Novel metabolic adaptation driven by glycoside hydrolase family 25 protein contributes to increasing trimethoprim-sulfamethoxazole resistance in clinical human Brucella melitensis isolates in China.

Brucellosis, caused by Brucella spp., is a globally zoonotic disease that results in substantial economic losses and public health concerns. Although antibiotic-resistant Brucella strains have been reported worldwide, the current status and underlying mechanisms of resistance among Chinese isolates remain poorly characterized. In this study, we analyzed 636 clinical human isolates of B. melitensis from China using genomic sequencing, transcriptomic sequencing, and neural network prediction to identify key determinants and mechanisms of antibiotic resistance. Functional validations were performed using gene editing and protein-protein interaction assays. We found a gradual increase in resistance to trimethoprim-sulfamethoxazole (SXT) among Chinese isolates in recent years, despite the absence of known antibiotic resistance genes. Comparative genomic analyses between high- and low-minimum inhibitory concentration (MIC) isolates revealed specific single nucleotide polymorphisms (SNPs) that were present only in high-MIC isolates. Transcriptomic analysis demonstrated that high-MIC and low-MIC isolates activated distinct metabolic pathways in response to SXT exposure. Notably, genes influenced by specific SNPs exhibited opposing expression patterns after SXT treatment. Gene-editing experiments revealed that deletion of the glycoside hydrolase family 25 (GH25) gene, which was identified through SNP analysis, was associated with SXT resistance and notably altered Brucella energy metabolism, although it did not impact virulence in host cells. Further, we identified a direct interaction between GH25 and XylF. Collectively, our study reveals a novel genetic mechanism driving SXT resistance in B. melitensis. These findings highlight the critical need for vigilant surveillance of antibiotic resistance to mitigate public health risks associated with the potential widespread emergence of antibiotic resistance.