Gut phages are linked to off-target antimicrobial resistance genes in orthopedic patients receiving peri-surgical antibiotics.

INTRODUCTION

Antibiotic prophylaxis alters the gut microbiome, potentially expanding antimicrobial resistance genes (ARGs). The role of bacteriophages in this process remains poorly understood. This study investigates the effects of predominantly flucloxacillin- and gentamicin-based perioperative antibiotic prophylaxis on phage and ARG dynamics in orthopedic trauma patients.

METHODS

Shotgun metagenomic analysis in two orthopedic cohorts: rib fracture fixation (n = 16) and frail hip fracture fixation (n = 22), versus frail (n = 18) and non-frail (n = 95) controls was performed. Phage abundance (viral sequence groups [VSGs] relative abundance) was quantified using MetaPhlAn 4.1, while ARG abundance using ResFinder 7 (v2022) with KMA (v2.3.0). ViroProfiler with CheckV for quality assessment was used to detect ARG-carrying viral contigs in antibiotic-treated patients.

RESULTS

Following antibiotics, seven VSGs were increased in rib and five in the frail hip fractures compared to controls (mean Log₂ Fold change: 16.89 and 17.21, respectively). In the frail cohort, 18 ARGs increased, including 14 off-target ARGs (resistance genes for antibiotics not administered; mean Log₂ Fold change >25 compared to controls). Five VSGs correlated with six off-target ARGs in antibiotic-treated patients (Rho = 0.612). Longitudinal analysis of rib fracture patients (n = 10) revealed off-target ARGs increased from discharge to follow-up, paralleling increased VSGs abundances, while on-target ARGs remained mostly stable. In antibiotic-treated compared to controls, phage-related and DNA-repair gene families were enriched, phage gene richness was associated with stress and biofilm formation pathways, and VSGs correlated with opportunistic species (e.g., E. coli). More frail antibiotic-exposed patients (81.5%) carried ARG-containing viral contigs than non-frail patients (41.4%).

DISCUSSION: Our findings suggest perioperative antibiotics may contribute to phage-associated dissemination of off-target ARGs. Understanding how ARGs spread in the gut microbiome is important for improving antibiotic stewardship and protecting vulnerable patients from difficult to treat infections.