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, Israel; 3 Toronto Centre for Antimicrobial Research & Evaluation, Department of Microbiology Mount Sinai Hospital, Toronto, Ontario, Canada
Received 23 June 2003; returned 24 July 2003; revised 8 October 2003; accepted 9 October 2003
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Abstract |
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Methods: The in vitro activities of 18 antibacterial agents against two strains of B. anthracis, the Sterne strain and the Russian anthrax vaccine strain ST-1, were tested by determining the MICs and by measuring the rates of antibiotic kill at 5x and 10x MIC.
Results: The fluoroquinolones ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, the ß-lactams penicillin G and amoxicillin, the macrolide clarithromycin, the ketolide telithromycin, as well as clindamycin, rifampicin and quinupristin/dalfopristin had MICs in the range of 0.030.25 mg/L. Minocycline had an MIC of 0.03 mg/L, as did penicillin, against the ST-1 strain. Ciprofloxacin had an MIC of 0.03 mg/L against both strains. Erythromycin, vancomycin and the oxazolidinone linezolid were less active (MIC 0.52.5 mg/L). Ceftriaxone was the least active, having an MIC of 8.0 mg/L. Chloramphenicol was inactive (MIC > 256 mg/L). Quinupristin/dalfopristin, rifampicin and moxifloxacin showed the most rapid bacterial killing, achieving a complete eradication of detectable organisms (2 log10 reduction within 0.53 h and 4 log10 reduction within 0.54 h for both strains at concentrations of 5x and 10x the MIC). The ß-lactams and vancomycin demonstrated a 24 log10 reduction within 515 h. Ceftriaxone had a similar effect to penicillin and amoxicillin against the ST-1 strain, but a slower effect than these two ß-lactams against the Sterne strain. The macrolides, tetracyclines and linezolid demonstrated a lower kill rate, while chloramphenicol did not kill at all.
Conclusions: These data expand on the spectrum of agents recommended for the treatment of anthrax (ciprofloxacin, penicillin G and tetracyclines) and add new options, such as other fluoroquinolones, amoxicillin, rifampicin and quinupristin/dalfopristin, as potential therapeutic agents.
Keywords: anthrax, fluoroquinolones, macrolides, ß-lactams
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Introduction |
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Methods of prevention of inhalational anthrax include: protective masks capable of filtering 15 µm particles, appropriate sheltering, use of pre- and post-exposure vaccination, and preventive and therapeutic antibiotic regimens. In unvaccinated individuals, antibiotics are the single most effective mode of management of this situation. Recently in the USA, antibiotic prophylaxis has been administered to 32 000 individuals suspected to have been exposed to anthrax.2 Up until the recent bioterror attack in the USA, the recommended antibiotics had not been tested in cases of human anthrax, but only in a monkey model with doses of ciprofloxacin, doxycycline and penicillin mimicking human pharmacokinetics.1 This publication did not study the pharmacodynamics of these agents, as in 1993 pharmacodynamics were at an early stage of development. The possible development of resistance was also not addressed in this study.
The importance of antibiotic prophylaxis received special interest, mainly because of the current shortage of vaccine and lack of toxin neutralizing agents (antitoxins). Although the in vitro susceptibility of representative B. anthracis strains has been tested using a wide variety of antibiotics, little is known about the rate of bacterial killing by these agents; only a single paper has described the kill activity of five antibiotics against seven B. anthracis clinical isolates.3 It is accepted that the likelihood of emergence of resistant mutants may depend on the rate of bacterial killing; the more rapid the rate the less likely the chance for the emergence of resistant strains.4,5 Moreover, rapid killing diminishes bacterial toxin formation, and thus may reduce resulting tissue damage.
In the present study we determined the susceptibility and the rate of kill of two B. anthracis strains by 18 antibacterial agents belonging to different antibiotic classes.
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Materials and methods |
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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), minocycline (obtained from Dexxon, Caesarea Or-Aqiva, Israel), tetracycline (obtained from Sigma, Rehovot, Israel), penicillin G (obtained from Rafa Laboratories, Jerusalem, Israel), amoxicillin (obtained from GlaxoSmithKline, Petach-Tiqva, Israel), ceftriaxone (obtained from Roche, Tel-Aviv, Israel), vancomycin (obtained from Eli Lilly, Indianapolis, IN, USA), erythromycin (purchased from Sigma, Rehovot, Israel), clarithromycin [obtained from Abbott (Promedico, Petach-Tiqva, Israel)], telithromycin and quinupristin/dalfopristin (a gift from Aventis, Paris, France), clindamycin and linezolid [a gift from Pharmacia (Agis, Bnei-Braq, Israel)], rifampicin (purchased from Sigma, Rehovot, Israel) and chloramphenicol (obtained from Teva, Jerusalem, Israel).
Penicillin G, minocycline, vancomycin, erythromycin, rifampicin, clindamycin, linezolid, ceftriaxone and quinupristin/dalfopristin were each received as a dry laboratory powder and were dissolved in phosphate-buffered saline (PBS) (pH 7.2). Amoxicillin was dissolved in distilled water. Clarithromycin was dissolved in analytical acetone. 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).
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. Ofloxacin, levofloxacin, moxifloxacin, ciprofloxacin and chloramphenicol were provided in a liquid form (as intravenous medications).
Bacterial strains and growth conditions
The bacteria used in this study were two strains of B. anthracis, the Sterne strain (a gift from the Colorado Serum Institute, Denver, CO, USA) and the Russian anthrax vaccine strain ST-1 (purchased commercially in Moscow, Russia). Bacteria were kept as spores in 30% glycerine in PBS at room temperature, and were grown to become vegetative forms for 1824 h at 37°C in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA).
Determination of MICs
The antibacterial agents were prepared as concentrated stock solutions of known concentration in distilled water. Two-fold dilutions were used in a concentration range from 0.015 to 1024 mg/L diluted in 100 µL of BHI broth and poured into wells of flat-bottomed microtitre plates (Nunc 96-well flat-bottomed microtitre plates; Nunc, Roskilde, Denmark). A 10 µL volume of culture containing 105 cfu/mL of B. anthracis ST-1 or Sterne strain was then added. Following incubation of the plates for 18 h at 37°C in ambient air, the MICs were determined. The MICs were recorded as the lowest concentration of an antibacterial agent that completely inhibited visible growth of the bacteria.6,7 Susceptibility was determined using broth microdilution according to NCCLS criteria for Staphylococcus aureus,6,7 as at the time this study was being carried out no NCCLS criteria for B. anthracis had been published.
Timekill
An overnight culture of B. anthracis was diluted 1:1000 with BHI in final volumes of 2 mL. Antibiotic solutions in concentrations of 5x and 10x MIC (of each strain tested) were added (12.525 µL according to the antibiotic) to the bacterial suspension (105 cfu/mL). As a control, similarly diluted bacterial inocula without any antibacterial agent were used. The test tubes were incubated at 37°C and samples (10 µL) removed at 0, 0.5, 2, 4, 6, 10, 12 and 24 h for viable bacterial count. Bacterial count was performed by diluting samples in sterile saline, of which 2050 µL was plated on brain heart infusion agar (BHA) (to improve detection in samples with low bacterial counts, 100 µL of undiluted specimen was also plated on BHA). After overnight incubation at 37°C, the number of cfu was counted. The lower limit of detection of bacteria was 200 cfu. All tests were performed in triplicate and average results used. Data were plotted as average log10 cfu/mL against time, and time required for 2 and 4 log10 reduction in viable count was interpolated from this graph.
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Results |
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The MICs of the antibacterial agents tested against the two strains of B. anthracis are shown in Table 1. All antibacterials tested had MICs below the accepted breakpoints for S. aureus except for chloramphenicol, which had an MIC above the susceptible range, and ceftriaxone, which had an MIC in the intermediate zone. Ciprofloxacin, minocycline and penicillin G had the lowest MICs, 0.03 mg/L.
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The killing rate of each of the antibacterial agents tested was similar for the two strains using 5x and 10x MIC antibiotic concentrations. However, some major differences did occur with time to reach 4 log10 kill with penicillin, amoxicillin and ceftriaxone.
Among the fluoroquinolones, moxifloxacin (Table 2) was found to be the most potent agent for bacterial killing: after 2 h no viable bacteria were recovered with both strains. Ciprofloxacin and ofloxacin had a similar killing effect, with a 23 log10 decrease in bacterial count at 45.5 h (Table 2).
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Vancomycin exhibited similar killing activity to the ß-lactams and the fluoroquinolones.
The macrolides, telithromycin, clindamycin and linezolid exhibited a slight reduction in bacterial viability within the first 10 h of exposure at both concentrations. Nevertheless, following 24 h of exposure clarithromycin, clindamycin and linezolid exhibited complete kill, whereas erythromycin and telithromycin were poorly bactericidal (Table 2).
Tetracycline and minocycline exhibited a similar pattern of killing. No reduction in the bacterial viability was observed within the first 15 h of exposure, while at 24 h these agents caused complete eradication (Table 2).
Quinupristin/dalfopristin was found to be the most potent agent, showing complete kill within 30 min with the ST-1 strain and within 4 h with the Sterne strain (Table 2).
Rifampicin also exhibited a rapid killing activity, with 100% of the bacteria killed within 13 h (Table 2).
Chloramphenicol caused no killing effect within 12 h, and only a 23 log10 reduction in bacterial count after 22 h (Table 2).
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Discussion |
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The B. anthracis strains used in the present study were highly susceptible to fluoroquinolones, penicillin G and amoxicillin, to the macrolides (except erythromycin), tetracyclines, quinupristin/dalfopristin and rifampicin, with MICs ranging between 0.03 and 0.25 mg/L. Similar results were obtained in other studies using different strains; the slight differences (e.g. one doubling dilution) are probably due to differences in strains tested, growth medium used and methods of MIC determination, and differences in cell cycle stage tested (spores versus vegetative cells).3,810
Vancomycin, erythromycin and linezolid showed an intermediate activity (MIC range 0.52 mg/L), as was reported in previous studies.6,10,11 Ceftriaxone possessed low activity (MIC 8 mg/L). It has been shown previously by Mohammed et al.,6 Coker et al.9 and Bryskier,10 and others, that B. anthracis may be resistant to penicillins and cephalosporins, either by the presence of a ß-lactamase or by a different mechanism, and that resistance may even develop during therapy. Thus ceftriaxone and other cephalosporins should not be indicated as therapeutic options for anthrax. Chloramphenicol was inactive (>256 mg/L), consistent with previous observations,10,12,13 and is therefore recommended neither for therapy nor as prophylaxis.
The fluoroquinolones showed a rapid killing effect, demonstrating a 34 log10 decrease within 46 h, with relatively low MICs. Among the fluoroquinolones, moxifloxacin demonstrated the most rapid effect, achieving a complete kill within 2 h. Drago et al.3 reported a similar killing effect of levofloxacin and gatifloxacin at similar concentrations to those used in the present study. Levofloxacin, on the other hand, needed longer to achieve a 4 log10 kill, and was less potent than ofloxacin. This latter phenomenon may be related to the biphasic response seen with fluoroquinolones14 rather than a real difference between levofloxacin and ofloxacin.
For rifampicin, a killing effect similar to the effect induced by the fluoroquinolones was observed. The ß-lactams and quinupristin/dalfopristin were found to possess the highest bacterial killing effect, achieving a 100% kill of the ST-1 strain within 30 min and a 100% kill of the Sterne strain within 2 h. The killing effects of rifampicin and quinupristin/dalfopristin have not yet been described.
The macrolides and tetracyclines exerted a slow killing effect, with a reduction of only 3 log10 within 12 h. Similar results were reported with rokitamycin, a 15-member macrolide.3 Thus, the ranking order of the efficacy of the tested antibiotics in bacterial kill was: quinupristin/dalfopristin and rifampicin were the best, ß-lactams and fluoroquinolones had an intermediate activity, while macrolides and protein synthesis-inhibiting agents were the weakest killers.
Some differences in bactericidal activity between the two strains tested in this study were observed, which underlines the versatility of B. anthracis strains, and draws attention to the need for individual study of antibiotic action against each strain.
To our knowledge, no previous studies have compared MICs with the rate of killing of B. anthracis. A rapid killing effect is believed to be associated with a decreased emergence of resistant strains, e.g. the reduced incidence of S. pneumoniae resistance to moxifloxacin and gatifloxacin has been suggested to be due in part to the rapid killing effect of those agents on that particular microorganism.15,16 In addition, rapid kill may diminish the host damage induced by the B. anthracis toxins released, such as the lethal factor, oedema factor and protective antigen, as has been shown with the action of ciprofloxacin on several exotoxins of Pseudomonas aeruginosa.17 On the other hand, rapid killing may release larger amounts of toxins and thus increase the damage, as has been suggested for some ß-lactams and the release of endotoxin from some Gram-negative bacteria.18 Reduction in bacterial toxin production and release is considered important as clindamycin, which has only a limited killing activity on B. anthracis, has been introduced into various therapeutic schemes in anthrax patients.
Further studies in B. anthracis need to establish the correlation between rapid killing and reduced resistance development.
In summary, our results point to additional antibiotics that might be tested as therapeutic agents against anthrax. The rapidity of bacterial kill should also be entertained as a therapeutic advantage in the treatment of clinical cases of anthrax.
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Acknowledgements |
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Footnotes |
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References |
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