A strain of Enterococcusfaecalis (A256) was isolated from the urine of a patient with urinary sepsis and was found to exhibit susceptibilities (micrograms per milliliter) to various glycopeptides as follows: vancomycin, 256; teicoplanin, 16; 62208, 512; 62211, 4; and 62476, 16. As judged by growth rates before and after exposure to sub-MICs of glycopeptides, vancomycin and 62476 induced self-resistance, 62208 and 62211 induced slight self-resistance, and teicoplanin did not induce self-resistance. Vancomycin induced cross-resistance to all other glycopeptides tested, as judged both in growth experiments and by direct measurement of inhibition of peptidoglycan synthesis in cells exposed to sub-MICs of vancomycin. Thus, the spectra of activity of the glycopeptides were not correlated with their patterns of induction. There was a correlation between the increased synthesis of a 39-kilodalton (kDa) protein located in the cytoplasmic membrane and the induction of resistance. Protoplasts of A256 were susceptible to inhibition of peptidoglycan synthesis by vancomycin at levels similar to those for susceptible strains. Vancomycin resistance was transferable on filters from the parent strain to E. faecalis JH2-2 at a frequency of about 10-7, and the 39-kDa protein was also inducible by glycopeptides in these transconjugants. We conclude that A256 is resistant to glycopeptides by virtue of the synthesis of a 39-kDa cytoplasmic membrane protein, that this protein is probably involved in preventing access of the glycopeptides to their peptidoglycan targets, and that this resistance is transferable, probably by conjugation.Enterococcus faecalis and E. faecium are common causes of infection in humans (6, 19). These infections can be difficult to treat because of the degree of antibiotic resistance normally conferred to these strains by chromosomal or plasmid-borne determinants (12,16). This appears to be especially true for E. faecium, which tends to be more resistant to aminoglycoside-beta-lactam synergy than E. faecalis (16). For strains with high levels of beta-lactam resistance or when beta-lactams are poorly tolerated by the patient, glycopeptide antibiotics, mainly vancomycin, have been used in treatment with favorable results. A new glycopeptide, teicoplanin, is in clinical trials in the United States and Europe (14). Recently, reports of glycopeptide-resistant enterococcal strains have appeared in the literature (11,13,22). Two such strains of E. faecium with transferable, plasmid-mediated, glycopeptide resistance have been described (13). In the latter study, for Streptococcuis sanquis strains carrying the plasmids but not for the enterococcal parent strains, glycopeptide MICs were shown to be increased after preexposure to low concentrations of glycopeptides, suggesting inducible resistance. We here present data regarding the mechanism of resistance in a glycopeptide-resistant strain of E. faecalis, A256. MATERIALS AND METHODSBacterial strains. A256 is an E. faecalis strain isolated from the urine of a patient wit...

Strain D366, a clinical isolate of Enterococcus faecium, is resistant (minimum inhibitory concentration [MIC] 32 mg/L) to vancomycin. When exponential-phase cultures were exposed to half the MIC of vancomycin, a lag of 3-4 h occurred before growth resumed. Cells preexposed to 1/2 MICs of vancomycin did not show any lag. Pregrowth of D366 with vancomycin caused resistance to all glycopeptides tested. Pregrowth in vancomycin resulted in synthesis of a 3.95-kDa cytoplasmic-membrane-associated protein. This protein was correlated with resistance in mutants with high-level resistance, in the presence of NaCl, which inhibited the activity of vancomycin, and when several glycopeptides with varying activities were tested. Vancomycin-grown cells appeared abnormal and lysed at a much slower rate than did normal cells. We conclude that (1) vancomycin resistance in D366 is inducible; (2) resistance is correlated with the synthesis of 39.5-kDa cytoplasmic membrane protein; and (3) this protein play an additional role in the inhibition of normal lytic functions.

Vancomycin was found to coinduce DD-carboxypeptidase activity, together with resistance, in eight low-or high-level glycopeptide-resistant strains of enterococci. The constitutively resistant mutant (MT10) of a low-level-resistant strain of Enterococcus faecium (D366) spontaneously expressed a level of carboxypeptidase similar to that of the induced strain D366. Pentapeptide, UDP-MurNac-pentapeptide, as well as D-alanyl-Dalanine were in vitro substrates for the carboxypeptidase which was not inhibited by penicillin. The level of vancomycin resistance correlated roughly with the level of carboxypeptidase activity. We infer from these results that the carboxypeptidase is one component of the glycopeptide resistance mechanism.