A reproducible GenBank-scale survey of antimicrobial resistance determinants and population structure in 30,327 Clostridioides difficile genomes

Background. Large collections of publicly available bacterial genomes can be used to examine antimicrobial resistance (AMR), but estimates from such collections may be influenced by over-representation of particular lineages and by the method used to identify resistance markers. These effects were examined in a large collection of Clostridioides difficile genomes.Methods. All Clostridioides difficile GenBank assemblies available on 20 August 2026 were identified. After exclusion of NCBI-flagged atypical or metagenome-assembled records, application of four assembly-quality thresholds adapted from the C. difficile EnteroBase framework, and BioSample deduplication, 30,327 assemblies were retained. Acquired AMR genes were screened with ABRicate against NCBI AMR and ResFinder. GyrA Thr82Ile and the causal PnimB^G promoter variant were called directly by a BLASTn-based procedure. Population structure was examined by seven-locus MLST, analysis of one representative genome from each sequence type (ST), and a maximum-likelihood phylogeny of 247 representative genomes based on UBCG core genes.Results. At least one NCBI-supported candidate acquired resistance gene was detected in 8,917/30,327 genomes (29.40%); erm(B) (18.19%) and tet(M) (16.56%) were the most frequent. Among 30,304 genomes callable for both focal variants, 2,960 (9.77%) carried both GyrA Thr82Ile and PnimB^G, 2,384 (7.87%) carried Thr82Ile without PnimB^G, 88 (0.29%) carried PnimB^G without Thr82Ile, and 24,872 (82.07%) carried neither. Of PnimB^G-positive genomes, 97.11% carried Thr82Ile, whereas only 55.39% of Thr82Ile-positive genomes carried PnimB^G. The proportion of Thr82Ile-positive genomes carrying PnimB^G was 99.26% in clade 2, 24.70% in clade 1 and 8.82% in clade 4; none was observed among the Thr82Ile-positive genomes in clades 3 or 5. Selection of one representative per ST reduced the frequencies of Thr82Ile from 17.63% to 5.98%, PnimB^G from 10.06% to 3.09%, and restricted multidrug resistance (MDR) from 10.00% to 2.90%. Thr82Ile-positive/PnimB^G-negative genomes occurred in 75 resolved STs and all five annotated MLST clades and did not form a single monophyletic group in the representative phylogeny.Conclusion. GyrA Thr82Ile and PnimB^G were strongly associated in this GenBank collection, but the association was asymmetric and differed markedly among lineages. Thr82Ile-positive/PnimB^G-negative genomes occurred in numerous sequence-type backgrounds rather than in a single sampled clone. Frequencies calculated from public genome collections should therefore be interpreted with regard to marker definition and the population structure of the genomes sampled.