| Summary: | 碩士 === 中國醫藥大學 === 醫學檢驗生物技術學系碩士班 === 99 === Stenotrophomonas maltophilia is an important opportunistic pathogen characterized by the phenotype of multidrug resistance (MDR). Overexpression of the resistance nodulation division (RND) efflux systems is a critical cause of the MDR phenotype in gram-negative bacteria. A chloramphenicol-selective S. maltophilia MDR mutant, KJ09C, was characterized in this study. In addition to chloramphenicol, KJ09C was cross-resistant to quinolones and tetracyclines. Surprisingly, mutant KJ09C increased aminoglycoside susceptibility compared to wild-type KJ. The results of qRT-PCR demonstrated that SmeVWX pump was overexpressed in mutant KJ09C. Inactivation of smeU1-V-W-U2-X operon of mutant KJ09C restored the antimicrobial susceptibility of KJ09C to the level as that of wild-type KJ, indicating that overexpression of SmeVWX pump contributes to the MDR phenotype of KJ09C. A LysR-type transcriptional regulator gene, smeRv, divergently located upstream of smeU1-V-W-U2-X operon. The results of transcriptional fusion assay showed that the SmeRv plays a positive role in the overexpression of smeU1-V-W-X-U2 operon, and that SmeRv has a characteristic of negative autoregulation in wild-type background and positive autoregulation in KJ09C background. To elucidate the role of each component of smeU1-V-W-U2-X operon in the resistance, a series of mutants were constructed, including KJ09C△SmeU1, KJ09C△SmeVW, KJ09C△SmeX, and KJ09C△SmeU2. The results showed the smeU1 of KJ09C had no effect on antibiogram resistance, and inactivation smeVW of KJ09C abolished SmeVWX pump activity for extrusion of chloramphenicol, quinolone, and tetracycline. To further clarify the role of smeU2 and smeX on the antibiotics resistance, smeU2 and smeX overexpression mutants were constructed in KJ09C△5, including KJ09C△5L2::SmeU2 and KJ09C△5L2::SmeX. The role of SmeU2 seems significant only when the SmeVWX pump is overexpressed. The smeX overexpression of KJ09C is responsible for the decreased aminoglycoside resistance of KJ09C.
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