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.

The role of the glycopeptide-inducible proteins of Enterococcus faecium D366 (39.5 kilodaltons) and Enterococcusfaecalis A256 (39 kilodaltons) in the mechanism of resistance to vancomycin and teicoplanin was examined. Crude cell walls from noninduced cells or from induced cells treated with sodium dodecyl sulfate to remove the inducible proteins were shown to bind vancomycin, in contrast to cell walls containing the cytoplasmic membrane-associated induced proteins, which did not bind vancomycin. Cytoplasmic membranes from vancomycin-induced cells did not inactivate (bind) vancomycin or teicoplanin, but they could protect the glycopeptides from being bound to the synthetic pentapeptide. This protection could be competitively abolished by D-alanyl-D-alanine. A decrease in glycopeptide binding to the pentapeptide was observed in a time-dependent fashion after treatment of the pentapeptide with the cytoplasmic membranes from induced cells. We hypothesize that the inducible proteins are responsible for glycopeptide resistance due to the binding to, and subsequent enzymatic modification of, the pentapeptide precursor of peptidoglycan, which is considered to be the natural target of glycopeptides.Recently, resistance to vancomycin has been found in clinical isolates of Enterococcus faecium and Enterococcus faecalis (5,6,13,15,16). This resistance was shown to be inducible, in most cases transferable (5, 6, 13), and sometimes plasmid mediated (5, 6). A correlation has been observed between resistance and the appearance or increased synthesis of proteins of either 39.5 (16) or 39 kilodaltons (kDa) (8, 13) which are also inducible by a variety of other glycopeptides (12,13,16). In this report, we experimentally confirm that the presence of these proteins in large quantities in the cytoplasmic membrane reduces the availability of the peptidoglycan pentapeptide which has been reported to be one of the main targets of the glycopeptides (1,3,4,9,10,14). MATERIALS AND METHODSBacterial strains and growth medium. E. faecium D366 and E. faecalis A256 are two vancomycin-resistant strains previously reported (13, 16). MICs of vancomycin and teicoplanin were, respectively, 64 and 0.5 ,ug/ml for E. faecium D366 and 256 and 16 ,ug/ml for E. faecalis A256. Cultures of these strains were grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich.).Antibiotics and bioassay. Vancomycin was provided by Eli Lilly & Co., Indianapolis, Ind., and teicoplanin was provided by Lepetit Research Center, Milan, Italy. Vancomycin and teicoplanin were quantified by a microbiological assay with Sarcina lutea ATCC 9341 as an indicator strain. For each experiment a reference curve was constructed with concentrations ranging from 0.2 to 200 ,ug/ml. Concentrations of 0.5 ,ug of each of the antibiotics per ml were reliably detected. All bioassays of glycopeptides were done in a final volume of 30 RI.Analysis of proteins from cell walls and cytoplasmic membranes. The cells from 200 ml of culture in the logarithmic phase of growth (optical density...

In this study, A. baumannii susceptibility to carbapenems showed a drastic reduction and represents a major epidemiological concern. The main carbapenem resistance mechanism is mediated by class D-OXA-type enzymes (oxa-23 and oxa-24/40) with Carbapenemase activity. Therapeutic options are exceedingly limited, relying on polymyxin combinations with other antibiotics. We are clearly missing new active agents against XDRAB.

Enterococcus faecium D399 was isolated from the blood and peritoneal abscess of a patient with intraabdominal sepsis. The patient had not been treated with vancomycin, but the strain was found to be resistant with a MIC of 1000 mg/l. Resistance was inducible and transferable (probably by conjugation) to JH2-2, and correlated with induction of synthesis of a 39 kDa protein. This mechanism appears to be identical to that previously described for E. faecalis A256, suggesting that dissemination of this form of glycopeptide resistance has already occurred. The resistance phenotype of D399, however, differed somewhat from that found in other enterococcal strains with inducible resistance.

Vancomycin-inducible proteins of 39.5 and 39 kDa from respectively, low-level and high-level resistant Enterococci were compared. Electrophoretic, immunoblot and peptide analysis revealed three types of protein, one in a low-level resistant strain of E. faecium, one in 2 high-level-resistant strains of E. faecium, and one in a high-level resistant strain of E. faecalis. The inducible proteins of E. faecium and E. faecalis, of 39.5 and 39 kDa respectively, which may function in a similar fashion (Al-Obeid et al. (1990) Antimicrob. Agents Chemother. 34, 252-256), are not related immunologically.

Mutants resistant to penicillin G were selected in a stepwise manner from nine different species of enterococci. Mutants with the highest level of resistance showed cross-resistance to all beta-lactams tested. For eight of the nine species, resistance correlated with increased production of a low molecular weight penicillin-binding protein (PBP). Two of these species produced a new PBP of low molecular weight, while two other species produced an additional PBP of high molecular weight. With the exception of Enterococcus faecium, no difference was observed in terms of lysis or bactericidal effect when the sensitive strains and their resistant mutants were tested at ten times their respective MICs of penicillin G. With E. faecium an increased lytic and bactericidal effect was observed for the resistant mutant.