Effect of clarithromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model

Katsunori Yanagihara,*, Kazunori Tomono, Yoshifumi Imamura, Yukihiro Kaneko, Misuzu Kuroki, Toyomitsu Sawai, Yoshitsugu Miyazaki, Yoichi Hirakata, Hiroshi Mukae, Jun-Ichi Kadota and Shigeru Kohno

The Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852, Japan

Received 10 September 2001; returned 4 December 2001; revised 3 January 2002; accepted 22 January 2002.


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Fourteen-membered macrolides (e.g. clarithromycin and erythromycin), but not 16-membered macrolides (e.g. josamycin), are effective in diffuse panbronchiolitis. However, there are no studies that have compared the effects of 14- and 16-membered macrolide antibiotics on biofilm formation. Treatment with high-dose clarithromycin (100 mg/kg) resulted in a significant decrease in the number of viable bacteria in an experimental murine model. Josamycin at a dose of up to 100 mg/kg had no effect on the number of viable bacteria in the lung. Our results may explain, at least in part, the clinical efficacy of 14-membered macrolide antibiotics in patients with chronic pneumonia caused by Pseudomonas aeruginosa.

Keywords: macrolide antibiotics, chronic respiratory infection, Pseudomonas aeruginosa, biofilm formation, murine model


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diffuse panbronchiolitis is a common and representative cause of chronic respiratory tract infections in Japan, treated effectively by a long course of macrolide antibiotics.1,2 The latter group of antibiotics is also efficacious against other airway inflammatory disorders such as cystic fibrosis.3 In this regard, 14-membered macrolides (e.g. clarithromycin and erythromycin), but not 16-membered macrolides (e.g. josamycin) are effective against diffuse panbronchiolitis (DPB). This difference has been reported by other investigations; josamycin failed to influence the proliferation of lymphocytes in a mouse model of chronic infection,4 and did not inhibit cytokine production from THP-1 cells.5 However, to our knowledge, there are no in vivo studies that have compared the effects of 14- and 16-membered macrolide antibiotics on biofilm formation.

Recently, we established a new murine model of chronic respiratory Pseudomonas aeruginosa infection produced by placement of a plastic tube precoated with a biofilm-forming organism in the bronchus.4,6 In the present study, we compared the effect of a 14- and a 16-membered macrolide on chronic respiratory infection with biofilm formation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Laboratory animals

Male, ddY, 7-week-old, 30–35 g body weight, specific pathogen-free mice were purchased from Shizuoka Agricultural Cooperative Association Laboratory Animals (Shizuoka, Japan). All mice were housed in a pathogen-free environment and received sterile food and water in the Laboratory Animal Center for Biomedical Science at Nagasaki University. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation at our institution.

Bacterial strain

Animals were infected with mucoid P. aeruginosa NUS10, a clinical isolate obtained from sputum of patients at Nagasaki University Hospital. The bacteria were stored at –70°C in Brain–Heart Infusion Broth (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 10% (v/v) gly-cerol and 5% (w/v) skimmed milk (Yukijirushi Co., Tokyo, Japan) until use.

Intubation tube

Disposable sterile plastic cut-down intravenous catheters (3 Fr, 1.0 mm diameter; Atom Co., Tokyo, Japan) were used for intubation/infection. The tubes were 3.0 mm long with a few slits made at the proximal end to prevent clogging by oral/airway secretions.

Preparation of bacterial precoated tube

A detailed description of the method used was reported previously by Yanagihara et al.6 In the first step, P. aeruginosa was cultured on Trypticase Soy agar (BBL Microbiology Systems) for 24 h. The bacteria were suspended in saline (0.9% NaCl), harvested by centrifugation (3000g, 4°C for 10 min), resuspended in sterile saline and adjusted to 1 x 109 cfu/mL as estimated by turbidimetry. The intubation tube was then im-mersed in the bacterial saline suspension for 3 days at 37°C. The number of bacteria on tubes after 3 days of incubation before intubation was 6.24 ± 0.43 log10 cfu/mL (mean ± S.D., n = 10). After immersing the tubes, they were subjected to scanning electron microscopy. These specimens were then fixed for 2 h at 4°C with 1% glutaraldehyde in 0.1 M phosphate-buffered saline, followed by refixation for 2 h at 4°C in 1% osmium acid in the same buffer, dehydration in a series of aqueous ethanol solutions (50–100%) and freeze drying. Samples were then coated with platinum/palladium using an ion sputter and observed with a JSC-35C scanning electron microscope (Nihon Dennshi Kogyo, Tokyo, Japan).

Experimental model of infection

The methods used were those described previously.4 The intubation procedure was carried out under anaesthesia. Briefly, the blunted end of the inner needle of an intravenous catheter (Angiocath; Becton Dickinson Vascular Access, Sandy, UT, USA) was inserted through the oral cavity of anaesthetized mice, with the outer sheath and the attached tube at the tip. The tube was advanced through the vocal cords into the trachea. The inner needle was pulled out followed by a gentle push of the outer sheath to place the pre-coated tube into the main bronchus. After intubation, the infected mice were provided with food and water.

Bacteriological examination

Both lungs were homogenized and cultured separately. Bacterial enumeration was carried out using serial dilutions on Trypticase Soy agar in NAC agar plates (BBL Microbiology Systems), incubating the plates at 37°C in air overnight and then counting colonies on plates to estimate cfu in lungs of mice.

Drug administration and MIC determination

Clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan) and josamycin (Daiichi Pharmaceutical Co., Tokyo, Japan) were dissolved in sterile water immediately before use. The MIC of each agent was determined by the agar dilution technique using Mueller–Hinton agar (Becton Dickinson Microbiology Systems), with an inoculum size of 104 cfu per spot. The MIC was defined as the lowest concentration of agent that inhibited visible growth of P. aeruginosa after 16 h of incubation at 37°C. The MICs of clarithromycin and josamycin were 250 and 500 µg/mL, respectively. Treatment commenced 7 days after intubation of the precoated tube with mucoid type P. aeruginosa. Thirty-five mice were allocated into seven treatment groups: clarithromycin (10 mg/kg/day), clarithromycin (50 mg/kg/day), clarithromycin (100 mg/kg/day), josamycin (10 mg/kg/day), josamycin (50 mg/kg/day), josamycin (100 mg/kg/day) and saline for the control group. Each drug was administered once a day for 10 days and the animal was watched carefully to ensure that it received the full dose.

Statistical analysis

Data were expressed as means ± S.E.M. Differences between groups were examined for statistical significance using the unpaired Student’s t-test. P < 0.05 denoted a statistically significant difference.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Scanning electron microscopy of intubated tube

A scanning electron micrograph of the surface of the catheter intubated for 7 days in mouse bronchus demonstrated in vivo formation of a biofilm containing blood cells, complex fibrous structures and bacteria (Figure 1).



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Figure 1. Scanning electron microscopy of the biofilm on the surface of a catheter that has been intubated in the mouse bronchus for 7 days (x3000).

 
Therapeutic effect of antibiotics against chronic respiratory P. aeruginosa infection

A significant number of viable bacteria were found in the lungs of control animals (5.50 ± 0.46 log10 cfu/mL, n = 5). Treatment with josamycin had no effect on the number of viable bacteria in the lung (Figure 2). In contrast, high-dose clarithromycin (100 mg/kg/day) resulted in a significant decrease in the number of viable bacteria compared with the control (3.20 ± 0.46 log10 cfu/mL, n = 5, P < 0.01).



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Figure 2. Effects of clarithromycin (CAM) and josamycin (JM) on viable bacteria in chronic respiratory infection. CAM 100 mg/kg resulted in a significant decrease in the number of viable bacteria compared with the control. JM had no effect on the number of viable bacteria. Each bar represents the mean ± S.D. of five animals. *P < 0.01 compared with the control.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Biofilm bacteria are a major concern for clinicians in the treatment of infectious diseases because of their resistance to a wide range of antibiotics. Biofilm has in fact been found on the surface of biomaterials and tissues in chronic bacterial infections characterized by resistance to chemotherapy and resistance to clearance by the humoral or cellular host defence mechanism.7 Recently, bacterial biofilms have been detected on a number of living and inert surfaces within the human body. Establishment of a biofilm is the prelude to the development of various chronic, refractory infections, such as biomaterial-associated infection and pulmonary infection in intubated patients and patients with cystic fibrosis or DPB.89 Macrolide antibiotics offer a new strategy for infection with biofilm formation.

In the present study, we demonstrated that high-dose clarithromycin was effective in treating biofilm-associated chronic respiratory P. aeruginosa infection without any antimicrobial agents against P. aeruginosa. It is as yet uncertain whether the activity of clarithromycin to remove glycocalyx is independent of the general modes of antibacterial activity of clarithromycin. With regard to bacterial biofilm, Yasuda et al.10 showed by using experimentally induced subcutaneous infection in rats in the presence of biofilm formed by P. aeruginosa that the quantities of alginate and hexose in which bacterial biofilm had been formed clearly decreased in a dose-dependent manner after treatment with clarithromycin. In our model, P. aeruginosa is expected to be eradicated by the mucocilial transportation system and phagocytic cells after the disappearance of biofilm formation.

We have already reported that 14-membered macrolides such as clarithromycin and erythromycin eradicated P. aeruginosa from patients with DPB without any antimicrobial agents against P. aeruginosa after 1–12 months of treatment.11 The present results add support to these clinical data. Moreover, the need for ‘long-term’ treatment for patients may be related to the ‘high dosage’ in our mouse model. The mechanisms of the differences in the effectiveness of 14- and 16-membered macrolides on biofilm formation are not clear at present. It would be worth looking at other 16-membered macrolides to see whether failure to effect biofilm formation is a ‘group’ effect of 16-membered macrolides. It is possible that such differences are due to differences in the fine structure of these compounds, such as substitutions on the lactone ring and/or sugar composition of these antibiotics.

In conclusion, we reported here that clarithromycin but not josamycin was effective against chronic respiratory infection with biofilm formation. The present data may explain, at least in part, the clinical efficacy of 14-membered macrolide antibiotics in chronic pneumonia caused by P. aeruginosa.


    Footnotes
 
* Corresponding author. Tel: +81-95-849-7276; Fax: +81-95-849-7285; E-mail: kyana-ngs{at}umin.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Kudoh, S., Uetake, T., Hagiwara, K., Hus, L.-H., Kimura, H. & Sugiyama, Y. (1987). Clinical effect of low-dose and long-term erythromycin chemotherapy on diffuse panbronchiolitis. Japanese Journal of Thoracic Disease 25, 632–42.

2 . Kudoh, S., Azuma, A., Yamamoto, M., Izumi, T. & Ando, M. (1998). Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. American Journal of Respiratory and Critical Care Medicine 157, 1829–32.[Abstract/Free Full Text]

3 . Jaffe, A., Francis, J., Rosenthal, M. & Bush, A. (1998) Long-term azithromycin may improve lung function in children with cystic fibrosis. Lancet 351, 420.[ISI][Medline]

4 . Yanagihara, K., Tomono, K., Sawai, T., Hirakata, Y., Kadota, J., Koga, H. et al. (1997). Effect of clarithromycin on lymphocytes in chronic respiratory Pseudomonas aeruginosa infection. American Journal of Respiratory and Critical Care Medicine 155, 337–42.[Abstract]

5 . Fujii, T., Kadota, J., Morikawa, T., Matsubara, Y., Kawakami, K., Iida, K. et al. (1996). Inhibitory effect of erythromycin on interleukin-8 production by 1{alpha}, 25-dihydroxyvitamin D3-stimulated THP-1 cells. Antimicrobial Agents and Chemotherapy 40, 1548–51.[Abstract]

6 . Yanagihara, K., Tomono, K., Sawai, T., Kuroki, M., Kaneko, Y., Ohno, H. et al. (2000). Combination therapy for chronic Pseudomonas aeruginosa respiratory infection associated with biofilm formation. Journal of Antimicrobial Chemotherapy 46, 69–72.[Abstract/Free Full Text]

7 . Gray, E. D., Peters, G., Verstegen, M. & Regelmann, W. E. (1984). Effect of extracellular slime substance from Staphylococcus epidermidis on the human cellular immune response. Lancet i, 365–7.

8 . Kobayashi, H. (1995). Airway biofilm disease: clinical manifestation and therapeutic possibilities using macrolides. Journal of Infection and Chemotherapy 1, 1–15.

9 . Koch, C. & Hoiby, N. (1993). Pathogenesis of cystic fibrosis. Lancet 341, 1065–9.[ISI][Medline]

10 . Yasuda, H., Ajiki, Y., Koga, T., Kawada H. & Yokota, T. (1993). Interaction between biofilms formed by Pseudomonas aeruginosa and clarithromycin. Antimicrobial Agents and Chemotherapy 37, 1749–55.[Abstract]

11 . Fujii, T., Kadota, J., Kawakami, Iida, K., Shirai, R., Kaseda, M. et al. (1995). Long-term effect of erythromycin therapy in patients with chronic Pseudomonas aeruginosa infection. Thorax 50, 1246–52.[Abstract]