The functional vanG Cd cluster of C lostridium difficile does not confer vancomycin resistance

Summary vanG Cd , a cryptic gene cluster highly homologous to the vanG gene cluster of E nterococcus faecalis is largely spread in C lostridium difficile . Since emergence of vancomycin resistance would have dramatic clinical consequences, we have evaluated the capacity of the vanG Cd cluster to confer resistance. We showed that expression of vanG Cd is inducible by vancomycin and that VanG Cd , VanXY Cd and VanT Cd are functional, exhibiting D ‐ A la :  D ‐ S er ligase, D , D ‐dipeptidase and D ‐ S er racemase activities respectively. In other bacteria, these enzymes are sufficient to promote vancomycin resistance. Trans‐complementation of C . difficile with the vanC resistance operon of E nterococcus gallinarum faintly impacted the MIC of vancomycin, but did not promote vancomycin resistance in C . difficile . Sublethal concentration of vancomycin led to production of UDP ‐ MurNAc ‐pentapeptide[ D‐Ser ], suggesting that the vanG Cd gene cluster is able to modify the peptidoglycan precursors. Our results indicated amidation of UDP ‐ MurNAc ‐tetrapeptide, UDP ‐ MurNAc ‐pentapeptide[ D‐Ala ] and UDP ‐ MurNAc ‐pentapeptide[ D ‐ S er]. This modification is passed on the mature peptidoglycan where a muropeptide Tetra‐Tetra is amidated on the meso ‐diaminopimelic acid. Taken together, our results suggest that the vanG Cd gene cluster is functional and is prevented from promoting vancomycin resistance in C . difficile .