Investigation of a Cryptic Aminoglycoside Resistance Gene in Salmonella Enterica Subspecies Enterica Serovar Typhimurium

Abstract

The gene aac(6’)-Iy is a cryptic aminoglycoside acetyltransferase (AAC) that is present in almost all Salmonella species and subspecies. Despite its ubiquitous presence in Salmonella, it does not confer Salmonella with aminoglycoside resistance. The reason why Salmonella maintains this cryptic gene is unclear. This thesis aimed to describe the role of aac(6’)-Iy in S. Typhimurium. Loss of aac(6’)-Iy in S. Typhimurium did not confer a significant difference in resistance to gentamicin, kanamycin, and amikacin, confirming its cryptic phenotype as an antibiotic resistance gene. However, the gene is not entirely cryptic as it has expression under in vitro conditions. In addition, aac(6’)-Iy is upregulated in intracellular S. Typhimurium during infection of both HeLa epithelial cells and RAW264.7 macrophages. These results suggested a role for the gene in intracellular survival, which was confirmed with infection assays in both HeLa epithelial cells and RAW264.7 macrophages. The pattern of survival in HeLa cells suggested a role for aac(6’)-Iy in survival within the Salmonella-containing vacuole (SCV). The survival of an aac(6’)-Iy mutant in both the cytosol and the SCV were measured to investigate whether aac(6’)-Iy specifically affected survival within the SCV. Loss of aac(6’)-Iy did not affect cytosolic survival within HeLa epithelial cells, but it did lead to a significant reduction in the percent of LAMP1-associated bacteria present in the SCV. This confirmed that aac(6’)-Iy has a role in S. Typhimurium survival within the SCV. Loss of aac(6’)-Iy also led to a significant competitive disadvantage in a murine model of systemic infection. Together these data demonstrate an alternative role for the antibiotic resistance gene in aac(6’)-Iy in Salmonella survival and infection in the host. This is the first chromosomally encoded AAC shown to play a role in survival within epithelial cells and within the SCV.