טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLee Shavit
SubjectThe Effect of Surfactin on Salmonella Typhimurium
Attachment and Biofilm Formation
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Yaron Sima


Abstract

Bacterial colonization and biofilm formation of foodborne pathogens on foods and food-contact surfaces pose a great threat to public health, with Salmonella being among the most predominant pathogens. Salmonella spp. are able to form biofilms, which are almost impossible to eradicate by conventional methods. Bacillus subtilis is a non-pathogenic rhizobacterium, which has long been used as a biocontrol agent against plant pathogens. Its ability to control phytopathogens has been attributed to the production of antimicrobial lipopeptides, such as surfactin. Although numerous studies have demonstrated the remarkable ability of B. subtilis to control phytopathogens, its activity against human pathogens has hardly been studied. In this study we aimed to investigate the ability of B. subtilis to control Salmonella enteric serovar Typhimurium.

            We demonstrated that B. subtilis exhibited various inhibitory activities against S. Typhimurium. B. subtilis efficiently inhibited bacterial growth and swarming motility on agar. Moreover, B. subtilis supernatant displayed anti-biofilm activity and was also able to hinder initial attachment to stainless steel and to parsley leaves. Additionally, bacterial colonization on mung bean sprouts was reduced after beans were treated with B. subtilis. To evaluate the role of surfactin in these control activities, we tested the control efficiency of a surfactin deficient mutant, srfAA.  We showed that B. subtilissrfAA filtrate failed to inhibit pellicle formation and bacterial attachment on stainless steel, indicating the importance of surfactin in the anti-adhesive and anti-biofilm activities against S. Typhimurium. B. subtilissrfAA was severely defective in swarming motility and failed in inhibiting the spreading of S. Typhimurium. Finally, B. subtilis srfAA was ineffective as biocontrol agent against S. Typhimurium on mung sprouts, demonstrating the crucial role of surfactin in controlling S. Typhimurium infections on sprouts. Nevertheless, colony diameter of S. Typhimurium was reduced in the presence of B. subtilissrfAA, and supernatants derived from B. subtilis wild-type and ∆srfAA exhibited a similar anti-adhesive effect on parsley leaves, suggesting that surfactin does not play a role in these inhibitory processes. Taken together our data, we assume that the anti-adhesive and anti-biofilm properties of surfactin might be directly involved in the ability of B. subtilis to inhibit plant colonization by S. Typhimurium. Yet, surfactin may also play an indirect role in the biocontrol activity through facilitating swarming motility in B. subtilis.

            Since the mechanisms allowing surfactin to inhibit biofilm formation are poorly understood, we further investigated which mechanisms may be involved in this process. We showed that purified surfactin was ineffective as antimicrobial agent against S. Typhimurium, and that colony morphology on Congo-red agar was unaffected by this biosurfactant, indicating that surfactin inhibits biofilm formation without affecting cells viability and cellulose and curli production. However, initial adhesion was reduced following preconditioning of stainless steel surfaces with purified surfactin, suggesting that inhibition of initial attachment could be related to the anti-biofilm activity of surfactin.

            Our findings may provide important clues for the development of novel bio-approaches for controlling S. Typhimurium adhesion and biofilm development in the food industry using B. subtilis or surfactin.