Identification of Clostridioides difficile Mutants with Increased Daptomycin Resistance

Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here we studied the effects of daptomycin on Clostridioides difficile, the leading cause of healthcare associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation and lysis phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. These mutants mapped to four genes: cdsA, ftsH2, esrR, and draS. Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane, or altering regulation of membrane processes. Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile.