The gut commensal Bacteroides thetaiotaomicron harbors prevalent class A and D β-lactamases with cross-taxa dissemination potential.

The abundant gut commensal Bacteroides thetaiotaomicron is repeatedly challenged by β-lactam exposure in the human intestine, yet its β-lactamase repertoire and dissemination potential remain incompletely characterized. Here, we screened 626 publicly available B. thetaiotaomicron genomes and identified 1059 putative β-lactamase homologs, all belonging to class A or class D families. Four highly prevalent representatives-BTA-1 and CfxA-3 (class A) and OXA-347 and OXA-1327 (class D)-were prioritized for experimental validation. Heterologous expression in Escherichia coli revealed heterogeneous resistance phenotypes, with ones conferring only modest MIC increases (e.g., OXA-347, ~2-fold for amoxicillin) and others producing strong resistance to penicillins (e.g., BTA-1caused a 256-fold increase in amoxicillin MIC). Purified BTA-1 and OXA-347 were active under gut-relevant conditions, with pH optima at 7 and 8 and temperature optima at 40 °C and 30 °C, respectively. Notably, OXA-347 hydrolyzed representatives of penicillins, cephalosporins, carbapenems, and monobactams, and mass spectrometry confirmed β-lactam ring opening. Beyond B. thetaiotaomicron, these β-lactamase alleles were detected across multiple gut commensal genera as well as taxa annotated as opportunistic pathogens. Genomic context analyses showed similar gene-cluster patterns in B. thetaiotaomicron, across gut commensal genera, and in opportunistic-pathogen-associated taxa. Collectively, our findings clarify the diversity, activity, and distribution of B. thetaiotaomicron-associated β-lactamases across the gut microbiome and clinically relevant taxa.