| Salmonella(salmonellosis) refers to those disease caused by all kinds of Salmonella in different clinical manifestations. The main clinical manifestations of human and animal infections including gastroenteritis, septicemia and local suppurative infection, which bring serious challenges to the livestock industry and public health. Recently, as a result of the abuse of antibacterials, drug resistance of Salmonella has become increasingly serious, even the formation of biofilms. Bacterial biofilm(BF) is defined as a structured community of bacterial cells,enclosed in a self-produced polymeric matrix and adherent to carrier surface. Studies show that biofilm formation are closely related to the resistance of Salmonella, and 80% of the clinical infections are related to bacteria biofilm. So it is urgent to development of new antimicrobial agents. Effects of Chinese herbal medicines on bacterial biofilm formation is more and more popular, since with little toxic and side effects, and not easy to produce drug resistance. Besides,when they combined with antimicrobials, it can reached Synergy effect. So in this study, one extract with strong anti-Salmonella effect was selected and discuss in vitro the antibiofilm activitiesits and the regulation of lux S, rpo E and omp R gene expression that related to Salmonella biofilm formation of the extract combined application with ciprofloxacin(CIP).Antimicrobial sensitivity of the 12 Salmonella clinical isolates to 10 antimicrobial agents were tested with Kirby-Bauer method. Results showed that, isolates resistant rate to ampicillin was the highest, up to 58.3%, the norfloxacin resistant rate was 50%; and resistance rates to kanamycin,gentamicin, cefotaxime cefazolin, tetracycline were also higher, 41.7%, 41.7%, 33.3%, 25%,respectively. The multidrug resistant rate was 41.7%(5/12) and the main resistance phenotypes were AMP-GEN-CFZ-KAN. The isolate 8 was resistant up to 9 kinds of antimicrobials and the resistant phenotype was the most complex, so would be used as experimental strain.The air-dried of Rhizoma coptidis, Scutellaria baicalensis, Clove leaf and Gallnut were pulverized and extracted with 75% EtOH by ultrasonic wave method; Determination of minimal inhibition concentration(MIC) values of ethanol extracts and ciprofloxacin were performed by micro dilution method in 96-well plates. Compared with other 3 kinds of extracts, the Rhizoma coptidis extract had a better effect of anti-Salmonella with a MIC value of 6 250 μg·mL-1. Further extraction was done, 5.31 g crude product was obtained and the extraction rate was 8.17%, and its MICs were 3 125 μg·mL-1. The fractional inhibitory concentration(FIC) of extract in combinationwith CIP was estimated by the checkerboard method, result showed that the FIC index was 0.75,indicating the antibacterial activity of the Rhizoma coptidis extract combined with CIP in vitro performanced as additive effect against Salmonella.The adhesion rate(B value) and the antibiofilm effect of antimicrobial agents(extract, CIP,their compounds) on Salmonella was tested quantified in 96-well plates by crystal violet(CV)assay for determining biofilm formation ability; Biofilm growth curve were evaluated using XTT assays; biofilm were cultured in 24-well plates, and microstructure of biofilms and the disruption of the antimicrobial agents on biofilm was analysed using scanning electron microscopy(SEM);Furthermore, the regulation of mRNA expression of rpo E, luxS and omp R genes by different antimicrobials agents were determined by qRT-PCR method. The results of the biofilm adhesion rates, biofilm growth curve and the SEM images all indicated that the CVCC528 strain showed a weak potential of biofilm formation, whereas isloate 8 was classified as strong biofilm producers,and formed a typical and complete biofilm and apparently growed between 18 h and 30 h, reached stagnate phase in 72 h. The compound agent showed stronger inhibition of biofilm adhesion rate,growth rate, and had a serious damage of the biofilm microstructure, and also restrain lux S, rpo E and omp R genes expression. The inhibition action and drug concentration was positively correlated,and with best effect(p<0.01) at 1/2 MIC concentration, which showed that the combined use of the two drugs on resistant Salmonella produce synergistic effect, and in a certain extent reduced the dose of ciprofloxacin.In conclusion, compared with other 3 kinds of extracts, the Rhizoma coptidis had a better effect on anti-Salmonella than that of Scutellaria baicalensis, Gallnut and Clove leaf. Rhizoma coptidis extract in combination with ciprofloxacin enhanced the antibacterial effect of ciprofloxacin. Rhizoma coptidis extract, CIP and their compounds all did inhibit biofilm adhesion rate, growth rate, and damage the microstructure, and in molecular level mechanism it is associated with the down-regulation of lux S, rpo E and omp R gene expression. The compound agent to a certain extent, showed a stronger effect than the single drug. Therefore, Rhizoma coptidis extract combined with ciprofloxacin in inhibition of bacteria biofilm has great application prospect. This study provides theory evidence for the development and prevention of Salmonella clinical infections and the drug resistance in molecular level mechanism associated with biofilm. |
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