Study On The Antibacterial And Inhibition Biofilm Formation Of Sanguinarine Hydrochloride On Staphylococcus Aureus

Staphylococcus aureus（S.aureus）,belonging to the genus Staphylococcus,is a gram-positive bacterium and an important conditional pathogen.In veterinary clinics,it is usually manifested as skin and soft tissue infection,which induces bovine mastis,bovine mastis,canine rhinitis,and other diseases.As S.aureus is highly infectious and has a strong biofilm-forming ability,its proportion in animal bacterial infection is increasing year by year,resulting in more and more serious economic losses.For example,S.aureus will cause mastitis in the dairy cattle breeding industry,resulting in a series of economic losses such as the decline of milk production and quality,the increase in elimination rate,and mortality.At the same time,animals may become the source of S.aureus infection.One of the reasons why S.aureus is difficult to treat is that it can form a biofilm.When the bacteria form a biofilm,the drug resistance of the bacteria increases greatly,resulting in the poor treatment effect of antibiotics.It is urgent to treat the animal infection caused by S.aureus.Based on the policies of reducing and limiting resistance in China,it is of great significance to develop drugs with a new antibacterial mechanism based on traditional Chinese medicine in the treatment of S.aureus infection.L-histidine is an important biological essential amino acid.The L-histidine biosynthesis pathway is one of the important nitrogen metabolism pathways,which is highly conserved in different species.Imidazole glycerophosphate dehydratase（IGPD）is one of the key enzymes in the biosynthesis of L-histidine and is considered an excellent target.Sanguinarine is a benzo phenanthridine alkaloid with a good antibacterial effect.This study found that sanguine hydrochloride can inhibit the growth of S.aureus and interfere with the formation of its biofilm.At the same time,sanguine hydrochloride can also bind to IGPD protein.Based on this,we explored the antibacterial and anti-biofilm mechanism of sanguinarine hydrochloride against S.aureus and evaluated the therapeutic effect of sanguinarine hydrochloride in animal models.The results are as follows:（1）The effects of sanguinarine hydrochloride on the minimum inhibitory concentration and growth of S.aureus were evaluated by microdilution and a time-germicidal curve.The results showed that the MIC value was 20μg/m L.At the same time,sanguine hydrochloride can change the permeability,fluidity,and membrane potential of the bacterial cell membrane and affect the energy metabolism of bacteria to play an antibacterial role.（2）Using the crystal violet staining method,analysis of biofilm matrix composition,adhesion ability analysis,and scanning electron microscopy to observe the effect of sanguine hydrochloride on the biofilm of Staphylococcus aureus.The results showed that:1/2 times MIC（10μg/m L）,1/4 times MIC（5μg/m L）,and 1/8 times MIC（2.5μg/m L）of sanguine hydrochloride can significantly interfere with the formation of S.aureus biofilm and destroy the integrity of biofilm.At the same time,sanguine hydrochloride can reduce the content of extracellular polysaccharides,extracellular proteins,and extracellular DNA in the biofilm matrix（p<0.01）;Inhibit the adhesion function of S.aureus.（3）Exploring the regulatory effect of sanguine hydrochloride on IGPD using q RT PCR,WB assay,and determination of L-histidine content.The results showed that sanguinarine hydrochloride could affect the synthesis of L-histidine by significantly down-regulating the transcription level of the his B gene and the expression of IGPD（p<0.01）.（4）The binding of sanguinarine hydrochloride with IGPD was analyzed by BLI,amino acid site directed mutagenesis,and enzyme activity IC₅₀ assay.The results showed that sanguinarine hydrochloride could directly bind with IGPD and inhibit the enzyme activity of IGPD.The binding sites might be Lys81,Lys83,Lys84,His85,and Lys81 might be the key amino acid for binding.（5）Using animal models to evaluate the therapeutic effect of sanguine hydrochloride.The results showed that sanguine hydrochloride could alleviate the pathological changes in the kidneys of mice with systemic infection,and significantly reduce the bacterial load in the kidneys of mice;Hematogenine hydrochloride can also interfere with the formation of biofilms on the surface of implants in mice,reducing the bacterial load on the implant surface by 1 log value and alleviating pathological changes in the skin at the implant site.In summary,this study revealed the antibacterial effect of sanguine hydrochloride on S.aureus and the mechanism of its intervention in biofilm formation.At the same time,a mouse in vivo biofilm model and a mouse systemic infection model were constructed.At the same time,a mouse model of systemic infection and a mouse model of biofilm were constructed to evaluate the therapeutic effect of sanguinarine hydrochloride on the infection of S.aureus.This study will provide a scientific basis for the treatment of animal infections caused by S.aureus in the future.