摘要
为研究体外诱导敏感嗜水气单胞菌耐药后,其敏感性变化与基因突变、外排作用的关系,实验选取对氟喹诺酮类药物敏感的养殖鱼源嗜水气单胞菌临床分离菌株为研究对象,分别在含亚抑菌浓度恩诺沙星(EN)和诺氟沙星(NF)的培养基上逐步诱导培养,以获得高耐药菌株;对诱导菌株gry A和par C基因进行扩增和测序分析;测定诱导菌对诱导药物和16种非诱导药物的最小抑菌浓度(MIC)及添加外排泵抑制剂N-甲基吡咯烷酮(NMP)后的MIC值变化;并对诱导菌交叉耐药情况进行比较分析。结果发现,诱导后菌株对EN和NF的MIC分别提高了409.6和4096倍,对非诱导氟喹诺酮类药物和其他类药物的MIC也有较大变化;药物诱导后各菌株gyr A基因和par C基因编码的氨基酸QRDRs区发生了典型的点突变:Gyr A发生Ser83→Ile变化,Par C发生Ser87→Ile/Arg变化;添加NMP后,所有诱导菌株对两种药物的MIC值均有不同程度的下降;诱导后菌株交叉耐药情况与菌株密切相关,其中3和8号诱导菌株对16种非诱导药物均无交叉耐药反应,而EN诱导菌株对氨基糖苷类和利福霉素类药物基本未产生交叉耐药反应,NF诱导菌株对除庆大霉素以外的氨基糖苷类和利福霉素类药物基本未产生交叉耐药反应,所有诱导后菌株均对四环素类和氯霉素类药物产生较严重的交叉耐药。研究表明,嗜水气单胞菌对氟喹诺酮类耐药存在靶基因位点突变及主动外排作用等多种耐药机制;且应慎重考虑在防治耐药菌株引发病害时,交叉耐药情况对选择治疗药物的影响。
To investigate the relationship between sensitivity varieties,mutations,efflux pump activities and crossresistance of selected strains after they were selected. The strains of Aeromonas hydrophila（Ah） from cultured fish sensitive to fluoroquinolone were selected in vitro stepwise exposure to increasing concentration of Norfloxacin（NF） and Enrofloxacin（EN） on solid medium, aiming to gain the fluoroquinolone-resistant isolates. Then genes of gry A and par C from selected strains were amplified and sequenced. The minimal inhibitory concentrations（MICs）of selected and non-selected antimicrobials as well as the MICs after efflux pump inhibition 1-Methyl-2-pyrrolidinone（NMP） was added to each strain were determined by broth microdilution method. Results shows the MICs of EN and NF increased 409.6-and 4096-fold after selection, respectively, while the MICs of non-selected fluoroquinolone and other antimicrobials also changed greatly. Mutations happened in the quinolone resistancedetermining region（QRDR） encoded by gyr A and par C genes after being selected： Ser83→Ile mutations of Gyr A and Ser87→Ile/Arg mutations of Par C were identified. After NMP was added, MICs of all selected strains have declined in different degrees. Cross-resistance of selected strains is closely related to the strain, among the selected strains No. 3 and 8 have no cross-resistance to16 kinds of non-selected antibiotics, and the strains selected by EN and NF have nearly no cross-resistance to aminoglycosides, rifamycins and aminoglycosides（except GM）,rifamycins, separately. All selected strains have great cross-resistance to tetracyclines and chloramphenicols. The resistant mechanisms of Ah to fluoroquinolone might include the target gene mutations and an active efflux system, and we should also pay more attention to the cross-resisance when choosing antimicrobials.
出处
《水产学报》
CAS
CSCD
北大核心
2016年第3期495-502,共8页
Journal of Fisheries of China
基金
"十二五"国家科技支撑计划(2012BAD25B10)
中国水产科学研究院基本科研业务费(2014A06XK05)~~
