1 Laboratoire de Bactériologie-Hygiène, Hôpital Calmette, Bd Pr J. Leclercq; 2 Unité de Gastroentérologie, Hépatologie et Nutrition, Clinique de Pédiatrie, Hôpital Jeanne de Flandre, 2 Av Oscar Lambret, CHRU, 59037 Lille, France
Keywords: mucoid strains, non-mucoid strains, solid-phase adherence assays
Sir,
Pseudomonas aeruginosa, an opportunistic pathogen, is the major pathogen in the airways of patients with cystic fibrosis (CF) and is currently associated with the morbidity and mortality seen in this disease. Macrolides, such as azithromycin, are normally not included in the anti-pseudomonal therapeutic arsenal because of the absence of bactericidal or bacteriostatic activity. However, several previous investigators have reported that long-term administration of azithromycin is effective in patients with pulmonary P. aeruginosa infections.1 The clinical benefits achieved could include the effects of an anti-inflammatory2 and/or modulation of the production of virulence factors of P. aeruginosa, such as bacterial exoproducts,3 the formation of biofilm,4 or the synthesis of flagella3 and some outer membrane proteins.5 Adherence of P. aeruginosa to respiratory mucins plays an important role in the colonization of the airways of these patients. This adherence process involves flagella and several non-pilus adhesins localized on the outer membrane of P. aeruginosa. Supporting the clinical observations, adherence of this organism to human salivary or airway mucins has been demonstrated in vitro using liquid- or solid-phase adherence assays.6
In order to investigate the action of azithromycin on the pathogenesis of P. aeruginosa, we evaluatedin a solid-phase adherence assaythe action of subinhibitory concentrations of the macrolide on the adherence of different bacterial strains to respiratory mucins of CF patients. The reference strain PAO-1, and 13 strains (five mucoid and eight non-mucoid strains) freshly isolated from the sputum of CF patients known to be infected by different strains and who were not treated with azithromycin, were tested. MICs of azithromycin were determined by an agar dilution method, following the recommendations of the NCCLS (2002), with powdered azithromycin supplied by Pfizer laboratories (Groton, CN, USA). Adherence assays were performed in 96-well microtitre plates (Linbro, INC laboratories, France), in which wells were coated with 10 µg of purified bronchial mucins prepared from tracheobronchial secretions of CF patients. As described by Vishwanath & Ramphal,6 100 µL of a suspension of 1 x 106 bacteria/mL, grown while agitating for 18 h with or without 2 mg/L or 4 mg/L azithromycin, was added into each well. Plates were incubated at 37°C for 30 min. They were then washed 10 times with sterile phosphate buffer and spread out on trypticase soy agar plates for enumeration; any bound bacteria were desorbed with 0.5% Triton X-100. All experiments were performed at least twice in three wells coated with mucins. For each strain, a set of three uncoated wells was used as a negative control. Results were analysed when the number of bacterial cells bound to plastic was at least 10-fold lower than that bound to mucins. They were expressed as the ratio of the mean number of cfu bound to mucins, to the mean number of cfu present in the initial bacterial suspension. Results were compared using a non-parametric Wilcoxon test, where the differences were considered significant at P < 0.05.
The results of the adherence assays of P. aeruginosa to mucins are summarized in Table 1. All bacterial strains were able to adhere to bronchial mucins in our assays, with a mean rate of adherence of 7.8% for non-mucoid clinical strains and 6.2% for mucoid strains. The effect of azithromycin on these adherence properties was studied at 2 and 4 mg/L. These concentrations were much lower than the MICs, which varied from 64 to >1024 mg/L, but they were compatible with clinically achievable concentrations.7,8 At these two concentrations, the action of azithromycin on adherence was efficient for the PAO-1 strain, but varied with clinical strains, since it was observed for only six of the eight non-mucoid and for four of the five mucoid strains, with a significant decrease of 47.5% (range 30%88%) and 56% (range 36%90%), respectively. The concentration of azithromycin required for inhibition appeared to be correlated to the phenotype of the strains. The decrease in adherence of non-mucoid isolates was observed especially with azithromycin 2 mg/L, whereas that of mucoid isolates was low or even non-existent with azithromycin 2 mg/L but obvious with azithromycin 4 mg/L. This observation agreed with numerous studies that describe a decrease in antibiotic susceptibility of mucoid strains. However, we could not establish whether the inhibition of adherence was dose-dependent, since only two concentrations of azithromycin were analysed. For the PAO-1 strain, an increase in the inhibition of adherence was observed between azithromycin 2 and 4 mg/L, whereas it was not confirmed for the non-mucoid clinical strains. The inhibition of the adherence properties of bacteria were in agreement generally with low MICs, but it was difficult to show an absolute correlation between low MICs of azithromycin and the effect of the macrolide on adherence of bacterial cells to mucins. Thus, no decrease was observed in one strain (strain mucoid 2) with an MIC of 128 mg/L, whereas an effect was seen in another (strain non-mucoid 6) for which the MIC was 1024 mg/L.
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Acknowledgements
We thank the association Vaincre la Mucoviscidose for their grant.
Footnotes
* Corresponding author. Tel: +33-320-44-49-44; Fax: +33-320-44-48-95; E-mail: mohusson{at}chru-lille.fr
References
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