Spoilage Ability And Drug Resistance Of Shewanella Baltica Isolated From Large Yellow Croaker In Eight Chinese Coastal Cities

As an important marine-cultured fish species in Southeast China,large yellow croaker（Pseudosciaena crocea）is desirable for its good taste and high nutritional values.The active and aggressive characteristics of large yellow croaker make it very easy to death during the transport process,so the fish is often transported and stored in cold storage.Due to the high levels of nutrients and moisture in the fish,it is very prone to spoilage during storage,which mainly results from microbial growth and metabolism in the product.Shewanella baltica is the specific spoilage organisms（SSOs）found in many refrigerated seafood.In this study,several strains of S.bacteria were isolated from large yellow croaker,and their spoilage ability and drug resistance were analyzed.The main results are as follows:（1）One hundred and twenty-two strains of spoilage strains were isolated from large yellow croakers in eight major coastal cities in China,97%of the strains were S.baltica.A batch of strains with strong or weak spoilage ability were selected from each site,and it was found that the strain with strong spoilage ability produced more total volatile basic nitrogen（TVB-N）and trimethylamine（TMA）,and as well as high protease activity.Low temperature promoted biofilm formation.Twenty-eight strains were further screened according to their spoilage ability,and their whole genomes were sequenced and analyzed.It was found that one strain was not S.baltica,and two strains were from the same colony,so twenty-six strains were for downstream experiments.The genome sizes of these 28 strains ranged from 4.7 Mb to 5.2 Mb,and their GC contents ranged from 46.01%to 46.42%.The annotated genome functions were enriched of amino acid transport and metabolism.Genetic diversity results showed that gene boxes presented open structures and contained a high proportion of species-specific genes,and gene diversity was resulted from the random acquisition or deletion of some genes in strains.The single-copy core genome was mainly for the conserved functions of housekeeping genes,such as metabolism,cytoplasm,and ribosome.The carbohydrate-active enzymes encoded by species-specific genes were mainly involved in the glycosyltransferase family,carbohydrate metabolism,glycan biosynthesis and metabolism,and signal transduction.Evolutionary selection pressure analysis showed that functional genes related to cell structure were subjected to strong purifying selection pressure,which drove the evolution of S.balticia.（3）Transcriptome sequencing of strong and weak spoilage strains in different growth stages showed that more genes were expressed in the log phase,when was also the metabolic activity of genes related to spoilage.The differential metabolic pathways of strong and weak spoilage ability strains were mainly focused on amino acid metabolism,carbohydrate metabolism,and flagellar assembly.Amino acid metabolism mainly included metabolism of valine,leucine,and isoleucine,metabolism of glycine,serine,and threonine,and metabolism of cysteine and methionine.Carbohydrate metabolism was mainly propanoate metabolism,which affected pyruvate metabolism by regulating the expression of succinate and also affected citrate metabolism,resulting in differences in bacterial energy acquisition.Flagellar assembly mainly affected the expression of hook protein and flagellin.Strongly spoiled strain expressed high gene encoding flagellin,which accelerated the formation of biofilm and enhanced the ability to resist external environmental stress,thereby forming stronger spoilage ability.（4）The drug resistance test of the twenty-six strains showed that each strain was resistant to at least one antibiotic,and the multidrug resistance rate reached 61.54%.The resistance rate of isolates to nalidixic acid,sulfisoxazole,and vancomycin was very high.The three strains isolated from Lianyungang were all multi-drug resistant,and could be resistant to up 11antibiotics.The resistance genes of bla _OXA,rsm A,and ade F were conserved in 26 strains of S.balticia.Genes of mcr-4.3,arr-3,qnr VC6,qnr A1,sul1,tet D,aad A16,dfr A27,cat,and EF-Tu（R234F）might be transferred to other bacteria by S.balticia.Only mcr-4.3 and tet D genes need to be induced by antibiotics for expression.