| As an important zoonotic pathogen,Streptococcus suis（S.suis）is widely prevalent worldwide and causes diseases such as meningitis,septicemia and arthritis,which are important causes of high mortality and culling rates in piglets.In addition to causing great economic losses to the swine industry,S.suis is also transmitted to humans through contaminated environments or diets,where it threatens human health.Sulfonamides are commonly used to treat bacterial meningitis in the veterinary clinical,in which sulfadiazine sodium has the highest concentration in cerebrospinal fluid and is more effective in the treatment of bacterial meningitis.However,the overuse of sulfonamides in recent years has caused widespread selective pressure on bacteria.Therefore,it is necessary to innovate therapeutic approaches with excellent antibacterial properties to address the sulfa drug resistance crisis.Recently,antibiotic-assisted strategies have emerged as promising approaches to prevent bacterial resistance or enhance antibiotic action.This strategy is considered more cost-effective than the development of novel antimicrobials.Meanwhile,it may overcome the limitations associated with the weak therapeutic effect of a single drug in severe infections.In addition,to address the problem of bacterial resistance of animal origin,the Ministry of Agriculture and Rural Affairs has carried out antibacterial drug reduction initiatives and issued Announcement No.194.The announcement pointed out the withdrawal of all growth-promoting drug feed additives in addition to Chinese medicine,which provides an opportunity for basic research and development of Chinese medicine in veterinary clinical“antibacterial reduction”.Therefore,from the epidemic trend of S.suis and China’s policy,the development of antibacterial potentiators mainly based on traditional Chinese medicine is of great significance in the prevention and treatment of clinical S.suis.In the present study,we first screened 30 monomeric compounds of traditional Chinese medicine from the constituents of 87 medicinal foods homologous traditional Chinese medicines by drug susceptibility testing.Among them,we found that gallic acid could reverse the resistance of S.suis to sulfonamides.Second,we constructed the Galleria Mellonella larvae infection model and the meningitis model in mice to demonstrate the in vivo therapeutic effects of gallic acid combined with sulfadiazine sodium.In addition,piglets clinically suffering from chronic S.suis disease were selected for treatment to evaluate the clinical efficacy of gallic acid combined with sulfadiazine sodium.Finally,based on the antibacterial characteristics of gallic acid,the mechanism of gallic acid to reverse sulfonamide resistance of S.suis was investigated.The specific findings of the study are as follows:（1）Screening and validation of sulfonamide antibacterial synergist in S.suisThe minimum inhibitory concentration（MIC）and graded inhibitory concentration index（FICI）of 30 monomers and sulfonamides were determined by micro-broth dilution method and checkerboard method using sulfadiazine sodium resistant S.suis as the test strain.It was found that the MIC values of monomeric compounds against S.suis ranged from 11 to 1280μg/m L,and the MICs of sulfadiazine sodium,sulfamethoxazole,sulfadimethoxypyrimidine,sulfadimethoxypyrimidine,and sulfisoxazole against S.suis were greater than 640μg/m L.The MIC values of gallic acid and sulfadiazine sodium against S.suis were 320μg/m L and 1280μg/m L,respectively.In addition,the MIC value of sulfadiazine sodium was reduced from 1280μg/m L to160μg/m L after the combination of gallic acid and sulfadiazine sodium,changing from resistant to sensitive level,which showed a synergistic effect with a FICI of 0.25.Meanwhile,the combination of gallic acid and sulfadiazine sodium had a synergistic or additive antibacterial effect on 87.1%（27/31）of the clinically resistant S.suis,with complete reversal of sulfadiazine sodium resistance in 70%of cases.In addition,after 21 consecutive days of in vitro induction,S.suis remained susceptible to sulfadiazine sodium in the presence of gallic acid.It showed that the combination of the two applications made S.suis less susceptible to develop resistance to sulfadiazine sodium.（2）Therapeutic effects of gallic acid combined with sulfadiazine sodium in S.suis infection modelTo evaluate the therapeutic effect of gallic acid on infections caused by S.suis using a survival rate model of the Galleria Mellonella larvae and a mice meningitis model.The survival rate of Galleria Mellonella larvae after gallic acid combined with sodium sulfadiazine was 70%,which was significantly higher than that of the drug-treated group alone（p<0.01）.Meanwhile,the combination treatment significantly reduced the level of inflammatory cytokines in the plasma of mice,decreased the colony number of S.suis in each organ,and effectively reduced the inflammatory damage in brain tissue（p<0.01）.（3）Clinical efficacy evaluation of gallic acid in combination with sulfadiazine sodiumThe clinical efficacy of gallic acid in combination with sulfadiazine sodium was evaluated in piglets clinically affected with chronic S.suis disease.The results showed that gallic acid combined with sulfadiazine sodium could effectively improve the clinical signs associated with the treatment of S.suis disease caused by sulfadiazine-resistant S.suis infection（p<0.01）.（4）Gallic acid disrupts the integrity of the cell membrane of S.suis and exerts a synergistic antibacterial effectThe effect of gallic acid in combination with sulfadiazine sodium on the ultrastructure of S.suis was analyzed using SEM and TEM observation methods.It was found that the surfaces of bacteria treated with gallic acid combined with sulfadiazine sodium appeared severely damaged.Subsequently,the effect of gallic acid on the cell membrane integrity of S.suis was assessed by measuring the extracellular DNA content and PI staining.The results showed that gallic acid combined with sulfadiazine sodium treatment significantly disrupted cell membrane integrity in S.suis（p<0.01）.（5）Gallic acid affects the cell membrane function of S.suis to exert a synergistic antibacterial effectThe effects of gallic acid on the cell membrane function of S.suis were investigated by measuring cell membrane fluidity,proton motive force（PMF）,intracellular ROS content and intracellular accumulation of sulfadiazine sodium in bacteria.It was found that gallic acid was unable to inhibit efflux pump function and increase the amount of intracellular sulfadiazine sodium accumulated.However,gallic acid combined with sulfadiazine sodium significantly reduced the cell membrane fluidity,dissipated the PMF of the cell membrane and induced excessive intracellular production of ROS in the bacteria to exert combined antibacterial effects（p<0.01）.（6）Gallic acid targets dihydrofolate reductase（DHFR）to exert combined antibacterial effectsThe direct binding of gallic acid to DHFR was corroborated using molecular docking technique and Bio-layer interferometry（BLI）.It was found that gallic acid directly binds to DHFR in a dose-dependent manner and inhibits DHFR activity,Kd=5.96×10-4 M.In parallel,Arg61 was demonstrated to be a critical amino acid for the interaction of gallic acid with DHFR using molecular dynamics simulations,site directed mutagenesis of amino acids,BLI,and enzymatic activity assays.（7）Gallic acid regulates folic acid biosynthesis to exert combined antibacterial effectsRegulation of the folate metabolic pathway by gallic acid was revealed using RT-PCR,Western blot as well as measurement of metabolite content in the folate metabolic pathway.It was found that gallic acid combined with sulfadiazine sodium could further affect the content of tetrahydrofolate,cysteine,homocysteine,S-adenosylmethionine,S-adenosylhomocysteine and S-ribose homocysteine by significantly down-regulating the transcription and translation of cys M,fol A,lux S,mtn N and met K（p<0.01）.In summary,this study confirmed that gallic acid combined with sulfonamides can effectively inhibit the growth of drug-resistant S.suis both in vitro and in vivo.Meanwhile,in vivo tests demonstrated that the gallic acid could enhance the therapeutic effects of sulfadiazine sodium in piglets with S.suis disease.It not only disrupts the integrity and function of cell membranes,but also block DHFR regulating tetrahydrofolate synthesis.In general,gallic acid can be used as an excellent antibacterial synergist for sulfonamides in the treatment of S.suis infectious diseases. |
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