Mechanism of intrinsic resistance to vancomycin in Clostridium innocuum NCIB 10674

Abstract

We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 microg/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 ddl(c. 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 ddl(c. 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 ddl(c. innocuum) and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.

FIG. 1.

Separation of C. innocuum cell wall muropeptides by RP-HPLC.

FIG. 2.

Sequence of the ddl_{c. innocuum} and C. innocuum racemase genes. The putative ribosome binding sites are underlined. The deduced amino acid sequence of Ddl_{c. innocuum} and C. innocuum racemase are shown above the nucleotide sequence. Start of the proteins is indicated by an arrow. The EcoRI (nt 781 to 786) and the DraI (nt 1923 to 1926) sites used for inverse PCR are boxed. The conserved motif in d-Ala:d-Ser ligases and the putative pyridoxal attachment site in alanine racemases are in boldface type and are underlined.

FIG. 3.

Phylogenetic tree derived from the alignment of d-Ala:d-Lac, d-Ala:d-Ser, and selected d-Ala:d-Ala ligases. The tree was constructed by the neighbor-joining method, taking into account the results of maximum-parsimony and bootstrapping analysis. Sequences of the ligases are from Amycolatopsis orientalis (AAD19835), Bacillus subtilis 168 (CAB12263), C. acetobutylicum [Ddl] (AAK80837), C. innocuum [Ddl _{c. innocuum}] (), C. perfringens [DdlA] (BAB81021), C. perfringens [DdlB] (BAB80525), C. tetani [DdlA] (AAO34934), C. tetani [DdlB] (AAO35288), D. hafniense [Ddl] (ZP_00099215), E. coli K12 [DdlA] (NP_414915), E. coli K12 [DdlB] (NP_414634), E. casseliflavus [VanC2] (AAA60990), E. faecalis V583 [VanB] (2007289A), E. gallinarum BM4174 [VanC1] (AAA24786), E. faecalis [VanD] (AAM09849), E. faecalis BM4405 [VanE] (AAL27442), E. faecalis WCH9 [VanG] (AAF71281), E. faecalis [Ddl] (AAC43218), E. faecium BM4147 [VanA] (AAA65956), E. faecium [Ddl] (ZP_00036460), E. gallinarum BM4174 [Ddl1] (AAN62561), E. gallinarum BM4174 [Ddl2] (AAK97387), E. hirae [Ddl] (Q47827), Lactococcus lactis (AAK04439), Leuconostoc mesenteroides [Ddl] (Q48745), Listeria monocytogenes (CAC98933), M. tuberculosis [Ddl] (CAB05431), P. popillae (AAF36803), Salmonella enterica serovar Typhi [DdlA] (AA070072), S. aureus [Ddl] (BAB43170), S. agalactiae [Ddl] (AAM99654), S. pneumoniae [Ddl] (CAB64467), Streptomyces coelicolor [Ddl] (NP_627790), and Streptomyces toyocaensis [Ddl] (AAC23582).