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cellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well mini- tube format under biosafety level 2 conditions.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1997, p. 570–574 0066-4804/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 41, No. 3

Rapid Screening of Natural Products for Antimycobacterial Activity by Using Luciferase-Expressing Strains of Mycobacterium bovis BCG and Mycobacterium intracellulare RIBHI M. SHAWAR,1* DEBBI J. HUMBLE,1 JILL M. VAN DALFSEN,1 C. KENDALL STOVER,1 MARK J. HICKEY,1 SHARON STEELE,1 LESTER A. MITSCHER,2 AND WILLIAM BAKER1 PathoGenesis Corporation, Seattle, Washington 98119,1 and Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-25062 Received 14 March 1996/Returned for modification 27 June 1996/Accepted 13 December 1996

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time. drug screening research for the discovery of antituberculosis compounds (1, 5, 15). However, such approaches have yet to be validated. The highly infectious nature of M. tuberculosis precludes its use in a high-throughput screen. Therefore, we have developed and evaluated a rapid, quantitative broth dilution method using recombinant strains of the bacillus Calmette-Gue´rin (Mycobacterium bovis rBCG) and Mycobacterium intracellulare expressing firefly luciferase. In this as in other luciferase reporter gene assays, the ability of a substance to inhibit the growth of a mycobacterial reporter strain is indicated by a decrease in luminescence. This paper describes the use of this method in a rapid, high-throughput broth microdilution assay to evaluate the activities of chemical extracts of plant substances. These results were compared to results from colorimetric endpoint detection with Alamar blue (3, 17, 19, 21–23).

An increase in the incidence of multiple-drug-resistant strains of mycobacteria has underscored the need to rapidly identify new drugs for chemotherapy. Current methods for determining the antimycobacterial activities of agents are based on the growth of organisms on solid and in liquid media. Methods such as agar proportion, BACTEC, and broth dilution are commonly used to determine the susceptibilities of Mycobacterium tuberculosis isolates (11). The agar proportion method requires large amounts of test substance and 3 weeks of incubation, and results are often hard to interpret (13). The BACTEC method, though somewhat faster (5 to 10 days), is costly and cumbersome and requires the use of radioactivity. Neither method is amenable to high-throughput testing of potential antimicrobial agents at various concentrations. Broth macro- and microdilution methods are widely accepted as standards for evaluation of activities of compounds against aerobic and anaerobic organisms. Such methods, employing both turbidimetric and colorimetric endpoints, have been described for rapid- and slow-growing mycobacteria, including M. tuberculosis (4, 9, 14, 20–23). Strategies for susceptibility testing of M. tuberculosis by a bioluminescence assay based on mycobacterial ATP have been successfully applied (16). Luciferase reporter gene assays have also been shown to have potential in expediting antimycobacterial drug susceptibility testing (1, 6, 7, 12). A rapid, accurate, quantitative, and less laborious technique is clearly needed to expedite the discovery of drugs against slow-growing mycobacteria. Because of the simplicity of testing these organisms, their rapid growth rate, and their low pathogenic potential, organisms such as Mycobacterium smegmatis and Mycobacterium aurum have been employed as markers in

MATERIALS AND METHODS Mycobacterial strains. The strains used in this study were the M. bovis BCG substrain Connaught (ATCC 35745) and M. intracellulare ATCC 35761. The integrating shuttle vector pMV361 was constructed and electroporated into rBCG and M. intracellulare as described previously (10). Culture and growth conditions. Stock strains of mycobacteria were maintained in 7H9 broth with 15% glycerol at 2808C. Subcultures of the microorganisms were made in Middlebrook 7H9 broth (Difco, Detroit, Mich.) containing 10% ADC (albumin-dextrose-catalase) enrichment (BBL, Cockeysville, Md.), 0.05% Tween 80 (BBL), and 20 mg of kanamycin (Sigma, St. Louis, Mo.)/ml. Cultures of rBCG and recombinant M. intracellulare were incubated in an ambient atmosphere for 48 and 24 h, respectively. Following incubation, the culture suspension was sonicated for 10 s with a VibraCell Sonicator (Sonics & Materials, Inc., Danbury, Conn.). To prepare the inoculum, the sonicated culture was diluted in Middlebrook 7H9 broth without kanamycin to an optical density at 540 nm of 0.05. This procedure yielded a suspension containing approximately 107 CFU/ml, as confirmed by a plate count on 7H11 agar (Remel, Lenexa, Kans.). This diluted suspension was used to inoculate test trays as described below, resulting in a final inoculum of 5 3 105 CFU/ml in the reaction tube. Antimycobacterial agents. Isoniazid (INH) (Sigma), ciprofloxacin (Miles Inc., West Haven, Conn.), ethambutol (Sigma), rifampin (Sigma), rifabutin (Pharmacia Inc., Columbus, Ohio), and clarithromycin (Abbott Laboratories, North Chi-

* Corresponding author. Mailing address: PathoGenesis Corporation, 201 Elliott Ave. W., Suite 150, Seattle, WA 98119. Phone: (206) 467-8100, ext. 3393. Fax: (206) 282-5065. E-mail: [email protected] .com. 570

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cago, Ill.) were used as drug controls and were included in test panels for quality control. INH, ciprofloxacin, and ethambutol were solubilized in water. Clarithromycin, rifampin, and rifabutin were solubilized in methanol. A series of four doubling dilutions of each drug was prepared at 100 times the desired final test concentration and stored at 2808C. Plant samples and preparation of extracts. Dried plant materials (roots, leaves, and stems, etc.) were pulverized with a Wiley mill. Approximately 100 g of the pulverized plant parts was allowed to stand in 100% ethanol (400 to 600 ml) for 24 h at room temperature. The ethanol solutions then were filtered and were concentrated under reduced pressure with a rotary evaporator, and the extract residue was stored at 2208C. Stock solutions of the extracts were prepared by dissolving preweighed samples of the extracts in 100% dimethyl sulfoxide to final concentrations of 10 and 30 mg/ml. These stock solutions were stored at 2808C. Test panel preparation and inoculation. The test was performed in 96-place racks containing 1.2-ml Autotubes (Elkay, Shrewsbury, Mass.). Each test was prepared by transferring 5 ml of extract stock to an Autotube containing 490 ml of enriched Middlebrook 7H9 broth. Five microliters of each doubling dilution of control drug stocks was added to a corresponding tube in the test panel. Eight Autotubes containing no drug and 1% dimethyl sulfoxide were included in each test panel and served as growth controls. Twenty-five microliters of the inoculum, prepared as described above, was added to the extract-, growth control-, and control drug-containing Autotubes, yielding a final inoculum of approximately 5.0 3 105 CFU/ml. At least four Autotubes received no drugs or inoculum and served as negative controls. The process was later automated with the Biomek 1000 (Beckman, Fullerton, Calif.) liquid handling system. Luciferase assay. The luciferase assay was performed by transferring 100 ml of each test solution to a corresponding well of a 96-well Microlite 1 tray (Dynatech Inc., Chantilly, Va.). With the MicroLumat LB 96P luminometer (Wallac Instruments, Gaithersburg, Md.), 100 ml of 1 mM luciferin (R&D Systems, Minneapolis, Minn.) was automatically dispensed to each well, and luminescence was measured for 15 s without preset delay. Luminescence was expressed as the number of relative light units (RLU) detected in the measurement period. The initial, or day 0, RLU was measured within 30 min of inoculation. The Autotubes were capped and incubated at 368C in an ambient atmosphere. The final RLU output was measured in the same manner after 3 days (recombinant M. intracellulare) and 5 days (rBCG) of incubation. The Autotubes were recapped and returned to the incubator for later colorimetric endpoint determination. Results were expressed as the relative change in luminescence as described below. Colorimetric assay. Colorimetric endpoints of the broth dilution assay were determined at day 7 for recombinant M. intracellulare and at day 12 for rBCG as previously described by Yajko et al. (22, 23). Samples of 200 ml each were removed from each Autotube and transferred into a corresponding well in a sterile, 96-well polystyrene microtiter tray. A 20-ml aliquot of 10% Alamar blue reagent (Alamar Biosciences, Inc., Camarillo, Calif.) and 12.5 ml of 20% Tween 80 were added to each well. The tray was incubated at 368C and read after 16 to 18 h. Results were recorded as pink (no inhibition of growth) or blue (inhibition of growth). Persistence of a blue color after addition of Alamar blue and appropriate incubation indicated the presence of activity. The lowest concentration resulting in persistence of blue color was considered the MIC. Determination of MICs by luciferase and colorimetric assays. To determine the length of incubation required to achieve accurate MICs by the luciferase assay, RLU output was measured daily for 5 and 7 days for recombinant M. intracellulare and rBCG, respectively, by using several panels containing growth controls, sterility controls, and serially diluted drug controls. Incubation of these panels was continued for an additional 4 and 5 days for recombinant M. intracellulare and rBCG, respectively, to determine colorimetric MICs, as previously described (22, 23). The optimal end of incubation period for the luciferase assay was considered the day on which there was no further change in the MIC for any of the drugs tested and on which there was agreement between methods. Definitions, calculations, and interpretation. A large number of extracts resulted in a nonspecific reduction in luminescence (i.e., interference), which affected the interpretation of changes observed in RLU output. Therefore, both the initial and final ratios between test and control RLUs had to be evaluated. The initial ratio was calculated as the ratio of test to control RLUs on day 0. The final ratio was calculated as the ratio of test to control RLUs on day 5 (day 3 for recombinant M. intracellulare). Percent interference was calculated as follows: (1 2 initial ratio) 3 100. To correct for the observed interference, results were expressed as the percent relative change in luminescence, which was calculated as follows: (final ratio/initial ratio) 3 100. A comparison of colorimetric (Alamar blue) and luminescence endpoints with results obtained with known control drugs revealed that a relative change in luminescence of #1% correlated with the presence of inhibitory activity. Therefore, the MIC of the control drugs was defined as the lowest concentration resulting in a relative change in luminescence of #1%. The MICs of an extract, fraction, and purified test compound were similarly defined. Validation of luciferase assay using extracts spiked with INH. The ability of the luciferase assay to detect active substances despite interference was evaluated by spiking extracts with INH. Ten extracts which were previously shown to cause interference ranging from 7 to 97% were tested at 300 mg/ml with and without spiking with 2 and 4 mg of INH/ml. Autotubes containing the unspiked and spiked extracts were inoculated with rBCG and tested by the luciferase and

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FIG. 1. Effect of incubation time on rate of change in RLU output from rBCG grown in 7H9 broth with and without drugs. Samples (0.1 ml each) were transferred at each time point, and the RLU was determined as described in the text. Concentrations plotted are MICs for each drug. See Table 1 for relative changes in luminescence and further details. CIP, ciprofloxacin; ETH, ethambutol; RIF, rifampin.

colorimetric assays. Activity was defined as the persistence of blue color after addition of Alamar blue. Results obtained colorimetrically were compared to the relative changes in luminescence to evaluate if the calculation method used could correctly predict activity despite the presence of interference. Isolation and purification of active compounds. The luciferase assay was used to test fractions and purified products from extracts that showed significant activity. Extracts or fractions that were active at 100 mg/ml were tested at lower concentrations in a twofold dilution scheme to identify the fraction(s) of interest for further workup. Extracts of interest were separated by silica gel chromatography. Chromatographic fractions were evaporated, weighed, and tested. Active fractions were pooled, separated again, and retested. The luciferase assay was used as a guide to identify the most active material that needed to be separated by silica gel chromatography. This process was repeated until a single active entity was isolated by preparative thin-layer chromatography. The separation process was repeated for a larger amount of the crude extract so that enough of the purified material would be available for analysis. The structure of the active compounds was determined by chemical analysis methods such as high resolution fast-atom bombardment mass spectrometry, electron impact mass spectrometry, proton and carbon-13 nuclear magnetic resonance spectrometry, and UV and infrared spectroscopy.

RESULTS Determination of MICs by the luciferase and colorimetric assays. Comparative studies on the length of incubation showed that the relative changes in luminescence obtained after 3 and 5 days of incubation for recombinant M. intracellulare and rBCG, respectively, matched the endpoints determined colorimetrically. Figure 1 shows the kinetic changes in RLU, and Table 1 shows the calculated relative changes in luminescence over 7 days with rBCG and known drugs. Relative changes in luminescence of #1% achieved on days 5 to 7 of incubation were in agreement with Alamar blue endpoints obtained on day 12. Assays performed with recombinant M. intracellulare showed that relative changes in luminescence of #1% achieved on days 3 to 4 of incubation were in agreement with Alamar blue endpoints obtained on day 5 (data not shown). There was no further change in the luciferase assay endpoint after day 5 (rBCG) or day 3 (recombinant M. intracellulare). As a result, screening of extracts was performed by measurement of initial (on the day of setup) and final (day 5 for rBCG and day 3 for recombinant M. intracellulare) RLU. The Alamar blue test was performed and read as described

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TABLE 1. Determination of the MICs of four antimycobacterial drugs for rBCG Drug

Concn (mg/ml)

TABLE 3. Summary and comparison of results obtained by luciferase and colorimetric broth dilution assays of plant extracts for activity against rBCG

Relative change in luminescence (%) at daya: 2

3

4

5

7

Ethambutol

1 2b 4

47.3 12.0 6.2

33.9 5.3 2.0

30.7 0.9 0.2

31.6 0.2 0.0

23.7 0.1 0.0

INH

1 2b 4

7.5 3.1 2.6

22.6 0.7 0.5

6.5 0.1 0.1

7.8 0.0 0.0

8.7 0.0 0.2

Ciprofloxacin

0.125 0.25b 0.5

28.8 3.4 0.4

51.3 5.0 0.2

39 0.4 0.0

10.8 0.0 0.0

34 0.0 0.0

Rifampin

0.008b 0.015 0.03

5.8 1.4 0.6

4.5 0.8 0.8

2.1 0.4 0.2

0.2 0.1 0.0

0.1 0.0 0.0

a Boldface type indicates days that the relative change in luminescence was #1%. b MIC.

above on day 12 for rBCG and on day 7 for recombinant M. intracellulare. Validation of the luciferase method with extracts spiked with INH. Nine extracts which were inactive both colorimetrically and by the luciferase assay were identified as active after being spiked with INH, despite interference ranging from 7 to 81%. One extract which exhibited 97 to 98% interference had a very high relative change in luminescence before being spiked and a relative change in luminescence of 2.5% after being spiked. This extract, when spiked with INH, was identified as active by Alamar blue. Table 2 shows a summary of results obtained with these extracts in the presence or absence of 2 mg of INH/ml. Results from screening. Screening results are summarized in Table 3. A total of 480 extracts were screened for activity against rBCG. Of these, 16 were considered active and 459

TABLE 2. Interference and relative change in luminescence for selected extracts with and without INHa Extractb

1031.07 1032.05 1033.05 1034.00 1035.04 1036.02 1039.13 1040.02 1041.10 1042.17

Interference (%)

Relative change in luminescence (%)c

Unspiked

Spikedd

Unspiked

Spiked

45 34 32 7 81 27 35 19 21 97

44 36 44 10 83 29 38 19 25 98

23 57 81 84 69 200 61 94 58 1,530

0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 2.5

a Extracts were also evaluated colorimetrically. In all unspiked samples, pink color developed after addition of Alamar blue, indicating no growth inhibition. In all spiked samples, blue color persisted after addition of Alamar blue, indicating the presence of an inhibitory effect on growth and metabolism. b 300 mg/ml. c Values of #1% indicate activity. Occasionally, values of .100% were obtained from inactive extracts; see text for explanation. d With INH (2 mg/ml).

Luciferase assay resulta

Active Inactive Total

Colorimetric assay result No. of active extracts

No. of inactive extracts

Total no. of extracts

16 2b 18

3 459 462

19 461 480

a Extracts with a relative change in luminescence of #1% were considered active; extracts with a relative change in luminescence of .1% were considered inactive. b These extracts showed relative changes in luminescence of 1.5 and 2.4%. Both extracts had high interference. If a cutoff value of 3% had been used, both of these samples would have been considered active. However, this would have resulted in 12 extracts being considered false-positive hits.

were inactive against rBCG by both the luciferase assay and the colorimetric assay. The luciferase assay resulted in two false-negative results and three false-positive results compared to the colorimetric assay. Compared to results obtained by the Alamar blue method, the calculated sensitivity, specificity, negative predictive value, and positive predictive value of the luciferase assay were 88.9, 99.4, 99.6, and 84.2%, respectively. There was an overall agreement of 99.0% between methods. Of the 16 extracts active against rBCG, 5 were also active against recombinant M. intracellulare at a concentration of 300 mg/ml and 2 were active at a concentration of 100 mg/ml. In order to examine the utility of this new methodology in screening and fractionation studies directed towards discovery of naturally occurring antitubercular agents, a number of active extracts were investigated further. Based on the luciferase assay results, fractionation and purification studies were performed on several active extracts and fractions until the active constituent was revealed. Figure 2 shows an example of a natural product identified from root extracts of Dulacia candida, a plant from South America. This compound, octadectrans-11-en-9-ynoic acid, also known as ximenynic acid (18), had MICs of 25 and .100 mg/ml for rBCG and recombinant M. intracellulare, respectively. DISCUSSION Our study demonstrates that a luciferase assay with recombinant mycobacteria containing the lux gene can rapidly and reliably identify antimycobacterial activity in crude extracts, fractions, and pure compounds. Bioluminescence decreases when such recombinant mycobacteria are tested against material having antimycobacterial activity. This method is amenable to screening a large number of samples in a quick and costeffective manner. In the first part of this study, we standardized the luciferase assay by examining the length of incubation and interpretive

FIG. 2. Structure of ximenynic acid.

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criteria for the observed reduction in RLU output. Ciprofloxacin, clarithromycin, ethambutol, INH, rifabutin, and rifampin were used to validate the assay. Using these agents, we determined that at least a 99% reduction in RLU compared to that of the growth control is required to indicate antimycobacterial activity. Accurate and predictive results were obtained by determining the RLU on day 5 when testing rBCG (Table 1 and Fig. 2) and on day 3 when testing recombinant M. intracellulare (data not shown). MICs obtained in this study are in essential agreement with an earlier study using rBCG (2). The MICs of INH were higher than would be expected for rBCG. A similar finding was reported by Arain et al. (2) and was attributed to the use of ADC enrichment obtained from BBL. Those authors obtained lower INH MICs when they used ADC from other commercial sources. It is noteworthy that we have used ADC from BBL in all of our studies, and although the test method formats are slightly different (macrodilution versus microdilution), our results are in essential agreement with this earlier report. In applying the luciferase assay to crude extracts, it was extremely important to address the fact that extracts can cause nonspecific reductions in luminescence (some as high as 98% interference) which could lead to erroneous interpretation. We made no attempt to study the reasons for this interference, although possibilities include the quenching of signal due to the color or opacity of substances and the effect of the extracts on the luciferase enzyme itself. A simple calculation method was devised to account for interference and to accurately predict the presence of activity. The validity of this approach was investigated by testing selected extracts with and without spiking with INH. Activity was revealed in all spiked extracts (with a relative change in luminescence of #1%), except for one which had extremely high interference (Table 2). On rare occasions, relative changes in luminescence of .100% were obtained with inactive extracts. Possible explanations for this include degradation of the interfering substances during incubation or the presence of growth factors in some extracts that stimulate growth beyond the level of the growth control. Extracts of plant products have been screened previously by agar dilution at concentrations of 100 and 1,000 mg/ml (15). However, based on our preliminary studies (data not shown), we chose to test concentrations (100 and 300 mg/ml) at which interference was minimized without the loss of our ability to detect activity. Using this assay, we evaluated the antimycobacterial activities of 480 extracts. Results were in agreement 99.0% of the time (475 of 480 extracts) with the colorimetric assay (Table 3). The luciferase assay proved to be a rapid and accurate screening method, with a negative predictive value of 99.6%. However, because the positive predictive value was 84.2%, we routinely confirm all positive results by another method. This assay has facilitated the rapid identification of a number of lead compounds, which are currently under investigation. An example of an active compound that was identified through testing a series of extracts, fractions, and purified material is shown in Fig. 2. Chung et al. (5) recently described a high-throughput radioactivity-based screen for detecting antimycobacterial agents by testing M. aurum as a marker. Our assay has the clear advantages of being nonradioactive and of using rBCG, which is likely to be more predictive of anti-M. tuberculosis activity than are rapidly growing mycobacteria. In comparison to BACTEC and agar-based proportion methods, the luciferase assay has the advantages of being simpler, faster, and amenable to highthroughput testing. The luciferase assay is also faster than the

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Alamar blue broth dilution and has the advantage of generating numerical results that can be helpful in interpretation. Methods of susceptibility testing and drug screening based on recombinant strains of M. tuberculosis expressing firefly luciferase have been previously described by Cooksey et al. (6, 8). Such methods require a biosafety level 3 facility in order to minimize the hazards of dealing with M. tuberculosis. The method we describe here has the advantage of requiring less stringent laboratory practices and facilities since biosafety level 2 recombinant organisms are used. Possible limitations of this and other bioluminescence-based methods in which recombinant strains are used include the requirement of a luminometer and the fact that only recombinant strains which are used as markers can be tested. In addition, differences between rBCG and M. tuberculosis require that all potentially active test substances be evaluated for activity against strains of M. tuberculosis to confirm the activity. ACKNOWLEDGMENTS The work at the University of Kansas was supported in part by NIH grants GM-13155 and AI-36650. We thank Jeff Ried for assistance in reviewing the writing style and Dave Gray for assistance with calculation methods. REFERENCES 1. Andrew, P. W., and I. S. Roberts. 1993. Construction of a bioluminescent mycobacterium and its use for assay of antimycobacterial agents. J. Clin. Microbiol. 31:2251–2254. 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. 3. Baker, C. N., S. N. Banerjee, and F. C. Tenover. 1994. Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of gramnegative bacteria. J. Clin. Microbiol. 32:1261–1267. 4. Brown, B. A., R. J. Wallace, Jr., and G. O. Onyi. 1992. Activities of clarithromycin against eight slowly growing species of nontuberculous mycobacteria, determined by using a broth microdilution MIC system. Antimicrob. Agents Chemother. 36:1987–1990. 5. Chung, G. A. C., Z. Aktar, S. Jackson, and K. Duncan. 1995. High-throughput screen for detecting antimycobacterial agents. Antimicrob. Agents Chemother. 39:2235–2238. 6. Cooksey, R., and G. Morlock. 1994. Minimum bactericidal concentrations (MBCs) of antimicrobial agents against Mycobacterium tuberculosis determined using a luciferase assay, abstr. E122, p. 263. In Abstracts of the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Washington, D.C. 7. 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. 8. 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. 9. Gomez-Flores, R., S. Gupta, R. Tamez-Guerra, and R. T. Mehta. 1995. Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method. J. Clin. Microbiol. 33:1842–1846. 10. 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. 11. Inderlied, C. B., and M. Salfinger. 1995. Antimicrobial agents and susceptibility tests: mycobacteria, p. 1385–1404. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C. 12. 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. 13. Kent, P. T., and G. P. Kubica. 1985. Public health mycobacteriology. A guide to the level III laboratory. Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Ga. 14. Mehta, R. T., A. Keyhani, T. J. McQueen, B. Rosenbaum, K. V. Rolston, and J. J. Tarrand. 1993. In vitro activities of free and liposomal drugs against Mycobacterium avium-M. intracellulare complex and M. tuberculosis. Antimicrob. Agents Chemother. 37:2584–2587.

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ANTIMICROB. AGENTS CHEMOTHER. method with 7H9 broth. J. Clin. Microbiol. 24:976–981. 21. Yajko, D. M., C. A. Sanders, P. S. Nassos, J. Madej, M. Lancaster, and W. K. Hadley. 1993. A colorimetric method for determining the antimicrobial susceptibility of M. tuberculosis, M. avium complex (sic) and rapid growing mycobacteria, abstr. U-20, p. 172. In Abstracts of the 93rd General Meeting of the American Society for Microbiology 1993. American Society for Microbiology, Washington, D.C. 22. Yajko, D. M., J. J. Madej, P. S. Nassos, C. A. Sanders, M. V. Lancaster, C. R. Sganga, and W. K. Hadley. 1994. A rapid colorimetric test for MICs of rifampin (RIF) against M. tuberculosis (MTB), abstr. U-142, p. 197. In Abstracts of the 94th General Meeting of the American Society for Microbiology 1994. American Society for Microbiology, Washington, D.C. 23. Yajko, D. M., J. J. Madej, M. V. Lancaster, C. A. Sanders, V. L. Cawthon, B. Gee, A. Babst, and W. K. Hadley. 1995. Colorimetric method for determining MICs of antimicrobial agents for Mycobacterium tuberculosis. J. Clin. Microbiol. 33:2324–2327.