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Journal of Clinical Microbiology, February 1999, p. 304-309, Vol. 37, No. 2
Department of Veterinary and Biomedical
Sciences1 and
Center for
Biotechnology,2 University of Nebraska,
Lincoln, Nebraska 68583-0905
Received 29 June 1998/Returned for modification 21 October
1998/Accepted 21 October 1998
Paratuberculosis (Johne's disease) is a fatal disease of ruminants
for which no effective treatment is available. Presently, no
drugs against Mycobacterium avium subsp.
paratuberculosis (M. paratuberculosis), the
causative agent of Johne's disease, are approved for use in livestock.
Additionally, M. paratuberculosis has been linked to a
human chronic granulomatous ileitis (Crohn's disease). To assist in
the evaluation of antimicrobial agents with potential
activity against M. paratuberculosis, we have developed a
firefly luciferase-based assay for the determination of drug susceptibilities. The microorganism used was M. paratuberculosis K-10(pYUB180), a clinical isolate carrying a
plasmid with the firefly luciferase gene. The MICs determined by the
broth macrodilution method were as follows: amikacin, 2 µg/ml; Bay y
3118, 0.015 µg/ml; clarithromycin, 1.25 µg/ml;
D-cycloserine, 25 µg/ml; ethambutol, 20 µg/ml; and
rifabutin, 0.5 µg/ml. The strain was resistant to isoniazid and
kanamycin. The results obtained by the luciferase assay were identical
or fell within 1 doubling dilution. These results suggest that a
combination of amikacin, clarithromycin, and rifabutin may be the most
efficacious therapy for the treatment of M. paratuberculosis infections and that the use of fluoroquinolone class of antibiotics deserves further consideration. We
demonstrate that the luciferase drug susceptibility assay is reliable
for M. paratuberculosis and gives results within 7 days, whereas the broth macrodilution method requires 14 days.
Paratuberculosis (Johne's disease)
is an incurable, fatal disease of domestic and wild ruminants.
Mycobacterium avium subsp. paratuberculosis
(M. paratuberculosis) is the etiologic agent of this
disease, which is manifested by chronic diarrhea and weight loss. After
months of diarrhea and wasting, the affected animals either die or are
culled (9, 11). In the United States, the prevalence of
M. paratuberculosis infection in dairy and beef cattle herds
has reached 34% in certain areas (12, 13) and causes
millions of dollars in lost revenue annually (35).
Furthermore, M. paratuberculosis has been tentatively linked
to Crohn's disease, a chronic granulomatous ileitis of humans. This
disease mimics other mycobacterial infections in both animals and
humans (31). Evidence supporting the possibility that
M. paratuberculosis is the etiologic agent of Crohn's
disease include culture of this organism from intestinal tissue
(11) and amplification of the subspecies-specific
IS900 sequence of M. paratuberculosis from biopsy
specimens by PCR (11, 21).
Currently, treatment of paratuberculosis in cattle is limited to
the extralabel use of therapeutic agents (29, 30), and no
antibiotic treatment is recommended for clinical cases of
Crohn's disease. Even with a prolonged drug regimen, paratuberculosis in cattle is invariably fatal. A significant problem hindering studies
of the antimicrobial susceptibilities of this organism is the long
generation time of M. paratuberculosis and the tendency of certain antibiotics to degrade during the evaluation period. Therefore, the objective of this study was to develop a new assay that
tests the drug susceptibilities of M. paratuberculosis
and that can be used to identify and screen a very large number of compounds in less time. This technology will facilitate the discovery of more powerful and less toxic drugs than those currently available for the treatment of these diseases. It is hoped that the development of this method, which is amenable to high-throughput screens, will
allow the identification of compounds which will eventually result in
shorter and more effective treatments for Johne's disease and possibly
Crohn's disease.
Recently, methods for the assessment of the antimicrobial
susceptibilities of mycobacteria have used the luciferase gene from Photinus pyralis, the American firefly; these methods have
been described elsewhere (1, 14, 15, 19, 20). This gene has
been introduced by transformation and phage infection into slowly
growing mycobacterial species, including M. paratuberculosis (14, 17, 20). The ability of an
antimicrobial agent to inhibit the growth of these strains can then be
measured by determining the decrease in bioluminescence. Here, we
report on the development of a firefly luciferase-based method for
determination of the drug susceptibilities of M. paratuberculosis. For the development of this assay, we tested
prototype drugs from various classes of antimicrobial agents. Since
there is no standardized method for determination of the drug
susceptibilities of mycobacteria except Mycobacterium
tuberculosis, we compared the luciferase-based assay with a
previously reported broth macrodilution assay used to determine the
drug susceptibilities of M. paratuberculosis (7).
Bacterial strains and growth conditions.
M.
paratuberculosis K-10(pYUB180) was grown in Middlebrook 7H9 broth
with 0.05% Tween 80 and 0.5 µg of mycobactin J (Allied Monitor,
Fayette, Mo.) per ml at 37°C as described previously (17).
The construction of the Escherichia coli-Mycobacterium spp.
shuttle plasmid pYUB180 has been described previously (20). This plasmid contains both the firefly luciferase gene downstream from
the Mycobacterium bovis BCG hsp60 promoter
(Phsp60) and a kanamycin resistance gene as a selectable
marker. All starter cultures used to inoculate test cultures were grown
in the presence of 50 µg of kanamycin per ml to an optical density at
600 nm of 0.3 to 0.4. To create an inoculum free of cellular clumping,
50 ml of cell culture was sonicated for 30 s with a Vibra-Cell
model VC600 disrupter (Sonics and Materials, Inc., Danbury, Conn.), passed through a 27-gauge needle three times, vortexed on high for
30 s, and allowed to sit for a minimum of 5 min. The top 5 ml was
then used for the inoculation of cultures for MIC assays. Cell
viability, as determined by BacLight Bacterial Viability staining (Molecular Probes, Eugene, Oreg.), demonstrated that 85% of
the bacteria were viable after being subjected to this procedure. A
total of 50 µl of this prepared culture (ca. 7.5 × 106 CFU) was then inoculated into each flask containing an
antibiotic, and the cells were grown at 37°C.
Antimicrobial agents.
Amikacin, D-cycloserine,
ethambutol, kanamycin, and isoniazid (all from Sigma Chemical Co., St.
Louis, Mo.) and Bay y 3118 (generously donated by L. E. Bermudez
and L. S. Young, Kuzell Institute, San Francisco, Calif.) were
prepared in sterile deionized water. Rifabutin (Pharmacia Inc.,
Columbus, Ohio) and clarithromycin (Abbott Laboratories, Abbott Park,
Ill.) were prepared in dimethyl sulfoxide (Fisher Scientific, St.
Louis, Mo.). All further dilutions of each antibiotic were prepared in
growth medium. The ranges of concentrations tested were as follows:
amikacin, 0.5 to 64 µg/ml; Bay y 3118, 0.008 to 100 µg/ml;
clarithromycin, 0.625 to 80 µg/ml; D-cycloserine, 0.8 to
100 µg/ml; ethambutol, 1.25 to 160 µg/ml; isoniazid, 1.95 to 250 µg/ml; kanamycin, 1 to 128 µg/ml; and rifabutin, 0.03 to 4 µg/ml.
Broth macrodilution assays.
Susceptibility testing was
performed in triplicate for each antimicrobial agent by following the
guidelines of the National Committee for Clinical Laboratory Standards
(22) with a total of eight twofold drug dilutions in 10 ml
of complete Middlebrook 7H9 broth with 0.05% Tween 80 and 0.5 µg of
mycobactin J per ml. Each culture was grown in sterile
25-cm2 tissue culture flasks, and for each antibiotic three
flasks without antimicrobial agent were included as controls. The MIC
was defined as the minimum concentration of the antimicrobial agent
which inhibited growth at day 14.
Luminescence assays.
Assays for bioluminescence were
performed by transferring 100 µl of each growing culture into a
separate 75-mm polystyrene test tube (Sarstedt, Newton, N.C.)
containing 250 µl of complete Middlebrook 7H9 broth without Tween 80. A total of 100 µl of 1 mM luciferin (Sigma) with 0.45 M sodium
citrate (pH 5.0) was automatically injected into each tube, and the
bioluminescence was measured for 20 s without preset delay with an
AutoLumat LB953 luminometer (Wallac Instruments, Gaithersburg, Md.).
Bioluminescence was expressed as the number of relative light units
(RLUs) detected in the measurement period. RLUs were obtained by
dividing the number of photoelectrons detected by 10, as specified by
the manufacturer. The initial (day 0) RLUs were measured within 30 min
of inoculation. Other RLU outputs were measured after 3, 7, and 14 days
of incubation.
Definitions, calculations, and interpretation.
The raw data
that were obtained were normalized by determining the initial and final
ratios of test and control RLUs for each antibiotic as described
previously (27). Briefly, the initial light output ratio was
calculated as the ratio of test RLUs to control RLUs on day 0. The
final ratio was calculated as the ratio of test RLUs to control RLUs
and was determined individually for days 3, 7, and 14. Results for each
time point were then expressed as the percent relative change in
bioluminescence, which was calculated as follows: (final ratio/initial
ratio) × 100. The MIC was defined as the lowest drug concentration
that gave a relative change in bioluminescence of Testing of susceptibility to selected antimycobacterial
agents by luciferase assays.
The changes in the
bioluminescence of strain M. paratuberculosis
K-10(pYUB180) in the presence of each antimicrobial agent are shown in
Fig. 1. Each graph
depicts the mean RLUs of three independent experiments for each
concentration of antibiotic after 3, 7, and 14 days of incubation.
Increases in RLUs could be observed by day 3 postinoculation in the
presence of subinhibitory drug concentrations, thus reflecting an
increase in the numbers of viable bacteria. This trend continued
through day 14 postinoculation, with total RLUs increasing by 100- to
1,000-fold. For those antibiotics with a discernible MIC, RLUs declined
to background levels and remained at this level for all higher drug
concentrations.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Development of a Firefly Luciferase-Based Assay for Determining
Antimicrobial Susceptibility of Mycobacterium avium
subsp. paratuberculosis
![]()
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
1.0% + 0.5%. At
these values, the bioluminescence of the sample is extinguished to less
than 1% of the original light output at day 0.
![]()
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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FIG. 1.
Bioluminescence of M. paratuberculosis
K-10(pYUB180) after incubation for 3 (circles), 7 (squares), and 14 (triangles) days in broth cultures containing different antimicrobial
agents (A and B). Bioluminescence (RLU × 0.001; y
axis) is plotted against the drug concentration (in micrograms per
milliliter; x axis). The value for each time point is the
mean of three independent experiments. Bars representing standard
deviations are indicated. The mean bioluminescence of the cultures
immediately after inoculation (day 0) was 340 ± 170.
1% + 0.5% (100-fold
extinction of bioluminescence compared with the bioluminescence for
controls containing no drug) were observed by day 7 for amikacin, Bay y
3118, clarithromycin, D-cycloserine, ethambutol, and
rifabutin, with corresponding MICs of 16, 0.015, 1.25, 25, 20, and 0.5 µg/ml, respectively. For day 14, identical MICs could be determined
for clarithromycin, D-cycloserine, and rifabutin. The MIC
of amikacin was significantly lower (2 µg/ml), while the MICs of Bay
y 3118 and ethambutol were 0.008 and 10 µg/ml, respectively, and were
within 1 doubling dilution of the MIC determined by day 7. Since for
kanamycin and isoniazid the percent relative change in bioluminescence
remained well above 1% for all dilutions through day 14, no MIC could
be determined for either of these antibiotics.
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Comparison of the luciferase and broth macrodilution assays.
The antimicrobial activities determined by the bioluminescence assay at
day 7 or 14 were closely correlated with those determined by the broth
macrodilution assay (determined at day 14; Table 2). The MICs obtained by the luciferase
assay at day 7 and by the broth microdilution assay were identical for
Bay y 3118, clarithromycin, D-cycloserine, ethambutol, and
rifabutin but differed by 2 doubling dilutions for amikacin. At day 14, the results of the luciferase assay were identical to those of the
broth macrodilution assay for amikacin, clarithromycin,
D-cycloserine, and rifabutin, but the results differed by 1 doubling dilution for Bay y 3118 and ethambutol. Strain K-10(pYUB180)
was resistant to isoniazid and kanamycin, and no MIC within the
concentration range tested could be determined by any of the methods.
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DISCUSSION |
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Presently, no drugs are approved for the treatment of Johne's disease in livestock. Therefore, antibiotic therapy is limited to the extralabel use of standard antimicrobial agents. Various treatment regimens for the control of Johne's disease have been proposed, and these have had various levels of success, but ultimately the disease is fatal to the animal. It is hoped that the development of the assay described here will allow the discovery of more effective and less toxic drugs. The most commonly prescribed treatment regimen consists of isoniazid in combination with rifabutin and/or ethambutol, followed by a daily dose of isoniazid for the duration of the animal's life (18, 30). Although isoniazid is used to treat infections caused by M. tuberculosis and M. bovis (23), the results presented here indicate that isoniazid therapy may not be the most effective treatment for M. paratuberculosis infections. Further in vitro screening of antimicrobial agents is warranted.
Our data indicate that the luciferase-based MIC assay may be applied for this purpose. It has the advantage that it requires less time to obtain MIC data. Furthermore, the other method commonly used to determine M. paratuberculosis susceptibility is the BACTEC assay (33). However, this assay requires a dedicated and specialized piece of equipment. In contrast, the firefly luciferase assay can be performed with a less expensive instrument such as a regular liquid scintillation counter or a one-well luminometer.
An important consideration in mycobacterial drug susceptibility assays is the use of Tween 80. Van Boxtel et al. (33) observed that at concentrations of 0.1 to 1%, Tween 80 affected colony morphology and caused cells to be more susceptible to the tested drugs. A similar effect was observed with strains of the M. avium complex (36). However, in a study with strains of M. tuberculosis, M. bovis BCG, M. avium, and Mycobacterium intracellulare, it was shown that Tween 80 had a significant effect on the MIC of rifampin but did not alter the activities of the other antimicrobial agents tested (2). In general, the absence of Tween 80 leads to considerable cell clumping, introducing additional sources of variation for MIC determinations. In our experiments, we used Tween 80 at a concentration of 0.05% so as to minimize both clumping of cells and detergent-induced changes in drug susceptibility.
To demonstrate the applicability of the firefly luciferase assay in the determination of M. paratuberculosis drug susceptibility, we selected eight prototype drugs inhibiting different targets involved in basic bacterial processes. These processes included inhibitors of cell wall biosynthesis (D-cycloserine, isoniazid, and ethambutol), protein synthesis (clarithromycin, amikacin, and kanamycin), DNA supercoiling (Bay y 3118), and RNA synthesis (rifabutin, a structural analog of rifampin with higher levels of antimycobacterial activity). Amikacin, isoniazid, and rifampin have been suggested as therapeutic agents for Johne's disease (34). D-Cycloserine, ethambutol, clarithromycin, and kanamycin are used as primary (ethambutol) or second-line antimycobacterial agents in human medicine to treat both M. tuberculosis and M. avium infections. Although Bay y 3118 has no therapeutic use for the treatment of mycobacterial diseases in animals or humans due to its phototoxicity (26), it was used as an example of the new types of fluoroquinolones that are being developed and that have extended activity against gram-positive organisms and high levels of inhibitory activity against mycobacteria (3, 4). Overall, M. paratuberculosis was sensitive to the majority of the antibiotics tested. The drug susceptibilities determined by the bioluminescence method were similar to those determined by the broth macrodilution method (Table 2). For most drugs, the MIC obtained on day 7 by the bioluminescence method was identical to the value obtained by the broth macrodilution method. Therefore, the bioluminescence assay provides a more rapid determination of a drug's effectiveness against M. paratuberculosis.
Individually, the activities of the antibiotics tested here correlated well with those described in previous reports on the susceptibilities of mycobacteria to antimicrobial agents. Of the studies reporting on the susceptibility of mycobacteria to D-cycloserine, M. tuberculosis was susceptible to the drug at 25 to 75 µg/ml (25), whereas Mycobacterium sp. isolated from patients with Crohn's disease were resistant to D-cycloserine at 5, 10, and 20 µg/ml (7). Our findings of a MIC of 25 µg/ml for M. paratuberculosis are in contrast to those of Thorel and coworkers (32), who reported that M. paratuberculosis strains are resistant to D-cycloserine at 30 to 50 µg/ml in Middlebrook 7H11 agar. The results from those researchers may not be comparable to our results due to methodological differences in drug susceptibility assays. Wallace et al. (34) demonstrated that for the M. avium complex, bacterial strains which are resistant to a single fixed drug concentration in agar dilutions are inhibited by lower concentrations of the same drug in broth culture.
For ethambutol, our reported MICs of 10 µg/ml correlate well with
previous findings of MICs of 5 µg/ml for M. avium and
M. paratuberculosis (10, 15). Ethambutol is
often prescribed in combination with isoniazid. Both M. tuberculosis and M. bovis are extremely sensitive
to isoniazid, but non-M. tuberculosis mycobacteria are
significantly more resistant to this drug. M. paratuberculosis K-10(pYUB180) was resistant to isoniazid at
250 µg/ml. In previous reports, animal isolates of M. paratuberculosis (8) and human isolates of
Mycobacterium sp. (10) were resistant to
isoniazid at the single concentration of 10 µg/ml. Similarly, Wallace
et al. (34) demonstrated that isolates of
Mycobacterium marinum, M. avium complex,
Mycobacterium smegmatis, and Mycobacterium chelonae were resistant to isoniazid at 16 to >32 µg/ml, the
highest concentrations tested. Further studies are needed to elucidate if the high level of resistance observed in our study is due to a
strain variation or if it is typical for most strains of M. paratuberculosis.
As expected, strain K-10(pYUB180) was resistant to kanamycin due to the production of the 3'-aminoglycoside phosphotransferase encoded by the Tn903-derived aph gene on plasmid pYUB180. Amikacin is another deoxystraptamine aminoglycoside which is structurally similar to kanamycin, but it is a poor substrate for the 3'-aminoglycoside phosphotransferase enzyme. Our MIC of 2 µg/ml and previous in vitro antimicrobial susceptibility results of 0.5 to 5 µg/ml for M. paratuberculosis (7) and Mycobacterium sp. (10) indicate that it is highly effective against this mycobacterial species. Our results are in contrast to those of Cooksey et al. (15), who reported that the MIC of amikacin is >100 µg/ml for a recombinant strain of M. avium carrying the same aph gene.
For Bay y 3118, our data indicated that M. paratuberculosis was very sensitive, with an MIC of 0.015 µg/ml. This agrees with other reports demonstrating MICs of 0.06 to 4 µg/ml for M. tuberculosis (28), M. avium complex (4, 28), and non-M. tuberculosis mycobacterial species (3). Clarithromycin is indicated for the treatment of human infections involving the M. avium complex, usually in combination with amikacin, rifabutin, and ethambutol. The MIC of 1.25 µg/ml that we found is slightly higher than the previously reported MICs of 0.25 µg/ml for M. paratuberculosis (24) and 0.5 µg/ml for M. avium (15). The rifamycin family of antibiotics has largely been reserved for use in human medicine, so that its value as a treatment in veterinary medicine may be underestimated. We report the MIC to be 0.5 µg/ml, consistent with values of 0.06 to 0.5 µg/ml for M. avium (14, 15) and other mycobacterial species (10).
Multiple-drug therapy is usually indicated for the treatment of mycobacterial infections. In vivo results of combination therapy against experimentally induced M. paratuberculosis infections in animals are unavailable, but Fattorini et al. (16) recently reported that monotherapy with isoniazid was ineffective in clearing M. avium infections in beige mice and that the triple-drug combination of isoniazid-amikacin-clarithromycin was the most effective. Of the drugs tested in our study, amikacin, Bay y 3118, clarithromycin, and rifabutin had MICs of 2 µg/ml or lower. On the basis of these results, we recommend that these classes of antibiotics be subjected to in vivo tests of their activities against M. paratuberculosis infections.
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ACKNOWLEDGMENTS |
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This research was supported by grant 95-37204-2148 from the U.S. Department of Agriculture NRI Competitive Grant Program, Cooperative State Research Project NEB 14-077, and Institute of Agriculture and Natural Resources (Agricultural Research Division) Interdisciplinary Research Program Project NEB 14-090.
We thank L. E. Bermudez and L. S. Young for providing compound Bay y 3118. We acknowledge L. E. Bermudez, J. D. Cirillo, G. E. Duhamel, and L. S. Young for critical review of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0905. Phone: (402) 472-8717. Fax: (402) 472-9690. E-mail for N. Beth Harris: bharris1{at}unl.edu. E-mail for Raúl G. Barletta: braul{at}crcvms.unl.edu.
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REFERENCES |
|---|
|
|
|---|
| 1. | Arain, T. M., A. E. Resconi, D. C. Singh, and C. K. Stover. 1996. Reporter gene technology to assess activity of antimycobacterial agents in macrophages. Antimicrob. Agents Chemother. 40:1542-1544[Abstract]. |
| 2. | Arain, T. M., A. E. Resconi, M. J. Hickey, and C. K. Stover. 1996. Bioluminescence screening in vitro (Bio-Siv) assays for high-volume antimycobacterial drug discovery. Antimicrob. Agents Chemother. 40:1536-1541[Abstract]. |
| 3. |
Bauernfeind, A.
1993.
Comparative in-vitro activities of the new quinolone, Bay y 3118, and ciprofloxacin, sparfloxacin, tosufloxacin, CI-960 and CI-990.
J. Antimicrob. Chemother.
31:505-522 |
| 4. | Bermudez, L. E., C. B. Inderlied, P. Kolonoski, M. Wu, L. Barbara-Burnham, and L. S. Young. 1996. Activities of Bay Y 3118, levofloxacin, and ofloxacin alone or in combination with ethambutol against Mycobacterium avium complex in vitro, in human macrophages, and in beige mice. Antimicrob. Agents Chemother. 40:546-551[Abstract]. |
| 5. | Chiodini, R. J., H. J. Van Kruiningen, and R. S. Merkal. 1984. Ruminant paratuberculosis (Johne's disease): the current status and future prospects. Cornell Vet. 74:218-262[Medline]. |
| 6. |
Chiodini, R. J.,
H. J. Van Kruiningen,
R. S. Merkal,
W. R. Thayer, and J. A. Coutu.
1984.
Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease.
J. Clin. Microbiol.
20:966-971 |
| 7. |
Chiodini, R. J.,
H. J. Van Kruiningen,
W. R. Thayer,
J. A. Coutu, and R. S. Merkal.
1984.
In vitro antimicrobial susceptibility of a Mycobacterium sp. isolated from patients with Crohn's disease.
Antimicrob. Agents Chemother.
26:930-932 |
| 8. | Chiodini, R. J. 1986. Biochemical characteristics of various strains of Mycobacterium paratuberculosis. Am. J. Vet. Res. 47:1442-1445[Medline]. |
| 9. | Chiodini, R. J., and H. J. Van Kruiningen. 1986. The prevalence of paratuberculosis in culled New England cattle. Cornell Vet. 76:91-104[Medline]. |
| 10. |
Chiodini, R. J.
1990.
Bactericidal activities of various antimicrobial agents against human and animal isolates of Mycobacterium paratuberculosis.
Antimicrob. Agents Chemother.
34:366-367 |
| 11. |
Cocito, C.,
P. Gilot,
M. Coene,
M. de Kesel,
P. Poupart, and P. Vannuffel.
1994.
Paratuberculosis.
Clin. Microbiol. Rev.
7:328-345 |
| 12. | Collins, M. T., D. C. Sockett, and W. J. Goodger. 1992. Herd prevalence, geographic distribution and risk factors for bovine paratuberculosis in Wisconsin. J. Am. Vet. Med. Assoc. 187:323-329. |
| 13. | Collins, M. T., D. C. Sockett, W. J. Goodger, T. A. Conrad, C. B. Thomas, and D. J. Carr. 1994. Herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in Wisconsin. J. Am. Vet. Med. Assoc. 204:636-641[Medline]. |
| 14. |
Cooksey, R. C.,
J. T. Crawford,
W. R. Jacobs, Jr., and T. M. Shinnick.
1993.
A rapid method for screening antimicrobial agents for activities against a strain of Mycobacterium tuberculosis expressing firefly luciferase.
Antimicrob. Agents Chemother.
37:1348-1352 |
| 15. | Cooksey, R. C., G. P. Morlock, M. Beggs, and J. T. Crawford. 1995. Bioluminescence method to evaluate antimicrobial agents against Mycobacterium avium. Antimicrob. Agents Chemother. 39:754-756[Abstract]. |
| 16. |
Fattorini, L.,
Y. Xiao,
M. Mattei,
Y. Li,
E. Iona,
M. L. Ricci,
O. F. Thorensen,
R. Creti, and G. Orefici.
1998.
Activities of isoniazid alone and in combination with other drugs against Mycobacterium avium infection in beige mice.
Antimicrob. Agents Chemother.
42:712-714 |
| 17. |
Foley-Thomas, E. M.,
D. L. Whipple,
L. E. Bermudez, and R. G. Barletta.
1995.
Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis.
Microbiology
141:1173-1181 |
| 18. | Gezon, H. M., H. D. Bither, H. C. Gibbs, E. J. Acker, L. A. Hanson, J. K. Thompson, and R. D. Jorgenson. 1988. Identification and control of paratuberculosis in a large goat herd. Am. J. Vet. Res. 49:1817-1823[Medline]. |
| 19. | Hickey, M. J., T. M. Arain, R. M. Shawar, D. J. Humble, M. H. Langhorne, J. N. Morgenroth, and C. K. Stover. 1996. Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice. Antimicrob. Agents Chemother. 40:400-407[Abstract]. |
| 20. |
Jacobs, W. R., Jr.,
R. G. Barletta,
R. Udani,
J. Chan,
G. Kalkut,
G. Sosne,
T. Kieser,
G. J. Sarkis,
G. F. Hatfull, and B. R. Bloom.
1993.
Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages.
Science
260:819-822 |
| 21. |
Mishina, D.,
P. Katsel,
S. T. Brown,
E. C. A. M. Gilberts, and R. J. Greenstein.
1996.
On the etiology of Crohn disease.
Proc. Natl. Acad. Sci. USA
93:9816-9820 |
| 22. | National Committee for Clinical Laboratory Standards. 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A3. National Committee for Clinical Laboratory Standards, Wayne, Pa. |
| 23. | Prescott, J. F., and J. D. Baggot. 1993. Antimicrobial therapy in veterinary medicine, p. 288-289. Iowa State University Press, Ames. |
| 24. |
Rastogi, N.,
K. S. Goh, and V. Labrousse.
1992.
Activity of clarithromycin compared with those of other drugs against Mycobacterium paratuberculosis and further enhancement of its extracellular and intracellular activities by ethambutol.
Antimicrob. Agents Chemother.
36:2843-2846 |
| 25. | Rastogi, N., V. Labrousse, and J. S. Goh. 1996. In vitro activities of fourteen antimicrobial agents against drug susceptible and resistant clinical isolates of Mycobacterium tuberculosis and comparative intracellular activities against the virulent H37Rv strain in human macrophages. Curr. Microbiol. 33:167-175[Medline]. |
| 26. | Schmidt, U., and G. Schluter. 1996. Studies on the mechanism of phototoxicity of Bay y 3118 and other quinolones. Adv. Exp. Med. Biol. 387:117-120[Medline]. |
| 27. | Shawar, R. M., D. J. Humble, J. M. Van Dalfsen, C. K. Stover, M. J. Hickey, S. Steele, L. A. Mitscher, and W. Baker. 1997. Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare. Antimicrob. Agents Chemother. 41:570-574[Abstract]. |
| 28. |
Sirgel, F. A.,
A. Venter, and H.-D. Heilmann.
1995.
Comparative in-vitro activity of Bay y 3118, a new quinolone, and ciprofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex.
J. Antimicrob. Chemother.
35:349-357 |
| 29. | St.-Jean, G., and A. D. Jernigan. 1991. Treatment of Mycobacterium paratuberculosis infection in ruminants. Vet. Clin. N. Am. Food Anim. Pract. 7:793-804[Medline]. |
| 30. | St.-Jean, G. 1996. Treatment of clinical paratuberculosis in cattle. Vet. Clin. N. Am. Food Anim. Pract. 12:417-430[Medline]. |
| 31. |
Thompson, D. E.
1994.
The role of mycobacteria in Crohn's disease.
J. Med. Microbiol.
41:74-94 |
| 32. |
Thorel, M.-F.,
M. Krichevsky, and V. V. Levy-Frebault.
1990.
Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov.
Int. J. Syst. Bacteriol.
40:254-260 |
| 33. |
Van Boxtel, R. M.,
R. S. Lambrecht, and M. T. Collins.
1990.
Effects of colonial morphology and Tween 80 on antimicrobial susceptibility of Mycobacterium paratuberculosis.
Antimicrob. Agents Chemother.
34:2300-2303 |
| 34. |
Wallace, R. J.,
D. R. Nash,
L. C. Steele, and V. Steingrube.
1986.
Susceptibility testing of slowly growing mycobacteria by a microdilution MIC method with 7H9 broth.
J. Clin. Microbiol.
24:976-981 |
| 35. | Wilson, D. J., C. A. Rossiter, H. R. Han, and P. M. Sears. 1996. Financial effects of Mycobacterium paratuberculosis on mastitis, culling and milk production in clinically normal dairy cattle, p. 151-158. In R. J. Chiodini, E. E. Hines II, and M. T. Collins (ed.), Proceedings of the Fifth International Colloquium on Paratuberculosis. International Association for Paratuberculosis, Inc., Madison, Wis. |
| 36. | Yamori, S., and M. Tsukamura. 1991. Paradoxical effect of Tween 80 between the susceptibility to rifampicin and streptomycin and the susceptibility to ethambutol and sulfadimethoxine in the Mycobacterium avium-Mycobacterium intracellulare complex. Microbiol. Immunol. 35:921-926[Medline]. |
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