Pre-existing β-lactamase gene diversity is associated with lower risk of ESBL-producing Enterobacterales colonization in patients exposed to ceftriaxone.

Exposure to broad-spectrum antibiotics, particularly to third-generation cephalosporins (3GC), increases the risk of colonization by extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E). While clinical risk factors for ESBL-E acquisition are well established, the role of the gut microbiome and resistome remains unclear. We conducted a prospective study of patients with suspected bacterial infections receiving ceftriaxone to identify microbiome and resistome features associated with ESBL-E acquisition. Rectal samples collected before antibiotic administration, during treatment, and 30 d after initiation were analyzed by shotgun metagenomic sequencing. Among 80 patients, 12 (15%) acquired ESBL-E colonization by day 30. Ceftriaxone exposure induced a profound and sustained reduction in microbial richness and diversity across all patients. However, no specific taxonomic signature predicted subsequent ESBL-E colonization. In contrast, patients who did not acquire ESBL-E displayed a significantly richer and more diverse repertoire of β-lactamase-encoding genes at baseline, which was independently associated with protection against colonization. Moreover, patients exposed to multiple antibiotics experienced greater and more sustained microbiome disruption compared with those receiving ceftriaxone alone. These findings provide the first real-world evidence that pre-existing β-lactamasome diversity may confer ecological protection against antibiotic-driven colonization by ESBL-E in infected patients, highlighting the importance of functional resistome diversity over taxonomic composition in colonization resistance.