טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentHollander Amit
SubjectAggregation Mechanisms in a Multi-Resistant Salmonella
Senftenberg Strain
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Professor Sima Yaron


Abstract

Following the 2007 basil outbreak in Europe, caused by Salmonella enterica serovar Senftenberg, it was important to comprehend the antimicrobial activity of basil essential oils and the mechanisms by which Salmonella may overcome them. Linalool, one of the major constituents of basil oil, perforates bacterial membranes, decreases cell motility and induces cell aggregation. A linalool adapted S. Senftenberg (LASS) strain, which developed through a selection pressure to linalool, better resists linalool, basil oil and several antibiotics, and better survives on harvested basil leaves. LASS has decreased permeability to ethidium bromide, altered fatty acid composition, impaired motility and ability to form larger aggregates in presence of linalool. The linalool-induced aggregation may indicate a plausible adaptation to linalool through formation of clustering similar to biofilm arrangement.

The aim of this study was to reveal the mechanism that lead to the linalool-induced aggregation in LASS and to evaluate its association with cell motility and membrane disruption. The second aim was to identify the related genetic and phenotypic differences between LASS and its wild-type.

Aggregation percentage was calculated from particle size distribution in comparison to planktonic cells. It was found that the aggregation was in common to other physical and chemical membrane-targeted treatments, which generated miscellaneous protein leakage. Cell aggregation was also observed with non-bacterial proteins (i.e. BSA), suggesting that the linalool-induced aggregation stemmed from general leaked proteins. Enzymatic degradation of proteins that leaked by linalool or by sonication restrained the aggregation. Aggregate disassemble was also possible by protein degradation. Whereas protein denaturation detained cell aggregation, reducing the disulfide bridges had no effect on protein capability to induce cell aggregation, although cell aggregation was partially stabilized by disulfide bridges.

Genome sequencing and bioinformatics analysis discovered six variations between LASS and the wild-type S. Senftenberg, located in wzx, rhlB, rbsR, in the promoter region of SBOV17141, and in ramA and its repressor binding site. The latter two mutations may be involved in the multi-drug resistance of LASS, since RamA regulates efflux/influx in Salmonella. The increased resistance of LASS may also be attributed to the mutated Wzx, a flippase involved in O-antigen synthesis, by changing cell surface properties. Defects in O-antigen synthesis can also be the reason for the extensive aggregation, the impaired motility of LASS and its impotence to form biofilms, a phenomenon which was first described here. Since both motility and biofilm formation are affected by cyclic di-GMP concentration, they may be influenced by the mutation in the promoter region of its regulator, SBOV17141.

Collectively, this study suggests a role for protein leakage from damaged bacteria in promoting bacterial cell aggregation. This finding may serve as a base for future studies in both aggregation investigations and antimicrobial activity, including the ability of bacteria to resist antimicrobial treatments via protein leakage and cell aggregation. In addition, the genetic analysis has gone some way towards enhancing our understanding of altered phenotypes in LASS resulted from selective pressure to linalool, hence, providing important insights for future research in acquisition of the multi-drug resistance phenotype.