Penicillin Binding Protein Substitutions Cooccur with Fluoroquinolone Resistance in Epidemic Lineages of Multidrug-Resistant Clostridioides difficile

Clostridioides difficile remains a key cause of healthcare-associated infection, with multidrug-resistant (MDR) lineages causing high-mortality (≥20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship is a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species, this is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins [PBPs]). Here, we investigated five C. difficile transpeptidases (PBP1 to PBP5) for recent substitutions, associated cephalosporin MICs, and co-occurrence with fluoroquinolone resistance. Previously published genome assemblies ( = 7,096) were obtained, representing 16 geographically widespread lineages, including healthcare-associated ST1(027). Recent amino acid substitutions were found within PBP1 ( = 50) and PBP3 ( = 48), ranging from 1 to 10 substitutions per genome. β-Lactam MICs were measured for closely related pairs of wild-type and PBP-substituted isolates separated by 20 to 273 single nucleotide polymorphisms (SNPs). Recombination-corrected phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1 to 4 doubling dilutions >wild-type, up to 1,506 μg/mL. Substitution patterns varied by lineage and clade, showed geographic structure, and occurred post-1990, coincident with the and/or substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. Their co-occurrence with fluoroquinolone resistance hinders attempts to understand the relative importance of these drugs in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. Fluoroquinolone and cephalosporin use in healthcare settings has triggered outbreaks of high-mortality, multidrug-resistant C. difficile infection. Here, we identify a mechanism associated with raised cephalosporin MICs in C. difficile comprising amino acid substitutions in two cell wall transpeptidase enzymes (penicillin binding proteins). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed t