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

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.