Department of Dermatology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho 2-5-1, Okayama 700-8558, Japan
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Abstract |
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Introduction |
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Materials and methods |
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Nine strains of S. aureus, isolated from furuncle lesions (coagulase type IV) and nine isolated from impetigo [five strains of coagulase type I (exfoliative toxin B producers) and four of coagulase type V (exfoliative toxin A producers)] were used to examine plasma coagulation and antimicrobial activities. Of the 18 strains of S. aureus, seven were methicillin susceptible (MSSA; oxacillin MIC 2 mg/L) and 11 were MRSA (oxacillin MIC
4 mg/L).
Bacterial suspension for inoculation
The strains of S. aureus were grown in 8 mL of tryptic soy broth (TSB; Nissui Pharmaceutical Co., Tokyo, Japan) at 37°C overnight without shaking. Following incubation, the bacterial cells were harvested by centrifugation at 6000g for 10 min at 4°C, then resuspended in sterile saline solution and centrifuged as described above. The process was repeated three times. The washed bacteria were resuspended in polypropylene microcentrifuge tubes (1 mL; Iuchi BioSystems, Tokyo, Japan) or tissue-culture dishes (35 x 10 mm2; Becton Dickinson, NJ, USA) and were used in the following experiments.
Plasma coagulation under various concentrations of tannic acid, gallic acid, ellagic acid, ()-epicatechin, ()-epicatechin gallate and ()-epigallocatechin gallate
Cell suspensions of S. aureus (c. 108 cfu) were used for inoculation into 0.5 mL of rabbit plasma (Denka Seiken, Tokyo, Japan) either alone (control) or supplemented with tannic acid (100 or 50 mg/L; Sigma, St Louis, MO, USA), gallic acid (5000 or 2500 mg/L; Sigma), ellagic acid (5000 or 2500 mg/L; Sigma), EC (1500 or 800 mg/L; Sigma), ECG (500 or 200 mg/L; Sigma) or EGCG (200 or 100 mg/L; Sigma) in microcentrifuge tubes. A clot (plasma coagulation) was looked for in the microcentrifuge tubes after incubation for 24 h at 37°C.
MICs of tannins and antibiotics
The MICs of tannic acid, gallic acid, ellagic acid, EC, ECG and EGCG against 18 S. aureus strains were examined in MuellerHinton agar (MHA; Difco, Detroit, MI, USA) using the agar plate method (inoculum size 106 cfu/mL). The MICs of oxacillin (Sigma) and cefdinir (Fujisawa Pharmaceutical, Osaka, Japan) for 18 strains of S. aureus were examined in MHA, MHA with 100 mg/L tannic acid, and MHA with 20 mg/L tannic acid. The MICs of tannic acid and oxacillin were also examined in MHA with 10% defibrinated rabbit blood (Japan Ram, Fukuyasu, Hiroshima, Japan) or 10% rabbit plasma.
Concentrations of ionic calcium and iron
The concentrations of ionic calcium of human serum (Sigma) either alone or supplemented with tannic acid (100 or 1000 mg/L) for 24 h at 37°C were measured by Scripps Reference Laboratories (SRL, Tokyo, Japan) using the ionic electrode method. The concentrations of iron in human serum either alone or supplemented with tannic acid (1000 mg/L) for 24 h at 37°C were measured by SRL using the International Standard Method.15 The precipitates were removed by centrifugation at 500g for 20 min at room temperature, and the concentrations of the supernatants were examined. Because the concentrations of these in plasma and TSB could not be technically measured, we examined those of human serum.
Effect of tannic acid on membranous structures
Cell suspensions of a strain of S. aureus from impetigo, containing 6 x 107 cfu and of a strain of S. aureus from a furuncle containing 1.1 x 108 cfu were each inoculated into 4 mL of a culture medium [plasma TSB (Pl-TSB); ratio of rabbit plasma:TSB, 1:1] covering 1.77 cm2 coverslips (Sumitomo Bakelite, Tokyo, Japan) in tissue culture dishes. The MICs of oxacillin, tannic acid and oxacillin with tannic acid (100 mg/L) for both strains were 32, 1000 and 0.06 mg/L, respectively. After incubation for 24 h at 37°C, membranous structures had formed on the coverslips. Coverslips with membranous structures were then placed into 4 mL of Pl-TSB either alone (control) or supplemented with oxacillin (40 mg/L), tannic acid (100 mg/L) or oxacillin (40 mg/L) and tannic acid (100 mg/L). We used tannic acid at a concentration of 100 mg/L, because tannic acid inhibited plasma coagulation of S. aureus cells and decreased the MIC of oxacillin at this concentration. After incubation for 24 h at 37°C, plasma coagulation was checked and the coverslips were gently washed five times with 1 mL of sterile saline. The coverslips were put in 5 mL of sterile saline and sonicated (Model M-225R, Ultrasonics Inc.) at 60% power for 60 s at 4°C. The number of organisms stripped from the coverslip was then counted (cfu).
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Results |
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Table 1 shows the MICs of tannic acid, gallic acid, ellagic acid, EC, ECG and EGCG for 18 S. aureus strains in MHA plates. The MIC of tannic acid for all 18 strains of S. aureus was >4500 mg/L in MHA with 10% defibrinated rabbit blood. The MIC of tannic acid was the same in the MHA both with and without 10% rabbit plasma.
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The colony counts of S. aureus cells in membranous structures in Pl-TSB containing oxacillin (40 mg/L) and tannic acid (100 mg/L) were c. 10-fold lower than those in Pl-TSB alone and Pl-TSB broth containing oxacillin (40 mg/L; P < 0.01; Table 3).
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Discussion |
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Ellagic acid was reported to accelerate blood clotting and has been used to control haemorrhage in animals.3 However, in the present study, ellagic acid (5000 mg/L) below the MIC inhibited plasma coagulation by S. aureus cells rather than accelerating it.
Catechins are a major component of green tea (1018% of the dry weight), with EGCG being the most important of the four classes of catechins.6,7 Ikigai et al.18 investigated the mode of antibacterial action of EGCG and EC and found that EGCG caused leakage of 5,6-carboxyfluorescein from phosphatidyl choline liposomes, while EC caused little damage to the membrane. They reported that bactericidal catechins primarily act on and damage the bacterial membrane.18 Yam et al.19 reported that gallocatechins and their gallates are the main chemical moieties responsible for the antibacterial activity of green tea extracts; the substances having epi configuration seem, in general, to be more active. Takahashi et al.8 reported that the colony counts of MRSA did not decrease in media with catechin (100 mg/L) or oxacillin (5 mg/L) compared with an untreated control, but decreased to between 1/1000 and 1/10 000 in media with catechin (100 mg/L) and oxacillin (5 mg/L) after incubation for 24 h. In the present study, we confirmed that the MICs of oxacillin and cefdinir for S. aureus were markedly decreased in the MHA plates with tannic acid (100 mg/L) below the MIC. However, the MIC of roxithromycin against S. aureus strains did not decrease in the MHA plates with tannic acid (100 mg/L), and the MIC of oxacillin did not decrease in the MHA plates with gallic acid (100 mg/L; data not shown). Therefore, tannic acid at a sub-MIC seemed to act on the membranes of S. aureus cells. These findings indicate that ß-lactam antibiotics have increased antistaphylococcal activity in the presence of tannic acid.
Staphylocoagulase is an extracellular protein produced by S. aureus. Thrombinstaphylocoagulase complexes appear to be formed immediately when prothrombin and staphylocoagulase are mixed. While this reaction is considered to be non-enzymic, thrombinstaphylocoagulase complexes do become enzymically active and initiate fibrin polymerization.20 The conversion of fibrinogen to fibrin first involves cleavage by thrombin of a specific AryGly bond in each of the -A and ß-B chains to release the fibrinopeptides A and B from the amino termini of the chains.21 Fibrin monomers are thus formed and aggregate to soluble fibrin. Soluble fibrin is then converted in a final step to insoluble fibrin by an activated cross-linking enzyme, factor XIIIa, the fibrin-stabilizing factor fibrinoligase. The presence of ionic calcium promotes the reaction.21 In the present study, all six tannins tested inhibited plasma coagulation of S. aureus at a concentration that was below the MIC. We suggest that inhibition of plasma coagulation by tannic acid is due to a decrease in the concentration of ionic calcium, inhibition of enzyme production and hindrance of the enzyme reaction. The mechanism of inhibition of plasma coagulation by tannic acid should be investigated further.
The presence of a fibrin-rich biofilm is a well-known factor responsible for prolonging S. aureus infections. The biofilm of S. aureus is reinforced with fibrin fibres, making it more resistant to physical effects than are other bacterial biofilms.10 We reported previously that the attachment of S. aureus to coverslips, the conversion of fibrinogen to fibrin and the abundant production of glycocalyx by S. aureus are minimum requirements for the production of a mature biofilm on coverslips after 72 h.22 Although the membranous structures in the present study following incubation for 24 h were similar to fibrin clots containing S. aureus and plasma components, the S. aureus in the membranous structures in plasma were resistant to imipenem at 40 x MIC and roxithromycin at 4 x MIC (data not shown). If plasma coagulation does not occur in the presence of some tannins, the formation of fibrin-rich membranous structures by S. aureus will probably be inhibited. The antistaphylococcal activity of oxacillin against membranous structures increased in Pl-TSB with tannic acid (100 mg/L) below the MIC. We suggest that these phenomena are due to inhibition of fibrin formation and a marked increase in the antistaphylococcal activity of oxacillin by the addition of tannic acid (100 mg/L). These results indicate that tannic acid may be a useful adjuvant agent for the treatment of S. aureus skin infections in addition to ß-lactam antibiotics, at least under in vivo conditions without blood. Given the apparent trend towards the evolution of resistant strains, further investigation of natural products having antistaphylococcal activities may reveal useful topical applications for clinical dermatology.
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Notes |
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References |
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2 . Miranda, C. M., Wyk, C. W., Bijl, P. & Basson, N. J. (1996). The effect of areca nut on salivary and selected oral microorganisms. International Dental Journal 46, 3506.[Medline]
3 . Chung, K.-T., Wong, T. Y., Wei, C.-I., Huang, Y.-W. & Lin, Y. (1998). Tannins and human health: a review. Critical Reviews in Food Science and Nutrition 38, 42164.[ISI][Medline]
4 . Chung, K.-T., Stevens, S. E., Jr, Lin, W.-F. & Wei, C. I. (1993). Growth inhibition of selected food-borne bacteria by tannic acid, propyl gallate and related compounds. Letters in Applied Microbiology 17, 2932.[ISI]
5 . Ikeda, I., Imasato, Y., Sasaki, E., Nakayama, M., Nagao, H., Takeo, T. et al. (1992). Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats. Biochimica et Biophysica Acta 1127, 1416.[ISI][Medline]
6 . Kono, K., Tatara, I., Takeda, S., Arakawa, K. & Hara, Y. (1994). Antibacterial activity of epigallocatechin gallate against methicillin-resistant Staphylococcus aureus (Japanese). Journal of the Japan Association for Infectious Disease 68, 151822.
7
.
Hamilton-Miller, J. M. T. (1995). Antimicrobial properties of tea (Camellia sinensis L.). Antimicrobial Agents and Chemotherapy 39, 23757.
8 . Takahashi, O., Cai, Z., Toda, M., Hara, Y. & Shimamura, T. (1995). Appearance of antibacterial activity of oxacillin against methicillin resistant Staphylococcus aureus (MRSA) in the presence of catechin. Journal of the Japan Association for Infectious Disease 69, 112634.
9 . Shiota, S., Shimizu, M., Mizushima, T., Ito, H., Hatano, T., Yoshida, T. & Tsuchiya, T. (1999). Marked reduction in the minimum inhibitory concentration (MIC) of ß-lactams in methicillin-resistant Staphylococcus aureus produced by epicatechin gallate, an ingredient of green tea (Camellia sinensis). Biological and Pharmaceutical Bulletin 22, 138890.[Medline]
10 . Nemoto, K., Hirota, K., Ono, T., Murakami, K., Murakami, K., Nagao, D. & Miyake, Y. (2000). Effect of varidase (streptokinase) on biofilm formed by Staphylococcus aureus. Chemotherapy 46, 1114.[ISI][Medline]
11 . Akiyama, H., Yamasaki, O., Kanzaki, H., Tada, J. & Arata, J. (1998). Effects of zinc oxide on the attachment of Staphylococcus aureus strains. Journal of Dermatological Science 17, 6774.[ISI][Medline]
12 . Akiyama, H., Yamasaki, O., Tada, J. & Arata, J. (1999). Calcium oxide and magnesium oxide inhibit plasma coagulation by Staphylococcus aureus cells at a lower concentration than zinc oxide. Journal of Dermatological Science 22, 625.[ISI][Medline]
13 . Akiyama, H., Yamasaki, O., Kanzaki, H., Tada, J. & Arata, J. (1998). Effects of sucrose and silver on Staphylococcus aureus biofilms. Journal of Antimicrobial Chemotherapy 42, 62934.[Abstract]
14 . Akiyama, H., Yamasaki, O., Tada, J. & Arata, J. (1999). Effects of acetic acid on biofilms formed by Staphylococcus aureus. Archieves of Dermatological Research 291, 5703.
15 . International Committee for Standardization Haematology. (1978). Recommendations for measurement of serum iron in human blood. British Journal of Haematology 38, 2914.[ISI][Medline]
16 . Sharquie, K. E., Al-Turfi, I. & Al-Salloum, S. M. (2000). The antibacterial activity of tea in vitro and in vivo (in patients with impetigo contagiosa). Journal of Dermatology 27, 70610.[Medline]
17 . Chung, K.-T., Lu, Z. & Chou, M. W. (1998). Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria. Food and Chemical Toxicology 36, 105360.[ISI][Medline]
18 . Ikigai, H., Nakae, T., Hara, Y. & Shimamura, T. (1993). Bactericidal catechins damage the lipid bilayer. Biochimica et Biophysica Acta 1147, 1326.[ISI][Medline]
19 . Yam, T. S, Shah, S. & Hamilton-Miller, J. M. T. (1997). Microbiological activity of whole and fractionated crude extracts of tea (Camellia sinensis), and of tea components. FEMS Microbiology Letters 152, 16974.[ISI][Medline]
20 . Kawabata, S., Morita, T., Iwanaga, S. & Igarashi, H. (1985). Enzymatic properties of staphylothrombin, an active molecular complex formed between staphylocoagulase and human prothrombin. Journal of Biochemistry 98, 160314.[Abstract]
21 . Orten, J. M. & Neuhaus, O. W. (1982). Blood. In Human Biochemistry, (Orten, J. M. & Neuhaus, O. W., Eds), pp. 434523. Mosby, St Louis, MO.
22 . Akiyama, H., Ueda, M., Kanzaki, H., Tada, J. & Arata, J. (1997). Biofilm formation of Staphylococcus aureus strains isolated from impetigo and furuncle: role of fibrinogen and fibrin. Journal of Dermatological Science 16, 210.[ISI][Medline]
Received 15 January 2001; returned 2 June 2001; revised 15 June 2001; accepted 4 August 2001