9th World One Health Congress

Abstract

Antimicrobial resistance has substantially influenced Clostridioides difficile epidemiology worldwide, particularly among strains shared across human, animal, and environmental reservoirs exposed to diverse antimicrobial pressures. Interpretation of tetracycline resistance in C. difficile remains challenging due to the lack of species-specific breakpoints, with studies often relying on generic anaerobe breakpoints or detection of tet(M) alone. In this study, we characterize tetracycline resistance phenotypes and genotypes of C. difficile strains and propose an epidemiological cut-off value (ECOFF). Whole-genome sequencing (WGS) was used to characterize tet-associated genes and respective mobile genetic elements (MGEs),
This study included C. difficile isolates from human (n = 643), animal (n = 119), and environmental (n = 51) sources, collected between 2020 and 2025.
Animal and environmental isolates were screened for the presence of the tet(M) gene by PCR, and, for tet(M)-positive isolates, minimum inhibitory concentrations (MICs) were determined by Etest®. A subset of human isolates (n = 385), collected between January 2024 and January 2025, were analysed using the same methodology. As a control group, 23 tet(M)-negative isolates from all sources were included for MIC testing.
From January 2025 onwards, tetracycline MIC testing was incorporated into the national antimicrobial surveillance panel, from which additional 258 human isolates were selected. In this group, tet(M) PCR was performed on isolates with MIC ≥1 mg/L (n=37). Six isolates with a MIC < 1 mg/L were also tested for tet(M) presence, as controls.
A subset of 161 isolates, representative of the overall collection, was selected for in-depth WGS analysis.
Among tet(M)-positive isolates (n = 61), tetracycline MICs ranged from 1–32 mg/L. In contrast, the 23 tet(M)-negative control isolates showed MICs ranging from 0.023–0.094 mg/L, except for two isolates with a MIC of 4 mg/L. MIC distribution was independent of isolate source. Based on these findings, a tetracycline ECOFF of 1 mg/L was proposed to distinguish non-wild-type (resistant) from wild-type (susceptible) C. difficile isolates.
Prospective application of this ECOFF to human clinical isolates collected from January 2025 onwards (n=258) identified 37 (14.3%) as resistant (MIC range: 1–12 mg/L). Of these, 26 were tet(M)-positive by PCR. Susceptible isolates (n=221) showed MICs ranging from 0.016–0.094 mg/L. No MICs were observed between 0.094 and 1 mg/L.Among isolates analysed by WGS, 59 were tet(M)-positive, corroborating PCR screening results. Of these, 19 carried additional tet genes, including tet(40) (n=11), tet(44) (n=5) and tet(O) (n=3), which may contribute to variability in MICs among resistant strains.
Among resistant tet(M)-negative isolates (n=13), nine were included in the WGS analysis and were found to harbour tetA(P) and tetB(P), supporting their independent role in mediating tetracycline resistance. Several transposons carrying tet genes were also identified.
An ECOFF of 1 mg/L showed strong correlation with tetracycline resistance , supporting its validity for standardized susceptibility interpretation in C. difficile. This species-specific breakpoint appears more appropriate than relying exclusively on tet(M) detection, as additional genetic determinants also contribute to the resistance phenotype. Validation across human, animal, and environmental strains underscores the interconnectedness of these reservoirs and reinforces the importance of a One Health approach for surveillance of community-circulating pathogens. Collectively, these findings highlight the importance of prudent antimicrobial stewardship, as shared resistance determinants, often located in MGE, may disseminate across ecological niches and shape C. difficile epidemiology.