Department of Dermatology, Okayama University Medical School, 2-5-1 Shikata-cho, 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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The strain of S. aureus (labelled X strain) was isolated from a furuncle. The X strain was of coagulase type II (latex agglutination method; Denka Seiken, Co., Tokyo, Japan). The MICs of oxacillin (MPIPC; Sigma, St Louis, MO, USA), roxithromycin (Eizai Co., Tokyo, Japan) and imipenem (Banyu Pharmaceutical Co., Tokyo, Japan) for X strain were 0.5, 1 and 0.25 mg/L, respectively. The fractional inhibitory concentration index of roxithromycin and imipenem against the X strain was below 0.5.
Bacterial suspension for inoculation
The X strain was grown in 8 mL of tryptic soy broth (TSB; Nissui Pharmaceutical Co., Tokyo, Japan) at 37°C for 24 h without shaking. Following incubation, the bacteria were harvested by centrifugation at 9000g for 10 min at 4°C. The bacterial cells were 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 Bio Systems, Tokyo, Japan) and tissue culture dishes (35 x 10 mm; Becton Dickinson Co., Lincoln Park, NJ, USA). They were used in the following experiments.
In vitro experiments on membranous structures
The X strain suspension containing 8.8 x 108 cfu was inoculated into 2.5 mL of rabbit plasma (Denka Seiken) on 1.77 cm2 coverslips (Sumitomo Bakelite Co., Tokyo, Japan) in tissue culture dishes.2 After incubation for 24 h at 37°C, a membranous structure had formed on the coverslips. The membranous structures (n = 5) were placed in 2.5 mL of rabbit plasma alone, or supplemented with roxithromycin at 2 x MIC, imipenem at 4 x MIC, imipenem at 40 x MIC, roxithromycin at 2 x MIC and imipenem at 4 x MIC or roxithromycin at 2 x MIC and imipenem at 40 x MIC. After incubation for 24 h at 37°C, the coverslips were gently washed with sterile saline solution (1 mL) five times and then suspended in 4 mL of sterile saline solution and sonicated (Model W-225R, Ultrasonics Inc., Plainview, NY, USA) at 60% power for 50 s at 4°C. The organisms stripped from the coverslips were counted as cfu using the 10-fold dilution method.
Production of skin infection in mice
Animal studies conformed to local guidelines for animal experiments, Okayama University Medical School. Five-week-old female mice of the ddy strain, weighing c. 20 g, were purchased from Japan SLC (Shizuoka, Japan). The animals were non-neutropenic. Strain X is expected to form aggregates in the presence of plasma and latex at the time and site of inoculation, allowing a prolonged infected condition. The inoculum suspensions were prepared by resuspending washed bacteria in 0.075 mL of rabbit plasma and 0.025 mL of latex beads (diameter 0.5 µm, 1.4 x 1014 pieces/mL; Sekisui Chemical Co., Tokyo, Japan) and incubated for 15 min. The bacterial inoculum ranged from 1.8 x 108 to 4.6 x 108 cfu/mL in the following experiments. The backs of the mice were shaved using a razor blade and 0.1 mL of the inoculum suspension was injected intradermally using a 27-gauge needle on a tuberculin syringe. The average white blood cell counts were 5400 ± 543 cells/µL (mean ± S.D.) in control mice (n = 3) and 6500 ± 720 cells/µL in the infected mice (n = 3) at 2 days after inoculation. This model was used in the following experiments: antibiotic therapy and effect of antibiotics in combination with colchicine.
Studies of the skin lesions using light microscopy and electron microscopy
Mice were killed at 1, 3, 5 and 8 days after inoculation. Autopsy samples were obtained aseptically from the skin lesions. Five mice were tested each time. Specimens for light microscopy were fixed in 10% formalin solution and embedded in paraffin, and sections stained with haematoxylineosin. Specimens for electron microscopical examination were prepared following a routine procedure and were examined using a JEM-100SX electron microscope (JEOL, Akishima, Japan).
Antibiotic therapy
Forty-eight hours after the inoculation, the mice were put on one of the following 6 day treatment regimens: roxithromycin 60 mg/kg given orally every 12 h, imipenem 200 mg/kg given im every 12 h, or roxithromycin 60 mg/kg given orally as above and imipenem 200 mg/kg given im as above. Control animals were left untreated. The Figure shows the experimental schedule. In addition, roxithromycin 0.6 or 6 mg/kg, and imipenem 200 mg/kg were also tested. Samples (1.0 x 1.0 cm2) taken from the experimental lesions (n = 5) on days 5 and 8 were cut into small pieces using scissors and were homogenized with a 2 mL saline solution using a pestle and mortar. Two aliquots of 0.1 mL were cultured on MuellerHinton agar at 37°C using the 10-fold dilution method. After 24 h of incubation, the cfu were counted and expressed as log10 cfu per lesion.
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Skin concentrations were measured in uninfected mice after a single administration of roxithromycin (0.6, 6 and 60 mg/kg) and imipenem (200 mg/kg). Full thickness skin samples were taken from mice. The concentrations of imipenem and roxithromycin were measured by the agar diffusion method (n = 3), using Bacillus subtilis ATCC 12432 and Micrococcus luteus ATCC 9341, respectively, as the indicator organisms. The limits of detection for these methods were 0.0625 µg/g for imipenem and 0.05 µg/g for roxithromycin.
Effect of antibiotics in combination with colchicine on experimental model
Colchicine (Sigma) at 0.5 mg/kg was administered to mice bd immediately after the inoculation whereas the regimens remained as describe above. Samples were obtained on day 8 after the inoculation (n = 5).
Effect of roxithromycin on biofilm model in neutropenic mice
In a previous biofilm model formed by inoculation with methicillin-resistant S. aureus (MRSA) (roxithromycin MIC 128 mg/L) on cut wounds1 in neutropenic mice treated by cyclophosphamide, we treated the mice with roxithromycin 60 mg/kg every 12 h and examined the biofilm lesions by light and electron microscopy.
Statistical methods
Data analysis was conducted using the t-test for unpaired comparisons.
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Results |
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The colony counts on membranous structures were not lower in rabbit plasma with imipenem, roxithromycin or imipenem plus roxithromycin than in the untreated control plasma after incubation for 24 h at 37°C (Table).
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After inoculation for 24 h, S. aureus cells formed clusters of bacterial colonies in the dermis. Fibrin-like homogeneous structures were seen around the clusters. On day 3 after the inoculation, numerous polymorphonuclear leucocytes and macrophages were seen around the clusters formed by S. aureus cells, which appeared to be separated from the clusters. Electron microscopic findings revealed that S. aureus cells had formed microcolonies, enclosed in membrane-like structures, by day 3. On days 5 and 8 after the inoculation, numerous polymorphonuclear leucocytes and macrophages did not invade the clusters in the untreated control group and imipenem-treated group (200 mg/kg), but did invade the clusters in the roxithromycin-treated (60 and 6 mg/kg) and roxithromycin/imipenem-treated groups (roxithromycin 60 mg/kg, imipenem 200 mg/kg).
Antibiotic therapy
The Figure shows the therapeutic effects of the combined use of imipenem and roxithromycin against the experimental infections in mice. Treatment with roxithromycin alone and imipenem alone did not result in a significant decrease in the number of viable bacteria compared with the control, wheras a combined treatment with roxithromycin and imipenem resulted in a significant decrease in the number of viable bacteria on day 8 after inoculation (P < 0.01). On day 8 after treatment with roxithromycin at 6 mg/kg and imipenem at 200 mg/kg, bacterial counts were about one-tenth of those in the untreated control (P < 0.05). However, on day 8 after treatment with roxithromycin at only 0.6 mg/kg and imipenem at 200 mg/kg, bacterial counts did not decrease compared with the control.
Antibiotic assay
After the intramuscular injection of imipenem at a dose of 200 mg/kg, the skin concentration of imipenem was 0.635 µg/g at 3 h. After oral administration of roxithromycin at a dose of 60 mg/kg, the mean skin concentrations of roxithromycin were 4.873 µg/g at 1 h, 1.413 µg/g at 3 h and 0.913 µg/mL at 6 h. After oral administration of roxithromycin at a dose of 6 mg/kg, the mean skin concentration of roxithromycin was 0.737 µg/g at 3 h. After oral administration of roxithromycin at a dose of 0.6 mg/kg, skin concentration was below the level of detection at 3 h.
Effect of antibiotics in combination with colchicine on the experimental model
After inoculation for 8 days, the colony counts (log10 cfu/ unit area; mean ± S.D.; n = 5) of strain X in the experimental mice model treated with colchicine at 0.5 mg/kg alone (control) were 6.47 ± 0.21, and those treated with roxithromycin 60 mg/kg, imipenem 200 mg/kg, or roxithromycin 60 mg/kg and imipenem 200 mg/kg in combination with colchicine were 6.56 ± 0.31, 6.17 ± 0.47 and 6.08 ± 0.14, respectively. The colony counts of strain X in mice treated with roxithromycin, imipenem or roxithromycin and imipenem in combination with colchicine were not lower than those of untreated controls.
Effect of roxithromycin on biofilm model in neutropenic mice
For 48 h after inoculation, inflammatory cells had not invaded the biofilm in the untreated control group. Inflammatory cells had invaded the biofilms and phagocytosed bacteria in the group after 2 days of treatment with roxithromycin.
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Discussion |
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Nemoto et al.3 reported that a S. aureus biofilm that formed in a culture medium on coverslips continued to thicken until 72 h after inoculation. Since a longer inoculation did not produce a marked change in the biofilm, they employed a 3 day inoculation when preparing their mature S. aureus biofilm.3 The membranous structures in our study, which employed a 24 h incubation, were considered to be immature biofilms containing S. aureus cells, fibrin and plasma components. We thought that these structures were a physical barrier, which did not permit the antimicrobial agents to reach the S. aureus.
In the present study, we demonstrated that the combination of roxithromycin and imipenem was effective in a non-neutropenic mouse model. Unlike other in vitro experiments on membranous structures or with the administration of colchicine to suppress neutrophil function, no such combined effect was observed. This indicates that PMNs have an important role in the treatment of S. aureus biofilm. PMNs also invaded MRSA biofilm with roxithromycin in the neutropenic mice model. This indicates that roxithromycin, which has no antibacterial activity against the strain, can enhance neutrophil activities against the biofilm. Further, these findings indicate that the in vivo combined effect of roxithromycin and imipenem in this non-neutropenic mice model was mainly attributable to invasion of neutrophils into the clusters.
There is disagreement in the literature regarding the effect of macrolide antibiotics on neutrophil function. Several studies have reported enhancement of neutrophil function by 14-membered ring macrolides.4,5 In contrast, others have demonstrated that macrolides suppressed neutrophil function.6,7 It is believed that the anti-neutrophilic mechanism may be one reason why macrolides are effective against diffuse panbrochiolitis where there is an excessive accumulation of neutrophils. Thus, the effect against neutrophils may differ depending on dose, duration, microbial target and the condition of the neutrophils in a given host.
Regarding the effect of macrolides on bacterial bio-films, several reports have indicated that a macrolide antimicrobial agent not only eradicated a biofilm produced by Pseudomonas aeruginosa, but also suppressed the production of the biofilm,8,9 and thereby enhanced the clearance of bacteria by PMNs and the therapeutic efficacy of other antibiotics against infections caused by P. aeruginosa.
There have been few investigations into the effect of macrolides on biofilms produced by S. aureus. While Dasgupta et al.10 reported that erythromycin demonstrates poor MRSA biofilm permeability and killing in a modified Robbins device compared with quinolone antibiotics, they did not discuss the influence of neutrophils against an S. aureus biofilm.
The present findings indicate that a combination treatment of roxithromycin and imipenem is potentially effective against biofilm-associated skin infections. However, further studies are necessary to elucidate the precise mechanism of these combined chemotherapeutic effects.
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Notes |
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References |
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2 . 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]
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4 . Anderson, R., Fernandes, A. C. & Eftychis, H. E. (1984). Studies on the effects of ingestion of a single 500 mg oral dose of erythromycin stearate on leucocyte motility and transformation and on release in vitro of prostaglandin E2 by stimulated leucocytes. Journal of Antimicrobial Chemotherapy 14, 4150.[Abstract]
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6 . Nelson, S., Summer, W. R., Terry, P. B., Warr, G. A. & Jakab, G. J. (1987). Erythromycin-induced suppression of pulmonary antibacterial defenses. A potential mechanism of superinfection in the lung. American Review of Respiratory Disease 136, 120712.[ISI][Medline]
7 . Kadota, J., Sakito, O., Kohno, S., Sawa, H., Mukae, H., Oda, H. et al. (1993). A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. American Review of Respiratory Disease 147, 1539.[ISI][Medline]
8 . Yasuda, H., Ajiki, Y., Koga, T., Kawada, H. & Yokota, T. (1993). Interaction between biofilm formed by Pseudomonas aeruginosa and clarithromycin. Antimicrobial Agents and Chemotherapy 37, 174955.[Abstract]
9 . Takeoka, K., Ichiyama, T., Yamasaki, T. & Nasu, M. (1998). The in vitro effect of macrolides on the interaction of human polymorphonuclear leukocytes with Pseudomonas aeruginosa in biofilm. Chemotherapy 44, 1907.[ISI][Medline]
10 . Dasgupta, M. K., Shishido, H., Salama, S., Singh, R., Larabie, M. & Micetich, R. G. (1997). The effects of macrolide and quinolone antibiotics in methicillin-resistant Staphylococcus aureus biofilm growth. Advances in Peritoneal Dialysis 13, 2147.[Medline]
Received 15 November 2000; returned 8 February 2001; revised 27 June 2001; accepted 29 June 2001