Evaluation Of Antibacterial Effect Of Antimicrobial Peptide Nisin Against Streptococcus Suis

Streptococcus suis(SS)is an important swine pathogen causing septicemia,arthritis,meningitis and endocarditis in pigs.SS is also an important zoonotic pathogen responsible for septicemia with or without septic shock,meningitis and death in humans.A total of 33 serotypes are defined based on the antigenicity of their capsular poly saccharide(CPS)and serotypes 1 to 9,14 and 16 can cause diseases in pigs.Generally,serotype 2(SS2)is considered to be the most virulent,and is frequently isolated from clinically-diseased piglets.During the past decades,the antibiotics had been extensively used to prevent and treatment of bacterial diseases in pigs industries.With the copiously used and abused antibiotics,the bacterial pathogens have been shown increasingly drug-resistance and multi-drug resistances.While most strains of SS are highly sensitive to penicillin and amoxicillin,resistance to macrolides,lincosamides,sulphonamides and tetracyclines,which have been reported that strains resistant are up to 85%.Given the rising concerns regarding resistance of SS to conventional antibiotics,the search for new and alternative antimicrobial compounds is of particular interest.Antimicrobial peptides,also known as Antimicrobial Peptides or Host Defense Peptides,have been extensively studied as broad-spectrum anti-bacterial and anti-viral agents and rarely caused the emergence of drug-resistant microorganism.The aim of this study was to evaluate the potential of Nisin as an antibacterial agent against the pathogen SS in vitro and in vivo.In addition,its ability to act in synergy with conventional antibiotics commonly used for the treatment of SS infections in vitro and in vivo were investigated.The results would be provide a basis for making protocols for prevention of the swine streptococcosis.Three parts of work related to the contents mentioned above were conducted.1.Antimicrobial activity of Nisin against the Streptococcus suis and its synergistic interaction with antibiotics in vitroThe antibacterial effect of Nisin against different serotypes and different strains of the same serotype of SS were determined with drug sensitivity test.Most SS strains were sensitive to Nisin and a few strains were extremely sensitive.The MICs of SS were ranged from 0.122?g/mL to 3.906?g/mL and the MBCs of SS were ranged from0.244?g/mL to 7.813?g/mL.The electron microscopy(TEM and SEM)shown that the SS chains were broken,the cell membranes were atrophied ruptured and pores were appeared in the cytoplasmic membrane resulting in spillage of the contents in the SS cells treated with different concentrations of nisin.The rapid killing effect of nisin on SS was determined by the time bactericidal curve method.The MICs and MBCs of several kinds antibiotics were tested for SS strains,the results showed that most of the SS strains were sensitive to penicillin,amoxicillin,cefotaxime sodium and tetracycline hydrochloride,but not to erythromycin,levofloxacin,and azithromycin.We then evaluated the synergistic activity of nisin in combination with above conventional antibiotics,the results showed that several synergistic effects were observed when Nisin was used in combination with the above antibiotics and a few antagonistic effects were observed by measuring the value of fractional inhibitory concentration index(FIC).2.Antimicrobial activity of Nisin against Streptococcus suis and its synergistic interaction with antibiotics in vivoAccording to the results of tests in vitro,we performed the protective assays by using BALB/c infection model to evaluate the antimicrobial activity of Nisin against SS virulent strains in vivo and its combination with antibiotic cefotaxime sodium.Determination of the optimal antimicrobial peptide Nisin dose by using different concentrations of Nisin in the treatment of BALB/c mice infected with SS virulent strain.The drug-prevention model was determined by measuring the prophylactic/therapeutic effect of Nisin against SS-infected BALB/c mice.Determination of the optimum amount of individual antibiotics Cefotaxime sodium with different concentrations was used to treat BALB/c mice infected with SS.Using a checkerboard method,cefotaxime sodium and Nisin were mixed at different concentrations to test whether Nisin can reduce the dosage of antibiotics or increase the clinical effects under the same dose of antibiotics.The results showed that the optimal concentration of Nisin alone in BALB/c mice infected with SS was 5 mg/kg.Nisin has certain therapeutic and preventive effects on BALB/c mice infected with SS2(SS2-1,GD19)or SS9(GZ0565)virulent strains,respectively,the therapeutic effect was better than the preventive effect.The optimal concentration of cefotaxime sodium alone in BALB/c mice infected with SS was 10 mg/kg.In vivo tests showed that the combined effect of Nisin and cefotaxime sodium had a synergistic effect.The usage of antimicrobial peptides and antibiotics was reduced to 1/8 of the single drug dose,respectively.Therefore,the combination of Nisin and antibiotics can reduce the usage of antibiotics and increase the treatment effect on the SS infection in BALB/c mice.3.The effect of Nisin on the formation of SS biofilmThe capacity for biofilm formation of 15 SS strains was tested in a 24-well plate assay by using crystal violet staining,the results showed that SS2-1,GD19,H6,X5,M51.SH040917,GZ0565 exhibited strong biofilm-forming ability,P1-7,HA0609,H8,2066,X2,and DX4-1 were less able to form biofilms,while 100802H30 and 1728-3 were unable to form biofilms.The disrupting effect of nisin on SS biofilm were confirmed by crystal violet staining and electron microscopy.The OD value of SS biofilms treated with Nisin was significantly lower than that of the untreated control group.Scanning electron microscopy observations revealed the SS biofilms were significantly degraded after treatment with Nisin.There were SS cells showed their destroyed traces by Nisin.These results showed that Nisin can reduce the formation of SS biofilms.In summary,our study provides new data in this area indicating that Nisin has antimicrobial activity against the SS and exerts synergy with conventional antibiotics in vitro and in vivo.In addition,our data indicate that the Nisin exerts strong antagonistic activity toward SS biofilms,which suggests that it may represent a probiotic of interest for the control of SS persistent infection.