Study On The Mechanism Of The Modulation Of Transfer-message RNA Regulating The Persistence Of Cefotaxime In Aeromonas Veronii

Persistent bacteria are a small number of phenotypic heterogeneous variants,which can survive instantly in high concentrations of antibiotics,causing repeated infections in humans and aquatic animals.Aeromonas veronii(A.veronii)is one of the most threatening pathogens in aquaculture.It causes a large number of deaths in aquaculture,causes catastrophic economic losses to the fish industry,and shows natural resistance to a variety of antibiotics.Transfer-messenger RNA(tmRNA),encoded by ssr A gene,is one of the elements of trans-translation and has been identified as a decisive regulator of β-lactam antibiotic retention.N-acetylglucosamine(GlcNAc)is one of the components of bacterial peptidoglycan,which is mainly involved in aminoglycoside metabolism and plays a role in bacterial pathogenicity.In this study,the growth curves of wild-type and tmRNA-deleted strains of Aeromonas veronii C4 tmRNA were detected,and it was found that there was no difference between the growth curves of wild-type and tmRNA-deleted strains,indicating that tmRNA is not necessary for growth in Aeromonas veronii.Then,the formation of cefotaxime retention bacteria of wild type and tmRNA deletion strains was detected,and it was found that the number of cefotaxime retention bacteria of tmRNA deletion strains increased significantly.In order to further explore the cause of the difference,by adding chloramphenicol pretreatment and simulating the condition of hindered protein synthesis,it was found that there was a significant increase in the number of persistent bacteria in the wild type and a slight increase in the number of tmRNA deletion strains,which may be related to the decrease of protein synthesis.Then,by analyzing the molecular basis of phenotypic changes by transcriptology and metabonomics,the results showed that the deletion of tmRNA significantly increased the level of intercellular metabolite GlcNAc,promoted the tolerance to Na Cl,and up-regulated the expression of synthetic genes in the upstream and downstream peptidoglycan biosynthesis pathways of peptidoglycan(PG)biosynthesis.Finally,the metabolite GlcNAc was studied separately.On the one hand,exogenous GlcNAc was added to the wild-type strain to detect its phenotype,and it was found that exogenous GlcNAc stimulated bacterial growth in a concentration-dependent manner,increased the content of GlcNAc in the cell wall,and improved the resistance to osmotic pressure and the retention to cefotaxime.On the other hand,its phenotype was detected by knocking out the GlcNAc transporterNag E.It was found that the transport ability of GlcNAc was impaired,the content of GlcNAc in cell wall was reduced,the stress osmotic ability to Na Cl and the retention to cefotaxime were reduced.The above results suggest that GlcNAc may be the reason for the phenotype of cefotaxime caused by tmRNA deletion strain.This study provides a new research perspective and breakthrough point for the formation mechanism of persistent bacteria,and is a broader evidence of the controversial retention mechanism.