZDHY95 and ZDHY372,two different specimens from the same patient,was performed by PFGE.3.The MIC values were determined using the agar dilution method and broth microdilution method to clarify the strains’ characterization.4.The location of the blaVIM gene was confirmed by S1-PFGE and Southern blot hybridization,5.The isolates’ conjugation experiment and the antimicrobial susceptibility testing of transconjugants confirm the plasmid’s transferability,the plasmid’s characteristics clarified by Plasmidfinder.6.The acquired resistance genes carried on the strains were analyzed by ResFinder.7.The annotation of whole-genome sequencing was conducted by RAST,and sequence alignment was performed using NCBI BLAST.Comparative genomics analysis of plasmid and genetic context of blaVIM was shown using BLAST Ring Image Generator(BRIG)and Easyfig 2.3,respectively.Result1.In this study,we identified a total of ten isolates of VIM-producing gram-negative bacilli from 1587 non-repetitive specimens from January 2019 to November 2019.All of the strains are belong to the Pseudomonas,except for the Providencia rettgeri,designated as ZDHY182.Among the isolates,three isolates carry blaVIM-1,five isolates harboring blaVIM-2,one isolate has blaVIM-24,and one isolate carries both blaVIM-1 and blaVIM-24.2.The PFGE electrophoresis results of the two strains,ZDHY95 and ZDHY372,showed that they are of the exact clone.Isolate ZDHY95 was selected for further detailed investigation.3.All the selected isolates are multidrug-resistant(MDR).They were resistant to amoxicillin-clavulanate,cefotaxime,ceftriaxone,imipenem,and trimethoprimsulfamethoxazole.And veried from intermediate to resistant for piperacillintazobactam,ceftazidime,cefepime,chloramphenicol,and gentamicin.Providencia rettgeri(ZDHY182)is resistant to tigecycline.4.All of 9 isolates harboring carbapenem,quinolones,sulfonamides,aminogly coside resistance genes.Other resistance genes,such as β-lactams,macrolides,tigecycline,fosfomycin,trimethoprim,and rifampicin,each strain’s carrying status is different.Only ZDHY354 carries the amide alcohol resistance gene.5.The genetic environment of the blaVIM gene is different,and it is mainly carried by different types of class I integrons,integrated into the transposon,and then contained on the plasmid.The plasmid carrying the class I integron embedded in blaVIM is the primary vector for disseminating blaVIM.6.There are seven types of the genetic environment of the blaVIM gene,and intl1-aacA4’-blaVIM-tniR is the conserved sequence.Conclusion1.In this study,the VIM-producing gram-negative bacilli have a diverse distribution among bacteria,a diversification of drug-resistant genes,and a broad spectrum of drug resistance.2.The plasmid carrying the class I integron embedded in blavVIM is the primary vector for disseminating blaVIM.3.The genetic environment of blaVIM is diverse,and intl1-aacA4’-blaVIM-tniR is the conserved sequence. |
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