Enterococcal NVE

Enterococci account for 5-15% of cases of NVE and is usually due to E. faecalis or E. faecium [4,12,111]. Treatment of enterococcal infections in general, and NVE in particular, is made difficult due to the mechanisms of resistance possessed by these pathogens, which can be divided into three categories: inherent (or intrinsic) resistance, tolerance, and acquired resistance. The inherent mechanisms of resistance are, by definition, species characteristics present in all or most of the strains of that species and are encoded on the chromosome [112]. Tolerance is defined as delayed or decreased bactericidal killing by growth-inhibiting concentrations of bactericidal compounds [113]. As mentioned before, a strain is defined as "tolerant" when the MBC/MIC ratio is > 32. Acquired resistance occurs either from a mutation in the existing DNA or, more clinically relevant, from acquisition of new DNA.

Enterococci are inherently resistant to certain P-lactams, specifically the semi-synthetic peni-cillinase-resistant penicillins (e.g., oxacillin, naf-cillin) and cephalosporins, as well as to lincosamides (e.g., clindamycin), traditional antimicrobial agents used for Gram-positive cocci [112,114]. Furthermore, enterococci are intrinsically resistant to trimethoprim-sulfamethoxazole (TMP/SMX) in vivo, aminoglycosides (low level), and aztreonam [112,115]. The mechanisms responsible for this natural resistance are diverse and have permitted the emergence of the enterococci as major pathogens.

Intrinsic resistance to the aforementioned P-lactams is due to the presence of specific penicillin-binding proteins (PBPs) with poor affinity to these antibiotics [112]. Low-affinity PBPs are multifunctional enzymes that can catalyze complete peptidoglycan synthesis under conditions in which all the other normal PBPs are inhibited by P-lactams [116]. In the enterococci, PBP-5 is the predominant low-affinity PBP. It is a normal component of the enterococcal PBP repertoire and is constitutively expressed, thereby allowing bacterial cell survival in the presence of semi-synthetic penicillins and cephems.

Lincosamide antibiotics include lincomycin, naturally produced by actinomycetes, and clin-damycin, a semi-synthetic derivative of lin-comycin. The enterococci are inherently resistant to clindamycin [112,115], although there are several mechanisms by which this occurs. For example, E. faecalis, the predominant clinical species, is characterized by the LSA phenotype, defined as resistance to not only the lincosamides, but also to streptogramins A (dalfopristin, pristinamycin II, virginiamycin M) [117]. This phenotype is mediated by the lsa gene, which encodes for a protein that has structural homology to antibiotic efflux pumps of other Gram-positive organisms [118]. There are two other major mechanisms by which the enterococci have developed lin-cosamide resistance. One method is by a riboso-mal methylase encoded by an ermAM-like gene. This enzyme leads to N6 dimethylation of a specific adenine in the 23S rRNA, which confers resistance to lincosamides, but also to macrolides and to streptogramin B antibiotics; this pheno-type is designated MLSb [119,120]. Acquired resistance can also occur via the dissemination of the linB gene, which encodes for lincosamide nucleotidyltransferase that leads to inactivation of such antibiotics [119].

TMP/SMX is considered to not be an effective antibiotic for the treatment of enterococcal infections, even though it demonstrates in vitro activity [121]. Treatment failures have been demonstrated in both animal models of endocarditis and in the clinical setting of urinary tract infections [122,123]. The proposed explanation as to why this combination is not effective is related to the ability of the enterococci to incorporate pre-formed folic acid, which enables them to bypass the inhibition of folate synthesis imposed by TMP/SMX [112].

Low-level aminoglycoside resistance (LLAR) is an inherent property of enterococci. Highlevel aminoglycoside resistance (HLAR) is an acquired characteristic and is discussed below. There are two major mechanisms conferring LLAR: First is decreased bacterial cellular uptake, seen in all enterococci [112]. The means by which enterococci are able to limit aminoglycoside uptake relate to the biochemical characteristics of the aminoglycosides, as well as to bacterial metabolism [124]. As aminoglycosides are charged, hydrophilic molecules, they are unable efficiently to cross the lipid-containing cell membrane of enterococci to reach their ribosomal target. Additionally, the anaerobic metabolism of enterococci results in poor active transport of these antibiotics into the cells.

The other method of LLAR is seen only in E. faecium and occurs via inactivation of certain aminoglycoside antibiotics (tobramycin, netilmicin, kanamycin, and sismicin) by a chro-mosomally encoded enzyme [112]. This additional method explains the differences in MICs of these aminoglycosides seen for E. faecalis when compared to E. faecium. The typical MIC of tobramycin for E. faecalis is in the range of 8-64 mg/L; that of kanamycin is in the range of 250 mg/L [112]. The MICs of tobramycin and kanamycin for E. faecium, however, are higher [112]. This resistance pattern is attributed to the production of an aminoglycoside 6'-acetyltrans-ferase (AAC-6') enzyme [124]. The clinical consequence of this enzyme is that combinations of a cell wall active agent with one of these amino-glycosides (tobramycin or kanamycin) will fail to demonstrate synergism against E. faecium. Synergism, or enhanced killing, for the entero-cocci is defined as a > 2-log10 increase in killing versus the effect of the cell-wall active agent alone when the aminoglycoside is used in a subinhibitory concentration [112]. However, synergy is maintained if the aminoglycoside that is used is either gentamicin or streptomycin.

As a consequence of these inherent mechanisms of resistance, the above-mentioned antibiotics possess no bactericidal or bacteriostatic activity against the enterococci. In addition, the majority of Enterococcus spp. demonstrate "tolerance" to various cell-wall active agents, whereby cell growth is inhibited at clinically achievable concentrations, but not cell death. The major antibiotics with such properties are penicillin, aminopenicillins (amoxicillin, ampicillin), and glycopeptides (teicoplanin, vancomycin). Ampicillin generally has lower MICs than penicillin, and thus may be the preferred agent [115,125]. Ampicillin MICs for E.faecalis generally are 0.5-4.0 |g/mL, whereas for E. faecium, the MICs are typically 4-8 |g/mL [115]. The ureidopenicillins (azlocillin, mezlocillin, piperacillin) have approximately the same activity against enterococci as penicillin and ampicillin [125]. This bacteriostatic effect is suboptimal in the management of infective endocarditis, which classically requires a bactericidal regimen. Such an effect can be achieved by the combination of gentamicin or streptomycin to one of these cell-wall active agents.

The mechanism of tolerance of enterococci to P-lactams remains unclear, but is clearly distinct from resistance, demonstrated by the fact that each feature can be elicited independently among E. faecalis strains exposed in vitro to penicillin [126]. It has been suggested that tolerance may be associated with changes in the autolysis system [127]. P-Lactam-induced lysis of bacteria is the consequence of inhibition of biosynthesis of peptidoglycan, as well as to hydrolysis of cell walls by bacterial autolytic enzymes. It has been shown that an increase in autolytic activity among clinical enterococcal isolates correlated with increased penicillin-induced lysis and killing [127]. Conversely, E. faecalis strains with reduced or absent autolytic activity were less susceptible to penicillin [128]. However, neither modification of one enterococcal autolysin gene, nor alteration of its expression, resulted in any significant change in MIC or in tolerance to P-lactams [129]. As such, tolerance to P-lactam remains a poorly understood phenomenon.

Because of the limited antimicrobial options, optimal management of ampicillin-susceptible enterococcal NVE should involve the addition of an aminoglycoside (i.e., gentamicin or streptomycin) to a cell-wall active agent (i.e., ampicillin or glycopeptides). This combination results in a synergistic bactericidal activity related to the fact that cell-wall active agents markedly increase the penetration of aminoglycosides into the bacterial cell, allowing binding to its ribosomal target [130]. Alternatively, if amino-glycoside therapy is contraindicated (e.g., potential worsening of renal insufficiency), prolonged treatment with a P-lactam, classically ampicillin, while maintaining the serum antibiotic concentration above the MIC of the isolate, may be sufficient (see Table 9.4).

Unfortunately, acquired antimicrobial resistance to aminoglycosides and to cell-wall active agents has complicated the management of this disease. High-level aminoglycoside resistance, currently defined by CLSI (NCCLS) as an MIC of streptomycin > 2,000 |g/mL or an MIC of gentamicin > 500 |g/mL, was first described in 1979 [131]. Rates have increased worldwide, with prevalence as high as ~75% [132], and it is particularly common among strains of E. faecium [133]. The mechanism of this resistance is related to the presence of aminoglycoside-mod-ifying enzymes, some of which are located on transferable plasmids [134,135]. A bifunctional enzyme (2''-phosphotransferase-6'-acetyltrans-ferase) mediates high-level gentamicin resistance, as well as resistance to tobramycin, amikacin, netilmicin, and kanamycin [114,125]. Streptomycin resistance, however, is mediated by completely different mechanisms. It occurs as a result of ribosomal resistance, in which there is alteration of ribosomal target sites, or by streptomycin adenyltransferase, which modifies and inactivates aminoglycosides [136]. Because gentamicin and streptomycin resistance may differ among Enterococcus spp., aminoglycoside screening should include tests for high-level resistance to both of these aminoglycosides. If one of these antimicrobials demonstrates lack of HLAR, it should be used, if the clinical situation permits. If NVE is due to an Enterococcus spp. with HLAR to both aminoglycosides, absence of synergism with a cell-wall active agent can be predicted. As there is no clinical efficacy to using such agent in these situations, and with the inherent risks of aminoglycosides, monotherapy with a cell-wall active agent should be employed.

Acquired ampicillin resistance has compromised the management of enterococcal infec tions. The two clinically major species each have their own mechanism mediating such resistance. P-lactamase production is exclusively described in E. faecalis; this enzyme is felt to have been acquired from S. aureus via a transferable plasmid [112,115]. P-Lactamase production occurs at low levels and produces an "inoculum effect," such that at low to moderate inocula (103-105 CFU/mL), there is only a minor increase in MIC and such penicillinase-produc-ing enterococci usually appear no more resistant than other enterococci [114]. However, at high inocula (> 107 CFU/mL), when sufficient enzymes are produced, such strains are highly resistant to penicillin, aminopenicillins, and ureidopenicillins [114]. As a result of this inoculum effect, P-lactamase-mediated penicillin resistance is not detected by routine disk susceptibility testing [112]. In the clinical laboratory, hydrolysis of the chromogenic cephalosporin, nitrocefin, is the definitive test for P-lactamase production [125]. The activity of the penicillinase is inhibited by P-lactamase inhibitors (i.e., clavulanic acid, tazobactam, sulbactam) [114]. Although there have been reports of clinical infection with P-lactamase-producing E. faecalis [133], it does not appear that this mechanism of resistance is a major virulence factor among enterococci [137,138].

Non-P-lactamase producing, ampicillin-resistant enterococci is usually E. faecium. The mechanisms of this resistance appear to be overproduction of the naturally present PBP5, as well as amino acid substitutions in PBP5 resulting in a further decrease in affinity to P-lactams [114,115,137,138]. Acquisition of this form of P-lactam resistance accounts for the majority of clinically relevant isolates.

In the face of P-lactam resistance, the only therapeutic options, until recently, were the glycopeptides (vancomycin, teicoplanin). These antibiotics function by binding to the terminal D-alanyl-D-alanine present on the pentapeptide side chains of the peptidoglycan precursors, inhibiting peptidoglycan synthesis. In North America, vancomycin is the only glycopeptide currently commercially available and it is recommended as the drug of choice for serious enterococcal infection only in cases of significant penicillin allergy or in the treatment of ampicillin-resistant strains. Vancomycin, when combined with gentamicin or streptomycin, does demonstrate synergism against Enterococcus spp. in vitro and in vivo

[125]. Vancomycin should not, however, be used for ampicillin-susceptible strains, as it usually has higher MICs against enterococci than ampicillin [139]. As well, there is concern that careless overuse of vancomycin contributes to the emergence of vancomycin-resistant pathogens.

Glycopeptide resistance is an emerging problem. First described in the 1980s, vancomycin-resistant enterococci (VRE) have become an important nosocomial pathogen globally. The most common phenotype of resistance, vanA, is associated with acquired, inducible, high-level resistance to vancomycin (MIC > 64 |g/mL) and to teicoplanin (MIC >16 |g/mL) [121]. The vanA phenotype is mediated by genetic elements that are carried on a transposon (Tnl546) and is transferable to other susceptible enterococci by conjugation [115]. Other acquired glycopeptide-resistant phenotypes have been also been characterized, including vanB, as well as vanD, vanE, and vanG, which are much less common. The vanB phenotype, which is chromosomally mediated, inducible, and transferable by conjugation, mediates inducible resistance to vancomycin, but not to teicoplanin [121]. However, the development of teicoplanin resistance occurs rapidly during antibiotic exposure. Bloodstream infection with VRE can be very difficult to treat because there may be concomitant ampicillin resistance, as seen with virtually all E. faecium [115]. Vancomycin-resistant E. faecalis, however, usually remains susceptible to ampicillin. Furthermore, a recent retrospective case-control study demonstrated that patients with bac-teremia caused by VRE were more likely to die than were those with vancomycin-susceptible enterococcal bacteremia, with a summary odds ratio for death of 2.52, and a 95% confidence interval of 1.9-3.4 [140].

In face of glycopeptide resistance, treatment of VRE poses significant challenge. Fortunately, VRE endocarditis remains relatively uncommon, with no local, national, or international incidence rates reported in the English literature. For VRE infections in general, two classes of antibiotics have been approved: the strep-togramins and the oxazolidnones.

Among the approved streptogramin class of antibiotics is quinupristin/dalfopristin (Q/D, Synercid®, Aventis Pharmaceuticals, Inc.). It is a parenteral antibiotic that is structurally related to the macrolides and lincosamides. Its mechanism of action is inhibition of early (peptide chain elongation) and late stages of bacterial protein synthesis [141]. Interestingly, Q/D demonstrates good in vitroactivity against E. faecium, with MIC90 of 1-2 |g/mL, but very poor activity against E. faecalis, the predominant enterococcal pathogen, with MIC90 of 8-16 |g/mL [141]. The reason for this difference in activity is likely due to decreased 50S bacterial ribosomal binding of Q/D in E. faecalis [141]. In in vitro studies, Q/D is bactericidal for VRE [141]. However, in time-kill studies, Q/D demonstrates only bacteriostatic activity; this difference in effect is due to the expression of the MLSb phenotype (described previously), which encodes for the methylation of the 23S ribosomal binding site [141,142]. Q/D-resist-ance has been reported among clinical VRE isolates, ranging from <10% to 22% [142]. Furthermore, emergence of Q/D-resistance while on therapy has also been described. Clinical failure with Q/D has been reported with VRE endocarditis [143,144].

Linezolid (LZL, Zyvox™, Pfizer, Inc.) is the only currently available oxazolidinone. It is prepared as a parenteral or as an oral formulation, with the latter having 100% bioavailability [145]. LZL functions by binding to the 23S ribosomal RNA of the 50S subunit on the bacterial ribo-some, thus inhibiting protein synthesis [145]. By virtue of its unique action, cross-resistance to LZL has not been reported among enterococci that have developed resistance to other antibiotics [146]. LZL has shown consistent bacterio-static activity against vancomycin-susceptible and vancomycin-resistant E. faecium and E. faecalis. In murine models [147] and in clinical reports [148], LZL was effective in the treatment of VRE bacteremia. It has also been reported to be effective for VRE endocarditis [149-152], although not consistently [153]. Furthermore, resistance to LZL has developed among VRE in patients receiving the drug for an extended period of time, typically >3 weeks [154-156]. This issue raises some concerns about its use as monotherapy in VRE endcarditis, which typically requires a prolonged course of antimicrobial therapy. Ideally, synergism can be achieved when combined with other antimicrobials. However, using the standard checkerboard assay to determine the fractional inhibitor concentrations (FIC) indices, LZL primarily demonstrated in vitro indifference (i.e., no synergy) against Enterococcus spp. when assessed in combination with other antimicrobials [157].

Consequently, the role of LZL in VRE endocarditis remains unestablished.

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