For patients with vancomycin-resistant enterococci in stool, treatment with antianaerobic antibiotics promotes high-density colonization. Limiting the use of such agents in these patients may help decrease the spread of vancomycin-resistant enterococci.

Resistance to linezolid has been associated with a G2576U mutation in domain V of the 23S rRNA. We analyzed nine clinical isolates of linezolid-resistant enterococci and showed a clear association between the number of 23S rRNA genes containing this mutation and the level of linezolid resistance expressed.Linezolid inhibits protein synthesis by interacting with the bacterial initiation complex (7). Resistance to linezolid has been selected in vitro and encountered in the clinical setting (3, 6, 9) and has been attributed to a G2576U (Escherichia coli numbering scheme) mutation in domain V of the 23S rRNA (6). The presence of multiple 23S rRNA genes in most species suggests that an increased percentage of genes with this mutation may be associated with increased levels of resistance expressed. We report an analysis of 23S rRNA genes from nine clinical Enterococcus faecium isolates and one Enterococcus faecalis isolate derived from patients during treatment with linezolid. Our results confirm previous reports regarding the importance of the G2576U mutation and suggest a direct correlation between the levels of resistance and the percentage of 23S rRNA genes possessing this mutation.E. faecium LS-1 and LR-1 were clinical isolates cultured from the same patient before and after 49 days of linezolid therapy. E. faecalis LR-2 was isolated from a fecal sample as part of a screening program for linezolid-resistant enterococci in patients undergoing linezolid therapy. Seven additional E. faecium isolates representing linezolid treatment failures were kindly provided to us by John Quinn and Paul Schreckenberger of the University of Illinois at Chicago. For ease of reference, these isolates were designated LR-3 through LR-9 (original designations of these strains were M56114, M67118, X25750, T64825, T70210, 1225, and H11553, respectively). Five of these strains were described in a previous report (3).Species determination for E. faecium LS-1 and LR-1 was performed by using an API Strep strip (bioMerieux, Inc., Hazelwood, Mo.). Species determination for E. faecium LR-3 through LR-9 was performed using the Vitek GPI card (bioMerieux, Inc.). E. faecalis LR-2 gave conflicting results with the API 20 Strep strip, and so definitive determination occurred by analysis of 16S rRNA sequence according to a previously published protocol (8). MICs were determined by broth macrodilution in brain heart infusion broth (5).Enterococcal DNA was extracted, digested with restriction enzyme MaeI (Roche Applied Sciences, Indianapolis, Ind.), transferred, and hybridized with digoxigenin-labeled probes as previously described (1). Pulsed-field gel electrophoresis of SmaI-digested genomic DNA from LS-1 and LR-1 was performed as previously described (2). 23S rRNA amplification primers were commercially synthesized (Sigma Genosys, St. Louis, Mo.) based on the E. faecium 23S rRNA gene (primer A, 5Ј-GCAGAAGGGAGCTTGACTGCGAG-3Ј; primer B, 5Ј-ACCCAGCAATGCCCTTGGCAG-3Ј) and used to amplify a 389-bp internal segment flanking the 2576 site in both E. faecali...

We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-toalanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all ␤-lactams. Affinity for penicillin generally correlated with ␤-lactam MICs for the mutants, but these associations were not strictly proportional.Ampicillin resistance in Enterococcus faecium is due to expression of the low-affinity class B penicillin-binding protein 5 (PBP 5) (15). Early studies suggested that higher levels of ampicillin resistance in Enterococcus hirae (similar to those in E. faecium) were achieved by increasing levels of PBP 5 expression (5). However, higher levels of resistance in clinical isolates are only rarely associated with increased levels of PBP 5 expression (12, 16). More commonly, mutations that are presumed to lower the affinity for ␤-lactam antibiotics have been identified within pbp5 genes of highly resistant clinical isolates (1,12,16). It has been presumed that these mutations serve to lower the affinity of the PBP 5 molecule for ␤-lactam antibiotics, yielding higher MICs as a result.The specific impacts of individual or multiple pbp5 mutations on the resistance level have been difficult to determine because most analyses have been performed with clinical isolates, in which factors other than the PBP 5 amino acid sequence may contribute to resistance. In a previous study (14) a single mutation (M485A) was introduced into a cloned pbp5, and the impact of this mutation was relatively minor. However, the plasmid used in those experiments contained only pbp5 and its promoter, and expression of resistance prior to introduction of the mutation was minimal (ampicillin MIC, 6 g/ml) (14). The role of PBP 5 in expression of ampicillin resistance in E. faecium C68, a clinical isolate resistant to high levels of ampicillin (MICs, 256 to 512 g/ml), was reported recently (11). pbp5 of C68 is located downstream of two open reading frames, designated ftsW Efm and psr. It was observed that expression of ampicillin resistance from a cloned version of the C68 pbp5 was higher when pbp5 was located downstream of ftsW Efm and psr (ampicillin MICs, 64 to 128 g/ml) than when it was cloned with only its own promoter (ampicillin MICs, 8 to 16 g/ml) (data not shown) (11). The role of the putative ftsW Efm gene product is unknown, although recent work suggests that its homologue in Escherichia coli may serve as a chaperone protei...

We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW Efm ) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW Efm , psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW Efm promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF-(pCWR558) and D344-SRF(pCWR583) were 64 g/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.Penicillin resistance in Enterococcus faecium is associated with production of low-affinity penicillin-binding protein PBP5. The presence of this penicillin-binding protein (PBP) in virtually all clinical E. faecium strains that have been investigated (including those susceptible to clinically achievable levels of penicillin [L. B. Rice, unpublished data]) suggests that it is intrinsic to this species, rather than an acquired gene. Supportive evidence for the role of PBP5 in penicillin resistance is derived from experiments indicating that PBP5-expressing cells replicate when incubated with penicillin at concentrations sufficient to saturate all of the other PBPs, as well as from studies demonstrating that E. faecium strains lacking PBP5 are highly susceptible to penicillin (10-12, 25, 26).Early studies on Enterococcus hirae 9790 (which until 1985 was considered to be a type strain for Enterococus faecalis [8,16]) reported that elevated levels of penicillin resistance (to ca. 64 g/ml) were associated with increased quantities of detectable PBP5. Increased PBP5 production in one resistant mutant (R40) was associated with deletion of the N-terminal portion and some upstream DNA of an open reading frame (ORF) located ca. 1 kb upstream of the pbp5...

In vitro linezolid resistance was selected at a higher frequency in Enterococcus faecalis JH2-2 than in recombination-deficient E. faecalis UV202. Resistance in JH2-2 was related to accumulated G2576T mutations in 23S rRNA genes, with the least resistance conferred by mutations in two of four copies. UV202 resistance was associated with a G2505A mutation present in a single copy in mutants with different MICs.