Screening and identification of genes associated with streptomycin resistance in Brucella melitensis

Abstract:

[Background] Brucella spp. are facultative intracellular pathogens causing a worldwide zoonosis, brucellosis. Streptomycin is the recommended antibiotic for treating brucellosis, while streptomycin-resistant isolates have been identified in China (according to the breakpoint recommended by CLSI). [Objective] To screen and identify new genes associated with streptomycin resistance in Brucella melitensis. [Methods] We employed RNA sequencing (RNA-seq) to mine new genes associated with streptomycin resistance in B. melitensis. Homologous recombination and molecular docking were employed to predict and identify the functions of the relevant genes. [Results] B. melitensis M5 restored its proliferation after 12 h of culture in the medium supplemented with 2×MIC of streptomycin. The results of RNA-seq revealed that cell membrane components played a role in the resistance to low-concentration streptomycin. Under streptomycin stress, the expression of genes involved in the ribosome pathway was up-regulated [|log₂FC|≥2.0, P<0.05], while that of genes involved in quorum sensing and type IV secretion system was down-regulated (|log₂FC|≥2.0, P<0.05). The MICs of streptomycin for virB3- and virB5-deleted strains were higher than that for M5, while the MICs of streptomycin for complemented strains were not significantly different from that for the wild-type strain. Molecular docking results demonstrated that VirB3 and VirB5 could bind to streptomycin through hydrogen bond. [Conclusion] Streptomycin mainly affected pathways related to cell membrane components of B. melitensis. B. melitensis develops resistance to streptomycin by down-regulating the expression of virB3 and virB5 of the type IV secretion system. This study provides new insights into the research on the antibiotic-resistant strains of Brucella spp. and offers candidate targets for the development of new agents for treating brucellosis.

Key words:Brucella melitensis;streptomycin;type IV secretion system;VirB operon