Journal of Antimicrobial Chemotherapy (2004) 53, 609 615 DOI: 10.1093/jac/dkh130 Advance Access publication 3 March 2004 In vitro post-antibiotic effect of fluoroquinolones, macrolides, β-lactams, tetracyclines, vancomycin, clindamycin, linezolid, chloramphenicol, quinupristin/dalfopristin and rifampicin on Bacillus anthracis A. Athamna 1,2, M. Athamna 1, B. Medlej 1,2, D. J. Bast 3 and E. Rubinstein 2,4 * 1 The Triangle Research and Development Center, Kfar-Qaraa; 2 Department of Human Microbiology, Tel-Aviv University, School of Medicine, Tel-Aviv; 4 Infectious Diseases Unit, Sheba Medical Center, Tel-Aviv University, School of Medicine, Tel Hashomer 52621, Israel; 3 Toronto Centre for Antimicrobial Research & Evaluation (ToCARE), Department of Microbiology, Mount Sinai Hospital, Toronto, Ontario, Canada Introduction Received 17 December 2003; accepted 22 December 2003 Objectives: The aim of this study was to investigate in vitro the post-antibiotic effect (PAE) of 19 antibacterial agents against two strains of Bacillus anthracis (ST-1 and Sterne strains). Methods: PAE was determined by calculating the time required for the viable counts of antibiotic-exposed bacteria (at concentrations of 10 MIC and exposure for 2 h) at 37 C to increase by 1 log 10 above the counts observed immediately after antibiotic removal compared with the corresponding time for controls not exposed to antibiotics. Results: The PAEs of the fluoroquinolones (ciprofloxacin, ofloxacin, levofloxacin, moxifloxacin and garenoxacin) were 2 5 h. The macrolide (erythromycin, clarithromycin and telithromycin) PAEs were 1 4 h, and that of clindamycin was 2 h. The PAEs induced by tetracycline and minocycline were 1 3 h. The PAEs induced by the β-lactams (penicillin G, amoxicillin and ceftriaxone), vancomycin, linezolid and chloramphenicol were 1 2 h. The PAE induced by rifampicin was 4 5 h. Quinupristin/dalfopristin had the longest PAE, lasting for 7 8 h. Conclusions: Our results indicate that the PAE is unrelated to the MIC but may be related to the rapidity of bacterial kill. These observations may bear importance on treatment regimens of human anthrax. Keywords: anthrax, PAE, susceptibility Post-antibiotic effect (PAE) is a well-established pharmacodynamic parameter that reflects an arrested bacterial growth following the removal of the active antibacterial agent from the growth medium. 1 4 The duration of the PAE is mainly influenced by the bacterial species, and the nature of the antibacterial drug and its concentration, but also by environmental factors such as temperature, ph, po 2, growth medium, the kind of body fluid, etc. 5 8 The clinical significance of the PAE pertains primarily to the impact it may have on the design of antimicrobial dosing regimens in clinical practice. 6 Antimicrobials that induce long PAEs may be administered at longer dosing intervals than determined by their pharmacokinetic properties such as t 1/2 values. The benefit of a prolonged PAE may thus allow for fewer daily drug administrations without reduced efficacy, and possibly a lower frequency of adverse events. 9,10 Since post-exposure prophylaxis and therapy of anthrax require prolonged antibiotic therapy extending to 60 days or more, 11 a reduction in the frequency of dosings by an increase in the administration intervals might be more efficient than the current recommended regimen. We have therefore examined the PAEs of 19 antibacterials belonging to different antibiotic classes against two strains of Bacillus anthracis (the Sterne strain and the Russian vaccine strain ST-1). Materials and methods Antibacterial agents The antibiotics tested in this study were: ofloxacin and levofloxacin (gifts from Aventis, Netanya, Israel, and Aventis, Paris, France, respectively), ciprofloxacin and moxifloxacin (a gift from Bayer Leverkusen, Germany), garenoxacin (a gift from Bristol-Myers Squibb, Waterloo, Belgium), minocycline (Dexxon, Haifa, Israel), tetracycline (Sigma,... *Corresponding author. Tel: +972-3-5345-389; Fax: +972-3-5347-081; E-mail: erubins@yahoo.com... 609 JAC vol.53 no.4 The British Society for Antimicrobial Chemotherapy 2004; all rights reserved.
A. Athamna et al. Table 1. MICs of 19 antibiotics against B. anthracis strains MIC (mg/l) Antibiotic B. anthracis ST-1 B. anthracis Sterne Ofloxacin 0.25 0.25 Ciprofloxacin 0.03 0.03 Moxifloxacin 0.125 0.06 Levofloxacin 0.125 0.125 Garenoxacin 0.03 0.03 Amoxicillin 0.06 0.125 Penicillin G 0.03 0.25 Ceftriaxone 8.0 8.0 Vancomycin 1.25 2.5 Tetracycline 0.125 0.125 Minocycline 0.03 0.03 Clarithromycin 0.125 0.125 Clindamycin 0.25 0.125 Erythromycin 1.0 0.5 Telithromycin 0.125 0.125 Quinupristin/dalfopristin 0.06 0.125 Rifampicin 0.25 0.125 Linezolid 2.0 2.0 Chloramphenicol 512 256 Rehovot, Israel), penicillin G (Rafa Laboratories, Jerusalem, Israel), amoxicillin (GSK, Petach-Tikva, Israel), ceftriaxone (Roche, Tel-Aviv, Israel), vancomycin (E. Lilly, Italy), erythromycin (Sigma, Rehovot, Israel), clarithromycin [Abbott (Promedico, Petach-Tikva, Israel)], telithromycin and quinupristin/dalfopristin (Aventis, Paris, France), clindamycin and linezolid [Pharmacia (Agis), Bnei-Braq, Israel and Pharmacia, Kalamazoo, USA], rifampicin (Sigma, Rehovot, Israel) and chloramphenicol (Teva, Petach-Tikva, Israel). Penicillin G, minocycline, vancomycin, erythromycin, rifampicin, clindamycin, linezolid, ceftriaxone, garenoxacin and quinupristin/dalfopristin were each received as a dry laboratory powder and were dissolved in phosphate-buffered saline (ph 7.2). Amoxicillin was dissolved in distilled water. Clarithromycin was dissolved in analytical acetone, and telithromycin and tetracycline were dissolved initially in two drops of acetic acid and ethanol (100%), respectively, and subsequently diluted in distilled water to the required concentration. The antibiotics were sterilized through 0.45 µm pore-size filters (Millipore S.A., Paris, France). Ofloxacin, levofloxacin, moxifloxacin, ciprofloxacin and chloramphenicol were obtained in a liquid form (as injectables). Bacterial strains and growth conditions Bacteria used in this study were two strains of B. anthracis, the Sterne veterinary vaccine strain (a gift from the Colorado Serum Institute, Denver, Figure 1. Induction of PAE by ofloxacin (a), ciprofloxacin (b), levofloxacin (c) and moxifloxacin (d) against B. anthracis ST-1 (control, filled circles; antibioticexposed, open circles) and Sterne (control, filled triangles; 610
Post-antibiotic effect against B. anthracis Figure 2. Induction of PAE by clarithromycin (a), erythromycin (b) and telithromycin (c) against B. anthracis ST-1 (control, filled circles; antibiotic-exposed, open circles) and Sterne (control, filled triangles; Table 2. PAEs of various antibiotics against B. anthracis strains ST-1 and Sterne Antibiotic ST-1 (h) Sterne (h) Ciprofloxacin 2 3 2 3 Moxifloxacin 3 3 Ofloxacin 4 5 3 Levofloxacin 4 5 2 3 Garenoxacin 4 5 2 3 Erythromycin 1 2 1 Clarithromycin 2 2 Telithromycin 4 4 Clindamycin 2 2 Linezolid 1 1 Penicillin G 2 1 Amoxicillin 1 1 Ceftriaxone 1 1 Vancomycin 1 2 1 Tetracycline 1 1 Minocycline 2 3 2 Chloramphenicol 1 2 1 2 Rifampicin 5 4 5 Quinupristin/dalfopristin 7 8 7 8 CO, USA) and the Russian anthrax vaccine strain Sanitary Technical Institute (ST-1) purchased commercially from a veterinary supply store in Moscow, Russia. Both strains are not human pathogens, as both lack a plasmid necessary to produce the capsule of the vegetative cells. Bacterial spores were stored in sterile 30% glycerol in PBS, and were spread on brain heart infusion (BHI) agar (Difco Laboratories, USA) and incubated for 18 24 h at 37 C to obtain single colonies (vegetative form). A single colony was inoculated into 20 ml of BHI broth and incubated overnight at 37 C. The grown bacteria were used in the experiments. Determination of minimum inhibitory concentrations (MICs) MICs were determined according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria for Staphylococcus aureus. 12,13 Figure 3. Induction of PAE by clindamycin against B. anthracis ST-1 (control, Determination of the PAE Twenty millilitres of 1:20 diluted overnight culture (OD of 0.1 at 600 nm = 10 6 cfu) in BHI broth was incubated for 2 h at 37 C with or without the antibiotics to be tested, at concentrations of 10 MIC. In order to remove the antibiotics, exposed bacteria were washed twice with phosphate-buffered saline (ph 7.2) by centrifugation for 10 min at 7000 rpm; controls were handled similarly. The pellets were resuspended in 20 ml of BHI broth followed by incubation at 37 C and samples (1 ml) were obtained at 2 h (2 h of exposure to antibiotics) for corrections that were made to ensure that all the cultures would start with the same bacterial count. Samples were obtained at time 0 (immediately after washing and after correction) and then hourly up to 7 h and OD values determined (reflecting bacterial growth). The OD values were converted into cfu (bacterial growth) by using a standard curve which was constructed relating the number of bacteria to the OD. The PAE was defined according to Craig & Gudmundsson 6 as PAE = T C, where T is 611
A. Athamna et al. the time required for the viable counts of the exposed bacteria to increase by 1 log 10 above the counts observed immediately after washing and C is the corresponding time for the antibiotic unexposed controls. Results MICs The MICs of the 19 antibacterial agents tested against the two strains of B. anthracis, reported previously, are shown in Table 1. 14 PAEs All five fluoroquinolones tested exhibited similar PAE patterns for both strains (Figure 1). Ciprofloxacin and moxifloxacin had PAE durations of 2 3 h for both strains, whereas ofloxacin and levofloxacin had PAE durations ranging from 2 to 5 h, with the Sterne Figure 4. Induction of PAE by linezolid against B. anthracis ST-1 (control, filled circles; antibiotic-exposed, open circles) and Sterne (control, filled triangles; strain having PAEs of 2 3 h and the ST-1 strain having PAEs of 4 5 h (Table 2). The PAE durations of the macrolides erythromycin and clarithromycin and the ketolide telithromycin ranged from 1 to 4 h (Figure 2 and Table 2). The longest PAE in this group was observed with telithromycin (4 h for both strains), and the shortest with erythromycin (1 2 h), whereas clarithromycin had a PAE of 2 h for both strains. Clindamycin exhibited a PAE equal to clarithromycin (2 h for both strains) (Figure 3 and Table 2). Linezolid PAE was found to be 1 h for both strains (Figure 4 and Table 2). The PAE induced by the three β-lactams (penicillin G, amoxicillin and ceftriaxone) was similar and short (1 2 h against both strains) (Figure 5). Vancomycin exhibited a similar effect to the β-lactams with a PAE of 1 2 h (Figure 6 and Table 2). The tetracyclines (tetracycline and minocycline) exhibited somewhat divergent PAEs with tetracycline having a short PAE of Figure 6. Induction of PAE by vancomycin against B. anthracis ST-1 (control, Figure 5. Induction of PAE by penicillin G (a), amoxicillin (b) and ceftriaxone (c) against B. anthracis ST-1 (control, filled circles; antibiotic-exposed, open circles) and Sterne (control, filled triangles; 612
Post-antibiotic effect against B. anthracis Figure 7. Induction of PAE by tetracycline (a) and minocycline (b) against B. anthracis ST-1 (control, filled circles; antibiotic-exposed, open circles) and Sterne (control, filled triangles; Figure 8. Induction of PAE by chloramphenicol against B. anthracis ST-1 (control, 1 h, whereas minocycline had a somewhat longer PAE of 2 3 h for both strains (Figure 7 and Table 2). Chloramphenicol exhibited a PAE of 1 2 h for both strains (Figure 8 and Table 2). Rifampicin exhibited a PAE ranging from 4 to 5 h (Figure 9 and Table 2) and quinupristin/dalfopristin exhibited the longest PAE amongst all the antibacterials tested, ranging from 7 to 8 h (Figure 10 and Table 2). Discussion The dosage and frequency of administration of an antibacterial agent are determined by bacterial susceptibility expressed as the MIC, and by the pharmacokinetic and pharmacodynamic properties of the agent. The MICs of the antibiotics tested in this study were determined previously (Table 1). 14 All antibiotics tested except chloramphenicol and possibly ceftriaxone were active against the tested B. anthracis strains according to NCCLS breakpoints for S. aureus. 13 Figure 9. Induction of PAE by rifampicin against B. anthracis ST-1 (control, The PAE refers to the time period after complete removal of an antibiotic, during which there is delayed regrowth of the bacteria. Although this phenomenon was first described by Bigger in 1944, it was only many years thereafter that the PAE was recognized as an important pharmacodynamic parameter. 6 In our study, the exposure of the tested B. anthracis organisms to the antibiotics was at concentrations of 10 MIC, the MICs having been determined in a previous study. The exposure lasted for 2 h. In an earlier publication, 14 we have found that the rapidity of kill of most antibiotics against these strains is rather slow. Indeed, only moxifloxacin, rifampicin and quinupristin/dalfopristin were able to reduce the initial inoculum substantially within the first 2 h of exposure. It was therefore necessary to carry out the necessary corrections after 2 h of exposure. The PAEs induced by the fluoroquinolones ranged between 2 and 5 h, in accordance with PAEs induced by this class of agents in other bacteria, e.g. Streptococcus pneumoniae showed a PAE of 0.5 6.5 h in response to fluoroquinolones and a PAE of 2.3 h 613
A. Athamna et al. Acknowledgements This study was supported by a grant from Aventis France and Bayer AG, and by partial support from the Israeli Ministry of Science and Technology. References Figure 10. Induction of PAE by quinupristin/dalfopristin against B. anthracis ST- 1 (control, filled circles; antibiotic-exposed, open circles) and Sterne (control, filled triangles; following exposure to ciprofloxacin. 15,16 Against S. aureus, the PAE of fluoroquinolones ranged between 1.13 h for ciprofloxacin, 1.75 h for levofloxacin and 1.1 2.4 h for trovafloxacin. 17,18 The macrolides, in this study, showed slightly shorter PAEs compared with the fluoroquinolones (1 4 h), similarly to PAEs described for S. pneumoniae (2.9 h for clarithromycin and 2.5 h for erythromycin). 19 The shortest PAEs (1 2 h) were found with the β-lactams, vancomycin, linezolid and chloramphenicol. Similarly short PAEs were found for β-lactams and vancomycin against S. aureus. 20,21 Quinupristin/dalfopristin was found to possess the longest PAE, lasting 7 8 h. A similar PAE (7.4 h) with quinupristin/dalfopristin was demonstrated with S. pneumoniae. 22 All antibiotics tested in this study appeared to induce in vitro a similar PAE in both strains of B. anthracis, suggesting that pathogenic B. anthracis may also show the same phenomenon. In a previous study, 14 we have demonstrated that moxifloxacin, quinupristin/ dalfopristin and rifampicin caused the most rapid bacterial killing, achieving a complete kill within 2 4 h. The β-lactams and vancomycin demonstrated a 2 4 log 10 bacterial kill within 4 6 h. The macrolides, tetracyclines and linezolid demonstrated a slower kill rate, whereas chloramphenicol did not kill at all. Thus, it appears that the PAE is unrelated to the MIC but may bear some relationship to the rapidity of bacterial kill. For example, quinupristin/dalfopristin had the most rapid kill and the longest PAE. Rifampicin had a 2 4 log 10 bacterial kill within 4 6 h and a PAE of 5 h. In contrast, chloramphenicol was a poor killer of B. anthracis and had a short PAE of 1 2 h. However, a linear relationship between the rapidity of kill and the length of the PAE could not be established, R=0.54 and 0.35 for the Stern and ST-1 strains, respectively. The clinical implication of long PAEs lies in the possibility of increasing the intervals between drug administrations thus allowing for fewer daily dosages, thereby potentially reducing treatment costs, increasing patient compliance and decreasing drug exposure. 23 In addition to the possible therapeutically beneficial long PAE caused by some antibiotics, the patients who will receive these agents may potentially benefit also from a rapid bacterial kill caused by these antibiotics, possibly allowing for shorter therapeutic regimens. 1. Bigger, J. W. (1944). 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