Commensal Bacillus From Milk Inhibits Staphylococcus Aureus Induced Mastitis And The Mechanism Of Action

Mastitis is one of the most expensive diseases that harm the aquaculture industry and cause great losses to the national economy.Mastitis is basically because pathogen enters mammary gland to cause mammary gland inflammation sex disease thereby.S.aureus is an important cause of mastitis.Currently,antibiotics are still the main treatment for mastitis.However,many studies have shown that antibiotic treatment of cow mastitis is closely associated with the emergence of multi-resistant strains.Therefore,there is an urgent need to find alternative strategies to prevent and treat S.aureus infection.S.aureus has a strong ability to form biofilms.The colonization and virulence of S.aureus mostly depend on its quorum sensing system and the formation of biofilms.Bacterial biofilms are the reason for the persistent infection of S.aureus.Therefore,the development of anti-biofilm drugs for S.aureus biofilm can be a research direction to solve the problem of S.aureus biofilm infection and drug resistance.Bacillus is a potential class of probiotics that are widely present in the environment and can colonize the gut.Different Bacillus strains display antibacterial,antioxidant and immunomodulatory activities in the host.Additionally,pathogen clearance by inhibiting bacterial biofilms is another potential property of Bacillus strains.It has been reported in the literature that Bacillus can inhibit the quorum sensing system of S.aureus,thereby reducing the colonization of S.aureus in the intestine.S.aureus can induce S.aureus mastitis,and Bacillus is a mammary symbiotic bacterium that can inhibit the colonization of S.aureus,so whether Bacillus can alleviate S.aureus mastitis.Therefore,in this study,we explored the preventive effect and mechanism of Bacillus symbiotica on S.aureus mastitis.Provide ideas for the prevention of S.aureus mastitis,and explore a healthier and more effective way for the treatment of mastitis.First,we sampled milk from healthy cows and cows suffering from mastitis in Baicheng City,Jilin Province,Chifeng City,Inner Mongolia,and Weifang City,Shandong Province,China,and analyzed milk samples from different sources by smearing the milk samples to isolate and culture bacteria.Differences in colonization between S.aureus and Bacillus.It was found that the colonization of Bacillus and S.aureus in cow mammary gland was negatively correlated.Second,the effect of Bacillus on S.aureus was analyzed in vitro.Through the detection of antibacterial effect;the determination of total bacterial biofilm;the count of S.aureus colonies and the detection of genes related to the regulation of biofilm formation,we further explored the specific pathway by which Bacillus affects the colonization of S.aureus in the mammary gland,and at the same time isolated and screened Bacillus with excellent performance.It was found that Bacillus did not affect the growth of S.aureus,but inhibited the biofilm formation of S.aureus.In response to this phenomenon,we tested 145 strains of Bacillus isolated from milk samples for their ability to resist S.aureus biofilms.The final screening found that B.subtilis H28 significantly inhibited the formation of S.aureus biofilms.Through crystal violet staining and RT-PCR detection,we found that B.subtilis H28 inhibits S.aureus biofilm formation in a dose-dependent manner,which is associated with the fact that B.subtilis H28 can inhibit gene expression of biofilm formation-related genes Agr A,RNAIII,and ica.Finally,a S.aureus-induced mice mastitis model and a S.aureus-stimulated mice mammary epithelial cell inflammation model were established.B.subtilis H28 was added to the above model,through histopathological observation;TNF-α and IL-1β inflammatory cytokine analysis;MPO activity detection;expression changes of tight junction proteins;detection of S.aureus load in mammary glands;Changes of nuclear factors downstream of NF-κB signal transduction pathway and expression changes of genes related to biofilm formation in a mice mammary epithelial cell inflammation model stimulated by S.aureus,to verify whether B.subtilis H28 can inhibit S.aureus-induced mastitis has a protective effect,and its mechanism is initially explored.The results showed that B.subtilis H28 could significantly alleviate S.aureus-induced mastitis in mice.IL-1β production;reduces MPO activity;enhances blood-milk barrier integrity;and reduces S.aureus load in mammary glands.Furthermore,B.subtilis H28 inhibited S.aureus-induced activation of NF-κB signaling in mice mammary epithelial cells(MMECs)in a dose-dependent manner and decreased TNF-α,IL-1β,IL-6 and CXCL3 m RNA levels.Furthermore,we found that B.subtilis H28 reduced the expression of biofilm formation-related genes ica,RNAIII and Sar A,thereby inhibiting S.aureus biofilm formation.Taken together,commensal Bacillus commensal by inhibiting S.aureus biofilm formation reduces S.aureus colonization in the mammary gland,reduces activation of NF-κB signaling and ultimately alleviates S.aureus-induced mastitis,suggesting that interference B.subtilis H28 formed by the pathogen S.aureus biofilm may serve as a potential and effective strategy for the treatment of S.aureus-related diseases.