Department of Microbiology, Medical School, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece
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Abstract |
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Introduction |
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Materials and methods |
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The antimicrobial agents tested were ciprofloxacin, clarithromycin, erythromycin, pefloxacin and rifampicin. They were purchased from commercial sources and stock solutions of each drug were prepared in concentrations of 2 g/L and stored at 20°C for a maximum of 1 month. Final antibiotic concentrations tested were 0.0158 mg/L for clarithromycin and erythromycin, 0.0154 mg/L for ciprofloxacin and pefloxacin, and 0.0071 mg/L for rifampicin.
The agar dilution method with two-fold antibiotic dilutions was used to determine MICs. L. pneumophila isolates were inoculated on BCYE agar, incubated for 48 h at 37°C with 2.5% CO2 and harvested in sterile distilled water, and turbidity was then adjusted to 0.5 McFarland standard. The suspensions were subsequently diluted 1:100 in distilled water. A multiprong replicator device (Becton Dickinson, Meylan, France) was used to inoculate approximately 104 cfu/spot from each bacterial suspension onto the BCYE agar plates containing appropriate antimicrobial concentrations. Plates were incubated at 37°C in ambient atmosphere and were examined for growth after 48 h. The MIC was recorded as the lowest drug concentration at which no growth occurred. Single colonies or a faint haze were ignored. All determinations were carried out in duplicate and the results were expressed as the mean of the two values. As little information has been published on the susceptibility of type isolates of legionellae, we used Staphylococcus aureus ATCC 25923 as a control organism. It was inoculated in parallel onto antimicrobial agent-containing MuellerHinton agar plates as well as BCYE plates, to determine whether BCYE inhibited the activity of the antibiotics.
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Results and discussion |
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A standard method for the determination of MICs of antibiotics for Legionella spp. is not available. It has been proposed that results can be affected by the inactivation of certain compounds by components of Charcoal Yeast Extract agar, especially charcoal.10 We chose BCYE for MIC determinations because the ability of Legionella spp. isolates to grow on charcoal-free media is controversial.8 On such media we also observed problems of inadequate growth with some isolates of L. pneumophila, with resulting difficulty in interpreting results. Although the MICs of the drugs tested may be higher on charcoal media, we found that all isolates were susceptible. Moreover, the MICs for the control S. aureus isolate on BCYE were within the published ranges for the NCCLS method and did not differ markedly from those obtained on MuellerHinton agar, being usually only two-fold lower.
Surveillance studies on the in-vitro activities of antibiotics used for legionnaires' disease are necessary, to identify changes in susceptibility rates. Although there have been indications of the inadequacy of oral erythromycin in clinical cases of legionnaires' disease,2,3 there was no resistance noted among the 82 environmental isolates tested. Late treatment may be the main cause of therapeutic failure in legionnaires' disease. Considering the fact that the disease is caused by exposure to aerosols of legionella-colonized water, it could be suggested that potential cases of legionellosis in our regions could be successfully treated with the antibiotics tested, if they were given early.
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Acknowledgments |
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Notes |
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References |
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8 . Pendland, S. L., Killian, A. D., Woodward, J. G. & Rodvold, K. A. (1997). In-vitro activity of a new oral streptogramin, RPR 106972, alone and in combination with rifampicin or ciprofloxacin against Legionella spp.Journal of Antimicrobial Chemotherapy 39, 6513.[Abstract]
9 . Reda, C., Quaresima, T. & Castellani Pastoris, M. C. (1994). In-vitro activity of six intracellular antibiotics against Legionella pneumophila strains of human and environmental origin.Journal of Antimicrobial Chemotherapy 33,757 64.[Abstract]
10 . Bornstein, N., Roudier, C. & Fleurette, J. (1985). Determination of the activity on Legionella of eight macrolids and related agents by comparative testing on three media.Journal of Antimicrobial Chemotherapy 15, 1722.[Abstract]
Received 23 March 1999; returned 15 June 1999; revised 25 June 1999; accepted 7 July 1999