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Antimicrobial Chemotherapy. In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against. Gram-positive ...
Antimicrobial Chemotherapy

In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria Sibel Dosler, A. Alev Gerceker Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Istanbul University, Turkey The in vitro activities of two antimicrobial cationic peptides, melittin and nisin alone and in combination with frequently used antibiotics (daptomycin, vancomycin, linezolid, ampicillin, and erythromycin), were assessed against clinical isolates of methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus and Enterococcus faecalis. Using the broth microdilution method, minimum inhibitory concentration (MIC) ranges of melittin and nisin against all strains were 2–8 mg/ml and 2–32 mg/ml respectively. In combination studies performed with the microdilution checkerboard method using a fractional inhibitory concentration index of (0.5 as borderline, synergistic interactions occurred more frequently with nisin– ampicillin combination against MSSA and nisin–daptomycin combination against E. faecalis strains. The results of the time-killing curve analysis demonstrated that the concentration dependent rapid bactericidal activity of nisin, and that synergism or early synergism was detected in most strains when nisin or melittin was used in combination with antibiotics even at concentrations of 0.56MIC. Keywords: Melittin, Nisin, In vitro activity, Combination, Time-killing curve

Introduction Gram-positive pathogens, primarily Staphylococcus aureus and Enterococcus species, remain as an important cause of nosocomial infections due to their increased resistance, especially in intensive care units, where the use of antimicrobial agents is highest and the host is most susceptible.1 S. aureus is one of the major human pathogens associated with a variety of infections, both in the community and in hospitals, and range from moderate to severe. This is especially true with methicillin-resistant S. aureus (MRSA), known to be a major cause of hospital and community associated infections.2,3 Infections with MRSA have led to a significant increase in the use of the antibiotic, vancomycin, with the consequential development of intermediate and resistant pathogens, such as vancomycin-intermediate S. aureus or vancomycin-resistant enterococci (VRE). The natural resistance of enterococci to penicillin, cephalosporins, and quinolones, and acquired virulence genes resulting in hospitaladapted clones, has encouraged its emergence in recent years.4 Enterococcus faecalis is the most common enterococcal species in human gastrointestinal flora, and has emerged as a multiresistant nosocomial Correspondence to: S. Do¨sler, Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 34116 Beyazit, Istanbul, Turkey. Email: [email protected]

ß 2012 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947812Y.0000000007

pathogen that may cause a range of different infections including urinary tract infections, sepsis, and endocarditis in immunocompromised and critically ill patients.5 Since levels of antibiotic resistance have been increasing at an alarming rate worldwide, natural bactericidal compounds such as cationic peptides seem to be one of the most preferable classes of antimicrobial substances for use in the near future as therapeutic agents.6,7 Among antimicrobial cationic peptides, melittin is a 26 amino acid, amphipathic, alpha-helical and hemolytic peptide which is the principal toxic component found in the venom of the European honey bee Apis mellifera. Melittin is active against a wide range of microorganisms including Gram-negative and Gram-positive bacteria. Melittin is probably the most extensively studied membrane-active lytic peptide for monitoring mechanisms of pore formation and lipid– protein interactions in membranes.8 Nisin, which is a 34-residue lantibiotic peptide, isolated from the nonpathogenic bacteria Lactococcus lactis, belongs to a special group of antimicrobial peptides called bacteriocins.9 Lantibiotics are gene-encoded, ribozomally synthesized and quorum-sensing controlled product peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues.10 Nisin has a rapid bactericidal activity against Gram-positive bacteria including multidrug resistant pathogens.

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In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria

In this study, we investigated the in vitro activities of two antimicrobial cationic peptides, melittin and nisin, either used alone or in combination with frequently used antibiotics such as daptomycin, vancomycin, linezolid, ampicillin, and erythromycin against clinical isolates of methicillin-susceptible S. aureus (MSSA), MRSA, and E. faecalis strains.

Materials and Methods Microorganisms Forty-one clinical isolates, consisting of 15 MSSA, 11 MRSA, and 15 E. faecalis strains, obtained from the Clinical Microbiology Laboratories of Istanbul University, Istanbul Faculty of Medicine, Turkey, were tested. The control strains, S. aureus ATCC 29213 and E. faecalis ATCC 29212, were used to verify the accuracy of the microdilution test procedure.11

Antimicrobial substances

Determination of the fractional inhibitory concentration index (FICI)

Cation-adjusted Mueller–Hinton broth (Difco Laboratories) was used for the minimum inhibitory concentration (MIC) determinations, the microbroth checkerboard technique and the time-killing curve (TKC) analyses. Tryptic soy agar (Difco Laboratories) was used for minimum bactericidal concentration (MBC) determinations and colony counts.

The effects of antibiotics in combination were assessed by using the microbroth checkerboard technique.16 Strains with MICs ranging from 0.25 to 2 mg/ml for vancomycin, 0.125 to 4 mg/ml for daptomycin, 1 to 4 mg/ml for linezolid, 0.5 to 8 mg/ml for melittin, 0.25 to 64 mg/ml for ampicillin and erythromycin, and 2 to 32 mg/ml for nisin were selected. Each microtitre well containing the mixture of antibiotics was inoculated with a 4- to 6-hour broth culture diluted to give a final concentration of approximately 56105 cfu/ml. After incubation at 37uC for 18–20 hours, the FICI was determined as the combined concentration of antibiotics divided by the single concentration. The combination value was derived from the highest dilution of the antibiotic combination that permited no visible growth. With this method, synergy was defined as an FICI of (0.5, no interaction as an FICI of .0.5 to 4 and antagonism as an FICI of .4.0.17 Experiments were performed in duplicate.

MIC and MBC determinations

Determination of TKCs

MICs of antibiotics were determined by the microbroth dilution technique as described by the Clinical and Laboratory Standards Institute (CLSI).12 Serial twofold dilutions ranging from 0.125 to 256 mg/ml for daptomycin and ampicillin, from 0.06 to 128 mg/ml for vancomycin and erythromycin, and from 0.03 to 64 mg/ml for linezolid were prepared in cation-adjusted Mueller–Hinton broth. The MICs of antimicrobial cationic peptides were determined by using a broth dilution assay modified from the method of CLSI, since cationic peptides bind polystyrene.13 Briefly, serial twofold dilutions ranging from 0.3 to 640 mg/ml were prepared in 0.2% bovine serum albumin with 0.01% acetic acid buffer in Eppendorf tubes (polypropylene). For each concentration, 11 ml was added to each corresponding well in 96-well polypropylene (Greiner 650261) microtitre plates. Each well was inoculated with 100 ml of a 4- to 6-hour broth culture that gave a

In order to observe the dynamic picture of the bactericidal activity of melittin and nisin alone and in combination with antibiotics, the TKC method was used, as described previously,15 by testing melittin and nisin at 0.56 and 16 the MIC and antibiotics at 16 the MIC against six clinical strains, consisting of two MSSA, two MRSA, and two E. faecalis. An antibiotic-free control was included for each strain. Experiments were performed in duplicate. TKCs were constructed by plotting mean colony counts (log10 cfu/ml) versus time. The lower limit of detection for time-kill assays was 1 log10 cfu/ml. Antimicrobial carry-over was controlled by the inhibition of colonial growth at the site of the initial streak according to NCCLS guidelines.15 The results were interpreted by the effect of the combination in comparison with the effect of the most active agent alone. Synergy and antagonism were defined as a 2

Two cationic peptides, melittin and nisin, were obtained from Sigma-Aldrich (Istanbul, Turkey) and antibiotics; daptomycin was provided by Novartis Pharmaceuticals (Istanbul, Turkey), vancomycin, linezolid and erythromycin by Kocak Pharmaceutical Inc. (Istanbul, Turkey) and ampicillin by Fako Pharmaceutical Inc. (Istanbul, Turkey). Stock solutions from dry powders were prepared at a concentration of 1280 mg/L for cationic peptides and 5120 mg/L for antibiotics and stored frozen at 280uC for up to 6 months.

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final concentration of 56105 colony-forming units per ml (cfu/ml) in the test tray.14 The trays were covered and placed in plastic bags to prevent evaporation and incubated at 37uC for 18–20 hours. The MIC was defined as the lowest concentration of antibiotic to produce complete inhibition of visible growth. Experiments were performed in duplicate. MBCs were determined at the end of the incubation period by removing two 10 ml samples from each well containing no visible growth, and these were then plated onto tryptic soy agar. Resultant colonies were counted after an overnight incubation at 37uC. The MBC was defined as the lowest concentration of antimicrobials producing at least 99.9% killing of the initial inoculums.15

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Table 1 In vitro activities of antimicrobial agents against clinical strains (mg/ml) MIC (mg/ml) Strains/antimicrobials MSSA (n515) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin Melittin Nisin MRSA (n511) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin Melittin Nisin E. faecalis (n515) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin Melittin Nisin

Percent inhibited at CLSI breakpoints

Range

50%

0.125–1 0.25–2 1–4 0.25–256 0.25–128 0.5–4 2–32

0.25 0.5 2 8 1 2 8

90%

Susceptible

MS

Resistant

– – – 93 33

0.5 1 4 64 64 4 16

100 100 100 7 47

– – – – 20

0.25–1 0.5–1 1–4 16–.256 32–.128 0.5–4 2–32

0.5 0.5 2 64 64 2 4

1 0.5 4 .256 .128 2 16

100 100 100 – –

– – – – –

1–4 0.25–2 1–2 0.5–256 0.25–.128 1–8 4–32

1 0.5 1 8 4 4 16

2 2 2 128 .128 8 32

100 100 100 53 20

– – – – 33

– – – 100 100

– – – 47 47

Note: CLSI breakpoints susceptible and resistant to daptomycin were (1 and – mg/ml, vancomycin were (2 and >16 mg/ml, linezolid were (4 and >8 mg/ml, ampicillin were (0.25 and >0.5 mg/ml and erythromycin (0.5 and >8 mg/ml, for S. aureus respectively; to daptomycin were (4 and – mg/ml, vancomycin were (4 and >32 mg/ml, linezolid were (2 and >8 mg/ml, ampicillin were (8 and >16 mg/ml and erythromycin were (0.5 and >8 mg/ml, for E. faecalis respectively. There is no any breakpoint on antimicrobial cationic peptides susceptibilities MS; moderately susceptible, n: number of strains.

log10 decrease or increase respectively; in the colony count at 24 hours. Bactericidal activity was defined as a >3 log10 cfu/ml decrease from the initial inoculum.

faecalis (Table 1). The MIC values of the antibiotics against the quality control strains were within the accuracy range of the CLSI throughout the study.11 There was no significant difference between the bactericidal and inhibitory concentrations of the bactericidal antibiotics and the antimicrobial cationic peptides. The MBC values were usually equal to or two times greater than the MIC values (data not shown).

Results Susceptibility The in vitro activities of the studied antibiotics and antimicrobial cationic peptides were examined against the 41 clinical isolates of MSSA, MRSA, and E.

Table 2 Per cents of synergisms and number of isolates showing the following FICIs of melittin in combination with antibiotics against clinical strains FICIs Bacteria/antibiotics MSSA (n515) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin MRSA (n511) Daptomycin Vancomycin Linezolid Erythromycin E. faecalis (n515) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin

Synergism (%)

0.25

0.375

0.5

0.75

1

1.25

1.5

– 13 – 36 14

– – – – –

– – – 2 –

– 2 – 3 2

5 4 5 5 5

4 8 6 4 7

2 – 1 – –

4 1 2 – –

– 18 – 25

– – – –

– – – –

– 2 – 2

4 4 2 3

3 3 3 3

2 – 3 –

2 2 3 –

7 33 13 31 17

– – – – –

– – – 1 –

1 5 2 3 2

7 5 7 4 –

5 3 5 3 7

– – – – 1

2 2 1 2 2

Note: n: number of strains.

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Figure 1 Time-kill determinations for two MSSA strains after treatment with nisin (A, B) or melittin (C, D) at 16 MIC and 0.56 MIC alone or in combination with daptomycin (dap), vancomycin (van), linezolid (lin), amphicillin (amp), and erythromycin (eryt) at 16 MIC. The x-axis represents the killing time, and the y-axis represents the logarithmic MSSA survival.

Checkerboard The results of the experiments in which melittin and nisin were used in combination with the studied antibiotics against the clinical isolates, are shown in

Tables 2 and 3 respectively. With an FICI of (0.5 as borderline, synergistic interactions occurred more frequently with the nisin–daptomycin and nisin– linezolid combinations against E. faecalis (80 and

Table 3 Per cents of synergisms and number of isolates showing the following FICIs of nisin in combination with antibiotics against clinical strains FICIs Bacteria/antibiotics

Synergism (%)

0.25

33 40 27 64 14

– – – 1 –

36 27 9 25 80 40 73 54 17

MSSA (n515) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin MRSA (n511) Daptomycin Vancomycin Linezolid Erythromycin E. faecalis (n515) Daptomycin Vancomycin Linezolid Ampicillin Erythromycin Note: n: number of strains.

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0.375

0.5

0.75

1

1.25

1.5

1 – 1 2 –

4 6 3 6 2

5 2 4 4 4

4 7 5 1 8

– – 1 – –

1 – 1 – –

– – – –

1 – – –

3 3 1 2

3 5 2 4

4 3 3 2

– – 4 –

– – 1 –

1 – – – –

4 – 2 2 –

7 6 9 5 2

3 4 2 2 1

– 5 2 2 6

– – – – –

– – – 2 3

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In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria

Figure 2 Time kill determinations for two MRSA strains after treatment with nisin (A, B) or melittin (C, D) at 16 MIC and 0.56 MIC alone or in combination with daptomycin (dap), vancomycin (van), linezolid (lin), amphicillin (amp), and erythromycin (eryt) at 16 MIC. The x-axis represents the killing time, and the y-axis represents the logarithmic MRSA survival.

73% respectively), than with the nisin–ampicillin combination against MSSA strains (64%). The FICI can help to quantify the degree or potency of synergism. As shown in Table 3, low FICI values which were ,0.5 occurred mostly with combinations that contained nisin. On the other hand, the combination studies where melittin was used, synergism was mostly observed at an FICI of 0.5 (Table 2). No antagonism was observed with any of the combinations.

Time-kill kinetics The results of the TKC analysis showed a rapid bactericidal activity with nisin in a concentrationdependent manner, with a 3-log kill determined within one h for the two E. faecalis strains and for each of the MSSA and MRSA strains with MIC concentrations (Figs. 1–3). Killing was also rapid with nisin at 0.56the MIC, but with melittin the bactericidal effects of the antibiotics were considerably slower. The TKC analysis also showed enhanced killing of most tested strains when melittin and nisin were used in combination with antibiotics. Synergisms were detected more frequently with time-kill studies than

by the checkerboard method. The synergistic interactions of these antimicrobial agents were also achieved with combinations of melittin and nisin used at 0.56 the MICs (Figs. 1–3). Earlier synergistic effects were observed with most of the strains at both 4 and 7 hours.

Discussion Antimicrobial cationic peptides are widely distributed in nature, existing in organisms from insects to plants to mammals and non-mammalian vertebrates as substantial components of the innate immune system. Cationic peptides have rapid action and a broad spectrum of activity against infectious agents including bacteria, fungi, parasites, and viruses. Furthermore, many of the mechanisms of antibiotic resistance that limit the use of other antibiotics do not affect cationic peptides.18,19 In our study, the two cationic peptides examined, melittin and nisin, showed good antibacterial activity against MSSA, MRSA, and E. faecalis strains. It is notable that, contrary to other antibiotics, there were no major differences in the MIC ranges and MIC50/MIC90 Journal of Chemotherapy

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Figure 3 Time-kill determinations for two E. faecalis strains after treatment with nisin (A, B) or melittin (C, D) at 16 MIC and 0.56 MIC alone or in combination with daptomycin (dap), vancomycin (van), linezolid (lin), amphicillin (amp), and erythromycin (eryt) at 16 MIC. The x-axis represents the killing time, and the y-axis represents the logarithmic E. faecalis survival.

values between the MRSA and MSSA strains. Other researchers20,21 have reported similar results. Although the MIC procedure is still the gold standard for determining the activities of antimicrobial agents, it does not provide any information about the time course of the antibiotic’s activities. This limitation can be overcome with the use of TKC analysis. The results of TKC analysis in our study showed that the two cationic peptides, and in particular with nisin, was rapidly bactericidal in a concentration-dependent manner against all the studied strains within 1–4 hours, which was followed by a regrowth at 24 hours. Nisin, having unusual amino acids, has emerged as one of the most studied lantibiotics. Importantly, there is little evidence of stable resistant mutants arising in food products that have been treated with nisin, which has been used for almost 50 years and it has low toxicity for humans.9 In 1988, the Food and Drug Administration evaluated nisin for its safety, and since then it has been used extensively as a safe food preservative.22 Nisin, like other bacteriocins, is

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different from antibiotics in respect to their synthesis, mode of action, toxicity, and resistance mechanisms.23 The lack of cross-resistance in nisin is probably due to its double mode of action, i.e. the binding to lipid II and the subsequent inhibition of cell wall biosynthesis, as well as its ability to form pores in the cytoplasmic membrane of bacteria.24 Melittin has a very effective and rapid antimicrobial activity against a wide variety of microorganisms, and recent efforts have focused on designing or synthesizing short analogs of naturally occurring antimicrobial peptides. For example, Cao et al.,25 designed, synthesized, and expressed the hybrid peptide analogs CA(1–7)M(4–11) and CB(1–7)M(4– 11) in Pichia pastoris X33, and then evaluated their antibacterial activities and toxicities to human erythrocytes. The hybrid containing portions of the amino acid sequences of cecropin-A and melittin with differing properties have demonstrated higher antimicrobial activity without introducing the hemolytic properties of melittin.

Dosler and Gerceker

In vitro activities of antimicrobial cationic peptides; melittin and nisin, alone or in combination with antibiotics against Gram-positive bacteria

Besides the antimicrobial characteristics, cationic peptides also possess an ‘enhancer’ activity, i.e. synergy with classical antibiotics; the more resistant an isolate is to a given antibiotic, the more profound the enhancement activity is by the particular cationic peptide. Thus, cationic peptides have the ability to serve as anti-resistance compounds.26 Therefore, we tried to determine whether melittin and nisin would show the synergistic interactions in their combinations. By using the microbroth checkerboard technique, synergistic interactions were seen more frequently with nisin. However, with melittin combinations, which generally have an additive effect, synergism was only observed in 7–36% of the strains. The results of the TKC studies demonstrated that synergism at 24 hours was obtained with almost all melittin and nisin combinations in all of the strains tested, even when they were used at low concentrations of 0.56 the MIC. While melittin combinations were found additive in the checkerboard studies, the frequency of synergism in the TKC analyses were similar to that of nisin. In addition, Giacometti et al.27 showed that CA(1–7)M(2–9)NH2 (a cecropin-A and melittin hybrid peptide) is active against MRSA, and its activity is enhanced when combined with several antimicrobial agents. Our data confirm that the cationic peptides have beneficial antimicrobial activities against Gram-positive bacteria and exhibit synergistic interactions with various antibiotics. These findings strengthen the possibility that cationic peptides may have a place as a new and active group of antimicrobial agents, either as a single agent or as an adjuvant for antimicrobial chemotherapy in the near future.

Acknowledgements This work was supported by the Research Fund of Istanbul University (project no. 578/14082006).

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