Overexpression of SmeGH Contributes to the Acquired Quinolones Resistance of Stenotrophomonas maltophilia

• Main Authors: Man-San Zhang, 張嫚珊
• Other Authors: Tsuey-Ching Yang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/kfk345

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 107 === Stenotrophomonas maltophilia, an important nosocomial infection pathogen, displays high-level resistance to a variety of unrelated antibiotics. Quinolone is one of the few treatment options for S. maltophilia infection; however, the quinolone resistance rate is increasing. Overexpression of SmeDEF and(or) SmeVWX efflux pumps is the main mechanism contributing to quinolone resistance of S. maltophilia. In this study, we aimed to elucidate the unidentified mechanisms responsible for the quinolone resistance, in addition to the overexpression of SmeDEF and SmeVWX pumps. The spontaneous quinolone-resistant mutants were selected by spreading KJDEF5, a smeDEF and smeU1VWU2X operons deleted double mutant, on the ciprofloxacin- or levofloxacin-containing medium. Of 34 selected quinolone-resistant mutants, 32 mutants exhibited the phenotype of multidrug resistance (MDR), implying the involvement of efflux pumps. Six mutants were randomly chosen from 32 MDR mutants for assessment of outer membrane protein profile. The intensity of a 45-KDa protein band was increased in four mutants compared to that in parental strain KJDEF5. The 45-KDa protein was verified to be TolCsm by liquid chromatography tandem mass spectrometry (LC-MS/MS). The tolCsm transcript overexpression in the four MDR mutants was further confirmed by real-time RT-PCR. To identify the TolCsm-conjugated efflux pump(s) response for the MDR phenotype, the transcripts of efflux pumps in MDR mutants C2206 and L1301 were quantified by real-time RT-PCR. The tested transcripts included smeH, smeJ, smeN, smeP, and smeY. Results demonstrated that smeY and smeH transcirpts were upregulated in C2206, and smeH transcripts increased in L1301. It is known that SmeYZ of S. maltophilia KJ mainly contribute to aminoglycoside resistance; therefore, SmeGH overexpression is highly to be the reason responsible for quinolone resistance of mutants C2206 and L1301. To verify whether smeGH overexpression contributes to quinolone resistance of C2206 and L1301, smeH allele was introduced into wild type KJ, mutants C2206 and L1301 respectivley, yielding KJsmeH, C2206smeH, and L1301smeH. The antibiotics susceptibility of all mutants were surveyed. Deletion of smeH from L1301 chromosome rendered the mutant more susceptible to quinolones, macrolides, chloramphenicol and tetracycline. The alteration in the antibiotic susceptibility of C2206smeH was generally consistent with that of L1301smeH, but the reduced extents of MICs in C2206smeH mutant were not as obvious as those in L1301smeH. The occurrence of smeGH overexpression in clinical quinolone-resistant isolates was surveyed. From 125 clinical quinolone-resistant isolates, we selected 4 isolates, whose smeDEF and smeU1VWU2X operons were not upexpressed, and determined their smeH transcript by real-time RT-PCR. Of four isolates tested, three isolates had an increased smeH transcript compared to quinolone-susceptible isolates. In summary, this study demonstrated that overexpression of SmeGH efflux pumps contributes to quinolone resistance in S. maltophilia.