Rapid, Standardized Method for Determination of Mycobacterium tuberculosis Drug Susceptibility by Use of Mycolic Acid Analysis

Organisms and antibiotics.All M. tuberculosis strains (n = 59 clinical strains and strain H37Rv) were maintained on Lowenstein-Jensen agar slants (Difco, Detroit, MI) at 37°C. All drugs (INH, RIF, ethambutol [EMB], pyrazinamide [PZA], and streptomycin [STR]) were purchased from Becton Dickinson (Sparks, MD). All assays were performed in duplicate.

Standard susceptibility testing.Susceptibility testing and determination of MICs were done by using the Bactec 460 radiometric growth system (Becton Dickinson) and/or the agar proportion method by the use of standard protocols (15, 19). Table 1 shows the profiles of susceptibility to INH, RIF, EMB, PZA, and STR of each of the strains used in this study. Briefly, for the Bactec 460 system, a standardized inoculum (a 1.0 McFarland standard) was prepared for each isolate and 0.1 ml was injected into each of the following vials: a control vial with no antibiotic representing a 1:100 dilution of the inoculum; a control vial with no antibiotic representing the inoculum used in the drug-containing vials; and vials each containing INH (0.1 μg/ml and 0.4 μg/ml), RIF (2.0 μg/ml), EMB (2.5 μg/ml), and STR (2.0 μg/ml). The growth index (GI) was monitored at 24-h intervals until the reading in the control diluted 1:100 reached a value of 30. The GI change was then calculated for a 1-day period for each drug tested. A GI change of <30 indicated susceptibility to a given agent. Resistance (GI > 30) to any of the anti-M. tuberculosis drugs (INH, RIF, EMB, or STR) was confirmed by repeat testing with the Bactec radiometric system and/or by the Middlebrook agar proportion method (15). The anti-M. tuberculosis drugs and concentrations tested by the agar proportion method were as follows: INH, 1.0 μg/ml; RIF, 1.0 μg/ml; EMB, 5.0 μg/ml; and STR, 2.0 μg/ml.

PZA susceptibility testing.PZA is active only at lower pH values compared with the pHs at which the other first-line agents are active. Thus, PZA susceptibility testing was done by using a commercially available medium with a lower pH (6.0) and a variation of the standard Bactec 460 procedure (19). Briefly, 0.1 ml from a “seed vial” (GIs = 300 to 499) was used to inoculate two new pH-adjusted vials: an undiluted control vial and a vial containing PZA (100 μg/ml). The vials were incubated until the GI in the control vial reached 200. Susceptibility and resistance were defined as follows: if the GI in the PZA-containing vial was <9% of the GI in the control vial, the strain was classified as susceptible. If the GI in the PZA-containing vial was >11% of the GI in the control vial, the strain was classified as resistant. Strains exhibiting GIs between 9% and 11% of the control GI were classified as borderline resistant.

HPLC susceptibility testing. (i) Time course determination and sample processing.The same standardized inoculum and procedure outlined above for the Bactec 460 system were used. An undiluted control vial was included for each strain tested and was used for comparison of the TMAPs with those of the antibiotic-containing vials. The vials were monitored at 24-h intervals for up to 72 h, and the GI obtained with the Bactec system was recorded. Aliquots (2 ml) were removed from the individual vials at 20 min and 24, 48, and 72 h following the introduction of each antibiotic and were mixed 1:1 with 50% potassium hydroxide, followed by autoclaving for 30 min. Once the mixtures were cooled to room temperature, 3.6 ml of 50% HCl was added and the contents of the tubes were mixed by inverting the tubes two to three times. Subsequently, 3 ml of CHCl₃ was added to each tube, the tubes were vortexed for 1 min, and the phases were allowed to separate. Equal volumes of each CHCl₃ layer from each strain tested were transferred to a clean glass tube and dried under N₂. Fluorescent derivatives of the mycolic acids were prepared as described previously (22). Briefly, the samples were dissolved in 200 μl of CHCl₃ containing 200 μg of 4-bromomethyl-6,7-dimethoxycoumarin and 200 μg of 18-crown-6 ether (both from Sigma) and transferred to a 2-ml amber vial containing 100 μl of 2% potassium bicarbonate solution which had previously been evaporated. The vials were heated at 60°C for 10 min and allowed to evaporate. Dried extracts were resuspended in 150 μl of HPLC-grade isopropanol (Fisher Scientific) and transferred to a vial containing two fluorescently derivatized mycolic acid reference standards (∼40 and ∼97 carbons) whose retention times do not overlap with those of the mycolic acids present in M. tuberculosis.

(ii) HPLC conditions.HPLC analysis was performed on an Agilent (Wilmington, DE) 1100 HPLC system consisting of an autosampler, quaternary pump, column heater (70°C), and fluorescence detector. The instrument was controlled by the use of Sherlock (MIDI, Inc., Newark, DE) software coupled with Agilent ChemStation software. The sample injection volume was 5 μl. Separation of the mycolic acids was achieved with an Agilent Zorbax SB-C₁₈ column (4.6 mm by 75 mm), a mobile phase of methanol-isopropanol (IPA), and a linear gradient of 23% IPA to 95% IPA over a 10-min period with a flow rate of 1.7 ml/min. Mycolates were detected with a fluorescence detector by using excitation and emission wavelengths of 345 nm and 425 nm, respectively. To maintain maximum sensitivity, the column effluent was cooled to 22°C before it was placed into the detector.

(iii) Chromatographic data analysis.Raw chromatographic data, expressed as the peak height response, were acquired with ChemStation software and processed by using Sherlock software for calculation of the TMAP. The TMAPs were determined for each isolate-drug combination and were compared to that for the corresponding control. The resulting ratio was used for all further data analysis.

Growth rate determination.For growth rate comparisons, a suspension of each strain to be tested was prepared from solid medium by using commercially prepared diluting fluid (Becton Dickinson) and glass beads. The suspensions were vortexed and allowed to settle for a minimum of 30 min, and a 0.1-ml aliquot of the supernatant was removed and used to inoculate a Bactec 12B “seed vial.” The seed vials were read at 24-h intervals until a GI of 999 was reached. Subsequently, 0.1 ml was removed from each seed vial and used to inoculate a new Bactec 12B vial. The GI was then recorded at 24-h intervals for up to 9 days, and the average daily change in the GI was calculated for each strain.

Time course determination.A time course analysis was done to determine the optimal time point at which quantitative differences in the total mycolic acids could be correlated with drug susceptibility or resistance to a given agent. This was done for first-line antimycobacterial drugs that act in both a mycolic acid-specific and a non-mycolic acid-specific manner. The chromatograms were analyzed for the total mycolic acid profile for each strain-antibiotic combination and compared to the profile for the corresponding untreated control. Figure 1 shows a characteristic chromatogram for a drug-susceptible strain of M. tuberculosis, strain H37Rv, with and without INH, and the specific region which comprises TMAP. As shown, significant differences in TMAPs were found at 72 h of exposure to the antibiotic. Differences were noted between antibiotics. For instance, the mycolic acid-inhibiting drug, INH, caused a nearly linear decrease in the percent change in TMAP relative to that for the corresponding control over a 72-h incubation period, ranging from a 34% reduction at 20 min to an 80% reduction at 72 h for strain H37Rv (Fig. 2). However, for a non-mycolic acid-specific inhibitor such as RIF for a susceptible strain, little change in the TMAP was observed at the earliest time points tested: 5.2% at 20 min and 18% at 24 h. At the later time points of 48 and 72 h, the decreases in the TMAPs were 25% and 63%, respectively (Fig. 2). Other first-line drugs, such as EMB and PZA, showed similar patterns for drug-susceptible strains, with the greatest reductions in the TMAPs observed at 72 h of incubation (76% and 67%, respectively). This time point was selected for evaluation because it was the time at which the largest reproducible difference in TMAPs was observed for all drugs, regardless of their mechanisms of action.

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FIG. 1.

Characteristic chromatogram and TMAPs of drug-susceptible M. tuberculosis strain H37Rv following 72 h of incubation with and without INH. (Top panel) Control, no INH; (bottom panel) INH (0.1 μg/ml). FU, fluorescence units.

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FIG. 2.

Percent change in TMAP following exposure to INH (0.1 μg/ml) versus RIF (2.0 μg/ml) over time in drug-susceptible M. tuberculosis strain H37Rv.

TMAP susceptibility testing and breakpoint determination.TMAP susceptibility assays were conducted at 72 h for all M. tuberculosis strain-antibiotic combinations (n = 60), the TMAP was calculated, and the ratio of the values for the treated strains to those for the untreated controls were determined. At 72 h, drug-susceptible strains demonstrated an average reduction in TMAP of >78% (range, 52% to 90%) compared with the values for the corresponding controls. These differences varied by antibiotic, with the greatest inhibition in susceptible strains being observed with INH (average, −89%; range, −76% to −100%) and RIF (average, −88%; range, −43% to −100%) compared with the values for EMB (average, −78%; range, −52% to −89%) and PZA (average, 59%; range, −39% to −94%). In contrast, drug-resistant strains demonstrated a change in TMAP which was either a reduction of <25% or a slight increase over the values for the controls (range, +28% to −24%).

Several breakpoints ranging from 20% to 30% were examined to determine which one provided the best agreement between the standard Bactec 460 susceptibility test method and HPLC. For each strain-antibiotic combination, the percent change in TMAP for each treated culture compared with that for the control culture was determined at 72 h and compared with the known susceptibility of that particular strain. Susceptibility was defined as a reduction in TMAP relative to that for the corresponding control of greater than or equal to breakpoints set at 20% or 30%. Strains exhibiting TMAP changes of less than or equal to the breakpoint (20% or 30) were defined as resistant. By using a 20% breakpoint, the concordances were 71%, 88%, 94%, and 82% for INH, RIF, EMB, and PZA, respectively. In contrast, the highest percent agreement for all drugs was obtained by using the 30% breakpoint, which resulted in 99.5% agreement for INH, EMB, and PZA and 98.7% agreement for RIF compared to the values obtained by the Bactec 460 susceptibility testing method. Figure 3 illustrates the mean percent change in TMAP for all drug-susceptible and -resistant strains to each of the first-line agents obtained by use of the 30% breakpoint. With a total of 240 susceptibility tests, the overall concordance was 97.5%. Discordant results indicating false susceptibility were observed for the following antibiotics: INH and EMB (one result each) and RIF (two results). False resistance was demonstrated for RIF and PZA (one result each). Intra- and interassay variabilities were assessed by testing the same strain (strain H37Rv) six times within the same day or individual replicates over 6 consecutive days. For each culture the coefficient of variation was calculated on the basis of the ratio of the TMAP for the control cultures to the TMAP for the treated cultures. The overall precision was 13.4%. However, the precision did vary by drug (INH, 9.17%; RIF, 11.2%; EMB, 11.4%; PZA, 21.9%).

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FIG. 3.

Mean percent change in TMAPs for all susceptible and resistant M. tuberculosis strains to each first-line antibiotic by using a breakpoint of 30%. TMAP was determined for the treated versus the control cultures, and the percent change was calculated. INH, 0.1 μg/ml; RIF, 2.0 μg/ml; EMB, 2.5 μg/ml; PZA, 100 μg/ml.

STR, a second-line antimycobacterial drug, was also evaluated by the TMAP-based method. The overall agreement was 93.3% between the standard Bactec 460 method and TMAP analysis, which was less than that observed for the first-line drugs at the corresponding time point of 72 h. Susceptibility to STR was correctly identified in all susceptible strains (n = 38) by the TMAP method. However, STR resistance was not detected in 15 of 22 resistant strains. For each strain with discrepant results, a reduction in TMAP of >30% (average, ∼62.4%; range, −42% to −83%) was observed in each of two separate experiments. The reduction in the TMAP was similar in STR-susceptible strain H37Rv (average, ∼88%). Resistance to STR (2.0 μg/ml) was confirmed by both the Bactec 460 radiometric method and the agar proportion method. However, differences in growth rates were observed between STR-susceptible strain H37Rv and the resistant strains producing discrepant results. Overall, these strains exhibited slower growth than drug-susceptible strain H37Rv, slowing an average of 54 GI units over 9 days (range, −38 to −386 GI units).