Susceptibility of Bacillus anthracis to eleven antimicrobial agents including novel fluoroquinolones and a ketolide

John Frean1, Keith P. Klugman2,3,*, Lorraine Arntzen1 and Stanley Bukofzer4

1 National Institute for Communicable Diseases, National Health Laboratory Service, Johannesburg; 2 MRC/University of the Witwatersrand/NHLS Respiratory and Meningeal Pathogens Research Unit, Johannesburg, South Africa; 3 Department of International Health, Emory University, Atlanta, GA; 4 Abbott Laboratories, Abbott Park, Chicago, IL, USA

Received 3 April 2003; returned 28 April 2003; revised 4 June 2003; accepted 10 June 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: To determine the susceptibility of southern African strains of Bacillus anthracis to new, investigational agents as well as conventional antibiotics.

Materials and methods: The MICs of 26 isolates of B. anthracis from South Africa and Zimbabwe, as well as the Sterne vaccine strain and a type culture strain, were determined by agar dilution.

Results: The most active antimicrobial agents were the novel ketolide ABT 773, new and conventional fluoroquinolones, and doxycycline; macrolides were intermediately active. The lack of activity of extended-spectrum cephalosporins against B. anthracis was confirmed.

Conclusions: Susceptibility to conventional antibiotics was in keeping with previous studies. Two new fluoroquinolones and a ketolide showed promising in vitro activity that would support their further evaluation in animal models of anthrax.

Keywords: anthrax, antibiotics, fluoroquinolones, macrolides, ketolides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The recent use of Bacillus anthracis as an agent of bioterrorism in the United States1 has generated renewed interest in the antimicrobial susceptibility patterns of this organism. Current recommendations for treatment and prophylaxis for anthrax are based on limited data from animal experiments and historical reports.2 The possibility exists that antibiotic-resistant strains of B. anthracis might be used as bioterrorism agents, although the recent attacks did not employ such strains. Another factor considered in treatment recommendations is the production of constitutive or inducible ß-lactamases. In vitro resistance to penicillin,3,4 cefuroxime3,5 and extended-spectrum cephalosporins46 has been reported, but penicillin resistance is not consistently associated with ß-lactamase production.3,4 At the present time, fluoroquinolones or doxycycline are advocated as first-line agents for prophylaxis or treatment following exposure to spores,1 in combination with additional agents for therapy of inhalational or other serious forms of anthrax. Therapy may be altered appropriately (for example, to include penicillin) when the antibiotic susceptibility profile of the infecting isolate becomes known. Other agents that are generally active in vitro against B. anthracis, but that are not necessarily clinically useful, include clindamycin, rifampicin, imipenem, chloramphenicol, vancomycin, cefazolin, tetracycline, linezolid, macrolides and aminoglycosides.2 We tested the susceptibility of southern African strains of B. anthracis to several conventional and newly developed antimicrobials.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
B. anthracis isolates and control organisms

Two B. anthracis isolates were obtained from human infections acquired in Zimbabwe; the Sterne vaccine strain, 34F2, was a gift of Dr M. Henton, Onderstepoort Veterinary Research Institute (OVRI), Pretoria; NCTC 2606 was a culture collection isolate from the UK; all the other isolates were from domestic or wild animals in southern Africa, donated by Dr M. Henton (OVRI). Isolates had been stored in semi-solid agar; purity was checked by plating onto horse blood agar. The control organisms used were Staphylococcus aureus ATCC 29213 and Bacillus cereus ATCC 11778.

Antibiotics tested

Clarithromycin, erythromycin, cefdinir, cefditoren, ciprofloxacin, temafloxacin, tosufloxacin and the experimental agents cethromycin (ABT 773, a ketolide), olamufloxacin (HSR 903) and ABT 492 (fluoroquinolones), were all obtained from Abbott Laboratories, Abbott Park, IL, USA. Doxycycline was supplied by Sigma (S. Africa), Midrand, South Africa.

Determination of antimicrobial susceptibility

We used the agar dilution method to determine MICs, according to the method of the National Committee for Clinical Laboratory Standards (NCCLS).7 All work with bacteria was done in a class 2 biosafety cabinet in the biosafety level 3 laboratory of the National Health Laboratory Service, Johannesburg, South Africa. Inocula of B. anthracis were prepared by subculturing several colonies of an overnight growth on blood agar into trypticase soy broth, incubating at 37°C for 4–6 h, and adjusting the turbidity to the equivalent of a 0.5 McFarland standard. A multipoint replicator with 3 mm diameter pins was used to inoculate Mueller–Hinton agar with an estimated final inoculum size of 104 cfu/spot.7 For each antimicrobial tested, growth control plates without incorporated antimicrobials were similarly inoculated. Incubation was at 37°C for 18–20 h. The MICs at which 50% and 90% of isolates were inhibited (MIC50 and MIC90, respectively) were determined.

ß-Lactamase detection

We used the chromogenic cephalosporin method (Nitrocefin: Oxoid, Basingstoke, UK) to test for constitutive ß-lactamase production. Nitrocefin solution (four drops) was added to overnight 1 mL nutrient broth cultures of the B. anthracis isolates; the presence of a red colour on inspection after 30 min of incubation at 37°C indicated a positive reaction. A ß-lactamase-producing Branhamella catarrhalis strain was used as a positive control.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The results of the antimicrobial susceptibility testing are shown in Table 1. The MICs for the S. aureus ATCC 29213 control strain were within the expected limits for previously characterized antibiotics.7 The MIC50 of erythromycin for B. anthracis was 1 mg/L, consistent with that reported for the US strain associated with the recent bioterrorism events.6 The enhanced activity of clarithromycin, an agent recommended for secondary use in combination with either ciprofloxacin or doxycycline, was confirmed in our group of isolates, with an MIC50 of 0.125 mg/L, similar to that of recent US isolates (0.25 mg/L).6 These data support the view that anthrax strains are of intermediate susceptibility to the macrolides. The activity of the ketolide cethromycin (ABT 773) was however similar to that of the most active agents tested (the fluoroquinolones). There are to date no other published reports of the activity of this class of agent against the anthrax bacillus. The high MICs of the cephalosporins were in keeping with previous reports of ß-lactamase production3 and poor activity of cephalosporins36 against B. anthracis. Five of our B. anthracis strains were constitutively ß-lactamase producing, but the range of cephalosporin MICs (1–16 mg/L) for these strains was essentially the same as for the ß-lactamase-negative isolates.


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Table 1. Antimicrobial susceptibilities of 28 strains of B. anthracis
 
The activity of the other antimicrobials tested was in line with that of ciprofloxacin—the only one that appeared to be active at higher dilution was the (now withdrawn) agent tosufloxacin. The limitations of the use of the fluoroquinolones for anthrax prophylaxis and therapy are the potential for the emergence of resistance,8,9 the potential for relapse after even 30 days of prophylaxis10 in a non-human primate model, and the potential for toxicity in long-term use of this class of agent. Recent data show evidence of the selection of quinolone, macrolide and tetracycline resistance after sequential exposures of anthrax organisms to these drugs.9 Doxycycline, a recommended first line antibiotic for anthrax, was highly active against these strains of B. anthracis (MICs of 0.063 mg/L). Shown separately in Table 1 are the MICs for the 34F2 Sterne strain; this is an attenuated live vaccine strain that has been used to study antibiotic resistance in B. anthracis8,9 and although of low virulence, it is a potential infectious risk.

The data in this report would therefore support the further evaluation of both cethromycin (ABT 773) and ABT 492 against anthrax in animal models. The priority may be justifiably given to ABT 773 on the basis that it represents a new class of agent with activity against this pathogen.


    Acknowledgements
 
This study was financially supported by Abbott Laboratories, Abbott Park, IL, USA. Some of these data were presented at the 41st Interscience Conference of Antimicrobial Agents and Chemotherapy, Chicago, IL, USA, December 2001.


    Footnotes
 
* Corresponding author. Tel: +1-404-712-9001; Fax: +1-404-727-4590; E-mail: kklugma{at}sph.emory.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Centers for Disease Control and Prevention. (2001). Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. Morbidity and Mortality Weekly Report 50, 889–93.[Medline]

2 . Inglesby, T. V., Henderson, D. A., Bartlett, J. G. et al. (1999). Anthrax as a biological weapon. Medical and public health management. Journal of the American Medical Association 281, 1735–45.[Abstract/Free Full Text]

3 . Lightfoot, N. F., Scott, R. J. D. & Turnbull, P. C. B. (1990). Antimicrobial susceptibility of Bacillus anthracis. Salisbury Medical Bulletin 68, Suppl., 95–8.

4 . Mohammed, M. J., Marston, C. K., Popovic, T. et al. (2002). Antimicrobial susceptibility testing of Bacillus anthracis: comparison of results obtained by using the National Committee for Clinical Laboratory Standards broth microdilution reference and Etest agar gradient diffusion methods. Journal of Clinical Microbiology 40, 1902–7.[Abstract/Free Full Text]

5 . Doganay, M. & Aydin, N. (1991). Antimicrobial susceptibility of Bacillus anthracis. Scandinavian Journal of Infectious Diseases 23, 333–5.[ISI][Medline]

6 . Centers for Disease Control and Prevention. (2001). Update: investigation of bioterrorism-related anthrax and interim guidelines for exposure management and antimicrobial therapy, October 2001. Morbidity and Mortality Weekly Report 50, 909–19.[Medline]

7 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

8 . Choe, C. H., Bouhaouala, S. S., Brook, I. et al. (2000). In vitro development of resistance to ofloxacin and doxycycline in Bacillus anthracis Sterne. Antimicrobial Agents and Chemotherapy 44, 1766.[Free Full Text]

9 . Brook, I., Elliott, T. B., Pryor, H. I., II et al. (2001). In vitro resistance of Bacillus anthracis Sterne to doxycycline, macrolides and quinolones. International Journal of Antimicrobial Agents 18, 559–62.[CrossRef][ISI][Medline]

10 . Friedlander, A. M., Welkos, S. L., Pitt, M. L. et al. (1993). Postexposure prophylaxis against experimental inhalation anthrax. Journal of Infectious Diseases 167, 1239–43.[ISI][Medline]