Screening Of Streptococcus Suis Genetic Loci Involved In Resistance To Daptomycin And Vancomycin

Antibiotic-resistant bacteria is becoming increasingly common.Antibiotic-resistant bacteria is threatening global health and sustainable development of breeding industry,mainly due to the irrational drug uses.There is a desire to the research of antibiotic resistant mechanisms,which provide significant insights into the exploring of new drugs.The resistance mechanism is complexed.The resistance mechanism of Gram-negative species is different from the Gram-positive species'.The resistance mechanism of bacteria can be roughly classified into intrinsic resistanse and acquire resistance.Daptomycin and vancomycin are clinically important antibacterial drugs against Gram-positive pathogens.The targets of daptomycin is the anionic phospholipids of membrane,and vancomycin target D-Ala-D-Ala peptides of cell wall.The acquired resistance of these two antibiotics usually develops in a step-wise fashion,with multiple genetic loci participating in resistance.The resistance mechanisms of these two antibiotics remained to be revealed.Screening transposon(Tn)library is efficient for functional genomics research.Resistome studies in many bacterial pathogens have commonly utilized to define collections of transposon(Tn)insertion mutants.In the present study,we have established an experimental platform for identifying genes that have the potential to contribute to antibiotic resistance in S.suis,which causes severe diseases in pigs and humans.The main research results are summarized as follows:1.S.suis SC-19 was mutagenized with Tn917 and a transposon library containing1918 mutants was constructed.The 1724 insertion sites were identified and most insertion sites were located within the region ranging from 800 kbp to 1200 kbp.The 1724 insertion distributed among 431 different genes and 54 KEGG pathways.Among these pathways,the genes were only enriched in galactose metabolism.The mutants pool deserved further functional genomics research.2.The minimal inhibitory concentrations(MICs)for daptomycin and vancomycin against S.suis SC-19 were 8 and 0.25?g/ml,respectively.We undertook screening of the mutagenesis library based on the MICs.115 transposants(63 inserted genes)and 92transposants(54 inserted genes)exhibited reduced susceptibility to daptomycin and vancomycin,respectively.Among the inserted genes of mutants exhibiting reduced susceptibility to daptomycin,21 genes(21 transposants)were functionally similar to known resistance genes.Among the transposants exhibiting reduced susceptibility to vancomycin,19 genes(23 transposants)were functionally similar to known resistance genes.Meanwhile,42 novel genes(57 transposants)were identified to be involved in daptomycin resistance,including peptidase(SSUSC84?0924)and so on.35 novel genes(39 transposants)were identified to be involved in vancomycin resistance,including glycogen branching enzyme(SSUSC84?0919)and so on.25 transposants(25 inserted genes)exhibiting reduced susceptibility to both of daptomycin and vancomycin,including permease(SSUSC84?1013)and so on.Based on scanning electron microscope and structured illumination microscope analysis,it was found that transposants exihibiting reduced sensibility to daptomycin and vancomycin increased in cell length.