The Pathogenicity Control Of Salmonella Based On Functional Nucleic Acid

Currently,the contaminations of eggs and meat products from poultry and livestock resulted from Salmonella have become a frequent food safety issue.Meanwhile,the infectious diseases and drug resistance caused by Salmonella have posed the adverse influence to human public health.The biofilms formed by Salmonella,which is common in nature,are exacerbating repeated attacks and difficulty of many chronic infections.Unfortunately,there is little hope to witness the rapid development of novel antibiotic molecules that would overcome multidrug resistance.Therefore,it is urgent to develop new antibacteria agents that is more efficient than current antibiotics against Salmonella.Aptamers are oligonucleotides with specific recognition to targets.They are screened through Systemiatic Evolution of Ligands by Exponential Enrichment(SELEX).Nowadays,aptamers have been generated as small drugs for targeted therapy.Moreover,with the technological advancement in aptamer selection,recent examples of antimicrobial properties of some aptamers are obtained.Those aptamers can be used as antimicrobial agents by passively binding to surface antigens(such as flagella or fimbria),targeting intracelluar key biochemical processes,activating human immunoreaction,or specifically inhibiting the invasion of pathogen.In consideration of antibiotic potential of aptamers and based on the above descriptions,the main contents about aptamers of the dissertation are listed as follows:(1)Based on the aptamers recognizing S.typhimurium achieved by Cell-SELEX,we optimized the structures of their sequences.The secondary structure of three aptamers with high affinity were analyzed by Mfold software,then the sequences were truncated and revised on the basic of keeping the major stem-loop portion.As a result,a serial of shorter aptamers named STY-1,STY-2 and STY-3 were achieved.After sequence optimization,these aptamers still keep high affinities for bacteria with the Kd ranging from 0 to 100 nM(35.2±4 nM,29.9±4 nM,19.3 ±4 nM,respectively).And the affinity of STY-3 was increased markedly by sequence optimization.Three aptamers also had excellent sensibilities and specificities to distinguish target bacteria,which could serve as small probes or molecules for detection or analysis of pathogenesis.(2)The application of STY-1,STY-2 and STY-3 as antimicrobial agents against the pathogen.When being detected alone,aptamer STY-3 with the concentration of 1 μM had the strongest inhibition for bacterial colony(inhibition rate was 74.66 ± 1.28%).While under combination way,STY2+3 with the concentration of 1 μM had the strongest inhibition for bacterial colony(inhibition rate was 87.84± 1.14%).Interestingly,when binding to bacteria cells,STY-1 competed with STY-2,which was revealed by competitive binding assay,declaring that they might interfere with each other or bound the same target on the surface of bacteria.It was also clarified that aptamer STY-3 alone had the strongest effect against biofilm formation with the concentration of 1 ±M(survival rate of biofilms was 50.04±3.71%)in contrast to STY-1 and STY-2.Moreover,the combination of STY-2 and STY-3 had the strongest efifect against biofilm formation specifically with the concentration of 1,M(survival rate of biofilms was 35.99 ± 2.26%)in contrast to STY 1+3.The aptamers and their combination demonstrated lower effect against formed biofilms in the initial stage(survival rate of biofilms were 61.31 ±4.26%by 1.5±M STY-3,43.50±4.26%by 1.5 μM STY2+3,respectively),illustrating the more difficulty in eliminating biofilms.Consequently,this research not only extended the application of aptamer,but also made it sense in avoiding drug resistance strains and combating infections.(3)The application of STY-1,STY-2 and STY-3 in inhibiting the adhesion of S.typhimurium on piglet intestinal epithelium cells IPEC-J2.It was illustrated by our work that three aptamers were active as well to weaken the adherence of the bactreia on IPEC-J2 cells.And STY-3 behaved the best anti-adhesion efficacy(adhensive inhibition rate was 49.98 ±1.93%by 2 gM STY-3).Besides,the combination of STY2+3 was more efficient to combat adherence of S.typhimurium(adhensive inhibition rate was 67.26 ±1.64%by 2 μM STY2+3).The difference among various ways of adhesion inhibition proved that the disturbance effects of aptamers against adherence of pathogen were closely ralated with the concentration and time point of treatment of aptamers.Finally,this research provided a new strategy involving pathogenicity control of pathogens by aptamers.(4)A bifunctional conjugate by linking aptamer SC-3 targeting S.choleraesuis with ampicillin,named SC3-Amp,was developed to restrain biofilms.In summary,the bifunctional approach resulted in a synergistic effect on control of S.choleraesuis biofilms.This conjugate had specific antibacterial activity that was superior to that of either of its component molecules(survival ratio of biofilms was only 28.36±0.57%in biofilm suppression and 40.00 ±2.70%in biofilm dissolution).However,the suppression effect of the conjugate was better than its effect on biofilm dissolution,indicating that treatment should be administered at an early stage of biofilm formation.If various antibiotics are combined with aptamers,more efficient,specialized agents may become available to treat biofilms in the future.