The in vitro susceptibility testing of M. tuberculosis
is seriously limited by the time required to obtain results (5
). We demonstrated previously that susceptibility testing of M. tuberculosis
, specifically an attenuated laboratory isolate (H37Ra), could be accomplished rapidly by using a flow cytometer (24
). Results of tests were available within 24 h after M. tuberculosis
organisms were incubated with antimycobacterial agents. Furthermore, multiplication of mycobacteria was not required to obtain susceptibility results. The flow cytometric susceptibility test method is based on the ability of M. tuberculosis
organisms exposed to FDA to rapidly hydrolyze the compound to fluorescein by intrinsic esterases. In nonviable mycobacteria or mycobacteria susceptible to antimycobacterial agents, hydrolysis of FDA is reduced due to the decreased metabolic activity of the organisms. The use of flow cytometry allows rapid measurement (1 min or less per sample) of differences in the amounts of accumulated fluorescein among susceptible organisms and those resistant to, or untreated with, antimycobacterial agents. Consequently, determination of the susceptibility or resistance of mycobacteria can be accomplished rapidly. In this investigation, the feasibility of using flow cytometry to obtain susceptibility results for clinical isolates of M. tuberculosis
24 h after initiation of testing procedures was demonstrated.
We tested 35 clinical isolates of M. tuberculosis obtained from the CDC for susceptibility or resistance to INH by the flow cytometric and proportion methods. Overall, there was agreement between the two methods for 100 of the 105 total tests (95%). For two of the isolates, 531 and 843, discrepancies in INH inhibitory concentrations obtained by flow cytometry were corrected at the next higher concentration of INH. Isolate 863 was resistant to 0.2 μg of INH/ml by the proportion method but susceptible to this concentration of INH by flow cytometry. When higher concentrations (1.0 and 5.0 μg/ml) were tested, the same susceptibility results were obtained by the proportion method and by flow cytometry. Isolate 322 was resistant to 5.0 μg of INH/ml by flow cytometry, but it was susceptible by the proportion method. This isolate, however, was resistant to the lower concentrations of INH tested by the proportion method. Discrepancies were also noted for the susceptibilities of two and three isolates to inhibitory concentration of EMB and RIF, respectively. We classified these isolates as resistant by flow cytometry because their susceptibility indices did not match our chosen susceptibility cutoff value of 0.75. Another isolate, 231, was resistant to 1.0 μg of RIF/ml by the proportion method but susceptible to this concentration by flow cytometry.
A possible explanation for the discrepancies is that the majority of the population of M. tuberculosis cells was not in the exponential growth phase when tested by flow cytometry. Hydrolysis of FDA is affected by the metabolic activity of the mycobacterial cells. Similar levels of hydrolysis would occur in the drug-treated suspensions of M. tuberculosis cells and the drug-free controls if neither were metabolically active. Discrepancies have been detected if non-log-phase mycobacteria were used for testing by flow cytometry. Another explanation is the selection of a susceptibility index cutoff value. We conservatively set the cutoff value at 0.75. The value could have been 0.90, 0.85, 0.80, 0.76, or any numerical value within these numbers. By increasing the cutoff value to 0.90, only seven discrepancies among all of the tests performed would have been reported. Further experiments with the flow cytometric susceptibility test may reveal that the cutoff value for detection of susceptibility should be higher. Finally, it is assumed that the proportion method is correct. However, selection of a subpopulation of resistant or susceptible organisms within the population of M. tuberculosis organisms being tested has yielded conflicting results for the proportion method.
The use of flow cytometry for antimicrobial susceptibility testing is increasing (9
). A major advantage, besides rapidity and objectivity, is the ability to analyze bacterial cells individually or in small groups or clusters (9
). Classically, susceptibility testing of M. tuberculosis
depends on detection of growth (5
) or formation of colonies (5
) to assess the effectiveness of an antimycobacterial agent. This may require weeks of incubation (5
). By contrast, individual mycobacteria are examined by flow cytometry within hours of testing. Changes in individual mycobacterial cells can be assessed by forward or side angle light scatter or through the utilization of fluorescent dyes. The changes usually occur within 24 h after mycobacteria have been exposed to antimycobacterial agents (4
). In support of this observation, Bardou et al. (2
) and Takayama et al. (34
) showed by electron microscopy that there were dramatic changes in the cellular morphology of the tubercle bacillus after exposure to INH for 24 h or less. In this study, contour plots of forward angle light scatter obtained 24 h after M. tuberculosis
cells were incubated in the presence or absence of INH also showed dramatic differences. This is consistent with the alterations in cellular morphology reported by Barbou et al. (2
) and Takayama et al. (34
). Furthermore, the ability to analyze individual mycobacteria accounts for the detection of lower concentrations of antimycobacterial agents that affect the population of mycobacteria than those detected by the standard methods (22
). Although not shown here, several isolates of M. tuberculosis
were determined to be susceptible to 0.2 μg of INH/ml by the proportion method, but they were shown to be susceptible to 0.02 μg/ml by the flow cytometric susceptibility test. Norden et al. (24
) and Bownds et al. (4
) also reported similar findings.
There are several concerns regarding the use of flow cytometry for susceptibility testing of M. tuberculosis. Biosafety is frequently considered the most important. Viable mycobacteria with or without incubation with antimycobacterial agents are presently processed by the instrument. Although the test is rapid, accurate, and reproducible, many clinical laboratories do not have the facilities to safely perform the procedure. However, the present format of the test could be utilized safely by public health laboratories or large reference laboratories that have a biosafety level-three tuberculosis laboratory. Safety is a primary concern, and it is being improved by developing procedures that kill the mycobacteria prior to testing without compromising their staining characteristics. Another concern is the cost of the flow cytometer. However, when the high costs of supplies for performing susceptibility testing with the radiometric instrument and increasing difficulties in disposing of radioactive materials are considered, the flow cytometer is less expensive. The reagents used for flow cytometry are also relatively inexpensive. Costs are restricted to the purchase of 7H9 broth, microtubes, FDA, and the antimycobacterial agents. Technician times for performing the radiometric and flow cytometric methods, however, are similar.
In conclusion, flow cytometry and FDA staining can be used to perform susceptibility testing of clinical isolates of M. tuberculosis. The assay is extremely simple to perform and, most importantly, can be completed in 24 h after initiation of testing.