MECHANISMS

MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2014.0221

Antimicrobial Activity of Plectasin NZ2114 in Combination with Cell Wall Targeting Antibiotics Against VanA-Type Enterococcus faecalis Elena B.M. Breidenstein, Patrice Courvalin, and Djalal Meziane-Cherif

Antimicrobial peptide plectasin targeting bacterial cell wall precursor Lipid II has been reported to be active against benzylpenicillin-resistant Streptococcus pneumoniae but less potent against vancomycin-resistant enterococci than their susceptible counterparts. The aim of this work was to test plectasin NZ2114 in combination with cell wall targeting antibiotics on vancomycin-resistant Enterococcus faecalis. The activity of antibiotic combinations was evaluated against VanA-type vancomycin-resistant E. faecalis strain BM4110/pIP816-1 by disk agar-induction, double-disk assay, determination of fractional inhibitory concentration (FIC) index, and time-kill curve. The results indicated that plectasin NZ2114 was synergistic in combination with teicoplanin, moenomycin, and dalbavancin but not with vancomycin, telavancin, penicillin G, bacitracin, ramoplanin, daptomycin, and fosfomycin. To gain an insight into the synergism, we tested other cell wall antibiotic combinations. Interestingly, synergy was observed between teicoplanin or moenomycin and the majority of the antibiotics tested; however, vancomycin was only synergistic with penicillin G. Other cell wall active antibiotics such as ramoplanin, bacitracin, and fosfomycin did not synergize. It appeared that most of the synergies observed involved inhibition of the transglycosylation step in peptidoglycan synthesis. These results suggest that teicoplanin, dalbavancin, vancomycin, and telavancin, although they all bind to the C-terminal D-Ala-D-Ala of Lipid II, might act on different stages of cell wall synthesis.

action is similar to that of teicoplanin and vancomycin: binding to the C-terminal D-Ala-D-Ala. However, telavancin can also interact with modified peptidoglycan precursors and with the cytoplasmic membrane leading to its depolarization and permeabilization.28 VanA, which is the most common type of glycopeptide resistance in enterococci, is characterized by inducible high level of resistance to both vancomycin and teicoplanin. It is due to acquisition of the vanA gene cluster carried by transposon Tn1546 and is associated with modified peptidoglycan precursors ending in D-Ala-D-Lac.9 Due to the difficulty of treating enterococcal infections with a single antibiotic, combination therapy could be an alternative approach to combat multidrug-resistant bacteria.29 Associations of vancomycin with b-lactams or with aminoglycosides have been shown to be synergistic17 and are efficient therapeutic options. The bacterial cell wall is an attractive and validated antimicrobial target.7 Several molecules acting on different steps of peptidoglycan synthesis (Fig. 1) are potent antibiotics. For example, moenomycin directly inhibits peptidoglycan glycosyltransferase,18 ramoplanin targets the pyrophosphate

Introduction

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nterococci are part of the gut microflora of humans but can be responsible for nosocomial infections in particular in immunocompromised patients.3 Treatment of enterococcal infections is challenging, as resistance to various classes of antibiotics is common, and glycopeptides vancomycin and teicoplanin are often used.2 However, an increasing number of enterococci are resistant to glycopeptides and constitute a serious public health problem. Glycopeptides inhibit peptidoglycan synthesis by binding to the C-terminal D-alanyl-D-alanine (D-Ala-D-Ala) of the pentapeptide in Lipid II blocking transglycosylation and transpeptidation reactions (Fig. 1).14,25 It has been suggested that teicoplanin predominantly acts on transglycosylation whereas vancomycin acts on transpeptidation.13 Resistance occurs through production of modified peptidoglycan precursors ending in D-Ala-D-lactate (D-Lac) or D-Ala-D-serine (D-Ser) to which glycopeptides exhibit low binding affinity. Telavancin19 and dalbavancin6 are semi-synthetic derivatives of vancomycin and teicoplanin, respectively. Their mode of

Department of Microbiology, Unite´ des Agents Antibacte´riens, Institut Pasteur, Paris, France.

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BREIDENSTEIN ET AL.

FIG. 1.

Schematic representation of the mode of action of cell wall targeting antibiotics.

domain of Lipid II,10 bacitracin interacts with undecaprenylpyrophosphate,11 fosfomycin targets the cytosolic UDP-Nacetylglucosamine enolpyruvyltransferase (MurA),12 and daptomycin, in the presence of Ca2 + , oligomerizes in micellelike structures and leads to membrane disruption.27 The antimicrobial peptide defensin plectasin, isolated from the saprophytic ascomycete Pseudoplectania nigrella, inhibits cell wall biosynthesis by directly binding the pyrophosphate moiety of Lipid II.26 It has potent activity against benzylpenicillin-resistant Streptococcus pneumoniae (minimum inhibitory concentration [MIC] 0.125–0.5 mg/L) but lower potency against Enterococcus spp. including vancomycin-resistant strains (MIC 32– ‡ 128 mg/L).20 Furthermore, plectasin was found to be synergistic with penicillin G and ceftriaxone against Staphylococcus aureus but not with erythromycin, gentamicin, ciprofloxacin, and vancomycin.22 A new plectasin variant NZ2114, which carries mutations: D9N, M13L, and Q14R, has been identified in a high-throughput mutation and screening approach,23 and it showed improved potency and better pharmacokinetic properties.1,30 To explore new alternatives against vancomycin-resistant enterococci, we investigated the activity of combinations of plectasin NZ2114 with glycopeptides and other cell wall targeting antibiotics against VanA-type vancomycin-resistant Enterococcus faecalis BM4110/pIP816-1.5 Although the strain is highly resistant to vancomycin and teicoplanin, plectasin

NZ2114 was synergistic with teicoplanin but not with vancomycin. Furthermore, we observed that dalbavancin and the structurally unrelated moenomycin were also synergistic with plectasin NZ2114. Materials and Methods Bacterial strains and growth conditions

VanA-type glycopeptide-resistant E. faecalis strain BM4110/pIP816-1, a JH2-2 transconjugant harboring plasmid pIP816-1 carrying Tn1546, was used.5 The strain was grown in brain heart infusion (BHI) broth or on agar at 37C. Antimicrobial agents and MIC determination

Plectasin NZ2114 was obtained from Novozymes, teicoplanin and telavancin from Sanofi-Aventis, dalbavancin from Pfizer, ramoplanin from Merrell Dow Research Institute, vancomycin was purchased from Merck, moenomycin from Hoechst AG, daptomycin ( + 50 mg/L CaCl2) from Selleckchem, fosfomycin, bacitracin, and penicillin G from Sigma. The antibiotics were diluted in deionized water except telavancin, which was diluted in dimethyl sulfoxide (DMSO). Telavancin and dalbavancin were supplemented with 0.002% polysorbate 80 as required by CLSI standards.8 Polysorbate 80 did not have any influence on plectasin

ANTIBIOTIC COMBINATIONS AGAINST VANA-TYPE E. FAECALIS

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activity. The broth microdilution method according to CLSI guidelines was used for MIC determination in BHI liquid. The inoculum size was 5 · 105 CFU/ml and growth inhibition was assessed after 24 hr at 37C.

mined in BHI broth according to CLSI standards with an inoculum of 5 · 105 CFU/ml. The antibiotics were tested in all combinations and growth inhibition was assessed after 24 hr at 37C. Synergism was defined as FIC £ 0.5.21

Synergy testing

Time-kill curves. Antibiotic activity was determined at subinhibitory concentrations (1/8 or 1/4 of the MIC) after 24 hr with an inoculum of 1 · 107 CFU/ml. Synergy was defined as a ‡ 2 log decrease of CFU/ml after 24 hr of incubation relative to the most active compound.4 Drug carryover was eliminated by broth dilution. The limit of detection was defined as 100 CFU/ml throughout all time-kill experiments.

Three methods were employed to determine synergism between combinations of cell wall acting antibiotics. All the experiments were repeated two to three times independently. Disk diffusion. Plectasin NZ2114 (200 mg), moenomycin (20 mg), vancomycin (4,000 mg), and teicoplanin (4,000 mg) disks were placed on agar plates at a distance, which was the sum of the two inhibition zones. An enhancement at the junction of the inhibition zones indicated synergy. The plates were inoculated with E. faecalis strain BM4110/pIP816-1 and incubated for 24 hr at 37C. Disks impregnated with plectasin NZ2114 (200 mg), ramoplanin (40 mg), bacitracin (130 mg), fosfomycin (50 mg), penicillin G (6 mg), teicoplanin (30 mg), vancomycin (30 mg), and moenomycin (20 mg) were placed on agar inoculated plates containing each of the above antibiotics at 1/4 or 1/8 MIC or no antibiotics as a control. An increase in the inhibition zone diameter compared to the control was a criterion for synergy. Checkerboard assays. The fractional inhibitory concentration (FIC) index of antibiotic combinations was deter-

Results Plectasin NZ2114 is synergistic with teicoplanin but not with vancomycin

The MICs of teicoplanin, vancomycin, plectasin NZ2114, dalbavancin, and telavancin against E. faecalis BM4110/ pIP816-1 were 1,024, 1,024, 256, 256, and 16 mg/L, respectively. The study of combinations of plectasin NZ2114 with glycopeptides was tested by double-disk diffusion. The fusion of the inhibition zones of teicoplanin and plectasin NZ2114 indicated synergy between the two drugs; in contrast, no synergy was observed with vancomycin (Fig. 2a–c, e, f). A plectasin NZ2114 disk (200 mg) had an increased inhibition zone on agar containing subinhibitory concentrations

FIG. 2. Synergy between plectasin NZ2114 and -teicoplanin, -moenomycin but not with vancomycin. (a) BHI agar; (b) BHI agar plus teicoplanin (256 mg/L); (c) BHI agar plus vancomycin (256 mg/L), (d) BHI agar plus moenomycin (0.05 mg/L) and (e–g) double-disk diffusion. BHI, brain heart infusion.

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Table 1. FIC Index of Combinations of Cell Wall Targeting Antibiotics Against Enterococcus faecalis BM4110/pIP8116-1 Antibiotic combination

FIC index

Teicoplanin-ramoplanin Teicoplanin-bacitracin Teicoplanin-fosfomycin Teicoplanin-plectasin NZ2114 Teicoplanin-vancomycin Vancomycin-ramoplanin Vancomycin-bacitracin Vancomycin-fosfomycin Vancomycin-plectasin NZ2114 Moenomycin-ramoplanin Moenomycin-bacitracin Moenomycin-fosfomycin Moenomycin-plectasin NZ2114 Moenomycin-teicoplanin Moenomycin-vancomycin Fosfomycin-plectasin NZ2114 Ramoplanin-plectasin NZ2114 Penicillin G-plectasin NZ2114 Bacitracin-penicillin G

0.29 – 0.01 0.46 – 0.16 0.39 – 0.03 0.22 – 0.04 1.03 – 0 0.96 – 0.11 1.04 – 0.01 1.04 – 0.01 0.71 – 0.06 0.45 – 0.06 0.5 – 0.08 1.11 – 0.06 0.2 – 0.05 0.29 – 0.08 0.93 – 0.2 0.82 – 0.35 0.95 – 0.11 1.06 – 0 1.03 – 0

FIC, fractional inhibitory concentration.

of teicoplanin (256 mg/L) but not of vancomycin (256 mg/L) (21 mm compared to 9 mm) (Fig. 2b, c). Similarly, plectasin NZ2114 showed synergy with dalbavancin but not with telavancin. Agar containing dalbavancin (4 mg/L) exhibited an inhibition zone for plectasin NZ2114 of 14 mm compared to 9 mm with 1 mg/L of telavancin or no antibiotic, respectively. The checkerboard assay indicated FIC indexes of 0.22, 0.26, 0.94, and 1.06 for, respectively, plectasin NZ2114-teicoplanin, -dalbavancin, -vancomycin, and -telavancin

combinations (Table 1) confirming the results of disk diffusion. For example, the concentrations of plectasin NZ2114 and teicoplanin inhibiting growth of BM4110/pIP816-1 were, each, reduced by 10-fold when combined, as opposed to the combination of vancomycin and plectasin NZ2114. Time-kill experiments at subinhibitory concentrations (1/4 or 1/8 MIC) showed that, unlike plectasin NZ2114-vancomycin combinations, those of plectasin NZ2114 with teicoplanin inhibited growth and led to killing (2 log reduction of CFU/ml compared to teicoplanin and plectasin NZ2114 alone after 24 hr) (Fig. 3a). Similarly, the combination of plectasin NZ2114 with dalbavancin resulted in > 2 log reduction of CFU/ml, whereas the combination of plectasin NZ2114 with telavancin led to no reduction in CFU/ml after 24 hr. It is surprising that teicoplanin, vancomycin, and their semi-synthetic derivatives, dalbavancin and telavancin acting on the same target, that is, the D-Ala-D-Ala terminus of the pentapeptide of Lipid II, have distinct synergistic behaviors with plectasin NZ2114. Plectasin NZ2114 is synergistic with moenomycin

To understand the basis of the synergy observed we studied combinations of plectasin NZ2114 with other cell wall targeting antibiotics, including moenomycin, ramoplanin, bacitracin, fosfomycin, penicillin G, and daptomycin. In addition to teicoplanin and dalbavancin, plectasin NZ2114 was synergistic with moenomycin (FIC index 0.2) whereas indifference was observed with all other combinations (FIC indexes close to 1) (Table 1). Using agar-induced disk diffusion assays, plectasin NZ2114 was confirmed to be synergistic with moenomycin (Fig. 2d, g) but not with the other antibiotics. When assayed by time-kill experiments using subinhibitory concentrations, plectasin NZ2114-moenomycin exhibited a 2 log decrease in CFU after 24 hr compared to the

FIG. 3. Activity of various combinations of cell wall targeting antibiotics. (a) Combinations with plectasin; (b) combinations with penicillin G or bacitracin.

ANTIBIOTIC COMBINATIONS AGAINST VANA-TYPE E. FAECALIS

FIG. 4. Summary of interactions between antibiotics targeting cell wall synthesis determined by FIC index. Connecting lines indicate synergy. FIC, fractional inhibitory concentration. most active compound alone indicating that the combination was synergistic (Fig. 3a). Combinations of cell wall targeting antibiotics

We also studied the interactions between other cell wall targeting antibiotics. Synergy was seen for all the drugs in combination with moenomycin and teicoplanin but not with

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vancomycin. However, unlike teicoplanin, moenomycin was not synergistic with fosfomycin (Table 1 and Fig. 4). Concentration-dependent synergy was studied by agarinduced disk diffusion. A disk of the tested antibiotic was deposited on an agar plate inoculated with BM4110/pIP8161 and containing increasing concentrations of either teicoplanin or vancomycin, the diameter of the inhibition zone was measured and compared with that on a plate devoid of antibiotic. For example, while at 32 mg/L (1/32 of the teicoplanin MIC) no synergy could be detected with bacitracin, at 256 mg/L (1/4 of the MIC) synergy was observed between the two antibiotics (Fig. 5). Moenomycin was also synergistic at 1/8 MIC (128 mg/L) of teicoplanin. Penicillin G was strongly synergistic with very low subinhibitory concentrations of teicoplanin (16 mg/L). Vancomycin was not synergistic with bacitracin, moenomycin, and ramoplanin but synergy with penicillin G was detected at concentrations as low as 1/32–1/16 of the vancomycin MIC (32 and 64 mg/L). For further studies we selected subinhibitory concentrations in the agar of 1/4–1/8 MIC of the antibiotics. We determined synergy, not only by agar-induced disk diffusion with teicoplanin or vancomycin in the agar but also by the reverse experiments to rule out potential limited diffusion of the antibiotics. Overall the two types of experiments gave similar results with only small changes (data not shown). Selected combinations of cell wall targeting antibiotics were further studied by time-kill experiments. The combinations teicoplanin with fosfomycin and teicoplanin with bacitracin led to a 3 log decrease in CFU after 24 hr but the same antibiotics in combination with vancomycin did not exhibit enhanced killing. However, vancomycin was synergistic

FIG. 5. Concentration-dependent synergy against Enterococcus faecalis BM4110/pIP816-1 assayed by agar-induction.

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with penicillin G (Figs. 3 and 5). We also investigated combinations of fosfomycin with bacitracin and ramoplanin with bacitracin but these were not synergistic as assayed by disk diffusion and the checkerboard assay (data not shown). Discussion

The increasing threat of glycopeptide resistance in Grampositive pathogens has led to the search for alternative strategies to treat infections due to these bacteria. Combinations of a high-dose of ampicillin plus -aminoglycosides, -daptomycin, -imipenem, or linezolid are possible alternatives for the treatment of severe vancomycin-resistant enterococci infections.16 Study of these combinations may provide additional insight on therapeutic options and could also contribute to better understanding of the mode of action of antibiotics and the selection of targets for the design of new antibacterials. Synergy between glycopeptides and b-lactams against glycopeptide-resistant enterococci was shown to be concentration dependent (Fig. 5) and attributed to the functional replacement of the low-affinity PBP5 by high-molecularweight penicillin-binding proteins (PBPs) that have high affinity for b-lactams.24 In these strains, the modified precursors ending in D-Ala-D-Lac cannot be cross-linked by PBP5, hence, high-molecular-weight PBPs (PBP1, PBP2, and PBP3) are required for cell wall synthesis. We investigated the activity of a new antimicrobial defensin, plectasin NZ2114, against VanA-type E. faecalis and found that it had little activity against E. faecalis BM4110/pIP816-1 as previously reported for plectasin.20 However, plectasin NZ2114 was synergistic in combination with teicoplanin, dalbavancin, or moenomycin (Fig. 2). For instance, the MIC of plectasin NZ2114 against BM4110/pIP816-1 was reduced from 256 to 16 mg/L in the presence of teicoplanin 128 mg/L. While these values are still high and cannot be used in clinical settings, they nevertheless indicate synergism of this combination highlighting the importance of Lipid II as an established molecular target. In contrast, we did not observe any synergism with vancomycin raising the question of the molecular basis for the observed synergy (Table 1 and Fig. 5). Plectasin and the glycopeptides teicoplanin and vancomycin, target Lipid II at different sites. Plectasin targets the pyrophosphate moiety of Lipid II26 whereas teicoplanin and vancomycin both bind the C-terminal D-Ala-D-Ala of the pentapeptide (Fig. 1).25 Thus, it is unclear why vancomycin, unlike teicoplanin, was not synergistic with plectasin NZ2114. Analysis of combinations of cell wall acting antibiotics revealed that teicoplanin is also synergistic with the majority of the antibiotics tested, whereas vancomycin was only synergistic with penicillin G. This difference in behavior against VanA-type E. faecalis suggests a difference in the mode of action of the two glycopeptides. Teicoplanin with a lipidated tail anchors in the bacterial membrane and, therefore, interacts preferentially with Lipid II in close proximity of the membranebound transglycosylase domain of penicillin-binding proteins and not with their transpeptidase domain.13 On the contrary, vancomycin inhibits predominantly transpeptidation as it does not anchor to the bacterial membrane and is therefore more broadly distributed in the peptidoglycan layers.13 Thus, it appeared that inhibition of transglycosylation is important for synergy to occur, which is strengthened by the fact that moenomycin, an efficient transglycosylation inhibitor, was like

BREIDENSTEIN ET AL.

teicoplanin synergistic with ramoplanin, bacitracin, penicillin G, daptomycin, and plectasin NZ2114 (Figs. 4 and 5). The differences observed for cell wall targeting antibiotics combinations reveal the mechanistic complexity of synergism and highlight the importance of further investigating the mode of action of antibiotics. We also tested second-generation lipoglycopeptides telavancin and dalbavancin, semi-synthetic derivatives of vancomycin and teicoplanin, respectively. In agreement with previous studies,6,15 VanA-type E. faecalis was resistant to dalbavancin and telavancin with MICs lower than those of vancomycin and teicoplanin. Like teicoplanin, dalbavancin synergized with plectasin NZ2114, which might be attributed to the presence of the lipophilic chain. On the contrary, no synergy was observed between telavancin and plectasin NZ2114. This further indicates that despite the common interaction of dalbavancin and telavancin with D-Ala-D-Ala of Lipid II, they might have differences in their mode of action that could account for the observed synergy. We also analyzed the effect of plectasin NZ2114 in combination with lipopeptide daptomycin, the drug of choice against vancomycin-resistant enterococci.16 Our data indicated no synergism, which further supports the importance of transglycosylation for synergy since daptomycin involves a Ca2 + -dependent membrane depolarization.27 In conclusion, combinations of teicoplanin, moenomycin, or dalbavancin with plectasin NZ2114 were synergistic against vancomycin-resistant E. faecalis BM4110/pIP816-1 and shed light on alternative strategies to design potential therapeutic options for infections caused by VanA-type enterococci. While we cannot provide a mechanism for synergy and can only speculate a possible inhibition of transglycosylation, further studies are required for a better understanding of the molecular basis of the synergy observed. Acknowledgments

This work was funded by an unrestricted grant from Reckitt Benckiser. E.B.M.B. was supported by the Fondation pour la Recherche Me´dicale and D.M.-C. by Reckitt Benckiser. We thank H.H. Kristensen from Novozymes for supplying plectasin NZ2114, F. Lebreton for daptomycin, P. Reynolds for critical reading of the article, and H.-J. Hong, M. Galimand, and T. Schneider for helpful discussions. Disclosure Statement

No competing financial interest exists. References

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Address correspondence to: Patrice Courvalin, MD, FRCP Department of Microbiology Unite´ des Agents Antibacte´riens Institut Pasteur 75724 Paris Cedex 15 France E-mail: [email protected]

Antimicrobial Activity of Plectasin NZ2114 in Combination with Cell Wall Targeting Antibiotics Against VanA-Type Enterococcus faecalis.

Antimicrobial peptide plectasin targeting bacterial cell wall precursor Lipid II has been reported to be active against benzylpenicillin-resistant Str...
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