טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSchlisselberg Dov
SubjectMechanisms of Antimicrobial Resistance in
Biofilm-Associated Salmonella
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Sima Yaron


Abstract

In this study we aimed to investigate the characteristics of Salmonella enterica serovar Typhimurium inside biofilms using two approaches.

First, we aimed to identify new ways to prevent bacterial colonization and biofilm formation of S. Typhimurium cells on stainless-steel. Our aim was to find correlation between surface finish and colonization, biofilm formation and susceptibility of S. Typhimurium to disinfection. We compared the surface roughness and composition of three different finishes of stainless steel surfaces and a not-treated control. Overall, we found a direct correlation between roughness parameters and initial bacterial attachment and a correlation between the kinetics of biofilm formation and surface composition. However, the most surprising finding was that chlorine treatment is highly effective against biofilm formed on the electro-polished surface than on the other surfaces. Based on these results, we suggest that careful selection of materials used for surfaces in production lines would improve microbial safety and quality of products.

In the second part, we aimed to identify genetic mechanisms that contribute to the high antimicrobial resistance of biofilm-associated S. Typhimurium cells. We screened and identified genetic elements that are induced in response to antimicrobials on a genome-wide scale with the use of a modified differential fluorescence induction (DFI) approach. In this approach, a fluorescence activated cell sorter (FACS) is used to sort random gDNA fragments cloned as promoters of GFP. We discovered 50 individual inserts that are presumably up-regulated in response to ciprofloxacin and/or triclosan treatment.

Next, we chose eight promoters for further analysis; one of the chosen promoters was of the marRAB regulon which controls 60 genes involved in a wide range of stresses. We found that two genes of the marRAB regulon, micF and soxR show a heterogeneous transcription profile following ciprofloxacin treatment of biofilm. This heterogeneity was found to be highly specific for a given compound and depends on its concentration. When analyzing a set of mutants, we showed that Rob, a transcriptional regulator of the AraC/XylS family has a role in MicF heterogeneity.

We concluded that while DFI allows the identification of some of the genes that show a change in transcription following exposure to antimicrobials, those genes might be not important for resistance. We observed that only when using single-cell resolution methods we were able to differentiate between seemingly identical responses of the biofilm to different stress inducers and to discover new genetic circuitries.