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

Abstract

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 vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-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 vanGCd. The substitutions impaired VanSR’s function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding d-alanine-d-serine ligase in the vanGCd 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 vanGCd, 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.

Chromosomal Resistance to Metronidazole in Clostridioides difficile Can Be Mediated by Epistasis between Iron Homeostasis and Oxidoreductases

“…Sanger sequencing confirmed genetic changes. The clinical strains 491858 and 490054 were mapped to the genome of C. difficile R20291, and genetic variations were identified, exactly as we recently reported (59).…”

Characterization of Clostridioides difficile Isolates Available through the CDC & FDA Antibiotic Resistance Isolate Bank

“…A total of 28 isolates contained genes belonging to the vanG -like gene cluster, which is highly conserved among C. difficile strains, but their presence is not indicative of vancomycin resistance ( 13 ). Recently, investigators suggested that mutations in the regulatory genes vanS and vanR may be associated with vancomycin resistance in RT027 isolates ( 14 ). No RT027 isolates in this panel contained known mutations in vanS or vanR ; however, 3/3 RT002 and 1/3 RT014 (AR-1090) isolates contained the VanS Thr349Ile mutation described by Shen et al ( 14 ) but had a vancomycin MIC of 0.5 μg/ml ( Table 1 ).…”

“…Recently, investigators suggested that mutations in the regulatory genes vanS and vanR may be associated with vancomycin resistance in RT027 isolates ( 14 ). No RT027 isolates in this panel contained known mutations in vanS or vanR ; however, 3/3 RT002 and 1/3 RT014 (AR-1090) isolates contained the VanS Thr349Ile mutation described by Shen et al ( 14 ) but had a vancomycin MIC of 0.5 μg/ml ( Table 1 ). Five isolates with a clindamycin MIC of >16 μg/ml harbored ermB genes, which are associated with macrolide-lincosamide-streptogramin B resistance ( 15 , 16 ).…”

Ebselen Not Only Inhibits Clostridioides difficile Toxins but Displays Redox-Associated Cellular Killing

“…MBCs were determined by the agar plating method, as described previously ( 41 ). Cultures were grown to OD 600 of ∼0.2 and exposed to various concentrations of ebselen.…”