טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentScher Keren
SubjectCaracterization of the Biofilm Produced by Salmonella
Typhimurium at the Air-Liquid Interface
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Sima Yaron
Full Thesis textFull thesis text - English Version


Abstract

Biofilms have developed into a significant issue for public health, since biofilm-associated microorganisms are less susceptible to antimicrobial agents and treatments.

The aim of this research was to characterize the biofilm produced by Salmonella enterica serotype Typhimurium at the air-liquid interface (pellicle). The biofilm is formed in standing culture in less than 24 hours and composed mainly from cellulose and thin aggregative fimbriae.

We focused mainly on the effect of cellulose on the morphology of young and old biofilms and the survival mechanisms of the biofilm cells to physical conditions (low pH, heat) and chemical conditions (sodium hypochlorite and triclosan).

We characterized the morphology of both sides of the biofilms using Atomic Force Microscope (AFM) and Scanning Electron Microscope (SEM). We analyzed changes during the maturation of the biofilm and evaluated the role of cellulose in the biofilm structure and morphology. Overall, there was a marked difference in the morphology of the water facing and air facing biofilm surfaces. During maturation of the biofilm, porousivic structures were created until breakup. We concluded that the disintegration of mature biofilms probably occurred by combination of self hydrolysis of the matrix components and physical forces. This phenomenon provides a potential new approach to combat microbial cells in biofilms.

In the second part of the research we investigated the resistance of Salmonella Typhimurium to stress conditions. Resistance of bacteria from the biofilm to heat, acidification, chlorination and triclosan was compared to resistance of planktonic cells (logarithmic and stationary phases). Biofilm cells were significantly more resistant to chlorination and triclosan treatment but not to heat or acidification. In order to investigate this phenomenon we quantified the transcription of several genes fabI, micF, marA, acrA, bcsA and bcsE mRNAs which are related to resistance to a variety of antimicrobial agents and genes related to biofilm production using quantitative RT-PCR. We observed a significant increase in transcription of the cellulose-synthesis coding genes, upon exposure to triclosan.

Our results demonstrated that the tolerance of Salmonella towards triclosan in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials. However, resistance of biofilm cells to sodium hypochlorite was attributed to low diffusion and reaction of the matrix polymers with chlorine. We concluded that every one of the physical and chemical condition that we have studied triggered different resistance mechanism in the biofilm.