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Antimicrobial Agents and Chemotherapy, June 2000, p. 1766-1766, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
LETTERS TO THE EDITOR
In Vitro Development of Resistance to Ofloxacin and
Doxycycline in Bacillus anthracis Sterne
 |
LETTER |
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.
 |
FOOTNOTES |
*
Phone: (301) 295-2698 Fax: (301)
295-6503 E-mail: brook{at}mx.afrri.usuhs.mil
 |
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| | | | |
Chong H. Choe
Selim S. Bouhaouala
Itzhak Brook*
Thomas B. Elliott
Gregory B. Knudson
Radiation
Medicine Department Armed Forces Radiobiology Research
Institute 8901 Wisconsin Ave. Bethesda, Maryland 20889-5603
|
Antimicrobial Agents and Chemotherapy, June 2000, p. 1766-1766, Vol. 44, No. 6
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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