Resistance to Glycopeptides in Enterococci

545 STATE-OF-THE-ART CLINICAL ARTICLE Resistance to Glycopeptides in Enterococci Roland Leclercq and Patrice Courvalin The development of resistance to glycopeptides (vancomycin and teicoplanin) is one of the best examples of the alternation between success and failure that spans the history of the antibiotic era. The introduction of penicillin G was revolutionary. However, the emergence in the 1950s of clinical isolates of Staphylococcus aureus that were resistant to penicillins and subsequently to erythromycin and tetracyclines stimulated the development of large-scale screening programs for new drugs. Under these circumstances, an American missionary, Reverend W. M. Bouw, provided Eli Lilly (Indianapolis) with a soil sample from Borneo [1]. The organism that produces vancomycin, Amycolatopsis orientalis, was isolated from this sample in 1954. Since vancomycin proved to be potent against grampositive bacteria, including penicillinase-producing staphylococci, it was licensed for human use in record time. However, the renal toxicity and ototoxicity associated with vancomycin therapy (due for the most part to impurities in the drug) together with the introduction of new penicillinase-resistant f3-lactams led to a rapid decline in the use of vancomycin. In the 1980s, interest in vancomycin revived because of several factors. During this period, methicillin-resistant and multiresistant staphylococci, against which vancomycin is often the only active antibiotic, were emerging worldwide. At the same time, medical advances had resulted in the prolongation of life for increasingly fragile, immunocompromised patients who are exposed to opportunistic pathogens such as multiresistant gram-positive organisms. In addition, it was determined that pseudomembranous colitis could be treated with oral vancomycin, and a new glycopeptide, teicoplanin, was introduced in 1984 in certain European countries. The apparent absence of resistance to vancomycin in grampositive bacteria and a decrease in the toxicity associated with vancomycin therapy (purified formulations of the drug had become available) led to a notable increase in the prescription of this antibiotic. In the United States, a 20-fold increase in the consumption of vancomycin was observed over a 10-year period at a university hospital [2]. Received 27 November 1996; revised 12 December 1996. Reprints or correspondence: Dr. Roland Leclercq, HOpital Henri Mondor, Service de Bactêriologie-Virologie, 51 Avenue de Lattre de Tassigny, 94010 Creteil cedex, France. Clinical Infectious Diseases 1997; 24:545-56 1997 by The University of Chicago. All rights reserved. 1058-4838/97/2404-0001$02.00 From the Service de Bacteriologie-Virologie, Hdpital Henri Mondor- Universite Paris XII, Creteil; and Unite des Agents Antibacteriens, Institut Pasteur, Paris, France The emergence in 1986 of transferable glycopeptide resistance in enterococci [3, 4] was unexpected and had several consequences: (1) glycopeptide resistance in enterococci that were already resistant to other antibiotics resulted in severe clinical problems in certain countries; (2) possible dissemination of resistance to other pathogens, such as methicillin-resistant S. aureus, became a concern; (3) it became difficult to control resistant infections because the multiple mechanisms by which resistance is disseminated in the community and the hospital had not (and still have not) been fully elucidated; and (4) isolation of glycopeptide-resistant enterococci from humans and animals in European communities suggested that there were dangers associated with the use of antibiotics (including a glycopeptide, avoparcin) for nonmedical purposes such as additives in animal feed. Finally, the biochemical mechanism and regulation of expression of acquired resistance to glycopeptides are one of the most sophisticated, perfect examples of the genetic adaptation of bacteria. Mechanism of Acquired Resistance to Glycopeptides Glycopeptides are inhibitors of cell wall synthesis. In contrast to /3-lactams, glycopeptides do not interact with cell wall biosynthetic enzymes. These drugs are large, rigid, hydrophobic molecules that form complexes with the peptidyl-d-alanyl- D-alanine termini of peptidoglycan precursors at the cell surface. The presence of these large complexes at the surface of the cytoplasmic membrane prevents cell wall synthesis by hindering the transfer of cytoplasmic precursors to the growing peptidoglycan chain and by blocking formation of interpeptidic bonds (figure 1) [5]. Resistance to glycopeptides is due to synthesis of modified precursors that display decreased affinity for vancomycin and teicoplanin (figure 1). This modification, which implies alteration of the peptidoglycan biosynthetic pathway, can result only from bacterial acquisition of a gene cluster encoding a coherent enzymatic machinery. VANA resistance is the type of resistance that has been most thoroughly studied [6]. In Enterococcus faecium strain BM4147, the resistance genes are carried by the 10.8-kb transposable element Tn1546 [7]. The transposon Tn1546 encodes seven polypeptides that act cooperatively to confer high-level vancomycin resistance [7]. Two of these polypeptides (VanR and VanS) are involved in the regulation of resistance-gene expression, three (VanH, VanA,

546 Leclercq and Courvalin CID 1997;24 (April) Figure 1. Schematic representation of peptidoglycan synthesis in glycopeptide-susceptible (left) or glycopeptide-resistant (left and right) Enterococcus species. S, D-alanine; MI, D-lactate; left: the chromosomal bacterial ligase synthesizes the dipeptide D-alanyl-D-alanine (D-Ala-D- Ala), which is then branched to a tripeptidic precursor that is translocated at the cell surface. Vancomycin (V) binds to the D-Ala-D-Ala terminus of the pentapeptide chain and prevents subsequent steps in synthesis of the peptidoglycan. Right: the VanH dehydrogenase of a glycopeptide-resistant enterococcus synthesizes D-lactate from pyruvate. The broad-substrate-range VanA ligase catalyzes the formation of the depsipeptide D-Ala-D-Lac, which is then branched to UDP-N-- acetyl muramyl tripeptide to form a pentadepsipeptide precursor. Vancomycin has a greatly reduced affinity for the precursors ending in D- Ala-D-Lac. As a consequence, enterococci can grow in the presence of glycopeptides. VanX and VanY are carboxypeptidases that prevent synthesis of the D-Ala-D-Ala dipeptide and the pentapeptide ending in D-Ala-D-Ala, respectively. and VanX) confer resistance to glycopeptides, and two (VanY and VanZ) are accessory proteins that are not essential for the expression of glycopeptide resistance. Coordinated expression of VanH, VanA, and VanX is necessary to confer glycopeptide resistance. Characterization of VanA provided the first clue as to the mechanism of resistance. VanA is homologous to bacterial ligases, which are chromosomally encoded enzymes that are involved in an early stage of the synthesis of the cell wall precursors. They synthesize a dipeptide D-alanyl-D-alanine (D- Ala-D-Ala), which is incorporated at the end of late peptidoglycan precursors that are then exported as monomers on the surface of the bacteria. VanA shares similar functions with the bacterial ligases but possesses a wider substrate specificity. It catalyzes the formation of ester bonds between D-alanine and D-lactate (D-Lac) to produce the depsipeptide D-Ala-D-Lac preferentially to the usual dipeptide D-Ala-D-Ala [8]. This change in product results in markedly reduced affinity between vancomycin and its target because of the loss of hydrogen bond critical for binding. However, VanA alone cannot confer resistance to vancomycin, probably because D-hydroxyacids such as D-Lac are neither natural products present in the environment of enterococci nor normally produced by enterococci. Thus, to synthesize D-Lac, enterococci must acquire the gene(s) within the vana operon required to produce the substrate for VanA. Structural and functional analysis of VanH revealed that it was a dehydrogenase that functioned in close cooperation with VanA, since VanH could reduce pyruvate in D-Lac, the substrate for VanA [9]. Cross-linking of the precursors to the growing peptidoglycan is processed in bacteria by the penicillin-binding proteins (PBPs), namely PBP5 in E. faecium [10]. The substitution of D-Ala by D-Lac does not impair cross-linking of the modified precursors to the growing peptidoglycan chain. However, PBPs other than PBP5, which are so far not known to have a role in cell wall synthesis, are probably required for processing of the altered precursors [11]. These high-molecular-weight PBPs display a higher affinity for /3-lactams. Since VANA resistance is inducible, the shift in PBPs occurs only in the presence of vancomycin and results in 13-lactam hypersusceptibility. This effect explains the synergy displayed by the combination of the two classes of drugs against vancomycin-resistant strains. The possible therapeutic implications of this synergy will be discussed below. On the basis of the mechanism detailed above, it would appear that production of VanH and VanA is sufficient to confer glycopeptide resistance; however, this is not the case because, as already mentioned, the additional VanX protein is required. In fact, the chromosomal enzymes that produce the usual susceptible cell wall precursors are still functional in the vancomycin-resistant enterococci, which should be considered as merodiploids for cell wall synthesis. Depsipeptides and dipeptides are coproduced and compete for the synthesis of modified and unmodified precursors.

CID 1997;24 (April) Enterococcal Resistance to Glycopeptides 547 Table 1. Phenotypes of glycopeptide -resistant enterococci. MIC (mg/l) Phenotype, species Vancomycin Teicoplanin Transferable resistance Acquired resistance VANA* E. faecium E. faecalis E. avium E. durans E. hirae 64 >1,000 16-512 Yes E. mundtii E. raffinosus E. gallinarum E. casseliflavus VANBt E. faecium E. faecalis 4-1,024 0.25 2 Yes VAND E. faecium E. faecalis 16-64 2-4 NT Intrinsic resistance VANC E. gallinarum E. casseliflavus 2-32 0.12-2 No E. flavescens NOTE. NT = not tested. * Also detected in Arcanobacterium haemolyticum and Cellulomonas turbata. t Also detected in Streptococcus bovis. The levels of vancomycin resistance depend on the respective proportions of each type of precursor [12]. Resistance will be phenotypically expressed only if the levels of precursors susceptible to vancomycin are sufficiently low. VanX is a D,D-dipeptidase that hydrolyzes D-Ala-D-Ala but not D-Ala-D- Lac and thus prevents synthesis of precursors ending in D- alanine [13]. The accessory protein VanY probably acts as a complementary lock: VanY is a D,D-carboxypeptidase that could contribute to resistance by cleaving the terminal D-Ala of late peptidoglycan precursors resulting from incorporation of D-Ala-D-Ala that escaped hydrolysis by VanX [14]. Finally, VanZ confers low-level resistance to teicoplanin by an unknown mechanism [15]. Diversity of Phenotypes and Genotypes for Acquired and Intrinsic Vancomycin Resistance Acquired enterococcal resistance to glycopeptides was first detected in 1986 [3, 4]. Three phenotypes VANA, VANB, and VAND can be distinguished on the basis of the antibacterial and resistance-inducing activity of vancomycin and teicoplanin (table 1). The VANA phenotype is characterized by inducible resistance to high levels of vancomycin (MIC, mg/l) and teicoplanin (MIC, mg/l) [3, 6]. This class of resistance is usually mediated by self-transferable plasmids that bear transposons that are structurally closely related to Tn1546. The vana genes were recently found on a mobile element, Tn5482 (composed of Tn1546 and insertion sequences 1S1251), that could direct its own transfer from the chromosome of one Enterococcus strain to that of another strain [16]. VANB strains are inducibly resistant to various amounts of vancomycin (MICs range from 4 mg/l to 1,000 mg/l), but they remain susceptible to teicoplanin [17, 18] because vancomycin acts as an inducer whereas teicoplanin does not. Lowlevel and high-level VANB resistance is transferable by conjugation in certain strains [18, 19]. Transfer of resistance is associated with transfer of large (i.e., from 90 kb to 250 kb) genetic elements from chromosome to chromosome [18]. The 250-kb element has recently been characterized [20]; it contains the composite transposon Tn1547, which is flanked by insertion sequences related to 1S256. VAND is a recently described phenotype [21] characterized by resistance to moderate levels of vancomycin and lowlevel resistance or susceptibility to teicoplanin. The vana and vanb clusters encode related dehydrogenases (VanH and VanH B), ligases (VanA and VanB), and D,D-dipeptidases (VanX and VanX B), which is consistent with the fact that they confer resistance to glycopeptides by means of similar mechanisms [22]. As discussed above, the resistance genotype can be inferred from the phenotype characterized for vancomycin and teicoplanin. However, two unusual modes of expression of the VANA and VANB types of resistance deserve further description. First, clinical isolates of vancomycin-dependent VANB E. faecium have been reported [23-25]. These organisms require vancomycin for growth. It has been hypothesized that the chromosomal D-Ala-D-Ala ligase is not produced or is impaired in these strains and that expression of the VanB D-Ala- D-Lac ligase that can be induced by vancomycin allows for cell wall synthesis and survival of the strains in the presence of vancomycin [26, 27]. The exact incidence of vancomycin-dependent strains is unknown; however, in a rabbit model of aortic endocarditis due to a VANB strain, vancomycin-dependent mutants could easily be selected by treatment with vancomycin or teicoplanin [28]. VANA-type dependent mutants could also be obtained in vitro [26, 27]. The importance of vancomycin-dependent strains is due to the fact that they can be missed when a standard agar medium is used to isolate them. In addition, a clinical isolate of VANB E. faecium that expressed cross-resistance to teicoplanin and vancomycin has been reported [29]. The strain was a constitutive mutant selected by vancomycin treatment in a patient infected with a VANB-inducible strain [29]. Similar VANB mutants can be selected by administration of teicoplanin in an animal model [28]. Strains belonging to Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens are intrinsically re-

548 Leclercq and Courvalin CID 1997; 24 (April sistant to vancomycin (MICs, 4-32 mg/l) but remain susceptible to teicoplanin; these organisms display the VANC phenotype (table 1). E. gallinarum and E. casseliflavus- E. flavescens (these organisms are thought to belong to the same species) contain the vanc1 and the vanc2 gene, respectively, which participates in the synthesis of peptidoglycan precursors ending in D-Ala-D-serine displaying reduced affinity for vancomycin [22, 30, 31]. Neither these bacteria nor other gram-positive organisms that are intrinsically resistant to glycopeptides (i.e., Lactobacillus species, Pediococcus species, and Leuconostoc species) appear to be the source of the genes encoding acquired resistance in enterococci [32]. Dissemination of Glycopeptide Resistance: Facts and Thoughts Resistance to glycopeptides has spread primarily in E. faecium strains that are already resistant to a broad range of antimicrobial agents. Strains of E. faecium that are resistant to every antibiotic that can be used for efficient treatment of systemic infection (i.e., aminoglycosides, penicillins, and glycopeptides) have been described [33]. However, glycopeptide resistance is not confined to a single species, and the vana gene cluster has been detected in a variety of enterococcal species (table 1). A high incidence of glycopeptide resistance (mainly VANA resistance in E. faecium) is observed in certain countries and certain hospitals. For example, a 20-fold increase in the incidence of such resistance in the United States from 1989 (0.4%) to 1995 (10%) was reported by the Centers for Disease Control and Prevention [34]. Although vancomycin resistance was first reported in the United Kingdom and France, the incidence remains low in Europe generally <2% [35, 36]. On the other hand, epidemics have been reported in certain hospitals, particularly in the United Kingdom [37]. The transfer of vancomycin resistance from enterococci to other gram-positive bacteria was predictable because there is no barrier against expression of the resistance genes in various gram-positive organisms including Streptococcus species, Listeria monocytogenes, and, of most importance, S. aureus [38, 39]. Corynebacterium-related bacteria (Arcanobacterium haemolyticum and Cellulomonas turbata) harboring vana-related genes were recently isolated from the stools of some patients in the United Kingdom, and a strain of Streptococcus bovis harboring a vanb-related gene cluster was isolated from a patient in France [40, 41]; these findings represent an in vivo demonstration of this transferability. Conjugative transfer of glycopeptide resistance from E. faecalis to S. aureus has been reported only under laboratory conditions; this transfer has been observed both in vitro and in vivo on the skin of mice [39]. Risk Factors for Colonization or Infection with Glycopeptide-Resistant Enterococci The risk factors for colonization or infection by glycopeptide-resistant enterococci have been investigated in numerous studies. There are often multiple factors involved; the role of a predominant factor is difficult to elucidate. Studies based or multivariate analysis are most suited to answer this question, but samples of a sufficient size are required. The results of several case-control studies have been published that establish the typical profile of a patient at risk for colonization or infection with glycopeptide-resistant enterococci (table 2). Such patients have received previous antibiotic treatment, mainly with third-generation cephalosporins (or other /3-lactams); have severe underlying conditions (e.g., they have undergone liver or bone-marrow transplantation or have renal failure, cancer, diabetes, or decubitus ulcers); are hospitalized in a renal unit, oncology (including hematology) unit, intensive care unit, or surgical unit; have been hospitalized for prolonged periods; have been treated with multiple antibiotics including vancomycin and/or cephalosporins; and have undergone invasive procedures. Most of the reported risk factors for colonization or infection are nonspecific, since they are the same for colonization or infection with vancomycin-susceptible enterococci [48]. Data from case-control studies in which nosocomial glycopeptideresistant enterococci and glycopeptide-susceptible enterococci have been isolated from patients are shown in table 2. These studies again showed that treatment with vancomycin or thirdgeneration cephalosporins as well as length of hospital stay were independent risk factors. In one study, administration of metronidazole was a significant risk factor for the development of bacteremia due to glycopeptide-resistant enterococci [43]. The authors of this study hypothesized that the elimination of anaerobes from the gut might confer an advantage to enterococci in terms of colonization that subsequently leads to bacteremia. Administration of other antibiotics, such as moxalactam, clindamycin, or imipenem, with activity against anaerobes has also been found to be a risk factor for colonization or infection due to enterococci or ampicillin-resistant enterococci [49-51]. However, these antibiotics have poor activity against enterococci and could also directly select for these microorganisms. In two studies, prior vancomycin therapy was associated with glycopeptideresistant enterococcal bacteremia [47, 52]. Clinical Experience The mortality associated with bloodstream infections due to enterococci, regardless of vancomycin resistance, is generally high from 34% to 46%; however, enterococci are paradoxically considered to be weak pathogens [53]. This high mortality is usually attributed to the underlying conditions of the patients, which predispose to the development of enterococcal bacteremia. The clinical outcome for patients infected with glycopeptide-resistant enterococci is similar overall, with mortality rates of >50%, although these rates have varied from 8% to 73% in recent studies [43, 45-47, 54-57].

CID 1997; 24 (April) Enterococcal Resistance to Glycopeptides 549 Table 2. Summary of data from case -control studies of patients infected with glycopeptide -resistant enterococci. Reference Methodology Ward Source of glycopeptideresistant enterococci (no. of cases) Glycopeptide resistance phenotype (no. of strains) Risk factors [42] Univariate analysis Medical-surgical/ ICU Blood (4), urine (2), stools (3) VANA Enterococcus faecium (9) [43] Univariate analysis Oncology Blood (11) VANA E. faecium (11) [44] Univariate analysis Various* Various (colonization + infection) (41) VANA E. faecium (6) VANB E. faecium (35) Duration of ceftazidime treatment, no. of days in ICU (no isolation) Intestinal colonization with glycopeptide-resistant enterococci, use of antibiotics active against anaerobes Previous exposure to antibiotics, use of third-generation cephalosporins, use of parenteral vancomycin [45] Multivariate analysis Varioust Various (20) NA Use of multiple antibiotics (ciprofloxacin, aztreonam, vancomycin), severity of illness [46] Multivariate analysis Various (mainly oncologyhematology, AIDS, surgical, ICU) [37] Multivariate analysis Hepatology (included transplant recipients) [47] Multivariate analysis Liver transplantation NOTE. ICU = intensive care unit; NA = data not available. * Seven hospitals, including both primary and tertiary care centers. One hospital. Based on APACHE II score. Blood (46) E. faecium (40, including 38 VANA), Enterococcus faecalis (2) Various (colonization + infection) (38) NA Hematologic malignancy, use of vancomycin, severity of illnessi Length of hospital stay Blood (54) VANA (54) Length of hospital stay Glycopeptide-resistant enterococci seem to more often cause life-threatening diseases than do their glycopeptide-susceptible counterparts [43, 54], although one study did not show any difference [57]. For instance, Linden et al. [47] conducted a casecontrol study that showed that vancomycin resistance played a critical role in the recurrence of bacteremia and in the associated mortality; shock and liver failure were other cofactors. Virulence factors specific to glycopeptide-resistant enterococci have not been identified so far. In fact, the high mortality rates could be due to the fact that the patients who have developed bloodstream infections with such strains have been severely ill and therefore have been at higher risk of dying. Indeed, a careful study of mortality due to enterococcal bacteremia showed that if glycopeptide-resistant enterococci are more likely to cause death (15 of 46 patients infected with resistant strains died) than are glycopeptide-susceptible enterococci (3 of 46 patients infected with susceptible strains died), the difference was no longer significant after controlling for severity of illness and gender [46]. Another study showed that immunocompromised patients are more likely to have bloodstream infections than are immunocompetent patients [55]. The lack of uniformly effective therapy for patients infected with glycopeptide-resistant enterococci is also an important consideration. Although the results with respect to the role of appropriate therapy in reducing the mortality associated with enterococcal infections have conflicted, Hoge et al. [58] showed that if the clinical significance of bacteremia is strictly defined, specific therapy is associated with improved outcome. Therapeutic Options Penicillin G, ampicillin, or amoxicillin combined with an aminoglycoside remains the therapy of choice for infections due to glycopeptide-resistant enterococci. For E. faecium infections, only gentamicin (or streptomycin) should be used in combination with a /3-lactam, since the remaining aminoglycosides are inactivated by a species-specific chromosomal acetylating enzyme (AAC(6')-I) and do not display synergism with 0-lactams [59, 60]. However, most glycopeptide-resistant E. faecium express resistance to one or both classes of antibiotics. As mentioned above, combinations of vancomycin and various /3-lactams have been shown in vitro to have bacteriostatic synergy but not bactericidal synergy against glycopeptide-resistant enterococci [61, 62]. However, mutants resistant to these

550 Leclercq and Courvalin CID 1997;24 (April', synergistic combinations can readily be observed both in vitro and in animal models [62, 63], and synergism is not observed for strains that are highly resistant to penicillins [64]. Finally, the clinical effectiveness of combined therapy is not known. In one study, the addition of gentamicin to the 0- lactam vancomycin combination prevented the emergence of mutant strains in animals, provided that the strain was not highly resistant to the aminoglycoside [63]. The combination of ampicillin and imipenem appeared efficient in an experimental model of endocarditis in rabbits [65]. Chloramphenicol, fluoroquinolones, tetracycline, and rifampin have been used against susceptible enterococci, but these antibiotics are not bactericidal [56, 66]. A limited clinical study showed that chloramphenicol was only partially efficient [56]. In an experimental model of endocarditis in rats, a 5-day course of treatment with ciprofloxacin/rifampin/gentamicin was efficient, although selection of rifampin-resistant mutants occurred in all animals [67]. Novobiocin and bacitracin have been used in attempts to suppress fecal carriage of glycopeptide-resistant enterococci, but these drugs have had limited effects [68, 69]. Quinupristin/ dalfopristin is an injectable streptogramin that has been used in a compassionate protocol and is under investigation [70]. Several other antimicrobial agents are at an early stage of development, but their usefulness has not yet been proven. Prevention: the Reservoirs and Routes of Dissemination Since there is no fully efficient therapy for infections due to glycopeptide-resistant enterococci, efforts to limit the spread of these microorganisms are now considered essential. Prevention is first based on the knowledge of the reservoirs and the routes of dissemination of glycopeptide-resistant enterococci. Hospital-associated infections due to glycopeptide-resistant enterococci can derive endogenously from the patient's own flora, or such infections can be exogenously acquired via the spread of an epidemic strain, or both. Several European studies have demonstrated that glycopeptide-resistant enterococci are part of the normal human fecal flora. These bacteria have been found outside hospitals, in waste waters [71-73], and in the feces of nonhospitalized patients [71, 74, 75] including healthy volunteers who were not health care workers, had never received glycopeptides, and had not been treated with antibiotics during the year preceding the study [76]. Incidences of 1.3% and 28% (Belgium) [75, 76], 2% (France) [77], and 12% (Germany) [74] have been reported for community-acquired infections, which contrast with the low incidence of infections in hospitals (see above). The wide variations in the incidences could be explained in part by differences in the sensitivity of the stool culture methods that were used. A possible source of glycopeptide-resistant enterococci is the food chain, since VANA E. faecium has been isolated from farm animals in the United Kingdom [72], Germany [78], and Denmark [79] and from animal-derived food products, including frozen poultry [72, 78]. German and Danish investigators have raised the possibility that the glycopeptide avoparcin, used for nearly 20 years as a feed additive in animal husbandry in numerous European countries (but not the United States or Canada), could have selected for glycopeptide-resistant enterococci in the digestive tracts of animals. It has been observed that the VANA strains are cross-resistant to vancomycin, teicoplanin, and avoparcin, a finding consistent with this possibility. VANA E. faecium has been isolated from pigs and chickens grown on German and Danish farms where avoparcin is used, whereas the enterococci isolated from the feces of animals raised on farms that do not use avoparcin are susceptible to vancomycin [74, 78, 79]. The enterococci isolated from animals raised on the Danish farms contained clusters of vana gene closely related to that found in enterococci isolated from humans [80]. However, other growth promoters among the numerous antibiotics used might have selected glycopeptideresistant enterococci because these organisms were resistant to several antibiotics (i.e., macrolides, tetracyclines, and aminoglycosides). In the United States, attention has focused on the epidemiology of glycopeptide-resistant enterococci, mainly in hospitals rather than in the community, since glycopeptide-resistant enterococci have most often been responsible for outbreaks of infection in the intensive care units of large hospitals. In a study in Texas, investigators failed to find any glycopeptideresistant enterococci in the feces or carcasses of chickens [81]. In addition, glycopeptide-resistant enterococci could not be isolated from healthy volunteers from the community in two studies [45, 81]. In hospitals in the United States, most outbreaks of glycopeptide-resistant enterococci are due to intrahospital or interhospital spread of clonal strains [52, 82], although unrelated bacteria can also be isolated [45, 82]. This circumstance suggests that patient-to-patient transmission is the major factor responsible for dissemination of glycopeptide-resistant enterococci in the United States. The reasons for the differences between the European and North American rates of resistance are still unknown. Hypothetically, these differences could be related to differences in the use of glycopeptides, resulting in distinct antibiotic selective pressures specific to individual countries. For example, in Europe, oral administration of avoparcin could have favored intestinal carriage of glycopeptide-resistant enterococci outside hospitals, initially in animals and then in humans, via the food chain, whereas in North America, heavy use of both intravenous and oral vancomycin in hospitals could have led to selection of resistant enterococci primarily in clinical settings. In any case, the intestinal reservoir is of major importance, and isolation of glycopeptide-resistant enterococci from clinical samples correlates with carrier status [57]. Glycopeptide-resistant enterococci have been isolated from the hands of health care workers [46]. However, the role of environmental contamination in the transmission of these

CID 1997;24 (April) Enterococcal Resistance to Glycopeptides 551 organisms has not been fully elucidated and has probably been underestimated so far. The results of surveillance screening during outbreaks have indicated that glycopeptide-resistant enterococci can be isolated from the inanimate environment in the wards where colonized or infected patients are located. Air cushions, automated medication dispensers, bed rails, blood pressure cuffs, control panels of pulse oximeters, doors, electrocardiographic monitors, electronic thermometers, glucose meters, headboards, linens, monitoring devices, overbed tables, stethoscopes, toilet surfaces, and ventilator tubing were found to be contaminated during recent outbreaks [37, 42, 43, 45, 46, 83]. Enterococci can survive for prolonged periods, both on the hands of health care workers and on environmental surfaces [84]. Prevention and Control of Infections Due to Glycopeptide-Resistant Enterococci Of the risk factors for acquisition of colonization or infection by glycopeptide-resistant enterococci, antibiotic consumption and duration of treatment are amenable to intervention. The administration of vancomycin is frequently reported as a risk factor [44-46, 84, 85]. It is noteworthy that when oral vancomycin or teicoplanin was administered to 22 healthy Belgian volunteers, selection of large numbers of glycopeptide-resistant enterococci in the fecal flora of 14 of these volunteers was observed [76]. Similar selection of high counts of glycopeptideresistant enterococci was reported when oral vancomycin was administered in an animal model [86]. Therefore, recommendations have been formulated by the Hospital Infection Control Practices Advisory Committee (HICPAC) that discourage the use of oral or parenteral vancomycin in many situations for instance, for routine prophylaxis, for primary treatment of antibiotic-associated colitis, and for selective decontamination of the digestive tract (for details see [87]). Oral metronidazole can be used in place of vancomycin to treat colitis due to Clostridium difficile. The impact of reducing the consumption of glycopeptides remains to be evaluated. Morris et al. [45] reported that in an attempt to control the spread of glycopeptide-resistant enterococci, reducing the consumption of parenteral and oral vancomycin by 59% and 85%, respectively, in combination with hygienic measures, did not result in changes in the rates of stool colonization and infection due to these strains. However, it must be stressed that at least no increase in the rates was observed and that the control study was done shortly after the implementation of an infection control program and thus should be repeated later. Clinicians should also remember that the use of third-generation cephalosporins is a common risk factor for infection due to glycopeptide-resistant enterococci and that prolonged therapy with these antibiotics should probably be avoided when possible. Microbiology laboratories play a crucial role in the detection and timely reporting of colonization or infection due to vancomycin-resistant enterococci. It is particularly important to detect the initial isolates before colonization becomes endemic and then difficult to control. For early detection, laboratories should use reliable conventional broth and agar methods rather than automated systems, which perform poorly in detecting glycopeptide-resistant enterococci [88]. In addition, a multiplex PCR technique has been proposed for rapid detection and characterization of glycopeptide resistance genes [89]. If a patient is found to be infected or colonized with multiresistant enterococci, fecal samples should be obtained to determine colonization status. If glycopeptide-resistant enterococci are detected, the next step is to limit the nosocomial spread of these organisms by instituting conventional rigorous infectioncontrol measures. These nonspecific measures include the use of barrier precautions and isolation or grouping of the infected or colonized patients. The barrier precautions should be strict and should include the use of gloves, masks, and gowns by persons who have contact with patients who are colonized or infected with glycopeptide-resistant enterococci. The importance of handwashing with an antiseptic (chorhexidine) soap after patient contact must be stressed. The optimal duration of isolation for carriers of glycopeptide-resistant enterococci has not been established. Asymptomatic carriage can last for several months [90], and glycopeptide resistant enterococci can be present in barely detectable numbers in the feces and can thus be missed by screening procedures [76]. The above measures have proved to be efficacious in controlling outbreaks of glycopeptide-resistant enterococci [91]. However, the relative efficiency of each measure remains to be evaluated. In one report, an outbreak could be controlled only when gowns were used in addition to gloves and the patients were placed in isolation [83]. In contrast, another study showed that the use of gowns in addition to gloves was no better than the use of gloves alone for controlling outbreaks of glycopeptide-resistant enterococci [92]. Barrier precautions alone might not be sufficient if multiple modes of reintroducing the organisms into hospitals (i.e., via the food chain or via the admission of healthy carriers from the community) exist. The usefulness of stool culture surveys remains to be studied. The HICPAC recommended that periodic culture surveys be performed for critically ill patients in hospitals where such patients are numerous [87]. However, Wells et al. [57] showed that during one outbreak, stool cultures were negative for nearly 50% of patients who acquired glycopeptide-resistant enterococci. Conclusion Glycopeptide-resistant enterococci have emerged at different rates in Europe and North America. Although first reported in Europe [3, 4], resistant enterococcal strains have not disseminated extensively in European hospitals so far. However, investigation of the epidemiology of glycopeptide-resistant entero-

552 Leclercq and Courvalin CID 1997; 24 (April) cocci has led investigators to question the use of antibiotics in animal feed and to stress the putative danger of this practice. By contrast, in the United States, rapid dissemination of resistance among enterococci led to investigations of the use of vancomycin in humans; the results of these investigations indicated that the routes by which dissemination of resistance occurs in the hospital ecosystem are more complex than initially thought. References 1. McGraw DJ. The antibiotic discovery era (1940-1960): vancomycin as an example of the era [thesis]. Corvalis, Oregon: Oregon State University, 1974:78-81. 2. Ena J, Dick RW, Jones RN, Wenzel RP. The epidemiology of intravenous vancomycin usage in a university hospital: a 10-year study. JAMA 1993; 269:598-602. 3. Leclercq R, Derlot E, Duval J, Courvalin P. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N Engl J Med 1988;319:157-61. 4. Uttley AHC, Collins CH, Naidoo J, George RC. Vancomycin-resistant enterococci [letter]. Lancet 1988; 1:57-8. 5. Barna JCJ, Williams DH. The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu Rev Microbiol 1984; 38: 339-57. 6. Arthur M, Courvalin P. Genetics and mechanisms of glycopeptide resistance in enterococci. Antimicrob Agents Chemother 1993; 37:1563-71. 7. Arthur M, Molinas C, Depardieu F, Courvalin P. Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147. J Bacteriol 1993;175:117-27. 8. Bugg TDH, Dutka-Malen S, Arthur M, Courvalin P, Walsh CT. Identification of vancomycin resistance protein VANA as a D-alanine:D-alanine ligase of altered substrate specificity. Biochemistry 1991;30:2017-21. 9. Bugg TDH, Wright GD, Dutka-Malen S, Arthur M, Courvalin P, Walsh CT. Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VANA. Biochemistry 1991; 30:10408-15. 10. Fontana R, Cerini R, Longoni P, Grossato A, Canepari P. Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin. J Bacteriol 1983;155:1343-50. 11. Al-Obeid S, Billot-Klein D, van Heijenoort J, Collatz E, Gutmann L. Replacement of the essential penicillin-binding protein 5 by high-molecular mass PBPs may explain vancomycin-/.3-lactam synergy in low-level vancomycin-resistant Enterococcus faecium D366. FEMS Microbiol Lett 1992; 91:79-84. 12. Arthur M, Depardieu F, Reynolds P, Courvalin P. Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide-resistant enterococci. Mol Microbiol 1996;21:33-44. 13. Reynolds PE, Depardieu F, Dutka-Malen S, Arthur M, Courvalin P. Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine. Mol Microbiol 1994;13:1065-70. 14. Arthur M, Depardieu F, Snaith HA, Reynolds PE, Courvalin P. Contribution of VanY D,D-carboxypeptidase to glycopeptide resistance in Enterococcus faecalis by hydrolysis of peptidoglycan precursors. Antimicrob Agents Chemother 1994; 38:1899-903. 15. Arthur M, Depardieu F, Molinas C, Reynolds P, Courvalin P. The vanz gene of Tn1546 from Enterococcus faecium BM4147 confers resistance to teicoplanin. Gene 1995; 154:87-92. 16. Handwerger S, Skoble J. Identification of chromosomal mobile element conferring high-level vancomycin resistance in Enterococcus faecium. Antimicrob Agents Chemother 1995;39:2446-53. 17. Williamson R, Al-Obeid S, Shlaes JH, Goldstein FW, Shlaes DM. Inducible resistance to vancomycin in Enterococcus faecium D366. J Infect Dis 1989;159:1095-1104. 18. Quintiliani R Jr, Evers S, Courvalin P. The vanb gene confers various levels of self-transferable resistance to vancomycin in enterococci. J Infect Dis 1993; 167:1220-3. 19. Quintiliani R Jr, Courvalin P. Conjugal transfer of the vancomycin resistance determinant vanb between enterococci involves the movement of large genetic elements from chromosome to chromosome. FEMS Microbiol Lett 1994;119:359-64. 20. Quintiliani R Jr, Courvalin P. Characterization of Tn1547, a composite transposon flanked by the 1516 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281. Gene 1996; 172:1-8. 21. Perrichon B, Reynolds PE, Courvalin P. A glycopeptide-resistant VAND Enterococcus faecium. In: Program and abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy (New Orleans). Washington, DC: American Society for Microbiology, 1996. 22. Billot-Klein D, Gutmann L, Sable S, Guittet E, van Heijenoort J. Modification of peptidoglycan precursors is a common feature of the low-level vancomycin resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. J Bacteriol 1994; 176:2398-405. 23. Fraimow HS, Jungkind DL, Lander DW, Delso DR, Dean JL. Urinary tract infection with an Enterococcus faecalis isolate that requires vancomycin for growth. Ann Intern Med 1994; 121:22-6. 24. Green M, Shlaes JH, Barbadora K, Shlaes DM. Bacteremia due to vancomycin-dependent Enterococcus faecium. Clin Infect Dis 1995;20: 712-4. 25. Dever LL, Smith SM, Handwerger S, Eng RHK. Vancomycin-dependent Enterococcus faecium isolated from stool following oral vancomycin therapy. J Clin Microbiol 1995; 33:2770-3. 26. Rosato A, Pierre J, Billot-Klein D, Buu-Hoi A, Gutmann L. Inducible and constitutive expression of resistance to glycopeptides and vancomycin dependence in glycopeptide-resistant Enterococcus avium. Antimicrob Agents Chemother 1995; 39:830-3. 27. Baptista M, Depardieu F, Courvalin P, Arthur M. Specificity of induction of glycopeptide resistance genes in Enterococcus faecalis. Antimicrob Agents Chemother 1996; 40:2291-5. 28. Aslangul E, Banos Baptista M, Fantin B, Arthur M, Courvalin P, Carbon C. Evaluation of the activity of vancomycin (Vm) and teicoplanin (Te) in experimental endocarditis due to Enterococcus faecalis with a VANB phenotype [abstract B48]. In: Program and abstracts of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy (San Francisco). Washington, DC: American Society for Microbiology, 1995. 29. Hayden MK, Trenholme GM, Schultz JE, Sahm DF. In vivo development of teicoplanin resistance in a VANB Enterococcus faecium isolate. J Infect Dis 1993; 167:1224-7. 30. Reynolds PE, Snaith HA, Maguire AJ, Dutka-Malen S, Courvalin P. Analysis of peptidoglycan precursors in vancomycin-resistant Enterococcus gallinarum BM4174. Biochem J 1994; 301:5-8. 31. Navarro F, Courvalin P. Analysis of genes encoding D-alanine-D-alanine ligase-related enzymes in Enterococcus casseliflavus and Enterococcus flavescens. Antimicrob Agents Chemother 1994;38:1788-93. 32. Evers S, Casadewall B, Charles M, Dutka-Malen S, Galimand M, Courvalin P. Evolution of structure and substrate specificity in D-alanine- D-alanine ligases and related enzymes. Journal of Molecular Evolution 1996;42:706-12. 33. Handwerger S, Raucher B, Altarac D, et al. Nosocomial outbreak due to Enterococcus faecium highly resistant to vancomycin, penicillin, and gentamicin. Clin Infect Dis 1993; 16:750-5. 34. Gaynes R, Edwards J, National Nosocomial Infection Surveillance (NNIS) System. Nosocomial vancomycin resistant enterococci (VRE) in the

CID 1997; 24 (April) Enterococcal Resistance to Glycopeptides 553 United States, 1989-1995: the first 1000 isolates [abstract 13]. Infect Control Hosp Epidemiol 1996; 17(5 pt 2)(suppl):P18. 35. Vandamme P, Vercauteren E, Lammens C, et al. Survey of enterococcal susceptibility patterns in Belgium. J Clin Microbiol 1996; 34:2572-6. 36. Schmit JL, Leclercq R, Scheimberg A, Landauer D. Approche epidemiologique et clinique des enterocoques: resultats d'une enquete. Medecine et Maladies Infectieuses 1994; 24(special):141-8. 37. Wade JJ. The emergence of Enterococcus faecium resistant to glycopeptides and other standard agents-a preliminary report. J Hosp Infect 1995; 30(suppl):483-93. 38. Leclercq R, Derlot E, Weber M, Duval J, Courvalin P. Transferable vancomycin and teicoplanin resistance in Enterococcus faecium. Antimicrob Agents Chemother 1989; 33:10-15. 39. Noble WC, Virani Z, Cree RGA. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol Lett 1992; 93:195-8. 40. Power EGM, Abdulla YH, Talsania HG, Spice W, Aathithan S, French GL. vana genes in vancomycin-resistant clinical isolates of Oerskovia turbata and Arcanobacterium (Corynebacterium) haemolyticum. J Antimicrob Chemother 1995;36:595-606. 41. Poyart C, Pierre C, Quesne G, Pron B, Berche P, Trieu-Cuot P. Emergence of vancomycin resistance in the genus Streptococcus: characterization of a vanb transferable determinant in Streptococcus bovis. Antimicrob Agents Chemother 1997 (in press). 42. Livornese LL Jr, Dias S, Samel C, et al. Hospital-acquired infection with vancomycin-resistant Enterococcus faecium transmitted by electronic thermometers. Ann Intern Med 1992; 117:112-6. 43. Edmond MB, Ober JF, Weinbaum DL, et al. Vancomycin-resistant Enterococcus faecium bacteremia: risk factors for infection. Clin Infect Dis 1995;20:1126-33. 44. Moreno F, Grota P, Crisp C, et al. Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium during its emergence in a city in southern Texas. Clin Infect Dis 1995;21:1234-7. 45. Morris JG Jr, Shay DK, Hebden JN, et al. Enterococci resistant to multiple agents, including vancomycin: establishment of endemicity in a university medical center. Ann Intern Med 1995; 123:250-9. 46. Shay DK, Maloney SA, Montecalvo M, et al. Epidemiology and mortality risk of vancomycin-resistant enterococcal bloodstream infections. J Infect Dis 1995;172:993-1000. 47. Linden PK, Pasculle AW, Manez R, et al. Differences in outcomes for patients with bacteremia due to vancomycin-resistant Enterococcus faecium or vancomycin-susceptible E. faecium. Clin Infect Dis 1996;22: 663-70. 48. Korten V, Murray BE. The nosocomial transmission of enterococci. Current Opinion in Infectious Diseases 1993; 6:498-505. 49. Boyce JM, Opal SM, Potter-Bynoe G, et al. Emergence and nosocomial transmission of ampicillin-resistant enterococci. Antimicrob Agents Chemother 1992; 36:1032-9. 50. Sexton DJ, Harrell LJ, Thorpe JJ, Hunt DL, Reller LB. A case-control study of nosocomial ampicillin-resistant enterococcal infection and colonization at a university hospital. Infect Control Hosp Epidemiol 1993; 14:629-35. 51. Yu VL. Enterococcal superinfection and colonization after therapy with moxalactam, a new broad-spectrum antibiotic. Ann Intern Med 1981; 94:784-5. 52. Montecalvo MA, Horowitz H, Gedris C, et al. Outbreak of vancomycin-, ampicillin-, and aminoglycoside-resistant Enterococcus faecium bacteremia in an adult oncology unit. Antimicrob Agents Chemother 1994; 38:1363-7. 53. Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev 1990; 3:46-65. 54. Centers for Disease Control and Prevention. Nosocomial enterococci resistant to vancomycin-united States, 1989-1993. MMWR Morb Mortal Wkly Rep 1993;42:597-9. 55. Montecalvo MA, Shay DK, Patel P, et al. Bloodstream infections with vancomycin-resistant enterococci. Arch Intern Med 1996;156: 1458-62. 56. Norris AH, Reilly JP, Edelstein PH, Brennan PJ, Schuster MG. Chloramphenicol for the treatment of vancomycin-resistant enterococcal infections. Clin Infect Dis 1995;20:1137-44. 57. Wells CL, Juni BA, Cameron SB, et al. Stool carriage, clinical isolation, and mortality during an outbreak of vancomycin-resistant enterococci in hospitalized medical and/or surgical patients. Clin Infect Dis 1995; 21:45-50. 58. Hoge CW, Adams J, Buchanan B, Sears SD. Enterococcal bacteremia: to treat or not to treat, a reappraisal. Rev Infect Dis 1991; 13:600-5. 59. Costa Y, Galimand M, Leclercq R, Duval J, Courvalin P. Characterization of the chromosomal aac(6')-ii gene specific for Enterococcus faecium. Antimicrob Agents Chemother 1993; 37:1896-903. 60. Moellering RC Jr, Korzeniowski OM, Sande MA, Wennersten CB. Species-specific resistance to antimicrobial synergism in Streptococcus faecium and Streptococcus faecalis. J Infect Dis 1979; 140:203-8. 61. Leclercq R, Bingen E, Su QH, Lambert-Zechovski N, Courvalin P, Duval J. Effects of combinations of beta-lactams, daptomycin, gentamicin, and glycopeptides against glycopeptide-resistant enterococci. Antimicrob Agents Chemother 1991;35:92-8. 62. Caron F, Lemeland J-F, Humbert G, Klare I, Gutmann L. Triple combination penicillin-vancomycin-gentamicin for experimental endocarditis caused by a highly penicillin- and glycopeptide-resistant isolate of Enterococcus faecium. J Infect Dis 1993; 168:681-6. 63. Caron F, Pestel M, Kitzis M-D, Lemeland J-F, Humbert G, Gutmann L. Comparison of different /3-lactam-glycopeptide-gentamicin combinations for an experimental endocarditis caused by a highly fi-lactamresistant and highly glycopeptide-resistant isolate of Enterococcus faecium. J Infect Dis 1995;171:106-12. 64. Handwerger S, Perlman DC, Altarac D, McAuliffe V. Concomitant highlevel vancomycin and penicillin resistance in clinical isolates of enterococci. Clin Infect Dis 1992;14:655-61. 65. Brandt CM, Rouse MS, Laue NW, Stratton CW, Wilson WR, Steckelberg JM. Effective treatment of multidrug-resistant enterococcal experimental endocarditis with combinations of cell wall-active agents. J Infect Dis 1996; 173:909-13. 66. Unal S, Flokowitsch J, Mullen DL, Preston DA, Nicas IT. In-vitro synergy and mechanism of interaction between vancomycin and ciprofloxacin against enterococcal isolates. J Antimicrob Chemother 1993; 31: 711-23. 67. Whitman MS, Pitsakis PG, Zausner A, et al. Antibiotic treatment of experimental endocarditis due to vancomycin- and ampicillin-resistant Enterococcus faecium. Antimicrob Agents Chemother 1993; 37:2069-73. 68. Montecalvo MA, Horowitz H, Wormser GP, Seiter K, Carbonaro CA. Effect of novobiocin-containing antimicrobial regimens on infection and colonization with vancomycin-resistant Enterococcus faecium [letter]. Antimicrob Agents Chemother 1995; 39:794. 69. O'Donovan CA, Fan-Havard P, Tecson-Tumang FT, Smith SM, Eng RHK. Enteric eradication of vancomycin-resistant Enterococcus faecium with oral bacitracin. Diagn Microbiol Infect Dis 1994; 18:105-9. 70. Low DE. Quinupristin/dalfopristin: spectrum of activity, pharmacokinetics, and initial clinical experience. Microbial Drug Resistance 1995;1: 223-34. 71. Torres C, Reguera JA, Sanmartin MJ, Perez-Diaz JC, Baquero F. vanamediated vancomycin-resistant Enterococcus spp. in sewage. J Antimicrob Chemother 1994; 33:553-61. 72. Bates J, Jordens JZ, Griffiths DT. Farm animals as a putative reservoir for vancomycin-resistant enterococcal infection in man. J Antimicrob Chemother 1994; 34:507-14. 71 Klare I, Heier H, Claus H, Witte W. Environmental strains of Enterococcus faecium with inducible high-level resistance to glycopeptides. FEMS Microbiol Lett 1993;106:23-30.

554 Leclercq and Courvalin CID 1997; 24 (April) 74. Klare I, Heier H, Claus H. Enterococcus faecium strains with vana-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microbial Drug Resistance 1995;1:265-72. 75. Gordts B, Van Landuyt H, Ieven M, Vandamme P, Goossens H. Vancomycin-resistant enterococci colonizing the intestinal tracts of hospitalized patients. J Clin Microbiol 1995; 33:2842-6. 76. Van der Auwera P, Pensart N, Korten V, Murray BE, Leclercq R. Influence of oral glycopeptides on the fecal flora of human volunteers: selection of highly glycopeptide-resistant enterococci. J Infect Dis 1996; 173: 1129-36. 77. Boisivon A, Thibault M, Leclercq R, and The College de Bacteriologie- Virologie-Hygiêne des HOpitaux generaux francais. Colonization by vancomycin-resistant enterococci of the intestinal tract of patients in intensive care units from French general hospitals. Clin Microbiol Infect 1997 (in press). 78. Klare I, Heier H, Claus H, Reissbrodt R, Witte W. vana-mediated highlevel glycopeptide resistance in Enterococcus faecium from animal husbandry. FEMS Microbiol Lett 1995; 125:165-72. 79. Aarestrup FM. Occurrence of glycopeptide resistance among Enterococcus faecium isolates from conventional and ecological poultry farms. Microbial Drug Resistance 1995; 1:255-7. 80. Aarestrup FM, Ahrens P, Madsen M, Pallesen LV, Poulsen RL, Westh H. Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the vana cluster. Antimicrob Agents Chemother 1996; 40:1938-40. 81. Coque TM, Tomayko JF, Ricke SC, Okhuysen PC, Murray BE. Vancomycin-resistant enterococci from nosocomial, community, and animal sources in the United States. Antimicrob Agents Chemother 1996; 40: 2605-9. 82. Sader HS, Pfaller MA, Tenover FC, Hollis RI, Jones RN. Evaluation and characterization of multiresistant Enterococcus faecium from 12 U.S. medical centers. J Clin Microbiol 1994; 32:2840-2. 83. Boyce JM, Opal SM, Chow JW, et al. Outbreak of multidrug-resistant Enterococcus faecium with transferable vanb class vancomycin resistance. J Clin Microbiol 1994;32:1148-53. 84. Wade JJ, Desai N, Casewell MW. Hygienic hand disinfection for the removal of epidemic vancomycin-resistant Enterococcus faecium and gentamicin-resistant Enterobacter cloacae. J Hosp Infect 1991; 18: 211-8. 85. Boyle JF, Soumakis SA, Rendo A, et al. Epidemiologic analysis and genotypic characterization of a nosocomial outbreak of vancomycinresistant enterococci. J Clin Microbiol 1993;31:1280-5. 86. Whitman MS, Pitsakis PG, DeJesus E, Osborne AJ, Levison ME, Johnson CC. Gastrointestinal colonization with vancomycin-resistant Enterococcus faecium in an animal model. Antimicrob Agents Chemother 1996; 40:1526-30. 87. Centers for Disease Control and Prevention. Recommendations for preventing the spread of vancomycin resistance: recommendations of the Hospital Infection Control Practices Advisory Committee (HICPAC). MMWR Morb Mortal Wkly Rep 1995; 44(RR-12):1-13. 88. Tenover FC, Swenson JM, O'Hara CM, Stocker SA. Ability of commercial and reference antimicrobial susceptibility testing methods to detect vancomycin resistance in enterococci. J Clin Microbiol 1995; 33:1524-7. 89. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995;33:24-7. 90. Noskin GA, Cooper I, Peterson LR. Vancomycin-resistant Enterococcus faecium sepsis following persistent colonization. Arch Intern Med 1995; 155:1445-7. 91. Boyce JM, Mermel LA, Zervos MJ, et al. Controlling vancomycin-resistant enterococci. Infect Control Hosp Epidemiol 1995; 16:634-7. 92. Slaughter S, Hayden MK, Nathan C, et al. A comparison of the effect of universal use of gloves and gowns with that of glove use alone on acquisition of vancomycin-resistant enterococci in a medical intensive care unit. Ann Intern Med 1996; 125:448-56. The "Conflict-of-Interest Policy" of the Office of Continuing Medical Education, UCLA School of Medicine, requires that faculty participating in a CME activity disclose to the audience any relationship with a pharmaceutical or equipment company which might pose a potential, apparent, or real conflict of interest with regard to their contribution to the program. The author reports no conflict of interest.