JAC Advance Access published online on February 26, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkl539
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Potent, synergistic inhibition of Staphylococcus aureus upon exposure to a combination of the endopeptidase lysostaphin and the cationic peptide ranalexin
Centre for Biomolecular Sciences, School of Biology, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK
* Corresponding author. Tel: +44-1334-463406; Fax: +44-1334-462595; E-mail: pjc5{at}st-andrews.ac.uk
Received 10 October 2006; returned 5 December 2006; revised 11 December 2006; accepted 12 December 2006
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Abstract |
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Objectives: Successful treatment of infections involving multiply drug-resistant methicillin-resistant Staphylococcus aureus (MRSA) is becoming increasingly difficult. In this work, we have investigated the potential of combining lysostaphin with cationic antimicrobial peptides to effectively inhibit Staphylococcus aureus.
Methods: S. aureus strains were grown in 96-well plates in the presence of increasing concentrations of lysostaphin and the peptides ranalexin, dermaseptin S3(1-16) or magainin 2. Growth was determined visually after 48 h and the plates imaged, or by automated optical density readings in a plate reader. Susceptibility to the combination of lysostaphin and ranalexin was also determined by viable cell counts. The efficacy of combined lysostaphin and ranalexin on a solid surface was tested via disc diffusion assays.
Results and conclusions: Combination of lysostaphin with ranalexin resulted in potent, synergistic inhibition of S. aureus MSSA476 and MRSA252. Synergistic inhibition was specific for lysostaphin-susceptible staphylococci and was not observed with clinical isolates of the Gram-negative Escherichia coli, or other Gram-positive organisms, such as Enterococcus faecalis. Lysostaphin was not specifically synergistic with ranalexin alone. Synergy was also observed with two other cationic antimicrobial peptides, magainin 2 and dermaseptin s3(1-16); although combination with ranalexin was most potent. Synergistic inhibition by ranalexin in combination with lysostaphin resulted in an enhanced bactericidal effect. Importantly, synergy between lysostaphin and ranalexin was also observed after impregnation and drying in filter paper discs that clearly inhibited growth of S. aureus on the surface of agar; a solid, porous matrix. Thus, the combination could represent a novel route to target wounds infected with drug-resistant MRSA via dressings impregnated with the two compounds.
Key Words: MRSA , magainin 2 , dermaseptin S3 , antimicrobial peptides
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Introduction |
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Methicillin-resistant Staphylococcus aureus (MRSA) strains are an important cause of hospital and community infection. A potential source of new antibiotics to target MRSA are cationic antimicrobial peptides that are produced by all living creatures as a defence against invasive microbial pathogens. Ranalexin is a 20 amino acid, cationic peptide originally isolated from the skin of the American bullfrog, Rana catesbeiana.1 Ranalexin is a member of a family of antimicrobial peptides produced by the Rana frog species that contain a single intramolecular disulphide bond which forms a heptapeptide ring at the carboxy terminus that plays a key role in the antimicrobial action of ranalexin.1 Ranalexin has potent activity against Gram-positive bacteria in vitro, particularly S. aureus, displaying MICs between 1 and 32 mg/L.2
Illustrating the potential of ranalexin to be developed for clinical application in vivo, ranalexin significantly reduced the growth of methicillin-resistant Staphylococcus epidermidis in a rat model of vascular graft infection.3 Furthermore, the combination of ranalexin with a range of clinically used antibiotics resulted in significant inhibitory synergy and enhanced efficacy against 40 MRSA strains.2 The use of combinations of antimicrobials is common in the clinical setting and expands the spectrum of organisms that can be targeted, prevents the emergence of resistant organisms, decreases toxicity by allowing lower doses of both agents and can result in synergistic inhibition. Here, we describe potent inhibitory synergy between ranalexin and the 27 kDa glycylglycine endopeptidase, lysostaphin on S. aureus. Lysostaphin is produced by Staphylococcus simulans and has potent antistaphylococcal activity because the enzyme specifically cleaves the abundant pentaglycine cross-bridges of the staphylococcal cell wall leading to cell lysis.4
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Materials and methods |
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Bacteria
S. aureus MRSA252 and MSSA476 were kindly provided by Dr Mark Enright, University of Bath. Extended spectrum ß-lactamase (ESBL) Escherichia coli, vancomycin-resistant Enterococcus faecalis, vancomycin-resistant Enterococcus faecium and Streptococcus sanguis were provided by Dr Cyril Lafong, Fife Area Laboratory, Victoria Hospital, Kirkcaldy, UK. All strains were maintained on Tryptone Soya Agar (TSA; Oxoid, Basingstoke, UK).
Ranalexin was synthesized according to the published sequence1 by Peptide Protein Research Ltd, Wickham, UK, to >95% purity and verified by HPLC and mass spectrometry. A stock solution of 50 mg/mL in water was used. Lysostaphin from S. simulans was purchased from Sigma (Saint Louis, Missouri, USA) as a lyophilized powder containing 60% protein (specific activity: 500 units/mg protein). A stock solution of 2 mg/mL in water was used.
Cells were grown to mid-exponential phase in Tryptone Soya Broth (TSB; Oxoid, Basingstoke, UK) and diluted in fresh medium to a starting optical density at 600 nm of 0.001 (approximately 1.0 x 106 cells/mL). Growth of 100 µL cultures was measured in 96-well plates (Greiner Bio-one Ltd, Stonehouse, UK), using TSB as a blank. Plates were incubated at 37°C with shaking for 24 h in a PowerWave XS automated microplate spectrophotometer (Bio-tek Instruments Inc., Winooski, VT, USA) with optical density (600 nm) readings taken every 11 min. Data were collected automatically using KC4 v.3.2 software (Bio-tek, Instruments Inc., Winooski, VT, USA) and analysed in Microsoft Excel.
The 96-well plates were set up exactly as above and incubated for 48 h at 37°C without shaking. Plates were scanned on an ImageScanner (GE Healthcare UK Ltd, Chalfont St Giles, UK) using ImageMaster Labscan v.3 software (GE Healthcare UK Ltd).
Mid-exponential cells (200 µL in TSB) were exposed to ranalexin, lysostaphin, or combinations of both, for 30 min in a 96-well microtitre plate. Viable cells were determined by serial dilution and plating on TSA. Colonies were counted after 24 h incubation at 37°C.
This method was as described in Kusuma and Kokai-Kun.5 Briefly, 6 mm paper discs were impregnated with ranalexin, lysostaphin, or combinations of both, and allowed to dry at room temperature overnight. TSA plates were spread with mid-exponential cultures, dried, and discs applied. Plates were incubated for 96 h at 37°C and images scanned.
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Results and discussion |
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The effect of combination of ranalexin with lysostaphin on growth over 48 h is shown in Figure 1(a). Visible growth of MSSA476 and MRSA252 was inhibited by approximately 35–40 mg/L ranalexin (Figure 1a). Lysostaphin alone had an MIC of approximately 0.75 mg/L on MSSA476 and approximately 0.45 mg/L on MRSA252 (Figure 1a). Significantly, combination of ranalexin with lysostaphin resulted in synergistic inhibition of growth because complete inhibition was achieved upon exposure to lower concentrations of the two compounds than when either was used alone, for example 5 mg/L ranalexin with 0.15 mg/L lysostaphin completely inhibited the growth of MRSA252, and 5 mg/L ranalexin with 0.3 mg/L lysostaphin inhibited MSSA476.
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Using lower concentrations of the two compounds, we defined the boundaries of growth (Figure 1b). Thus, 1 mg/L ranalexin with 0.15 mg/L lysostaphin and 5 mg/L ranalexin with 0.15 mg/L lysostaphin represented the minimum concentrations that abolished growth of MRSA252 and MSSA476 respectively. Synergistic growth inhibition was demonstrated more precisely using an automated spectrophotometer (Figure 1c). The growth curves clearly show concentrations of ranalexin and lysostaphin that had no effect on growth of either S. aureus strain alone, but when they were combined, completely inhibited growth over a 24 h period.
Synergy has also been observed between lysostaphin and polymyxin B, a peptide with some structural resemblance to ranalexin.1,6 Also, combination of mammalian cationic peptides and the human innate defence protein lysozyme resulted in synergistic inhibition.7 This implies that combination of membrane-active peptides with cell wall-degrading enzymes is a general mechanism of antimicrobial synergy.
A potential barrier to clinical development of lysostaphin is the ease with which resistance can be selected in vitro.8 However, resistance is less likely to be selected by combining ranalexin and lysostaphin because bacteria are exposed to two compounds with differing mode of action.
We tested the effect of the combination versus a range of Gram-negative and -positive human pathogens (data not shown). No synergistic inhibition by ranalexin and lysostaphin was observed on hospital isolates of Escherichia coli ESBL, vancomycin-resistant Enterococcus faecalis, vancomycin-resistant Enterococcus faecium and Streptococcus sanguis (data not shown). Lysostaphin cleaves between the second and third glycines of the pentaglycine cross-bridges present in S. aureus peptidoglycan.4 No synergistic inhibition was detected because Gram-negative bacteria, such as E. coli, or the Gram-positive bacteria tested do not contain glycine residues in their cross-bridges. Thus, the potent, synergistic inhibition that occurs upon combination of ranalexin and lysostaphin is specific for lysostaphin-susceptible staphylococci.
Next, we investigated the importance of the antimicrobial peptide component of the synergistic combination by combining lysostaphin with two other amphibian cationic antimicrobial peptides, magainin 2 and dermaseptin S3(1-16) (data not shown).9,10 Alone, neither magainin 2, or dermaseptin S3(1-16), had any inhibitory effect on either strain of S. aureus (up to 50 mg/L). However, combination of each peptide with lysostaphin resulted in significant synergistic inhibition, for example the MICs of the combined compounds were 5 mg/L dermaseptin S3(1-16), or magainin 2, and 0.05 mg/L lysostaphin. Thus, the combination of cationic antimicrobial peptides with lysostaphin results in synergistic inhibition that must be due to a general inhibitory action of these peptides that either enhances, or is enhanced by lysostaphin.
A 30 min exposure to ranalexin (up to 10 mg/L) had no effect on viable cell numbers (data not shown). Identical duration of exposure to increasing concentrations of lysostaphin (from 0.01 to 0.1 mg/L) resulted in some decrease in viability, for example 0.06 and 0.1 mg/L lysostaphin resulted in a 0.5 log and a 2 log reduction (>99%) respectively. Notably, combination of 8 mg/L ranalexin with increasing concentrations of lysostaphin resulted in a dramatic reduction in viability after a 30 min exposure, for example 8 mg/L ranalexin with 0.08 mg/L lysostaphin resulted in >4 log reduction in cell numbers. Thus, the synergistic inhibition by ranalexin and lysostaphin results in an enhanced bactericidal effect. The inhibitory action of the combination may be due to lysostaphin cleaving the cell wall peptidoglycan, allowing greater access of the membrane-active peptide ranalexin to the surface of the plasma membrane.
Next, we determined whether the combination was still synergistic on a porous surface (data not shown). Exposure of lawns of S. aureus MRSA252 and MSSA476 on agar to sterile, filter paper discs impregnated with 10 µg ranalexin, resulted in no evident zones of inhibition even after 96 h exposure. Lysostaphin impregnated discs showed only minor zones of inhibition at the higher concentrations tested of 1 and 2 µg. However, exposure to discs impregnated with combinations of 10 µg ranalexin with 1 and 2 µg of lysostaphin clearly revealed synergistic inhibition, with larger, more significant zones of inhibition after 96 h exposure. Thus, we have demonstrated that the synergistic combination effectively kills MRSA growing on a porous surface even after the compounds are allowed to dry on an absorbent matrix beforehand. This implies that wounds infected with drug-resistant MRSA could be effectively treated with dressings impregnated with the two compounds.
In summary, we have demonstrated potent, synergistic inhibition of S. aureus, including MRSA, by combination of the endopeptidase, lysostaphin, with cationic, antimicrobial peptides, such as ranalexin. The combination could represent a novel route to target infections by multiply drug-resistant strains of S. aureus.
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The work described is the subject of a UK patent application (No. 0615500.6), in which the authors have no financial interest at present.
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Acknowledgements |
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The authors acknowledge the support of the BBSRC for this work under the Structural Proteomics of Rational Targets initiative (Grant No. BBS/B/14426).
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References |
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