| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術研究所 === 94 === Using in vitro stepwise induction, a new strain named R200 of Salmonella enterica serovar Typhimurium with high resistance to ceftriaxone (MIC over 256 mg/l) was generated (Cheng 2003). Following, we applied transposon mutagenesis on the R200 strain to generate the mutants with a more-than-four-fold reduction to ceftriaxone resistance (Li 2003).
Among these mutants, a strain 5B7 has a transposon inserted in the rfaG gene and results in susceptible to ceftriaxone. This strain was proved to be the deep rough LPS phenotype using combined hot-phenol water extraction of LPS and SDS-PAGE analysis. The results revealed the reduction of LPS and outer membrane proteins. Nine spots in 2-DE image of outer membrane protein profile were found to be altered. The protein levels of five out of nine spots were significantly increased and those of the other four spots were significantly decreased. All these protein spots change in the strain 5B7 might be related to ceftriaxone (CRO) susceptibility. The other mutant 4H11, a transposon insertion in the rfbX gene was also selected for study. It was proved to have a rough LPS phenotype with reduction of LPS and part of outer membrane proteins, but its MIC to CRO is intermediate. It was found that nine spots in 2-DE image of outer membrane protein profile to be altered. The protein levels of six out of nine spots were significantly increased and those of the other three spots were significantly decreased. The permeability assay proved that the highest permeability in strain 5B7 and reduced efflux pump capacity in both strains 4H11 and 5B7 may contribute to reduction of antibiotics resistance. I also chose strain 5G8 which carried a transposon inserted in tolC gene for study. Since TolC protein associated with multiple drug resistance such as SDS and lipophilic ��-lactams antibiotics, it resulted in reduction of CRO resistance (MIC < 2.5 mg/l). In our previous study, we found a putative outer membrane protein STM3031 correlated with CRO resistance (Cheng 2003). The expression level of STM3031 in strains 5B7 and 4H11 were also found to be reduced. We concluded that a gene disruption in LPS biosynthesis could affect outer membrane protein expression, membrane permeability and the transporters or efflux pumps, all of these may contribute to the susceptibility to ceftriaxone.
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