Molecular Characteristics Of Enterobacteriaceae Producing NDM-1&CTX-M-15 And Non-targeted Metabonomics Of Mouse Infection

In the first part,we detected the molecular characterization of 3 isolates carrying blaCTX-M-15 and blaNDM-1.The MICs of the strains were determined by agar dilution method,the antimicrobial resistance genes were detected by PCR amplifications,strain types were determined by MLST,plasmid contents and gene locations on the plasmids were visualized by Sl-PFGE followed by Southern blot analysis,and the fitness costs of carrying the resistance genes in the transconjugants were evaluated by growth rate determination.In the second part,LC-MS was applied to non-targeted metabonomics study of mice infected with K.pneumoniae ATCC(?)BAA 2146 to discover potential biomarkers and metabolic pathways associated with sepsis.Blood samples were collected after infection for white blood cell counting as well as procalcitonin and C-reaction protein assays.Univariate analysis(UVA)and multivariate analysis(MVA)were applied to select changed metabolites.In addition,commercial databases including KEGG and MetaboAnalyst was utilized to search for the pathways of metabolites.The results showed that 3 isolates were highly resistant to aztreonam and meropenem.PCR analysis showed that all isolates carried resistance genes of different categories encoding β-lactamases,aminoglycoside modifying enzymes(AMEs),together with plasmid-mediated quinolone resistance(PMQR).Multilocus sequence typing assigned the 3 strains to type ST449(K.pneumoniae 1705 and 1706)and ST2(E.coli C600).S1-PFGE and Southern blot analysis demonstrated that the blaCTx-M-15 and blaNDM-1 located on different plasmids.Conjugation analysis demonstrated that the two genes cannot be cotransferred to one recipient,and usually only one type of transconjugants can be obtained.The plasmid didn’t cause obvious fitness cost in transconjugants with E.coli C600 as the recipient strain,while high fitness cost can be observed when E.coli J53 was the recipient strain.In the second part,58 metabolites were identified by principal component analysis(PCA)and partial least-squares discriminant analysis(OPLS-DA),combined with variable projection importance(VIP)and nonparametric test.18 of the 58 metabolites were further found to be involved in 8 metabolic pathways,including nicotinate and nicotinamide metabolism,pyrimidine metabolism,vitamin B6 metabolism,taurine and hypotaurine metabolism,arginine and proline metabolism,alanine,aspartate and glutamate metabolism,D-Glutamine and D-glutamate metabolism and glycerophospholipid metabolism.