Inhibitory Effect Of Berberine On Salmonella Type Ⅰ Fimbriae And Its Biofilm

Salmonella Typhimurium is a Gram-negative bacteria belonging to the Enterobacteriaceae family and the main cause of food contamination and human diseases.As a common food-borne pathogenic bacterium,it can cause serious diseases,including gastroenteritis,sepsis,and partial purulent infections,as well as pose serious challenges to food safety and public health.Its pathogenicity is closely related to its complex virulence factors,such as pathogenic islands,virulence plasmids,flagella,and type Ⅰ fimbriae.As one of the main appendages of bacteria,type Ⅰ fimbriae play key roles in bacterial movement and invasion and adhesion to the host.In addition,S.Typhimurium often form a biofilm to enhance its pathogenicity after infecting the host.At present,traditional sterilization methods have many problems in combating bacteria and their biofilm.Moreover,antibiotic abuse leads to an increase in resistant bacteria.Therefore,developing or finding an effective antibacterial agent has become an urgent requirement.Natural active products have attracted much attention due to their powerful effects and low side effects.Among them,berberine has good antibacterial applications.Therefore,to deeply explore the possible internal connections among type Ⅰ fimbriae,biofilm,and berberine,this study was conducted based on the following two aspects.(1)The main structure of type Ⅰ fimbriae is encoded by the fim A and fim H genes.Homologous recombination was used to construct two mutants of the fimbriae genes(Δfim A,Δfim H).Then,their effects on the biofilm formation were analyzed at different levels.Growth curve and transmission electron microscopic experiments revealed that the mutants(Δfim A,Δfim H)had the same growth curve as the wild-type bacteria;however,they did not have complete type Ⅰ fimbriae.Analysis of the expression of the fim gene cluster via fluorescence quantitative PCR confirmed the complete deletion of fim A and fim H genes.Adhesion experiments of different strains to the IPEC-J2 cell line proved that Δfim A and Δfim H decreased the cell adhesion ability.Subsequent bacterial movement experiments proved that Δfim A swarming was inhibited.Crystal violet staining and laser confocal microscopy imaging were further used to evaluate the biofilm formation ability of different tested bacteria at different times.The results showed that at 24 and 48 h,the biofilm of Δfim A and Δfim H were less than those of the wild-type.However,there was no difference in biofilm formation after 72 h.These results indicate that type Ⅰ fimbriae is essential for biofilm.(2)One of the keys to prevent and control biofilm is to prevent the formation of biofilm.Therefore,inhibiting the formation of type Ⅰ fimbriae is considered a new strategy to prevent S.Typhimurium infection.Therefore,by exploring the relationship between berberine and the mutants(Δfim A,Δfim H),the mechanism of action of berberine and type Ⅰ fimbriae can be explained.The broth microdilution method was used to determine the antibacterial activity of berberine against the selected strains(WT,Δfim A,and Δfim H).The results showed that the minimum inhibitory concentration of Δfim A was higher than that of the other test groups.Subsequent cell agglutination experiments showed that the number of type Ⅰ fimbriae of berberine-treated S.Typhimurium decreased;this result is consistent with that of transmission electron microscopy.Fluorescence quantitative PCR experiments also confirmed that berberine can decrease the expression of the fim A gene,indicating that there is an interaction between berberine and the fim A gene.In addition,confocal laser imaging of the biofilms showed that berberine can prevent biofilm formation by decreasing the number of type Ⅰ fimbriae.Overall,it is well speculated for us that berberine could be a excellent combating-biofilm drug in clinical microbiology and food preservation.