Ampicillin Enhances Daptomycin- and Cationic Host Defense Peptide-Mediated Killing of Ampicillin- and Vancomycin-Resistant Enterococcus faecium

Abstract

We studied an ampicillin-and vancomycin-resistant Enterococcus faecium (VRE) isolate from a patient with endocarditis and bacteremia refractory to treatment with daptomycin (6 mg/kg of body weight) plus linezolid. Blood cultures cleared within 24 h of changing therapy to daptomycin (12 mg/kg) plus ampicillin. We examined the effects of ampicillin on daptomycin-induced growth inhibition and killing, surface charge, and susceptibility to several prototypical host defense cationic antimicrobial peptides. MICs and time-kill curves with daptomycin were assessed in the presence and absence of ampicillin. The impact of ampicillin on surface charge was assessed by flow cytometry and a poly-L-lysine binding assay. The effects of ampicillin preexposures upon VRE killing by five distinct cationic peptides of different structure, charge, origin, and mechanism of action were analyzed using the epidermal cathelicidin LL-37, thrombin-induced platelet microbicidal proteins (tPMPs), and a synthetic congener modeled after tPMP microbicidal domains (RP-1), human neutrophil peptide-1 (hNP-1), and polymyxin B (bacteria derived). Fluoroscein-Bodipy-labeled daptomycin was used to evaluate daptomycin binding to VRE membranes in the presence or absence of ampicillin. In media containing ampicillin (25 to 100 mg/liter), daptomycin MICs decreased from 1.0 to 0.38 mg/liter. Based on time-kill analysis and an in vitro pharmacodynamic model, ampicillin enhanced daptomycin activity against the study VRE from a bacteriostatic to a bactericidal profile. VRE grown in ampicillin (25 to 150 mg/liter) demonstrated an incremental reduction in its relative net positive surface charge. When grown in the presence (versus absence) of ampicillin (25 and 100 mg/ liter), the VRE strain (i) was more susceptible to killing by LL-37, tPMPs, hNP-1, and RP-1 but not to polymyxin B and (ii) exhibited greater binding to Bodipy-labeled daptomycin. We conclude that ampicillin induces reductions in net positive bacterial surface charge of VRE, correlating with enhanced bactericidal effects of cationic calcium-daptomycin and a diverse range of other cationic peptides in vitro. While the mechanism(s) of such ␤-lactam-mediated shifts in surface charge remains to be defined, these finding suggest a potential for ␤-lactam-mediated enhancement of activity of both daptomycin and innate host defense peptides against antibiotic-resistant bacteria.

Fosfomycin Enhances the Activity of Daptomycin against Vancomycin-Resistant Enterococci in an In Vitro Pharmacokinetic-Pharmacodynamic Model

“…Similar effects have been seen in methicillin-resistant S. aureus, where the addition of fosfomycin to daptomycin delayed the emergence of resistance to daptomycin in vitro (43). Fosfomycin exposure appeared to reduce cell surface charge, which suggests that the mechanism of synergy is similar to that seen with beta-lactams and daptomycin against enterococci and Staphylococcus aureus (27,44,45). Support for this hypothesis comes from recent data, which demonstrate that fosfomycin appears to reduce PBP1 expression in S. aureus (10).…”

“…Synergy between daptomycin and beta-lactams is believed to be mediated through beta-lactam-induced alterations in surface charge that facilitate an increase in daptomycin binding, leading to enhanced membrane depolarization (23)(24)(25)(26)(27)(28). Since fosfomycin also disrupts cell wall synthesis, it could be theorized that there is a similar mechanism that drives the synergy that has been observed with daptomycin and fosfomycin.…”

Ceftaroline Restores Daptomycin Activity against Daptomycin-Nonsusceptible Vancomycin-Resistant Enterococcus faecium

“…Our group has previously shown a perhaps counterintuitive effect of ampicillin in converting daptomycin from a bacteriostatic to a bactericidal antibiotic against an ampicillin-resistant bloodstream VRE, with successful clearance of refractory bacteremia by this organism using daptomycin plus ampicillin combination therapy (5). In this study, ampicillin also conferred increased susceptibility of VRE to killing by innate cationic host defense peptides (HDPs) such as cathelicidin LL37.…”

“…Transmission electron microscopy (TEM) was used to determine cell wall thickness and septation, and membrane fluidity was analyzed by fluorescence polarization using 6-diphenyl-1,3,5-hexatriene as previously described (6). Fluorescein isothiocyanate (FITC)-labeled poly-L-lysine (PLL) binding assays were performed using flow cytometry methods previously described (5). PLL is a polycationic molecule used to study the interactions of cationic peptides with charged bacterial envelopes.…”

Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy

“…Fluorescein isothiocyanate (FITC)-labeled poly-L-lysine (PLL) binding studies were performed to evaluate surface charge in the presence of ␤-lactams. Assays were performed using a flow cytometric method as previously described (27). PLL is a polycationic molecule used to study the interactions of cationic peptides with charged bacterial envelopes.…”