Monitor Of S.suis Resistance In Some Pig Farms From Heilongjiang Province And The Role Of CysM In S.suis Multidrug Resistance

Streptococcus suis is an important zoonotic bacterium that can cause swine meningitis,arthritis,endocarditis and sepsis,which could bring huge economic losses to the pig breeding industry.In clinical,antibacterial drugs are mainly used to prevent and treat Streptococcus suis.However,the long-term use of antibacterial drugs has gradually increased the multi-drug resistance of Streptococcus suis to the extent that the therapeutic effect of antibacterial drugs has continued to decrease.Therefore,exploring its multi-drug resistance mechanism is a prerequisite for solving the problem of bacterial resistance and rational drug use.Macrolide and tetracycline are commonly used drugs in veterinary clinic.However,the resistance of Streptococcus suis to macrolides,tetracyclines and fluoroquinolones are getting worse,and even showing multiple drug resistance.The use of fluoroquinolones has been restricted in China.Why does clinically isolated Streptococcus suis show multiple resistance to three types of drugs?Therefore,in this study,we carried out the clinical monitoring of drug resistance in Streptococcus suis on the one hand.And on the other hand,we took Streptococcus suis ATCC700794 as the research object,and obtained the multi-drug resistant Streptococcus suis which induced by tylosin in vitro.The multi-drug resistant Streptococcus suis were resistant to macrolides,tetracyclines and fluoroquinolones.After that bioinformatics was used to analyze the proteomics data that Streptococcus suis was treated by tylosin,we found that O-acetylserine?thiol?-lyase?CysM?,which belonged to a cysteine biosynthetic pathway,might be related to multiple drug resistance.In order to prove the relationship between the protein and drug resistance,gene knockout and replenishment techniques were used to construct cysM gene deletion strains and complement strains.We clarified the relationship between CysM-regulated cysteine synthesis and multi-drug resistance of Streptococcus suis from four levels:resistant phenotype,bacterial biofilm,metabolite content changes and gene levels.Finally,due to the molecular simulation and molecular interaction technology,we analyzed whether tylosin also binds to CysM protein.The specific research results are as follows:?1?Conducting drug resistance surveillance for Streptococcus suis in 7 different farms of Heilongjiang Province from 2017 to 2019.It is determined that the current clinical Streptococcus suis in Heilongjiang Province has regarded serotype 4 as the dominant serotype.These clinical Streptococcus suis were multi-drug resistant.The most severe resistances to tetracycline and macrolides were 97.5%and 92.5%,respectively.The MIC₅₀ and MIC ₉₀ of macrolides were exceed128?g/m L,the mainly resistance genes of macrolide was erm B,and the mainly resistance genes of tetracyclines was tet M.The resistance rate of fluoroquinolones was reached 60%.Using the crystal violet staining method to measure the biofilm formation ability,the results showed that the clinical isolated Streptococcus suis has the ability to form biofilms.Since the coincidence rate of clinical isolated Streptococcus suis resistance phenotypes and resistance genes were not 100%,it was speculated that the biofilm may be an important factor for bacteria to form multiple resistance.?2?Based on the early proteomics in the laboratory,Heatmap and PPI analysis were used to reanalyze the 171 differential proteins of Streptococcus suis which under 1/4 MIC?0.125?g/m L?tylosin selection pressure.We found that CysM protein was in the middle of these differential proteins,and it was speculated that it may play an important role in bacterial resistance.?3?Using tylosin as a test drug,the tylosin-resistant Streptococcus suis was obtained by means of strong induction in vitro from the laboratory,and the drug-resistant phenotype was determined.Tylosin-resistant Streptococcus suis has increased the MIC of tetracyclines and fluoroquinolones.Among them,the MIC of tylosin increased 128 times,the MIC of tilmicosin increased 128 times,the MIC of chlortetracycline increased 64 times,the MIC of tetracycline increased 4 times,the MIC of ofloxacin increased 32 times and the MIC of enrofloxacin increased4 times,while the MIC of florfenicol and penicillin K didn't change.At the same time,tylosin-resistant Streptococcus suis was measured by RT-PCR and Western Blot technology for the expression of the cysM gene and CysM protein.It was found that it was significantly up-regulated,proving that CysM protein might be related to the production of multi-resistant.?4?The cysM gene deletion strain was obtained by homologous recombination combined with flow cytometry screening.The cysM gene complement strain was obtained by plasmid backfilling.The drug resistance phenotype of tylosin-resistant Streptococcus suis cysM gene deletion strain and cysM gene complement strain were determined by microdilution method?MIC method?.In the cysM gene deletion strain,the MIC results showed that the MIC of tylosin decreased 4 times,the MIC of tetracycline decreased 4 times,the MIC of enrofloxacin decreased 4 times,the MIC of telmicoxin decreased 128 times,the MIC of chlortetracycline decreased 8 times and the MIC of ofloxacin decreased 32 times.In the complement strain,the phenotypic of MIC didn't return to the level of the tylosin-resistant Streptococcus suis although the MIC of these six drugs increased.The MIC of tylosin increased 2 times,the MIC of ofloxacin increased 2 times,the enrofloxacin increased 2 times,the chlortetracycline increased 2 times,the tetracycline increased 2 times and the MIC of telmicoxin increased 32 times.The drug resistance phenotype experiment initially proved the role of CysM in resistance to Streptococcus suis.?5?By using crystal violet staining and scanning electr on microscopy,we explored the relationship between CysM and the amount of biofilm or biofilm formation.We found that in the cysM gene deletion strain,the biofilm formation ability and amount of biofilm formation were also significantly reduced,which proved that cysM may be involved in the biofilm formation of tylosin-resistant Streptococcus suis.?6?Using the cysM gene deletion strain and the complement strain,the content of intermediate metabolites and related genes of the cysteine biosynthesis pat hway were measured.The results showed that compared with tylosin-resistant Streptococcus suis,the contents of cysteine,homocysteine and S-adenosylmethionine were significantly reduced in the cysM gene deletion strain.While in the supplementary strains,there was no significant difference in the contents of cysteine and homocysteine that compared with the content before knockout.Although the content of S-adenosylmethionine was partially restored,but didn't restore the expression level of tylosin-resistant Streptococcus suis,it was still a significant difference.The gene expression of met I,met K,mtn N and lux S were significantly reduced in the cysM gene deletion strain.In the complement strain,the expression level of met I gene was not significantly different from that of tylosin-resistant Streptococcus suis,while the gene expression levels of met K,mtn N,and lux S were partially restored,but not recovered to the expression level of tylosin-resistant Streptococcus suis,and they still has significant differences.?7?Based on molecular docking and BLI technology,to clarify whether tylosin can bind to CysM protein and lead to the development of multiple drug resistance of Streptococcus suis.The tylosin drug failed to interact with the amino acid res idues of the CysM protein.The affinity dissociation curve of the tylosin and CysM protein didn't change.All the results proved the tylosin didn't bind to CysM protein.In summary,the multi-drug resistance of Streptococcus suis to tylosin,tilmicosin,enrofloxacin,ofloxacin,chlortetracycline,and tetracycline were regulated by cysM gene that mediated by cysteine biosynthesis pathway.