Validation of the Use of Middlebrook 7H10 Agar, BACTEC MGIT 960, and BACTEC 460 12B Media for Testing the Susceptibility of Mycobacterium tuberculosis to Levofloxacin

Bacterial strains.Fourteen fluoroquinolone-resistant strains of M. tuberculosis were either selected from clinically resistant isolates submitted to the California Department of Health Services for susceptibility testing or obtained from a collection of well-characterized isolates provided by the Public Health Research Institute (PHRI) Tuberculosis Center, New York City, N.Y. (14). Six of the 16 M. tuberculosis isolates tested were strains identified by the California Department of Health Services as fluoroquinolone resistant by both BACTEC radiometric and AP methods and were from patients who were culture positive despite a clinical history of fluoroquinolone treatment for TB. The remaining eight resistant isolates were part of a collection of strains obtained from PHRI, which have a mutation in their gyrA gene region associated with fluoroquinolone resistance. The isolates from PHRI included four strains that are IS6110 type W and one strain that is type W1. Thirty-two pan-susceptible strains of M. tuberculosis were selected from isolates submitted to the California Department of Health Services for susceptibility testing between January 1998 and February 2000 from the Pacific Basin—Kwajalein, Marshall Islands (8); Palau (8); American Samoa (5); Saipan, Northern Mariana Islands (4); Yap, Federated States of Micronesia (FSM) (4); Majuro, Marshall Islands (1); Pohnpei, FSM (1); and Chuuk, FSM (1)— where treatment for M. tuberculosis or other infectious agents with fluoroquinolones is unlikely.

LVX.LVX stock solution (1,000 μg/ml) was prepared according to the manufacturer's recommendations and stored at −20°C until use.

LVX stability studies.For the mycobacterial growth indicator tube (MGIT) method, dilutions of LVX were prepared in sterile water, and then 0.1 ml of each drug dilution was added to sets of tubes containing 7.0 ml of MGIT broth supplemented with 0.8 ml of oleic acid-albumin-dextrose-catalase. Two subinhibitory concentrations of LVX, 0.5 and 0.25 μg/ml, were tested. Each tube was then inoculated based on the protocol of Bergmann and Woods (1) with 0.1 ml of a suspension of M. tuberculosis H37Rv equivalent to a McFarland turbidity standard of 0.5. One set of MGIT tubes containing drug dilutions was stored at 4°C for 14 days prior to inoculation with M. tuberculosis H37Rv. A second set of tubes containing drug dilutions was incubated for 14 days at 37°C prior to inoculation. A third set of tubes received added drug dilutions on the same day as inoculation. Five replicates were run per analysis. Stability comparisons were determined based on the number of days that were required to detect a positive result by the MGIT instrument. Results were compared for statistical significance using the one-way analysis of variance.

For the BACTEC 460 method, dilutions of LVX were prepared in sterile water such that when 0.1 ml was added to 12B medium, concentrations of 1.0 and 2.0 μg/ml were obtained. These concentrations were selected to provide sufficient inhibition of growth of the test strain to yield usable endpoint values. To each drug-containing vial, 0.1 ml of a suspension of M. tuberculosis H37Rv adjusted to a McFarland turbidity standard of 1 was added. A drug-free control was inoculated with the same H37Rv suspension (McFarland turbidity standard of 1) diluted 1:100 as recommended by the manufacturer. To compare drug stability, one set of 12B vials (containing 1.0 and 2.0 μg of LVX/ml and a drug-free control) was stored at 4°C for 14 days prior to inoculation. A second set of vials was stored at 37°C for 14 days prior to inoculation. A third set of vials was prepared on the same day they were inoculated. Once inoculated, the 12B vials were analyzed with the BACTEC 460 instrument according to the manufacturer's recommendations for the standard nonweekend reading schedule. Five replicates were run per analysis. When the growth index (GI) of the control was ≥30 U, the GI values of the sets of drug-containing vials were compared using the one-way analysis of variance test.

Critical test concentration value determination using the BACTEC 460 radiometric method.Serial twofold dilutions of LVX were prepared with sterile water. For fluoroquinolone-resistant strains, LVX test concentrations were 4, 2, 1, 0.5, and 0.25 μg/ml. For susceptible strains, LVX test concentrations were 2, 1, 0.5, 0.25, and 0.125 μg/ml. The test concentrations selected were expected to be satisfactory, based on previously published MIC results of LVX for M. tuberculosis (5, 7, 12). BACTEC 460 radiometric MIC determinations were done by standard methods. The critical concentration for the BACTEC 460 radiometric method for LVX was the concentration that inhibited 100% of susceptible strains of M. tuberculosis while permitting growth of all LVX-resistant strains. Once critical concentration values were determined, the values were tested against a subset of susceptible and resistant strains for which MICs were near the critical values to validate the selected critical concentration, since those strains would be the most sensitive indicators of the validity of the selected critical test concentration. The results were then analyzed for statistical significance using chi-square analysis.

Critical test concentration value determination by the AP method.Middlebrook 7H10 agar quadrant plates were prepared by standard methods (6). For fluoroquinolone-resistant strains, the plates were configured such that quadrant I was the drug-free control, and quadrants II, III, and IV contained LVX at 4, 2, and 1 μg/ml, respectively. For fluoroquinolone-susceptible strains, quadrant I was the drug-free control, and quadrants II, III, and IV contained LVX at 1, 0.5, and 0.25 μg/ml, respectively. The test concentrations selected were expected to be satisfactory based on previously published MIC results of LVX for M. tuberculosis (5, 7, 13). Plates were inoculated by standard methods (6). Plates were inoculated according to previously published recommendations (6) with 0.1 ml of 10⁻² and 10⁻⁴ dilutions of inoculum adjusted to the turbidity equivalent to a McFarland standard of 0.5. After 21 days of incubation, the colonies on the plates were counted with the aid of a Quebec colony counter and/or microscope. Susceptibility to LVX was determined by comparing the growth in the drug-containing quadrants to the growth in the drug-free control quadrant. Quadrants showing 1% growth or more compared to growth in the control quadrant were considered resistant to that concentration of LVX. The critical concentration for the AP method was the LVX concentration that inhibited 100% of susceptible strains of M. tuberculosis while permitting growth of all LVX-resistant strains (2). Once critical concentration values were determined, the values were tested against a subset of susceptible and resistant strains for which the MIC was near the critical values to validate the selected critical concentration.

Critical test concentration value determination for the nonradiometric BACTEC MGIT 960 method.Serial twofold dilutions of LVX were prepared in sterile water. For fluoroquinolone-resistant strains, LVX test concentrations were 4, 2, 1, 0.5, and 0.25 μg/ml. For susceptible strains, LVX test concentrations were 2, 1, 0.5, 0.25, and 0.125 μg/ml. MIC determinations were done with the BACTEC MGIT 960 instrument and were performed according to the method of Bergmann and Woods (1). Briefly, serial twofold dilutions of LVX were prepared in sterile water, and then 0.1 ml of each drug dilution was added to sets of tubes containing 7.0 ml of MGIT broth supplemented with 0.8 ml of oleic acid-albumin-dextrose-catalase. Each MGIT tube was then inoculated with 0.1 ml of a suspension of the M. tuberculosis isolate equivalent to a McFarland turbidity standard of 0.5. At the time of inoculation, a drop of the inoculum was streaked on a Middlebrook 7H10 plate and incubated at 37°C to check for purity. Once inoculated, the MGIT tubes were then entered into the instrument. The BACTEC MGIT 960 instrument monitors the tubes for oxygen utilization, which results in an increase in fluorescence as an indirect indicator of growth. The criterion for drug resistance was based on the time to positivity of a drug-containing tube compared to the time to positivity of the drug-free control. A reading was considered resistant if the tube was positive within 2 days or less of the day the drug-free control tube became positive for that isolate. MGIT tubes showing drug resistance were smeared and stained with Ziehl-Neelsen stain to verify the presence of acid-fast bacilli. The critical concentration for the nonradiometric BACTEC MGIT 960 method for LVX was the concentration that inhibited 100% of susceptible strains of M. tuberculosis while permitting growth of LVX-resistant strains. Once critical concentration values were determined, the values were tested against a subset of susceptible and resistant strains for which MICs are near the critical values to validate the selected critical concentration.

Lot-to-lot variability testing.Each selected critical testing concentration of LVX for each of the three methods was tested in more than one lot of medium. Comparisons were made between expected and observed susceptible or resistant results.

LVX is stable over the course of testing for each of the methods examined.For the BACTEC 460 and BACTEC MGIT 960 methods, a critical testing concentration of 2 μg of LVX/ml appears to be a valid testing concentration, because that drug level reliably tested pan-susceptible strains as susceptible and clinically resistant strains and strains with known gyrA mutations as resistant. Results also show that a critical testing concentration of 1 μg/ml appears to be a valid testing level for the AP method. Conveniently, this testing level of LVX can easily be obtained in agar by using one commercially available 5-μg LVX disk per 5-ml quadrant of Middlebrook medium, which simplifies the testing process. The findings suggest that results are unaffected by lot-to-lot variability of media by any of the three methods.