Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates

Objectives: To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027. Methods: Clinical isolates and Laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanG(Cd), the vancomycin resistance operon encoding Lipid II D-a nine-D-serine that is Less bound by vancomycin than native Lipid II D-alanine-D-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis. Results: Vancomycin-resistant Laboratory mutants and clinical isolates acquired mutations to the vanSR two-component system that regulates vanG(Cd). The substitutions impaired VanSR's function, resulting in constitutive transcription of vanG(Cd). Resistance was reversed by silencing vanG, encoding D-alanine-D-serine Ligase in the vanG(Cd) operon. In resistant cells, vancomycin was Less bound to the cell wall septum, the site where vancomycin interacts with Lipid II. Vancomycin's bactericidal activity was reduced against clinical isolates and Laboratory mutants (64 and >= 1024 mg/L, respectively) compared with WT strains (4 mg/L). Truncation of the potassium transporter TrkA occurred in Laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations. Conclusions: Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanG(Cd), which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are warranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-Level resistance, which would be devastating.