Previous Article | Next Article 
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
| |
REFERENCES |
| 1.
|
Davies, T. A.,
G. A. Pankuch,
B. E. Dewasse,
M. R. Jacobs, and P. C. Appelbaum.
1999.
In vitro development of resistance to five quinolones and amoxicillin-clavulanate in Streptococcus pneumoniae.
Antimicrob. Agents Chemother.
43:1177-1182[Abstract/Free Full Text].
|
| 2.
|
Liu, H. H.
1999.
Antibiotic resistance in bacteria: a current and future problem.
Adv. Exp. Med. Biol.
455:387-396[Medline].
|
| 3.
|
Pile, J. C.,
J. Malone,
E. Eitzen, and A. Friedlander.
1998.
Anthrax as a potential biological warfare agent.
Arch. Intern. Med.
158:429-434[Abstract/Free Full Text].
|
| | | | |
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.
This article has been cited by other articles:
-
Heine, H. S., Bassett, J., Miller, L., Bassett, A., Ivins, B. E., Lehoux, D., Arhin, F. F., Parr, T. R. Jr., Moeck, G.
(2008). Efficacy of Oritavancin in a Murine Model of Bacillus anthracis Spore Inhalation Anthrax. Antimicrob. Agents Chemother.
52: 3350-3357
[Abstract]
[Full Text]
-
Heine, H. S., Bassett, J., Miller, L., Hartings, J. M., Ivins, B. E., Pitt, M. L., Fritz, D., Norris, S. L., Byrne, W. R.
(2007). Determination of Antibiotic Efficacy against Bacillus anthracis in a Mouse Aerosol Challenge Model. Antimicrob. Agents Chemother.
51: 1373-1379
[Abstract]
[Full Text]
-
Yoong, P., Schuch, R., Nelson, D., Fischetti, V. A.
(2006). PlyPH, a Bacteriolytic Enzyme with a Broad pH Range of Activity and Lytic Action against Bacillus anthracis.. J. Bacteriol.
188: 2711-2714
[Abstract]
[Full Text]
-
Grohs, P., Podglajen, I., Gutmann, L.
(2004). Activities of Different Fluoroquinolones against Bacillus anthracis Mutants Selected In Vitro and Harboring Topoisomerase Mutations. Antimicrob. Agents Chemother.
48: 3024-3027
[Abstract]
[Full Text]
-
Turnbull, P. C. B., Sirianni, N. M., LeBron, C. I., Samaan, M. N., Sutton, F. N., Reyes, A. E., Peruski, L. F. Jr.
(2004). MICs of Selected Antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides from a Range of Clinical and Environmental Sources as Determined by the Etest. J. Clin. Microbiol.
42: 3626-3634
[Abstract]
[Full Text]
-
Athamna, A., Athamna, M., Abu-Rashed, N., Medlej, B., Bast, D. J., Rubinstein, E.
(2004). Selection of Bacillus anthracis isolates resistant to antibiotics. J Antimicrob Chemother
54: 424-428
[Abstract]
[Full Text]
-
Frean, J., Klugman, K. P., Arntzen, L., Bukofzer, S.
(2003). Susceptibility of Bacillus anthracis to eleven antimicrobial agents including novel fluoroquinolones and a ketolide. J Antimicrob Chemother
52: 297-299
[Abstract]
[Full Text]
-
Elliott, T. B., Brook, I., Harding, R. A., Bouhaouala, S. S., Shoemaker, M. O., Knudson, G. B.
(2002). Antimicrobial Therapy for Bacillus anthracis-Induced Polymicrobial Infection in 60Co {gamma}-Irradiated Mice. Antimicrob. Agents Chemother.
46: 3463-3471
[Abstract]
[Full Text]
-
Cavallo, J.-D., Ramisse, F., Girardet, M., Vaissaire, J., Mock, M., Hernandez, E.
(2002). Antibiotic Susceptibilities of 96 Isolates of Bacillus anthracis Isolated in France between 1994 and 2000. Antimicrob. Agents Chemother.
46: 2307-2309
[Abstract]
[Full Text]
-
Mohammed, M. J., Marston, C. K., Popovic, T., Weyant, R. S., Tenover, F. C.
(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. J. Clin. Microbiol.
40: 1902-1907
[Abstract]
[Full Text]
-
Inglesby, T. V., O'Toole, T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Friedlander, A. M., Gerberding, J., Hauer, J., Hughes, J., McDade, J., Osterholm, M. T., Parker, G., Perl, T. M., Russell, P. K., Tonat, K., for the Working Group on Civilian Biodefense,
(2002). Anthrax as a Biological Weapon, 2002: Updated Recommendations for Management. JAMA
287: 2236-2252
[Abstract]
[Full Text]
