1 Department of Biological and Chemical Sciences, Faculty of Pure and Applied Sciences, The University of the West Indies, Cave Hill Campus, PO Box 64, Bridgetown, Barbados; 2 Département de Biochimie et Microbiologie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada
Received 4 May 2005; returned 11 June 2005; revised 12 July 2005; accepted 26 July 2005
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Abstract |
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Methods: Intraperitoneal infection was induced with a methicillin-susceptible Staphylococcus aureus strain in mice. Some of the mice were simultaneously injected intraperitoneally with mutacin B-Ny266, some with the vehicle only and some with vancomycin.
Results: While there was 70 and 100% mortality in the control groups of mice, no mortality was observed in the mice injected with vancomycin or mutacin B-Ny266.
Conclusions: The results presented here show, for the first time, the in vivo efficacy of a mutacin (B-Ny266) against an experimental intraperitoneal infection by S. aureus in a mouse model.
Keywords: antimicrobial peptides , lantibiotics , Streptococcus mutans , MIC , MBC
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Introduction |
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In order to eventually be used as an antibiotic, an inhibitory substance has to be evaluated in pre-clinical and clinical tests. As part of these tests, we present here results showing that the lantibiotic mutacin B-Ny266 is active in vivo against Staphylococcus aureus in a mouse model infection. This is the first time that the efficacy of a mutacin has been demonstrated in vivo.
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Materials and methods |
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The mutacin-producing strain, S. mutans Ny266 (ATCC 202022) was a kind gift from J. S. van der Hoeven (Department of Preventive Dentistry, University of Nijmegen, Nijmegen, The Netherlands) and was originally isolated by A. H. Rogers in South Australia (Department of Dentistry, University of Adelaide, South Australia) under the designation of T2.18 Escherichia coli 0025 (hyperpermeable strain), Pseudomonas aeruginosa 0028, Candida albicans 0030, vancomycin- resistant Enterococcus faecalis (VREF) 0032, methicillin-resistant S. aureus (MRSA) 0034, and methicillin-susceptible S. aureus (MSSA) Smith are IntraBiotics Pharmaceutical Inc. (Palo Alto, CA, USA) strains.
Mutacin B-Ny266 production and purification
Mutacin B-Ny266 was produced in supplemented cheese whey permeate (SWP) which consists of 6% cheese whey permeate (kind gift from Agropur, Granby, QC, Canada) to which 3% yeast extract (Rossel, Montreal, QC, Canada) is added19 and purified by hydrophobic chromatography as previously described15 with the following modifications: the activity of the culture supernatant was first concentrated using Amberlite (XAD-7) columns and HCl was used instead of trifluoroacetic acid in the HPLC chromatographic steps.20 The mutacin B-Ny266 preparation was evaluated to be >99.9% pure by capillary electrophoresis.15
In vitro activity
Pure mutacin was suspended in Clark and Lubs buffer, pH 2 at 640 mg/L. The antibacterial control compounds were suspended in double-distilled water at 6.4 mg/mL. The antifungal control, amphotericin B, was suspended in DMSO at 6.4 mg/mL. Two-fold serial dilutions were made in the corresponding diluent and further diluted 1:10 in MuellerHinton broth (MHB; Difco, Detroit, MI), when necessary, to achieve a concentration range of 0.640640 mg/L. The dilutions were added to MHB inoculated with 5 x 103 yeast cells or 5 x 105 bacterial cells. The cultures were incubated at 35°C and the MIC determined after appropriate incubation time. The MBC was determined by plating 10 µL from test wells (
1 x MIC) on blood agar plates, then incubated for 24 h (yeasts 48 h) at 35°C. MIC and MBC tests were done in triplicate.
In vivo activity
This study was designed to evaluate the protective effect of the mutacin B-Ny266 when administered by intraperitoneal injection to mice intraperitoneally infected with methicillin-susceptible S. aureus (MSSA) Smith strain. Methicillin was not tested to conserve the limited stock of mutacin B-Ny266.
Preparation of inoculum
An overnight culture was initiated by adding MSSA Smith strain to 50 mL of brain heart infusion broth (BHI; Difco, Detroit, MI). The culture was maintained at room temperature in a shaker-incubator overnight and was brought to a temperature of 37°C in the morning. Approximately 4.5 h later, the culture was centrifuged and the pellet was washed with sterile PBS. The washed pellet was then reconstituted in MHB to achieve the desired density of bacteria (4.0 x 107 cfu/mL).
Test article and test formulation
Mutacin B-Ny266 was formulated in Clark and Lubs buffer as described above. The pH of the buffer was 2.0. Vancomycin was formulated in 5% dextrose in water. The vehicle group received Clark and Lubs buffer at the same dose volume as the other groups.
Regimen
Ten mice of each group received 0.5 mL of the inoculum via intraperitoneal administration. Immediately after infection, mice were treated intraperitoneally with a single dose of vehicle (Clark and Lubs buffer), vancomycin (1 or 3 mg/kg), or mutacin B-Ny266 (1, 3 or 10 mg/kg).
All animal experiments were approved by the Committee of Laboratory Animal Care (IntraBiotics Inc.) and carried out according to US Animal Welfare Act Regulations, and the US Public Health Service Policy. All animals were housed and kept according to the rules and regulations of IntraBiotics Pharmaceutical Inc. at their animal facilities.
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Results |
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For the in vivo tests, no mortality was observed in vancomycin- or mutacin B-Ny266-treated mice (Table 2). Mortality in the vehicle-control mice was 100 and 70% for the high and low inoculum levels of MSSA Smith strain, respectively. All three concentrations of B-Ny266 tested are protective, and to produce doseresponse data would necessitate further experiments using lower concentrations of B-Ny266 than those presented in this paper.
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Discussion |
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The fact that the hyperpermeable E. coli strain was susceptible to mutacin B-Ny266 could indicate that this compound acts like the other related lantibiotics. They are active against Gram-negative bacteria only after treatments altering the outer membrane thus giving access to the cytoplasmic membrane.17
The results of the in vivo experiments show that when administered intraperitoneally as a single dose immediately after infection, mutacin B-Ny266 (1 mg/kg) is 100% protective to mice challenged with the MSSA Smith strain.
Although not designed to assess mutacin B-Ny266 toxicity, the present results indicate that intraperitoneal injection of 10 mg/kg of the substance did not apparently affect the health of the mice. These results will however have to be extended in more formal toxicity tests. Whether intravenous injection of mutacin B-Ny266 could protect from bacterial infection will also have to be assessed before considering eventual clinical studies. Nevertheless, it is shown for the first time, in the present results, that mutacin B-Ny266 is efficient in vivo against a bacterial infection.
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Acknowledgements |
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References |
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