Mechanism of Intrinsic Resistance to Vancomycin in Clostridium innocuum NCIB 10674

We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 μg/ml). Analysis by high-pressure liquid chromatography (HPLC) and mass spectrometry of peptidoglycan nucleotide precursors pools revealed the presence of two types of UDP-MurNac-pentapeptide precursors constitutively produced, an UDP-MurNAc-pentapeptide with a serine at the C terminus which represented 93% of the pool and an UDP-MurNAc-pentapeptide with an alanine at the C terminus which represented the rest of the pool. C. innocuum cell wall muropeptides containing pentapeptide[Ser], either dialanine substituted on the epsilon amino group of lysine or not, were identified and represented about 10% of the monomers while only 1% of pentapeptide[d-Ala] monomers were found. The sequence of a 2,465-bp chromosomal fragment from C. innocuum was determined and revealed the presence of ddlc. innocuum and C. innocuum racemase genes putatively encoding homologues of d-Ala:d-X ligases and amino acid racemases, respectively. Analysis of the pool of precursors of Enterococcus faecalis JH2-2, containing cloned ddlc. innocuum and C. innocuum racemase genes showed in addition to the UDP-MurNAc-pentapeptide[d-Ala], the presence of an UDP-MurNAc-pentapeptide[d-Ser] precursor. However, the expression of low-level resistance to vancomycin was observed only when both genes were cloned in E. faecalis JH2-2 together with the vanXYc gene from Enterococcus gallinarum BM4174 which encodes a d,d-peptidase which eliminates preferentially the high affinity vancomycin UDP-MurNAc-pentapeptide [d-Ala] precursors produced by the host. We conclude that resistance to vancomycin in C. innocuum NCIB 10674 was related to the presence of the two chromosomal ddlc. innocuum and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.