ORIGINAL ARTICLE. Clin Microbiol Infect 2002; 8:

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1 ORIGINAL ARTICLE In vitro activity of levofloxacin against contemporary clinical isolates of Legionella pneumophila, Mycoplasma pneumoniae and Chlamydia pneumoniae from North America and Europe I. A. Critchley 1, M. E. Jones 2, P. D. Heinze 3, D. Hubbard 3, H. D. Engler 3, A. T. Evangelista 4, C. Thornsberry 1, J. A. Karlowsky 1 and D. F. Sahm 1 1 Focus Technologies Inc. (formerly MRL), Herndon, VA, USA, 2 Hilversum, The Netherlands, 3 Cypress, CA, USA and 4 Ortho-McNeil Pharmaceutical, Raritan, NJ, USA Objective To assess the activities of levofloxacin and the comparator agents erythromycin, clarithromycin, azithromycin and doxycycline against atypical respiratory pathogens. Methods One hundred and forty-six Legionella pneumophila, 41Mycoplasma pneumoniae and nine Chlamydia pneumoniae isolates were procured from various culture collections in North America and Europe and tested for susceptibility to the above agents by broth microdilution. The isolates came primarily from clinical sources and were collected from patients between 1995 and Results Against L. pneumophila, levofloxacin was the most active agent, with an MIC 90 of 0.03 mg/l, twofold more active than clarithromycin (0.06 mg/l), 16-fold more active than erythromycin and azithromycin (0.5 mg/l) and 64-fold more active than doxycycline. Against M. pneumoniae, azithromycin (MIC mg/l) was the most active agent. However, two isolates of M. pneumoniae, one from the USA and one from Finland, were macrolide resistant (MIC 4 mg/l), but levofloxacin susceptible (MIC 0.25 mg/l). The geographic origin of L. pneumophila and M. pneumoniae did not affect the MIC range for any antimicrobial agent tested. Against C. pneumoniae, clarithromycin was the most active agent, with an MIC range of mg/l. Conclusions Levofloxacin had comparable activity to the other agents tested against the atypical respiratory pathogens, confirming its potential as an alternative for empirical therapy of community-acquired pneumonia. Keywords Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, atypical pathogens, levofloxacin Accepted 21 November 2001 Clin Microbiol Infect 2002; 8: INTRODUCTION It is estimated that there are three to four million cases of community-acquired pneumonia (CAP) in the United States each year, resulting in Corresponding author and reprint requests: I. A. Critchley, Focus Technologies Inc., Dulles Technology Drive, Suite 200, Herndon, Virginia, 20171, USA Tel: þ Fax: þ icritchley@focusanswers.com hospitalizations [1]. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are common etiologic agents; however, the atypical respiratory pathogens Legionella pneumophila, Mycoplasma pneumoniae and Chlamydia pneumoniae have also recently been shown to be important etiologic factors in CAP [2,3]. In many cases, initial therapy for CAP is empirical, and in 30 50% of cases the causative organism is never established [4]. Diagnosis of atypical pathogens is further complicated by a relative absence of routine diagnostic testing, making it difficult to estimate their ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 Critchley et al Levofloxacin activity against atypical respiratory pathogens 215 true prevalence in CAP. Oral b-lactam antibiotics are becoming less attractive as therapy because of increasing resistance in S. pneumoniae; also, they do not provide coverage against Legionella and Chlamydia (intracellular pathogens) due to poor intracellular penetration, and M. pneumoniae, because it lacks a cell wall. The spectrum of activity of macrolides includes both typical and atypical respiratory pathogens; however, a recent TRUST surveillance study undertaken during the respiratory season in the USA [5] found azithromycin (22.7%) and clarithromycin (23.2%) to have the second highest resistance rates in S. pneumoniae after trimethoprim sulfamethoxazole (27.3%). Studies in Europe [6] have shown similar levels of macrolide resistance in this species, with an aggregate 20.8% recorded in , although regional variations do occur. The newer fluoroquinolones have attracted increasing attention as agents for empirical therapy of respiratory tract infections because of their potent activity against both typical and atypical pathogens. Levofloxacin is highly active against S. pneumoniae, including penicillin-resistant isolates, and against H. influenzae and M. catarrhalis, including b-lactamase-producing isolates [7]. Reports have shown that levofloxacin and other fluoroquinolones have activity against L. pneumophila, M. pneumoniae and C. pneumoniae as well [8 14]. The objective of the current study was to determine the antimicrobial activity of levofloxacin, comparator macrolides and doxycycline against isolates of L. pneumophila, M. pneumoniae and C. pneumoniae from collections in North America and Europe. MATERIALS AND METHODS The 146 isolates of L. pneumophila were derived from culture collections in the following countries: USA (86), UK (14), France (10), The Netherlands (10), Finland (10), Germany (9) and Denmark (7). The 41 isolates of M. pneumoniae were derived from collections in the USA (28), Denmark (5), Spain (3), Finland (3), and Canada (2). The nine isolates of C. pneumoniae included in the study were derived from collections in the USA (6), Finland (2) and the UK (1). Most of the isolates were derived from clinical sources and were collected from patients between 1995 and All isolates were maintained at 80 8C until the time of MIC testing. L. pneumophila isolates were propagated on buffered charcoal yeast extract (BCYE) agar plates (Hardy Diagnostics, Santa Maria, CA, USA). M. pneumoniae isolates were grown in SP-4 with glucose broth (Remel, Lenexa, KS, USA) prior to testing. C. pneumoniae isolates were propagated in HEp-2 cells (BioWhittaker, Walkersville, MD, USA) [15]. Levofloxacin was supplied by Ortho-McNeil Pharmaceutical (Raritan, NJ, USA), azithromycin was provided by Pfizer (Groton, CT, USA), clarithromycin was provided by Abbott (Abbott Park, IL, USA) and erythromycin and doxycycline were purchased from the Sigma Chemical Company (St Louis, MO, USA). Legionella pneumophila MIC testing was conducted by broth microdilution according to NCCLS guidelines [16] for aerobic bacteria (M7-A5, 2000) using 96-well microtiter plates containing the test antimicrobial concentrations. The bacterial inoculum was prepared from an overnight culture grown on BCYE agar at 35 8C in a humidified chamber. A suspension of organisms equivalent to a 0.5 McFarland standard was prepared in buffered yeast extract (BYE) broth and was diluted to give a concentration of CFU/mL in a final volume of 100 ml. Plates were incubated at 35 8C in air, and the MIC was read as the first well showing no visible growth at 48 h. Control strains were L. pneumophila ATCC and Staphylococcus aureus ATCC Mycoplasma pneumoniae MIC testing was based on a broth microdilution method described by Waites et al. [17]. Briefly, the method uses a chromogenic substrate, SP-4 with glucose (SP-4G; Remel) as an indicator of growth. Fermentation of glucose, as a result of growth, produces an acid byproduct which changes the medium from red to yellow. Serial 10-fold dilutions of each isolate were prepared in tubes containing SP-4G to determine the number of colorchanging units (CCU). The CCU method is a dilution method for determining the number of mycoplasmas in a broth culture, the endpoint being the reciprocal of the highest dilution in which growth was present as evidenced by a color change in the medium. These titers were used to adjust the inoculum used in the MIC test to approximately 10 4 CCU/mL. Antibiotics were

3 216 Clinical Microbiology and Infection, Volume 8 Number 4, April 2002 serially diluted in antibiotic-free SP-4G. The inoculated plates were incubated aerobically at 35 8C and examined daily until a color change was detected in the drug-free (growth control) wells. The MIC endpoint was determined as the lowest concentration of antimicrobial agent at which the metabolism of the organism was inhibited, as evidenced by a color change, 24 h after the growth control changed color. Any isolate that was resistant to an agent was re-tested on another day to ensure reproducibility of the results. Control strains were M. pneumoniae ATCC and S. aureus ATCC Chlamydia pneumoniae Susceptibility testing of C. pneumoniae was conducted with HEp-2 cells, using a slight modification of the methods described by Gieffers et al. [18] and Hammerschlag et al. [19]. The HEp-2 cells were grown in 24-well microtiter plates and inoculated with 100 ml of the test organism diluted in Eagles Minimal Essential Medium (EMEM) to contain between 10 3 and 10 4 inclusion-forming units/ml. Prior titration studies established this optimum concentration. The microtiter plates were centrifuged for 1 h at 1500 g and then incubated for 1 h at 35 8C. The inoculum-containing wells of each plate were then aspirated and the cells washed with ml of Hanks Balanced Salt Solution. The Hanks medium was then aspirated and the cell monolayer was overlayed with 500 ml of chlamydia overlay medium (COM) without antimicrobial agents. Serial dilutions were made by adding 500 ml of the antimicrobial agent to be tested to the first well and serially diluting 500 ml through the rest of the wells. One well without antibiotic was used as the growth control. The plates were incubated at 35 8C for 72 h in 5% CO 2. COM was aspirated, and the monolayer was fixed and stained with a Chlamydia-specific fluorescent monoclonal antibody (Merifluor Chlamydia; Meridian Diagnostics, Cincinnati, OH, USA). The MIC was determined as the lowest concentration at which no inclusions were observed. Control strains were C. pneumoniae ATCC VR-1360 and S. aureus ATCC RESULTS The results in Table 1 show the activity of levofloxacin and comparator agents against L. pneumophila Table 1 Antimicrobial susceptibility of L. pneumophila isolates from the USA and Europe MICs (mg/l) for all countries (n ¼ 146) MIC ranges (mg/l) for individual countries USA (n ¼ 86) UK (n ¼ 14) The Netherlands (n ¼ 10) Germany (n ¼ 9) France (n ¼ 10) Finland (n ¼ 10) Denmark (n ¼ 7) Antimicrobial Range MIC 50 MIC 90 Mode Levofloxacin Erythromycin Clarithromycin Azithromycin Doxycycline

4 Critchley et al Levofloxacin activity against atypical respiratory pathogens 217 Table 2 Antimicrobial susceptibility of 41 isolates of M. pneumoniae MIC (mg/l) Antimicrobial Range MIC 50 MIC 90 Mode Levofloxacin Erythromycin to Clarithromycin to Azithromycin to Doxycycline isolates from the USA and Europe. Levofloxacin was the most active agent (MIC mg/l), closely followed by clarithromycin (MIC mg/l). Azithromycin and erythromycin were the least active agents (MIC mg/l). Geographic origin of the isolates had no significant impact on susceptibility to levofloxacin, the comparator macrolides, or doxycycline, as there was little variation in the MIC ranges for each antimicrobial agent against the isolates from each country. The isolates that were most susceptible to levofloxacin (MIC mg/l) originated in Germany and The Netherlands. Analysis of MIC distributions for all five agents against all 146 isolates tested showed that levofloxacin was the most active agent, with a modal MIC of mg/ L, and this activity was maintained within a narrow range from to 0.03 mg/l. The modal MICs for clarithromycin, azithromycin and erythromycin were 0.03, 0.12 and 0.25 mg/l, respectively, with MICs that spanned wide ranges: mg/l for clarithromycin, mg/l for azithromycin, and mg/l for erythromycin. Doxycycline MICs ranged from 1 to 4 mg/l, the highest of all agents tested. The results in Table 2 show the activity of levofloxacin and comparator agents against 41 isolates of M. pneumoniae from the USA, Canada, Denmark, Finland, and Spain. Azithromycin demonstrated remarkable potency (MIC mg/ L) and was eightfold more active than clarithromycin (MIC mg/l) and 30-fold more active than erythromycin (MIC mg/l). Doxycycline and levofloxacin were the least active agents, with MIC 90 s of 0.5 and 1 mg/l, respectively. Interestingly, in the case of levofloxacin, all isolates were inhibited at concentrations of 1 mg/l. This is in contrast to the macrolides, where two isolates were not inhibited at concentrations of 4 mg/l of azithromycin, clarithromycin and erythromycin. The two macrolide-resistant isolates, which originated in Finland and the USA, were inhibited by levofloxacin at a concentration of 0.25 mg/l, and by doxycycline at 0.5 mg/l (Table 3). Table 3 Antimicrobial susceptibility of two macrolide-resistant isolates of M. pneumoniae MIC (mg/l) Isolate no. Country of origin Levofloxacin Erythromycin Clarithromycin Azithromycin Doxycycline 1 USA 0.25 >8 > Finland 0.25 >8 >8 >8 0.5 Table 4 Antimicrobial susceptibility of nine C. pneumoniae isolates MIC (mg/l) Isolate no. Country of origin Levofloxacin Clarithromycin Azithromycin Doxycycline 1 Finland Finland UK USA USA USA USA USA USA

5 218 Clinical Microbiology and Infection, Volume 8 Number 4, April 2002 Levofloxacin and comparator agents were also tested against nine isolates of C. pneumoniae, of which seven originated in the USA and two in Finland. The MICs for all antimicrobial agents tested against all nine isolates are shown in Table 4. The most active agent was clarithromycin, with MICs of mg/l for three isolates, mg/ L for four isolates and 0.03 mg/l for two isolates. Azithromycin was the next most active agent, with MICs ranging from 0.06 mg/l (six isolates) to 0.12 mg/l (three isolates). Doxycycline MICs were 0.06 mg/l (three isolates), 0.12 mg/l (four isolates) and 0.25 mg/l (two isolates). Levofloxacin was also active against C. pneumoniae, with MICs of 0.25 mg/l (four isolates), 0.5 mg/l (four isolates) and 1 mg/l (one isolate). The fact that all agents were tested against C. pneumoniae cultivated in a HEp-2 cell line also confirms their ability to penetrate mammalian cells and retain antibacterial efficacy. DISCUSSION L. pneumophila is a facultative intracellular pathogen that has the ability to multiply within the phagosome of alveolar macrophages and blood monocytes [20]. As a consequence of its intracellular location, L. pneumophila is capable of evading the inhibitory effects of agents such as b-lactams, which are unable to penetrate host cell membranes. Macrolides have the ability to penetrate mammalian cells, so erythromycin is first-line therapy for L. pneumophila infections [8,9,11]. Unfortunately, there are a number of issues associated with the use of erythromycin, including the large volume required for intravenous infusion, ototoxicity and gastrointestinal intolerance [21]; the recent introduction of parenteral azithromycin has partially addressed these issues. The fluoroquinolones have also shown potent efficacy against L. pneumophila in broth microdilution studies [8,9,22] and against intracellular L. pneumophila cultivated in mammalian cell lines [8 10]. In the present study, the fluoroquinolone levofloxacin demonstrated potent activity against 146 isolates of L. pneumophila from the USA and Europe. Levofloxacin was 20-fold more active than erythromycin, based on MIC 90 values of 0.03 mg/l and 0.5 mg/l, respectively. Levofloxacin at 1 mg/ L has been shown to have bactericidal activity against intracellular organisms grown in guinea pig alveolar macrophages by reducing the viable counts of two isolates by 2 3 log 10 CFU/mL, and was significantly more effective than erythromycin in the same model [9]. Furthermore, the fluoroquinolones have also been shown to have a significant postantibiotic effect against L. pneumophila [23,24]. Uncontrolled clinical trials have shown that the newer fluoroquinolones, including levofloxacin, were effective in treating both community-acquired and nosocomial infections [25,26]. As a consequence, agents such as levofloxacin may prove to be as efficacious as the macrolides for the therapy of L. pneumophila infections, with the advantage of fewer sideeffects. The results of this study also demonstrated the potent efficacy of the macrolides against M. pneumoniae, confirming the observations of other investigators [12,27]. However, two isolates of M. pneumoniae tested in this study had reduced susceptibility to erythromycin, clarithromycin and azithromycin (MICs 4 mg/l). There are numerous reports of macrolide-resistant isolates recovered from patients following therapy with macrolides [28 31]. In addition, erythromycin-resistant isolates can also be generated in the laboratory [32]. Such isolates have been shown to have point mutations in their 23S rrna genes, resulting in ribosomes having reduced affinity for the macrolide [33]. Although levofloxacin was less active than the macrolides based on MIC 90 ( mg/l for azithromycin compared with 1 mg/l for levofloxacin), it did retain activity against the two macrolide-resistant isolates (MICs of 0.25 mg/l for both isolates), indicating that it may have potential in the therapy of infections caused by macrolideresistant isolates. Levofloxacin was also active against the obligate intracellular pathogen C. pneumoniae, with MICs ranging from 0.25 to 1 mg/l. These results were in agreement with results generated by other investigators using Hep-2 cell lines for organism cultivation and susceptibility testing [13,14,34]. The fact that levofloxacin is active against C. pneumoniae in this model indicates that this agent has the ability to penetrate host cells and retain antimicrobial activity. No standards have been adopted for antimicrobial susceptibility testing of C. pneumoniae, although the C. pneumoniae workshop group on Detection, Culture, Serology and Antimicrobial Susceptibility Testing has made

6 Critchley et al Levofloxacin activity against atypical respiratory pathogens 219 a strong recommendation for the adoption of standardized procedures [35]. The choice of cell line used for susceptibility testing can affect the susceptibility test results. For example, Roblin et al. [15] have shown that McCoy cells used by various investigators [36] are 10- and 100-fold less prone to infection with C. pneumoniae than HEp-2 cells, which may result in lower MIC endpoints. In conclusion, MIC studies have shown that the fluoroquinolone levofloxacin is active against both L. pneumophila and M. pneumoniae. Furthermore, evaluating the efficacy of levofloxacin against C. pneumoniae in an intracellular model confirms the ability of this agent to penetrate mammalian cells and retain intracellular antibacterial activity. Levofloxacin is rapidly bactericidal and may be administered once a day for mild-to-severe infections [37 39]. There are also clinical data to demonstrate the efficacy of levofloxacin in the treatment of adults with CAP, achieving greater than 98% success in patients infected with atypical pathogens, including L. pneumophila, M. pneumoniae and C. pneumoniae [25,40]. The current study showed that geographic origin of the atypical pathogen (North America or Europe) did not affect its susceptibility to levofloxacin. Collectively, these results confirm the potential for levofloxacin as therapy for CAP caused by atypical respiratory pathogens. ACKNOWLEDGMENTS The authors thank the following individuals for contributing valuable isolates to this study: Dr Maureen Davidson, University of Florida, Gainesville, FL, USA; Professor Jerome Etienne, Hospital Eduard Harriot, Lyon, France; Dr Bill Graham, Scottish Legionella Reference Laboratory, Glasgow, UK; Dr Tim Harrison, CPHL, London, UK; Dr Maarjana Klimola, National Public Health Institute, Helsinki, Finland; Dr Maria Antonia Meseguer, Hospital Ramon y Cajal, Madrid, Spain; Dr Mika Paldanius, National Public Health Institute, Helsinki, Finland; Professor Marcel Peeters, St Elizabeth Hospital, Tilburg, The Netherlands; Riitta Räty, National Public Health Institute, Helsinki, Finland; Professor Gotthart Ruckdeschel, Germany; Dr Jørgen Skov-Jensen, Statum Serums Institute, Copenhagen, Denmark; Dr Hannele Somer, National Public Health Institute, Helsinki, Finland; Dr Joseph Tully, National Institutes of Health, Frederick, MD, USA. REFERENCES 1. Ashby BL. Treatment of pneumonia: new strategies for changing pathogens. Clin Ther 1991; 13: Ruiz-Gonzalez A, Felguera M, Nogues A, Rubio- Caballero M. Is Streptococcus pneumoniae the leading cause of pneumonia of unknown etiology? A microbiologic study of lung aspirates in consecutive patients with community-acquired pneumonia. Am J Med 1999; 106: Plouffe J. Importance of atypical pathogens of community-acquired pneumonia. Clin Infect Dis 2000; 31(suppl 2): S Plouffe JF, Herbert MT, File TM et al. Ofloxacin versus standard therapy in the treatment of community-acquired pneumonia requiring hospitalization. Antimicrob Agents Chemother 1996; 40: Sahm DF, Karlowsky JA, Kelly LJ et al. Need for annual surveillance of antimicrobial resistance in Streptococcus pneumoniae in the United States: 2-year longitudinal analysis. Antimicrob Agents Chemother 2001; 45: Jones ME, Staples AM, Critchley IA et al. Benchmarking the in vitro activity of moxifloxacin against recent isolates of Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae. A European multi-centre study. Diagn Microbiol Infect Dis 2000; 37: Thornsberry C, Ogilvie P, Kahn J, Mauriz Y, the Laboratory Investigation Group. Surveillance of antimicrobial resistance in Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the United States in the respiratory season. Diagn Microbiol Infect Dis 1997; 29: Stout JE, Arnold B, Yu VL. Comparative activity of ciprofloxacin, ofloxacin, levofloxacin and erythromycin against Legionella species by broth microdilution and intracellular susceptibility testing in HL-60 cells. Diagn Microbiol Infect Dis 1998; 30: Edelstein PH, Edelstein MA, Lehr KH, Ren J. In vitro activity of levofloxacin against clinical isolates of Legionella spp., its pharmacokinetics in guinea pigs, and use in experimental Legionella pneumophila pneumonia. J Antimicrob Chemother 1996; 37: Jonas D, Engels I, Friedhoff C, Spitzmuller B, Daschner FD, Frank U. Efficacy of moxifloxacin, trovafloxacin, clinafloxacin and levofloxacin against intracellular Legionella pneumophila. J Antimicrob Chemother 2001; 47: Baltch AD, Smith RP, Franke MA, Michelsen PB. Antibacterial effects of levofloxacin, erythromycin and rifampin in a human monocyte system against Legionella pneumophila. Antimicrob Agents Chemother 1998; 42:

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