1 Department of Infection, Chungshan Medical University Hospital; 2 Institute of Nutritional Science, Chungshan Medical University, No. 110, Sec. 1, Chien Kuo N. Rd., Taichung City, Taiwan, ROC
Received 31 March 2003; returned 18 July 2003; revised 26 August 2003; accepted 16 September 2003
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Abstract |
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Methods: Garlic extract at 100% and 50%; diallyl sulphide (DAS) at 10% and 5%; diallyl disulphide (DADS) at 1% and 0.5% were used in this study. Sixteen clinical MRSA isolates obtained from infected patients were used in this study (n = 16). Mice were infected by injecting 200 µL MRSA-PBS solution, which contained 107 cfu, via the tail vein. At 16 h post-infection (p.i.), garlic extract, DAS or DADS at 200 µL was administrated orally. At 24 h p.i., mice were killed and blood, liver, kidney and spleen of each mouse were collected. Plasma and the filtrate from each organ and serial dilutions were used to determine colony count. Plasma fibronectin level was determined by rabbit anti-rat fibronectin antibody and quantified by ELISA. Interleukin-6 levels were determined by commercial kit. Lipid oxidation was determined by measuring malondialdehyde levels.
Results: The oral administration of these agents significantly decreased the viability of MRSA, in plasma, liver, kidney and spleen (P < 0.05). MRSA infection significantly increased fibronectin and interleukin-6 levels in plasma of MRSA-infected mice (P < 0.05); however, the oral administration of garlic extract and two diallyl sulphides significantly reduced both fibronectin and interleukin-6 levels (P < 0.05). MRSA infection also significantly enhanced lipid oxidation in plasma and three organs (P < 0.05). The treatments of garlic extract and two diallyl sulphides significantly decreased the malondialdehyde level and showed antioxidant protection (P < 0.05).
Conclusions: These data strongly supported the conclusion that garlic extract, diallyl sulphide and diallyl disulphide possessed multiple protective functions against MRSA infection, in which diallyl sulphide and diallyl disulphide could be considered as novel therapeutic agents for the treatment of MRSA infection.
Keywords: survival rate, fibronectin, interleukin-6, lipid oxidation
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Introduction |
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The in vitro anti-MRSA activity of garlic extract and its two diallyl sulphides (diallyl sulphide, DAS; diallyl disulphide, DADS) has been studied previously in our laboratory.7 Furthermore, our earlier study noted that the intake of fresh garlic extract by humans resulted in the presence of antimicrobial compounds in plasma, which further exhibited a marked inhibitory zone against MRSA.8 Therefore, we designed an animal study to evaluate the in vivo inhibitory effects of garlic extract, DAS and DADS against MRSA infection.
In addition, bacterial infection often results in increased interleukin-6 (IL-6) secretion, and elevated oxidative stress and/or altered fibronectin levels in infected animals or humans.913 Therefore, the influence of potential anti-MRSA agents upon the levels of fibronectin, IL-6 and lipid oxidation in MRSA-infected BALB/cA mice was also examined.
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Materials and methods |
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Peeled fresh garlic (100 g) was chopped and homogenized in 100 mL sterile distilled water in a Waring blender. After filtration through Whatman No. 1 filter paper, the filtrate was further sterilized by passing through a 22 µm pore-size filter. The filtrate was collected in a sterile vial and stored at 4°C until used. Diallyl sulphide (purity 97%) and crude diallyl disulphide (purity 80%) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Diallyl disulphide was further purified by fractional distillation and its final purity was 98%, as determined by HPLC.
Animals
Eight- to nine-week-old male BALB/cA mice (National Laboratory Animal Center, Science Council, Taipei City, Taiwan) were used in this study. Mice were housed on a 12 h light, 12 h dark schedule, and fed with rat and mouse standard diet no.1120 and water ad libitum. Use of the mice was reviewed and approved by the Chungshan Medical University Animal Care Committee. Various concentrations of garlic extract, DAS and DADS were tested to deduce sub-lethal levels in mice, and the survival rate was measured. Twenty mice were used for each agent at each concentration.
Bacterial strains
Sixteen clinical MRSA isolates were obtained from infected patients in Chungshan Medical University Hospital (Taichung City, Taiwan). All isolates were identified by Vitek (Vitek AMS; BioMérieux Vitek, Inc., Hazelwood, MO, USA) and API 20E (API-BioMérieux, La Balme Les Grottes, France). Antibiotic resistance profiles using vancomycin, methicillin, penicillin, cefotaxime and tetracycline were determined by disc diffusion. The antibiotic discs were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Discs were placed on the surface of MuellerHinton agar plates supplemented with 2% NaCl and seeded with MRSA. Inhibition zones were measured after 24 h incubation at 35°C. Interpretation of resistance was based on the National Committee for Clinical Laboratory Standards (NCCLS) criteria.14 The 16 MRSA isolates were susceptible to vancomycin and resistant to four other test antibiotics. All cultures were routinely maintained on MuellerHinton agar plates at 25°C until used. Overnight MRSA cultures in broth were diluted with PBS and adjusted to an optical density at 600 nm of 0.3 (about 1010 cfu/mL).15 Before infection, MRSA were then diluted with PBS to 108 cfu/mL, which gave approximately 107 cfu per mouse in a volume of 200 µL.
Experimental design
Mice at 2226 g were used. After overnight fasting, mice were infected by injecting 200 µL MRSA-PBS solution, which contained 107 cfu, via the tail vein. At 16 h post-infection (p.i.), garlic extract, DAS or DADS at 200 µL was administered orally, in which mineral oil was used as solvent for DAS and DADS. Vancomycin at 1% (200 µL of a 10 mg/L aqueous solution) was given orally as a comparator. At 24 h p.i. (8 h after administration of agents), mice were killed with carbon dioxide. Blood, liver, kidney and spleen from each mouse were collected. Plasma was separated from erythrocytes immediately after blood collection. Of each tissue, 200 mg was homogenized with 2 mL PBS (pH 7.2) in a motor-driven Teflon glass homogenizer (Glas-Col Co., CA, USA). The filtrate was used for analysis.
Culture
Plasma and the filtrate from each organ and serial dilutions at 100 µL were cultured on MuellerHinton agar plates supplemented with 2% NaCl. After incubation for 24 h at 35°C, colonies were counted and calculated as log10 cfu/mL or log10 cfu/g. The limit of detection was 200 cfu/mL.
Fibronectin and interleukin-6 assay
Plasma fibronectin level was determined by rabbit anti-rat fibronectin antibody and quantified by ELISA.16 Plasma level of IL-6 was measured by ELISA using the Cytoscreen Immunoassay Kit (BioSource International Camarillo, Camarillo, CA, USA) according to the manufacturers instructions.
Lipid oxidation determination
The concentration of malondialdehyde (MDA, nmol/mL) in plasma and the filtrate from each organ was determined by an HPLC method.17 In brief, 0.2 mL plasma or filtrate was mixed with 0.8 mL PBS. Then, 0.5 mL trichloroacetic acid (30%) was added. After vortexing and standing in ice for 2 h, samples were centrifuged at 1500g for 15 min. Then 1 mL supernatant was mixed with 0.25 mL thiobarbituric acid (TBA, 1%) and the mixture was kept in a boiling water bath for 15 min. The concentration of MDATBA complex was assayed using HPLC equipped with a reversed phase Shodex KC-812 column with a UVVis detector at 532 nm.
Statistical analysis
Sixteen clinical MRSA isolates obtained from infected patients were used in this study. Each isolate was used for one run experiment including all the above analyses. In every experiment, two mice were used for each agent at each concentration. Data were expressed as mean ± S.D. of 16 experiments (n = 16). Data were treated by analysis of variance (ANOVA) and computed using the SAS General Linear Model procedure.18 Difference among means was determined by the Least Significance Difference Test with significance defined at P 0.05.
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Results |
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Discussion |
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Fibronectin is an extracellular matrix protein and responsible for the adherence and internalization of pathogens to host cells.9,19 Several studies have reported that MRSA infection elevated fibronectin-binding protein production, which enhanced the invasion of MRSA into host cells.1922 We have also found that MRSA infection markedly increases plasma fibronectin level, but garlic extract, DAS and DADS effectively suppressed fibronectin production to a greater extent than vancomycin. This suppression may reduce the fibronectin available to react with fibronectin-binding proteins, which consequently decreases the adherence and/or internalization of MRSA into host cells. Therefore, the suppressive effect from garlic extract, DAS and DADS on fibronectin production could effectively protect host cells against pathogen invasion.
It has been reported that MRSA infection resulted in the increased production of several cytokines such as interleukin-6 and these elevated cytokines consequently enhanced the pathogenic development not only in MRSA-associated diseases such as glomerulonephritis and nephritis10,11,23 but also in other autoimmune/inflammatory diseases such as rheumatoid arthritis.2426 The results of our present study confirm that MRSA infection increased the IL-6 level in mice. Furthermore, our present study found that garlic extract, DAS and DADS markedly reduced the IL-6 level in these MRSA-infected mice, unlike vancomycin. This finding suggests that these agents are able to decrease immune complex formation and alleviate infection-induced inflammation reactions. Since these agents could markedly suppress IL-6 production, their use might be beneficial for patients with infection-related inflammatory diseases.
Oxidative stress resulting from bacterial infection such as Streptococcus pneumoniae meningitis has been reported.13,27 Our present study is the first report regarding MRSA-induced oxidative damage. This infection-induced oxidation could further worsen host self-defence systems. In addition, the combined effects of infection, IL-6- related inflammation and elevated oxidation could enhance the development of inflammatory diseases and increase therapy difficulty. Several studies have indicated that garlic extract, DAS and DADS possessed both enzymic and non-enzymic antioxidant activities such as scavenging superoxide ions and enhancing glutathione peroxidase activity.2830 Thus, it was highly likely that these agents contributed to the observed oxidation alleviation in this present study via their antioxidant protection. This finding suggests that, based on their anti-oxidative capability, these agents could provide benefit in anti-infective therapy.
In conclusion, garlic extract, DAS and DADS could effectively inhibit MRSA infection, suppress infection-induced elevation of fibronectin and interleukin-6, and decrease MRSA-induced oxidative damage in MRSA-infected mice. These data strongly suggest that these agents possess multiple protective functions against MRSA infection.
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Acknowledgement |
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Footnotes |
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References |
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