Microbiota-based Antimicrobial resistance Risk Screening (MARS): a novel PCR method for dynamic assessment of AMR colonization risk and ESBL carrier identification

Background The gut microbiome serves as a reservoir for antimicrobial-resistant (AMR) bacteria, making the prevention of silent colonization and transmission an urgent public health priority. Colonization risk often precedes established AMR colonization, yet no rapid method currently exists to identify individuals at risk. Results We developed a microbiota-based AMR risk screening (MARS) method, comprising two complementary real-time PCR assays to quantify the bacterial loads of Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae (LRB; indicators of colonization resistance) and Enterobacteriaceae (ETB; indicator of colonization permissiveness). Assay performance was evaluated in an antibiotic-treated mouse model and validated in clinical samples from 80 patients with diarrhea and 20 healthy controls. Both assays demonstrated high specificity and correlated with 16S rRNA gene sequencing. In mice, LRB assay cycle threshold (Ct) values measured just before inoculation with extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Eco) correlated with fecal bacterial load and shedding duration. In patients, LRB bacterial loads declined during antibiotic treatment and recovered after discontinuation. Combined ETB and LRB assay results identified both high-risk individuals and ESBL carriers, with strong discriminatory performance (AUCs = 0.86 and 0.76). Conclusions The MARS method provides a novel, rapid, and clinically applicable approach to assess AMR colonization risk and detect actual colonization, offering a valuable tool for early intervention in high-risk individuals before AMR carriage becomes established.