In Vitro Development of Resistance to Ofloxacin and Doxycycline in Bacillus anthracis Sterne

Anthrax is an infection caused by Bacillus anthracis. It occurs endemically and could also be used for biological warfare with devastating effects (3). The worldwide increase in the development of drug resistance in bacteria is a major concern. This phenomenon can develop after in vitro challenges (1) and/or following extensive clinical use of antibiotics (2). This prompted us to determine if B. anthracis could also develop resistance to the currently recommended antibiotics for the treatment of anthrax infection.

B. anthracis Sterne spores were harvested from batch fermentation in Schaeffer's sporulation medium using live veterinary vaccine as seed (Colorado Serum Co., Denver, Colo.). Spores were stored at −70°C in 10% glycerol–water. The spores were thawed and incubated in brain heart infusion broth (Becton Dickinson & Co., Rutherford, N.J.) at 37°C for 24 h. The antimicrobials used were ofloxacin (Ortho Pharmaceutical Corp., Raritan, N.J.) and doxycycline (Abbott Laboratories, Chicago, Ill.).

MICs were determined by macrodilution methodology in brain heart infusion broth inoculated with 10⁷ CFU from an overnight culture per ml and incubated for 24 h at 37°C. Glass tubes containing 4.5 ml of brain heart infusion with doubling (ratio of 1:2) antibiotic dilutions were each inoculated with 0.1 ml of suspension containing approximately 10⁵ CFU of B. anthracis Sterne per ml. The range of inoculated dilutions was from 2 below to 11 above the MIC. A total of 18 passages were performed where the inoculum for each successive passage was taken from the first tube below the MIC. The cells were subcultured before each passage and at the end of the 18 passages on sheep blood agar medium without antibiotics and visually verified.

Repeated subculturing of B. anthracis led to an increase in the MIC of ofloxacin on the 13th passage from an initial MIC of 0.2 to 0.8 μg/ml. The MIC of 0.8 μg/ml was stable for the next five passages. The MIC of doxycycline did not change after 18 passages. The MIC of ofloxacin for B. anthracis from passage 1 was 0.2 μg/ml, and the MIC of ofloxacin for B. anthracis of passage 13 was 0.8 μg/ml. This was repeated five times with the same results.

This pilot study shows the development in vitro of increased levels of resistance of B. anthracis to a quinolone. Although the increase in the MIC of ofloxacin was only fourfold, from 0.2 to 0.8 μg/ml, which still makes the organism susceptible to ofloxacin, it signifies the potential for emergence of resistance. The development of decreased susceptibility to ofloxacin raises a serious concern for public safety since quinolones are currently the recommended therapy for anthrax (3). It was reassuring, however, to find that resistance to doxycycline did not develop.

The study has several limitations. It included only one strain of B. anthracis, Sterne (the strain used for animal vaccination), and only a limited number of passages were done. Further studies are therefore necessary with a lethal strain of B. anthracis.

The mechanism by which B. anthracis develops resistance is unknown. However, there are currently two well-recognized ways in which other bacteria have developed resistance to quinolones: first, by mutations in genes encoding topoisomerase IV and DNA gyrase, with most mutations being present in parC, parE, gyrA, and gyrB (2), and secondly, by an efflux pump mechanism (2). Further studies should explore the mechanism of resistance to quinolones by B. anthracis.

In summary, this pilot study indicates that sequential subculture in subinhibitory concentrations of ofloxacin led to an increase in the MIC. In order to help minimize the potential dangers of anthrax, further studies are needed to determine whether drug resistance could develop in B. anthracis after exposure to antimicrobial agents during the recommended treatment.