云南土壤宏基因组文库中替加环素耐药基因的筛选与分析

Screening and analysis of tigecycline resistance genes from Yunnan soil metagenomic library

摘要:

【背景】随着抗生素的广泛应用以及滥用，出现了多重耐药的“超级细菌”，并且在临床上已出现对治疗“超级细菌”感染的最后一线抗生素——替加环素耐药的细菌。【目的】对土壤环境中存在的替加环素耐药基因进行筛选调查，探讨替加环素耐药机制，为临床抗生素治疗提供借鉴。【方法】利用功能宏基因组学技术对土壤宏基因组文库进行替加环素耐药阳性克隆的筛选和耐药亚克隆的构建，对构建成功的亚克隆进行测序和生物信息学分析探知其功能，同时进行最低抑菌浓度(Minimum Inhibitory Concentration，MIC)的测定。【结果】筛选得到一个四环素抗性Major Facilitator Superfamily (MFS)外排泵基因，携带该基因的菌株对四环素类抗生素均耐药，对替加环素和四环素的MIC值分别为16 μg/mL和32 μg/mL。当MFS外排泵抑制剂羰基氰化物间氯苯腙(Carbonyl Cyanide 3-Chlorophenylhydrazone，CCCP)浓度为4 μg/mL时可以抑制其对四环素类抗生素的外排作用。生物信息学结果表明该基因编码483个氨基酸，编码的蛋白质属于疏水稳定蛋白；其含有的14个跨膜区构成MFS外排泵蛋白典型的14次跨膜螺旋保守结构域；系统进化树分析表明其与其他替加环素耐药基因编码的蛋白质位于不同的分支上，相似性较低。【结论】利用功能宏基因组学技术筛选得到一个有替加环素抗性的MFS外排泵基因，编码的蛋白质属于14次跨膜螺旋MFS外排泵家族，为今后研究替加环素耐药机制提供参考。

Abstract:

[Background] With the wide application and abuse of antibiotics, the “super bacteria” resistant to almost all antibiotics emerged. And clinically, bacteria resistant to tigecycline, the current last line of defense to treat “super bacteria” infections, had emerged too. [Objective] To screen tigecycline resistance genes from the soil and study the resistant mechanism, providing the reference for clinical antibiotic treatment. [Methods] Functional metagenomics technology was used to obtain tigecycline resistance clone, and subcloning and sequencing were used to identify the tigecycline resistance gene. The characteristics of physicochemical properties, protein secondary structure, transmembrane domain, and system evolution analysis of the resistant gene were analyzed by bioinformatics methods. Besides, the determination of minimum inhibitory concentration was performed. [Results] A gene encoding tetracycline resistance MFS efflux pump was identified and the E. coli harboring this gene showed resistance to tigecycline and tetracycline with the MIC values of 16 μg/mL and 32 μg/mL, respectively. The efflux activity of the protein was inhibited by 4 μg/mL of MFS efflux pump inhibitor CCCP. The predictive protein encodes 483 amino acids and belongs to hydrophobins and stable proteins. And it contained 14 transmembrane regions which formed a typical 14-times transmembrane spiral MFS efflux pump conserved domain. Phylogenetic tree analysis showed that the protein was located on different branches and showed low homology with other protein encoded by tigecycline resistance genes. [Conclusion] A tigecycline resistance MFS efflux pump gene was obtained using functional metagenomics technology. The protein encoded by the gene belonged to 14-times transmembrane spiral MFS efflux pump family, and this study provided reference on future study of the mechanism of tigecycline resistance.