טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentOfir Menashe
SubjectThe Response of Intracellular and Extracellular Salmonella
enterica Serovars to Aminoglycosides Treatment
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Yaron Sima
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Aminoglycosides are antibiotics that inhibit the translation process of a broad range of bacteria. Aminoglycosides are highly effective against planktonic Salmonella and have a minor bactericidal effect on intracellular Salmonella.  The main goal of this study was to gain a better understanding regarding the aminoglycoside effect on intracellular Salmonella within macrophage and epithelial hosts and to determine the reasons for the unsatisfactory activity.

The efficacy of aminoglycosides against intracellular and extracellular Salmonella enterica was analyzed by a viability test and by using the luciferase system as a reporter. Our results indicated that the effect of aminoglycosides on intracellular S. enterica serovars Typhimurium and Virchow within a macrophages and Caco-2 epithelial cells is physiologic rather than bactericidal (0.5-1 log reduction). This effect occurred rapidly (within minutes) and its intensity was affected by the aminoglycoside type and concentration and by the host type. In contrast, when using the same aminoglycoside concentration (100-150µg/ml) against planktonic Salmonella, we observed a 6-log reduction in viable counts.  Proteomic analysis of intracellular S. Typhimurium revealed that aminoglycosides such as gentamicin induce the expression of stress response systems and ribosomal RNA proteins, indicating a direct antibiotic effect. Since we showed that aminoglycosides can penetrate the macrophage and even inhibit the translation process of the host too, we tried to explain why a significant bactericidal effect is not observed.

Previous studies suggested that an acidic environment may inhibit aminoglycoside uptake into the bacteria, and since the environment in the phagosome within the macrophages is acidic, we hypothesized that low acidity reduces the efficacy of aminoglycosides against intracellular Salmonella. This hypothesis was supported by showing that S. Typhimurium within Nramp-1 deficient macrophages (RAW 264.7_R21), which have a higher pH in the phagosome, is more susceptible to gentamicin compared to S. Typhimurium associated with macrophages (RAW 264.7_R37), which express functional Nramp-1.

S. Typhimurium in colon epithelial cells was less susceptible to gentamicin in comparison to S. Typhimurium in macrophages. Confocal microscope observations indicated the possibility of intra-nucleus pathogen loci. This unique observation may explain the low efficacy of gentamicin on intracellular Salmonella, since the antibiotic must surmount a number of obstacles before reaching the intracellular pathogen.

The current study enhances our knowledge about the ability of aminoglycosides to penetrate cells, and their effects on planktonic and intracellular bacteria. In order to improve the effectiveness of aminoglycosides in clinical use we recommend adjusting the aminoglycoside formulation or carrier to match the type of the host cell.