Journal of Antimicrobial Chemotherapy (1999) 44, 693-695
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
In-vitro activity of antibiotics against Legionella pneumophila isolates from water systems
Department of Microbiology, Medical School, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece
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Abstract |
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The activity of five antibiotics was tested against 82 isolates of Legionella pneumophila obtained from water systems in nine European countries. All isolates were susceptible to clarithromycin (MICs 0.03–0.5 mg/L), erythromycin (MICs 0.125–2.0 mg/L), ciprofloxacin (MICs 0.06–0.25 mg/L), pefloxacin (MICs 0.06–0.5 mg/L) and rifampicin (MICs
0.007–0.015 mg/L). It seems that antibiotics used for legionellosis
continue to exhibit
good activity against L. pneumophila isolates from environmental sources in European
countries. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Legionella pneumophila is a facultative intracellular pathogen causing cases of pneumonia known as legionnaires' disease. The infection is believed to be acquired by inhalation of aerosols from environmental sources and carries a mortality rate of 10–30%.1 Antimicrobial agents used for therapy should be able to penetrate eukaryotic cells, and clinical experience supports the choice of erythromycin, with fluoroquinolones and rifampicin as suitable alternatives.2 Although several studies2,3 have reported treatment failure in patients with legionnaires' disease receiving such antibiotics, increased resistance in clinical isolates of L. pneumophila has not been documented,4,5 and there are only in-vitro studies describing antibiotic-resistant variants.6 However, susceptibility testing of L. pneumophila to appropriate antibiotics should be undertaken to monitor the possible emergence of resistant isolates in different regions. As legionellosis is acquired from water systems, we examined isolates of L. pneumophila obtained from such environments in various European countries for susceptibility to five antimicrobials currently used for treatment.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Eighty-two isolates of L. pneumophila (all serogroup 1), obtained from water systems (tap water, shower water, cooling towers and spa water) in hotels and hospitals in nine European countries (Denmark, France, Germany, Greece, Italy, Spain, Sweden, Switzerland, UK) were tested. Isolates were not epidemiologically related and in only a few cases were from environments associated with human infection. They were stored at –70°C, after one subculture on buffered charcoal yeast extract (BCYE) agar (Oxoid, Basingstoke, UK), following receipt at our laboratory.
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 0.015–8 mg/L for clarithromycin and erythromycin, 0.015–4 mg/L for ciprofloxacin and pefloxacin, and 0.007–1 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 Mueller–Hinton 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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Susceptibility testing of L. pneumophila is not usually performed, as the species remains susceptible to antibiotics commonly used for treatment. However, susceptibility trends of these pathogens should be monitored periodically both in clinical and environmental isolates. In this study we compared the in-vitro activity of five antibiotics against L. pneumophila isolates from water systems in nine European countries. Percentages of isolates inhibited by different concentrations of the antibiotics tested are shown in the Table. MICs were in the following ranges: erythromycin 0.125–2 mg/L; clarithromycin 0.03–0.05 mg/L; rifampicin
0.007–0.015 mg/L; ciprofloxacin 0.06–0.25 mg/L; pefloxacin
0.06–0.5 mg/L. Thus, rifampicin was found to be the most active agent against L.
pneumophila. All isolates were inhibited by low concentrations of the two quinolones
tested, with ciprofloxacin being approximately two-fold more active than pefloxacin. They were
also susceptible to macrolide antibiotics, although MICs were somewhat higher. Clarithromycin
was found to exceed the activity of erythromycin by a factor of four (MIC90 0.25 and
1 mg/L, respectively). These results are in accordance with those of previous studies, regarding
the very high activity of clarithromycin against clinical isolates of L. pneumophila.7
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Susceptibility data based on environmental L. pneumophila isolates are of interest because legionellosis occurs after exposure to aerosols of legionella-colonized water and there is no carrier state for the microorganism. The results of the present study showed that antimicrobials used against legionnaires' disease continue to show excellent antimicrobial activity against environmental isolates obtained in various regions of Europe.Biotype diversity associated with a low level of intrinsic drug resistance may be responsible for the somewhat different MICs observed. However, when we compared isolates from hospital environments with isolates from hotel environments, differences in sensitivity to antibiotics were not detected (data not shown). Furthermore, our environmental isolates were almost four-fold more susceptible to erythromycin and ciprofloxacin and eight-fold to rifampicin in comparison with clinical isolates of L. pneumophila tested with the same culture medium and under the same conditions.8 Similarly, in a recent study,9 rifampicin was found to be 100-fold less active for human isolates than for environmental isolates.
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 Mueller–Hinton 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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We thank members of the European Working Group on Legionella Infections (EWGLI) for providing isolates from their culture collection.
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Notes |
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* Corresponding author. Tel:+30-31-99-91-49; E-mail: atsakris{at}med.auth.gr
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Received 23 March 1999; returned 15 June 1999; revised 25 June 1999; accepted 7 July 1999
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