1 Department of Microbiology, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414; 2 Department of Environmental Dermatology, Nagoya University School of Medicine, 1-1-20 Daikominami, Higashi-ku, Nagoya 461-0047, Japan
Received 3 July 2002; returned 12 September 2002; revised 27 September 2002; accepted 8 October 2002
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Abstract |
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Keywords: ß-thujaplicin, staphylococci, MRSA, atopic dermatitis, zinc oxide
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Introduction |
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Recently, we have found that (i) ß-thujaplicin has antimicrobial activity against both bacteria and fungi; (ii) ß-thujaplicin shows a paradoxical zone phenomenon (i.e. the phenomenon of inhibition of the growth of bacteria at a low concentration, followed by promotion of growth at a higher concentration and then complete inhibition of growth at an even higher concentration)9 against staphylococci; and (iii) the simultaneous use of ß-thujaplicin and Zn2+ inhibits the paradoxical zone phenomenon, and enhances antimicrobial activity towards various bacteria.10
Staphylococcus aureus has been shown to colonize in high density and at high frequency in eczematous lesions of atopic dermatitis (AD),1118 and is thought to be one of the factors exacerbating dermatitis.19,20 It has also been reported that S. aureus colonies on the skin surface of patients with AD easily changed from methicillin-sensitive S. aureus (MSSA) to methicillin-resistant S. aureus (MRSA), when antibacterial agents were administered over a long period of time.20 These facts suggest that if topical application of ß-thujaplicin reduces the number of bacterial cells (especially S. aureus cells) on the skin surface of patients with AD, this reduction in the number of bacterial cells may lead to elimination of one of the factors exacerbating dermatitis, and to improvement in eczematous lesions of patients with AD. The purpose of this study was to determine whether ß-thujaplicin alone, or in combination with zinc oxide (ZnO), acts on clinical isolates from patients with AD. Another purpose was to elucidate the relationship between changes in the number of viable bacterial cells, and clinical improvement in an eczematous lesion due to the topical application of ß-thujaplicin.
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Materials and methods |
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ß-Thujaplicin (synthetic product, purity 100%) was obtained from Takasago International Corporation (Tokyo, Japan). Methicillin sodium, ofloxacin, gentamicin sulphate, erythromycin and minocycline were purchased from Banyu Seiyaku (Osaka, Japan), Daiichi Seiyaku (Osaka, Japan), Wako Pure Chemical Industries (Tokyo, Japan), Shionogi Seiyaku (Osaka, Japan) and Lederle Japan (Tokyo, Japan), respectively. Triton X-100 was obtained from Nacalai Tesque (Kyoto, Japan). Heart infusion broth (HIB), heart infusion agar (HIA) and mannitol salt agar (MSA), were purchased from Nissui Seiyaku (Kyoto, Japan). Two kinds of film stamp check (6 x 6 cm2) were purchased from Bio Medical Laboratory (Kyoto, Japan); soy casein digest agar (SCDA) plates and MSA plates enabled the detection of viable bacterial cells and S. aureus cells. All other chemicals used in this study were commercially available products of analytical reagent grade. ß-Thujaplicin was dissolved in ethanol and diluted with sterile water to various concentrations. The final concentration of ethanol was <4%. The addition of 4% ethanol showed no remarkable effect on these assay systems. Methicillin sodium, ofloxacin, gentamicin sulphate, erythromycin and minocycline were dissolved in sterile water, and ZnO was suspended in sterile water or dissolved in 0.1 M HCl solution.
Subjects
A total of 43 outpatients (15 males and 28 females, aged 22.0 ± 9.8 years) who fulfilled the criteria of the Japanese Dermatological Association21 for AD, and a total of 30 healthy volunteers (14 males and 16 females, aged 22.5 ± 2.1 years), were selected for this study. Informed consent was obtained from all participants (or from parents in the case of patients <20 years old) prior to treatment. No patients with an apparent secondary infection were included in this study.
Antimicrobial treatment
Body shampoo containing 0.03% ß-thujaplicin in the detergent, and with a main component of aminomethyl-ß-alanine, and ZnO ointment containing 0.05% ß-thujaplicin, were used for test preparations. In order to evaluate the effect of each ß-thujaplicin preparation, the 43 patients were divided into two groups: a control group of 21 patients and a ß-thujaplicin group of 22 patients. The control group was further divided into two groups: a non-soap-user group, in which patients who had not used soap before treatment continued not to use soap until the completion of treatment, and a soap-user group, in which patients who had been using soap continued to use the same soap. The patients in the soap-user group used various commercial soaps that did not contain antibacterial agents. In the ß-thujaplicin group, the previously used face and body soaps were changed to ß-thujaplicin body shampoo. ß-Thujaplicin ointment was applied, if necessary, to the lesions in adequate amounts two or three times a day. The ß-thujaplicin group (n = 22) was further divided into two groups: one group of 18 patients who used body shampoo alone (body shampoo group) and one group of four patients who used both body shampoo and ointment (ointment group). The period of application was from 4 to 12 weeks, unless symptoms deteriorated or serious side effects occurred. There were no differences in the therapeutic procedures, except for the use of ß-thujaplicin preparations. The use of other oral or topical antimicrobial agents, antiseptics or oral corticosteroids that might affect the efficacy of ß-thujaplicin was avoided, and topical corticosteroids were not used in involved areas where bacteriological stamp sampling had been performed. The lifestyle of the patients was otherwise the same as it had been before the study.
The severity of each symptom (dryness, desquamation and pityriasis, erythema, epidermal exfoliation and erosion) was classified into five grades (+++, severe; ++, moderate; +, mild; ±, light; , none) and judged by the attending doctor before treatment and then at 2 or 3 week intervals. At the completion of treatment, the therapeutic efficacy was assessed from the results of objective judgement of skin condition and the patients judgement of subjective symptoms and side effects. Overall improvement was classified into six grades: resolved, markedly improved, improved, slightly improved, poor and worsened.
The involved area was stamped before and during treatment by the contact-plate technique described above, and the colonies on each plate were counted.
Assay of viable bacterial cells on the skin surface
Assays of viable cells on the skin surface were carried out on 23 randomly selected patients (eight males and 15 females, aged 25.4 ± 7.1 years), and on all of the healthy volunteers using the scrub technique. Assays using the contact-plate technique were also carried out on all participants. The involved area of AD was classified as normal skin, or dry or wet lesions, as judged by the conditions of dryness, desquamation, pityriasis, erythema and exudation.
Scrub technique
The technique used in this experiment was a modified procedure of Akiyama et al.20 A glass tube (22 mm in internal diameter) (Daiichi Kikai, Tokushima, Japan) was placed above the involved area and filled with 0.1% Triton X-100/0.01 M phosphate-buffered saline 1 mL, and mixed with a Teflon rod for 1 min. This procedure was then repeated. After mixing the 2 mL mixture, an 0.1 mL aliquot of the 10-fold diluted specimen was cultured at 37°C on HIA and MSA plates. After 24 h of culture at 37°C, viable cells on the HIA plates were counted for total colonies, and mannitol-degrading cells on the MSA plates were counted for S. aureus colonies. These counts were converted to the corresponding density of bacteria originally present in the area. The addition of 0.1% Triton X-100 showed no remarkable effect on this assay.
Contact-plate technique
MSA plates were stamped for 10 s on the severest site in the involved area, and SCDA plates were stamped for 10 s on a neighbouring site. After 48 h of culture at 37°C, viable cells on the SCDA plates were counted for total colonies, and mannitol-degrading cells on the MSA plates were counted for S. aureus colonies. When a very large number of colonies was observed, the viable cells per 1 cm2 were counted, and these counts were converted to the corresponding density of bacteria originally present.
Organisms
Forty-six staphylococcal strains tested were isolated from eczematous lesions of 23 patients for MIC determination. A few predominant colonies were randomly selected from each culture plate. Organisms were identified by Grams stain, coagulase-positivity and Staphaurex (rapid latex test kit for the identification of S. aureus; Murex Diagnostics Ltd, Dartford, UK) using standard procedures. To avoid duplication, only one isolate per patient in each group [S. aureus or coagulase-negative staphylococci (CNS)] was examined.
Susceptibility testing
The MIC of ß-thujaplicin and related compounds for clinical isolates was determined by an agar dilution method.22 All isolates were incubated overnight at 37°C in HIB and diluted to a final inoculum of 1 x 106 cfu/mL; the plates were incubated for 24 h at 37°C. MIC was defined as the lowest concentration of the compounds tested that completely prevented visible growth of the inoculum on the surface of HIA plates.
Disc diffusion tests
The interaction between ß-thujaplicin and ZnO was studied by an agar diffusion method23 using an HIA plate. ZnO was suspended in sterile distilled water. Discs containing 60 µL of each solution obtained after dilution, were placed on a plate that had been inoculated at 106 cfu/mL, in the usual fashion, with the organism to be tested. After overnight incubation at 37°C, the results were interpreted according to the zone diameters (including the 6 mm disc), which were measured on the undersurface of the Petri dish with a ruler, or whether, according to the paradoxical zone phenomenon, they had disappeared.
Timekill curve
The bactericidal activity of ß-thujaplicin, and the effect of the combination of ß-thujaplicin and ZnO, were determined, as described previously.24 Briefly, overnight bacterial cultures in HIB were diluted with fresh HIB to 107 cfu/mL, and incubated with the test compound at 37°C. The number of bacteria was counted after 2, 4 and 6 h incubation, by spreading 0.1 mL of the solution, obtained after diluting the culture 10-fold in sterile water, on to drug-free HIA plates. The colonies grown on the plates were counted after incubation for 24 h.
Statistical analysis
Statistical analysis was conducted as follows: the numbers of bacterial cells on skin surfaces were compared using the KruskalWallis rank test, and Scheffe correction for multiple testing. Wilcoxons signed rank test was used to compare numbers of viable bacterial cells in the same lesional site before and after treatment. Two-tailed tests were performed, and correlations with a level of P < 0.05 were considered to be significant.
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Results |
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The scrub and contact-plate techniques were used to evaluate the relationship between the number of viable bacterial cells on the skin surface and the skin conditions of AD. The results obtained by using the scrub technique (Figure 1) showed that the number of viable bacterial cells on the forehead was larger than that on the forearm of the healthy volunteers. S. aureus colonies were not detected in the involved areas of the healthy volunteers, but large numbers of colonies were detected on the skin of the patients with AD. In the AD group, the average number of total colonies or S. aureus on normal skin was less than that in dry lesions, which was less than that in wet lesions (P < 0.001, KruskalWallis rank test). In both total and S. aureus colonies, there was a significantly larger number of viable cells in wet lesions than in other lesions (P < 0.001, Scheffes test). This was also the tendency in more specific areas such as the forearm.
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MICs of ß-thujaplicin and related compounds were examined for 46 clinical isolates (23 S. aureus and 23 CNS), and the results are summarized in Table 1. Three methicillin-resistant staphylococci (methicillin MIC of 16 mg/L) were included in the 46 isolates. The paradoxical zone phenomenon, i.e. the phenomenon of inhibition of the growth of bacteria by an antimicrobial agent at a low concentration (1.563.13 mg/L), followed by promotion of growth by the agent at a higher concentration, and then complete inhibition of growth at an even higher concentration (50 mg/L), occurred in every isolate treated with ß-thujaplicin. The MIC of ß-thujaplicin was 1.563.13 mg/L, with regrowth at higher concentrations between 6.25 and 50 mg/L for all of the 46 clinical isolates (21 MSSA, two MRSA, 22 MSCNS and one MRCNS), while other tested antimicrobial agents showed a wide range of MICs. In terms of MIC50 value, the inhibitory effect of ß-thujaplicin was lower than that of the five other test compounds, which are all well-known antimicrobial agents.
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The results for clinically isolated S. aureus A-1, as representative examples, are shown in Figure 3. Prevention of the paradoxical zone phenomenon, and enhancement of the original inhibitory effect of ß-thujaplicin against S. aureus, are observed at and near the junction of the two zones of inhibition. In other isolates, the same results were obtained.
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The timekill curves are shown in Figure 4. The combination of ß-thujaplicin and ZnO enhanced the killing effect for both S. aureus A-5 and A-52, which were selected randomly. There was no significant decrease in the number of bacteria with the use of either ß-thujaplicin (100 mg/L) or ZnO (400 mg/L) alone over a 6 h period, while their use combined resulted in a reduction in the number of viable cells for both strains at the end of the 6 h period (Figure 4a and d). The combination of ß-thujaplicin (12.5 mg/L) and ZnO (100 or 400 mg/L) resulted in a dramatic decrease in the number of viable cells after 6 h, while exposure of both strains to ß-thujaplicin (12.5 mg/L) or ZnO (100 or 400 mg/L) alone, did not result in a reduction in the number of bacteria during the 6 h period (Figure 4b, e, c and f).
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Background data of the patients, sites of involvement and treatment durations are shown in Table 2. No statistical differences between the ß-thujaplicin group and control group were found for objective estimation. A comparison of the numbers of bacterial cells before and after treatment is shown in Figure 5. In the non-soap-user group, no relationship was found between treatment duration and number of total or S. aureus colonies. In the soap-user group, there was a slight decrease in the number of total colonies in the involved area (P = 0.108), but no significant change in the number of S. aureus colonies was observed. On the other hand, in the ß-thujaplicin groups, both the number of total and S. aureus colonies decreased significantly (P < 0.001). In the ointment group, although no significant difference was found before or after treatment due to the small number of patients, the difference between the mean numbers of viable bacterial cells of total and S. aureus colonies was greater than that in the other groups. ß-Thujaplicin preparations, including ß-thujaplicin alone or in combination with ZnO, resulted in a significant reduction in the number of viable bacterial cells after treatment.
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Discussion |
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The effects of ß-thujaplicin alone, and combinations of ß-thujaplicin and ZnO on 46 strains of staphylococci isolated from eczematous lesions of AD, were investigated. In terms of MIC50, the antimicrobial activity of ß-thujaplicin was lower than that of the other five antimicrobial agents tested (Table 1). However, susceptibilities to the other five antimicrobial agents showed a wide range of MICs, while the MIC of ß-thujaplicin was 1.563.13 mg/L, with regrowth producing the paradoxical phenomenon for all of the 46 clinical isolates, including three methicillin-resistant staphylococci (two MRSA and one MRCNS). In other words, ß-thujaplicin acts on MRSA, which was used in the present study, at the same concentration as it does on MSSA. There have been no reports of ß-thujaplicin-resistant strains, and no resistant strains were found in the present study. The paradoxical zone phenomenon, the mechanisms of which are still not clear, occurred in all of the strains of staphylococci treated with ß-thujaplicin in the present study. For example, S. aureus A-46 cells stopped proliferating when treated with 3.13 mg/L of ß-thujaplicin, then began to regrow when the concentration of ß-thujaplicin was increased to 12.5 mg/L, and was finally inhibited once more when the concentration of ß-thujaplicin was further increased to 50 mg/L (Figure 3a). Since this phenomenon has been shown previously to disappear when ZnO is added,10 the combined effect of ß-thujaplicin and ZnO on the paradoxical zone phenomenon was investigated with the clinical isolates. It was found that a combination of ß-thujaplicin and ZnO inhibited the paradoxical zone phenomenon, and reduced the number of clinically isolated staphylococci, even at the concentrations that had no inhibitory effect when the agents were used alone. Their action on the clinical isolates was the same as that on the standard strain, S. aureus 209 JP (data not shown).
Since the bactericidal effect of ß-thujaplicin on clinical isolates was confirmed by tests in vitro, use-tests were carried out in order to determine whether the number of bacterial cells, especially that of S. aureus, on the skin surface of patients with AD is reduced by topical application of ß-thujaplicin, and whether topical application of ß-thujaplicin is clinically effective for eczematous lesions of AD. Because the contact-plate technique is sufficiently reliable, accurate and simple, it was used to count the numbers of viable bacterial cells on the skin surface before and after ß-thujaplicin treatment, and the results were compared with those in the control groups (Figure 5). The use of ß-thujaplicin preparation significantly reduced the number of bacterial cells, especially S. aureus cells, on the skin surface. The degrees of reduction in the number of viable bacterial cells in the four subgroups were in the following order: non-soap-user group < soap-user group < ß-thujaplicin body shampoo use and ointment non-use group < ß-thujaplicin body shampoo and ointment use group.
The degree of reduction in the number of viable bacterial cells on the skin was found to be related to the degree of improvement in skin condition; the improvement rate was higher in groups with greater reductions in the number of viable bacterial cells on the skin. Moreover, in cases that did not show a noticeable improvement in skin condition (i.e. cases classified as slightly improved or worse), the number of viable bacterial cells (especially S. aureus cells) on the skin at the end of the study period had not significantly decreased compared with the number before treatment was commenced.
S. aureus has been shown to colonize in high density and at high frequency in eczematous lesions of AD, and is thought to be one of the factors exacerbating dermatitis. With regard to patients with AD colonized with S. aureus, Akiyama et al.25 reported that the attachment of S. aureus cells to coverslips was suppressed in the presence of 5% ZnO. Leyden & Kligman26 reported that patients with AD colonized with S. aureus responded better to a combination of topical corticosteroid and antibiotic therapy than to topical steroid therapy alone. The efficacy of ß-thujaplicin ointment may be due not only to the antimicrobial action of ß-thujaplicin and ZnO, but also to ZnOs inhibitory action27 on the attachment of S. aureus bacteria.
The results of this study indicate that the use of an antibacterial agent, such as ß-thujaplicin, which has a mild action, alone or in a preparation combined with ZnO, can reduce the number of viable bacterial cells on the skin surface of AD, and this reduction in the number of bacterial cells may be clinically effective for treating eczematous lesions of AD.
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Acknowledgement |
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Footnotes |
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References |
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