Epidemiological Investigation Of Animal-derived Salmonella In Parts Of Jiangsu And Anhui And The Inhibitory Effect Of Compound Pulsatillae On Its Biofilm Formation

Salmonella is an important pathogen in humans and animals,and can not only cause a variety of diseases in livestock and poultry,but also a very prominent problem in human food safety.At present,the problem of drug resistance of Salmonella is increaseing worldwide,and the ability of biofilm formation of Salmonella in addition to the inappropriate use of drugs in production are the main reasons.In order to reduce the generation of drug-resistant bacteria,reduce the use of antibiotics,and look for green standard drugs,traditional Chinese medicine begins to show its role in anti-infection and inhibiting the formation of bacterial biofilm.In this paper,we will investigate the epidemiology of Salmonella from animals in some areas of Jiangsu and Anhui,and use compound pulsatillae to preliminarily explore its mechanism of inhibiting the formation of Salmonella typhimurium biofilm.1.Epidemiological Investigation and Drug Resistance Analysis of Salmonella from Animals in some areas of Jiangsu and Anhui provincesFrom October 2018 to September 2020,the liver,gallbladder and cecum of animal samples from Jiangsu and Anhui were collected for Salmonella enrichment culture,and the suspected positive strains screened out were identified by multiplex PCR.The results showed that a total of 207 Salmonella strains were isolated and identified from 1618 samples collected,with a total isolation rate of 12.79%.Among common livestock and poultry,the isolation rate of Salmonella was geese(27.53%,109/306),pigs(18.46%,12/65),ducks(10.98%,18/173),chickens(6.42%,58/904),and sheep(0%,0/38),and the isolation rate of Salmonella in monkeys was significantly higher than that in other animals(P<0.001).The isolation rate of Salmonella in liver(14.9%)was significantly higher than that in gallbladder(9.3%)(P<0.05),there was no significant difference in the rate of Salmonella isolation between gallbladder and cecum(14%)(P>0.05).In addition,the risk of Salmonella infection in animals within 30 days of age(25.94%)was significantly higher than that in animals within 100 days of age(6.48%)and above(P<0.001).The isolation rate of Salmonella accoding to seasonal factors was spring(15.32%),winter(12.71%),autumn(12.61%)and summer(10.57%),respectively.The risk of Salmonella infection in spring and winter was significantly higher than that in summer(P>0.05),the isolation rate of Salmonella in autumn was not significantly different from that in other seasons(P>0.05).Multiplex PCR combined with slide agglutination test was used to identify Salmonella serotype.The results showed that seven serotypes were identified.Among them,Salmonella typhimurium(68.6%,142/207)was the dominant serotype in Jiangsu and Anhui province,and Salmonella pullorum(40%,23/58)was the dominant serotype in infecting chickens.The results of drug resistance analysis showed that Salmonella isolates showed high resistance rate to trimethoprim/sulfamethoxazole(67%),amoxicillin(47%)and doxycycline(45%),and high sensitivity to cefoperazone sulbactam(95%)and butamicarina(90%).Among 191 positive Salmonella strains,75.92%of the strains were resistant to one or more class of antibiotics,and the multiple drug resistance(three or more classes of antibiotics)rate was 62.83%.2.Detection of Salmonella biofilm formation and compound pulsatillae inhibiting Salmonella typhimurium biofilm formationThe biofilm formation ability of Salmonella typhimurium,Salmonella enteritidis,Salmonella pullorum,Salmonella indiana and Salmonella kentakii identified in the first chapter was detected.The results showed that all of these serotypes had the biofilm formation ability,and the formation rate from high to low was Salmonella indiana(100%,7/7),Salmonella indiana(100%,7/7),Salmonella typhimurium(92.45%,98/106),Salmonella kentucky(87.5%,7/8),Salmonella pullorum(75%,15/20)and Salmonella enteritidis(75%,6/8),respectively.The biofilm formation rate of Salmonella typhimurium was significantly higher than that of Salmonella pullorum and Salmonella enteritidis(P<0.05),and had no significant difference with Salmonella indiana and Salmonella kentucky(P>0.05).In addition,the biofilm formation rates of isolates from gallbladder,cecum and liver were 92%(37/40),100%(7/7)and 89%(89/100),respectively.The biofilm formation rates of gallbladder and cecum were slightly higher than those of liver,but there was no significant difference(P>0.05).According to the observation of light microscope and confocal microscope,the standard strain S162 of Salmonella typhimurium did not have the ability of biofilm formation.However,bacterial adhesion aggregation(6-12 h),biofilm matrix generation(12-24 h)and the biofilm structure maturation and bacterial spread(24-48 h)was successively observed in Salmonella typhimurium wild strain following the extension of culture time.After being treated with Compound pulsatillae,there was no obvious biofilm structure formation in Salmonella typhimurium wild strain in addition to a small amount of bacterial dispersion,and the bacterial morphology changed into the shape of bulbous rod in multiple stages.3.Effects of Compound pulsatillae on expression of biofilm forming genes and virulence genes of Salmonella typhimurium and on adhesion and invasion of Caco-2 cells of Salmonella TyphimuriumIn this study,several functional genes of Salmonella typhimurium in vitro were induced to downregulate by Compound pulsatillae,including flagella,fimbria genes flhA,fliF,fimA and pefA related to bacterial movement and adhesion;bssR,sdiA,and bapA related to biofilm formation;hilA,invA,sipB/C,rcsC and cheZ were associated with invasion and virulence.At the same time,motility could significantly inhibited by the Chinese herbal medicine using 1/4 and 1/2 MIC concentrations(P<0.001),and the swimming distance was inversely proportional to the concentration of traditional Chinese medicine.In the experiment of adhesion and invasion of Salmonella typhimurium to Caco-2 cells,the Chinese medicine group supplemented with compound pulsatillae could significantly reduce the adhesion ability of Salmonella typhimurium to Caco-2 cells(P<0.001)and significantly reduced the invasion of Salmonella typhimurium on Caco-2 cells(P<0.05).To sum up,the prevalence of Salmonella from animals in Jiangsu and Anhui was 12.79%,and the highest isolation rate of Salmonella was found in geese.Animals less than 30 days of age were more susceptible to Salmonella,and the isolation rate of Salmonella was significantly higher in spring than in other seasons,Salmonella typhimurium and Salmonella pullorum were the dominant serotypes in Jiangsu and Anhui.The drug resistance analysis of the 191 Salmonella isolates revealed 75.92%of the strains were resistant to more than one class of antibiotics,and the multiple drug resistance rate was 62.83%.Salmonella typhimurium,Salmonella enteritidis,Salmonella pullorum,Salmonella kentucky and Salmonella indiana have the ability of biofilm formation,and the biofilm formation rate of Salmonella typhimurium is significantly higher than that of Salmonella enteritidis and Salmonella pullorum.In addition,compound pulsatillae can down-regulate the expression of Salmonella genes associated with flagellum,fimbrium and virulence genes,inhibited the biofilm formation of Salmonella typhimurium wild strain,and reduced the motility of Salmonella typhimurium,and the adhesion and invasion ability of Salmonella typhimurium to Caco-2 cells.Therefore,this study provides a new theoretical basis and experimental basis for the anti-Salmonella typhimurium infection and biofilm formation of traditional Chinese medicine.