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

Brucellosis, caused by <i>Brucella</i> spp., is a globally zoonotic disease that results in substantial economic losses and public health concerns. Although antibiotic-resistant <i>Brucella</i> 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 <i>B. melitensis</i> 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 <i>Brucella</i> 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 <i>B. melitensis</i>. 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.