Identification of Clostridioides difficile mutants with increased daptomycin resistance

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• * Clostridioides difficile is a major cause of healthcare-associated diarrhea and recurrent infections.
• * Daptomycin, a lipopeptide antibiotic, targets Gram-positive bacteria by disrupting cell wall synthesis via membrane interaction.
• * Understanding C. difficile's cell envelope is crucial for developing novel therapeutic strategies.

Purpose of the Study:

• * To investigate the effects of sub-inhibitory daptomycin concentrations on C. difficile growth and morphology.
• * To identify and characterize mutations conferring daptomycin resistance in C. difficile.
• * To elucidate the mechanisms underlying daptomycin resistance and its relation to cell envelope biogenesis.

Main Methods:

• * Culturing C. difficile strain R20291 with sub-minimum inhibitory concentrations (sub-MIC) of daptomycin.
• * Phenotypic analysis of daptomycin-treated cells, including chaining, minicell formation, and lysis.
• * Selection and genetic characterization of daptomycin-resistant mutants.
• * Mapping resistance mutations to specific genes (cdsA, ftsH2, esrR, draS).

Main Results:

• * Sub-MIC daptomycin induced morphological changes in C. difficile, including cell chaining, minicell formation, and lysis.
• * Eight daptomycin-resistant mutants were isolated and their mutations mapped to four genes: cdsA, ftsH2, esrR, and draS.
• * Mutations in cdsA reduced phosphatidylglycerol levels, while mutations in ftsH2, esrR, and draS altered membrane process regulation.

Conclusions:

• * Daptomycin treatment perturbs cell membrane and peptidoglycan synthesis in C. difficile.
• * Identified mutations confer resistance through altered phospholipid synthesis or membrane regulatory pathways.
• * These findings enhance understanding of phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile, highlighting potential drug targets.