| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 103 === Abstract
Salmonella enterica serovar Typhimurium is a bacterial pathogen that causes gastroenteritis in humans. Resistance to multiple antimicrobial agents is common among S. enterica serovar Typhimurium strains, recently the resistance to expanded-spectrum cephalosporins, such as ceftriaxone has increased in Salmonella. Previously, we established a highly ceftriaxone-resistant strain R200 of S. enterica serovar Typhimurium by performing sequential subcultures in medium with a subinhibitory concentration of ceftriaxone. A series of insertion mutants generated by transposon mutagenesis in R200 strain with different MICs were established.
In this study, we investigated the mutant 4B1 with a transposon inserted in the lon gene, which encodes a protease in cytosol and named R200(∆lon). The ceftriaxone MIC of R200(∆lon) strain has a 16-fold reduction compared R200 strain (256 μg/ml versus 16 μg/ml). To confirm that lon gene was involved in resistance to ceftriaxone, complementation assay was performed by the introduction of plasmid pBAD-Myc-His-lon into strain R200(∆lon) to construct the strain R200(∆lon)/pBAD-Myc-His-lon. In the presence of arabinose induction, the MIC of the R200(∆lon)/pBAD-Myc-His-lon strain was shown 8-fold increase (128 μg/ml versus 16 μg/ml).
To test whether penicillin-binding proteins are involved in the ceftriaxone resistance, we analyzed the RNA expression levels in all types of penicillin-binding proteins by real-time qRT-PCR. The result showed that lon gene can influence stm1910 (putative penicillin-binding protein 2) gene expression. However the introduction of plasmid pBAD-Myc-His-stm1910 into strain R200(∆lon), MIC showed no significant difference between R200(∆lon) and R200(∆lon)/pBAD-Myc-His-stm1910. Moreover, we used proteomic analysis to compare protein profiles of outer membrane, inner membrane and periplasm among strains 01-4, R200, R200(∆lon) and R200(∆lon)/pBAD-Myc-His-lon, two proteins STM3031 and STM3030, which previously identified to associate with ceftriaxone resistance, were decreased in R200(∆lon) compared to R200. The restoration of STM3031 and STM3030 protein levels were found in the complementary strain R200(∆lon)/pBAD -Myc-His-lon. The mRNA levels of stm3031 and stm3030 were correlated with their protein levels in these strains.
Although how Lon protease regulates the expression of stm3030 and stm3031 remains unknown, these results suggest lon gene is indeed associated with ceftriaxone resistance in S. enterica serovar Typhimurium.
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