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Journal of Clinical Microbiology, June 1998, p. 1568-1573, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Flow Cytometric Testing of Susceptibilities of
Mycobacterium tuberculosis Isolates to Ethambutol,
Isoniazid, and Rifampin in 24 Hours
Scott M.
Kirk,1,2
Ronald F.
Schell,2,3,4,*
Andrea V.
Moore,2,3
Steven
M.
Callister,5,6 and
Gerald H.
Mazurek7
Wisconsin State Laboratory of
Hygiene3 and
Departments of Medical
Microbiology and Immunology2 and
Bacteriology,4 University of
Wisconsin, Madison, Wisconsin 53706;
Microbiology Research
Laboratory5 and
Department of
Infectious Diseases,6 Gundersen Lutheran
Medical Center, LaCrosse, Wisconsin 54601;
Bio-Rad
Laboratories, Hercules, California 945471;
and the
Centers for Disease Control and Prevention, Atlanta,
Georgia 303337
Received 7 November 1997/Returned for modification 22 December
1997/Accepted 24 February 1998
 |
ABSTRACT |
Susceptibility testing of Mycobacterium tuberculosis is
seriously limited by the time required to obtain results. We show that
susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy
by using flow cytometry. The susceptibilities of 35 clinical isolates
of M. tuberculosis to various concentrations of isoniazid,
rifampin, and ethambutol were tested by the agar proportion method and
by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin,
respectively. Only 11 discrepancies were detected among 155 total
tests. The results of flow cytometric susceptibility tests were
available within 24 h of inoculation of drug-containing medium,
while the proportion method required 3 weeks to complete. The flow
cytometric method is also simple to perform.
| |
INTRODUCTION |
In response to the resurgence of
tuberculosis and increases in resistance to antituberculosis drugs
(3, 6-8, 28) the Centers for Disease Control and Prevention
(CDC) have stated that rapid and accurate susceptibility testing of
Mycobacterium tuberculosis is essential and should be
performed for the control of the disease (6). Classically,
susceptibility testing for M. tuberculosis has been
performed by growing the tubercle bacillus on medium in the presence or
absence of antimycobacterial agents for 2 or 3 weeks of incubation
before obtaining results (5, 21, 22). A number of methods
are practiced or have been proposed that greatly decrease the time
required to obtain susceptibility test results. The most frequently
used method, BACTEC-460, requires 4 to 12 days of incubation before
results are available (13, 17, 22, 29, 32). Recently other
methods, including a bioluminescence assay for detection of
mycobacterial ATP (1, 23), the Gen-Probe DNA hybridization
system (15, 18), the luciferase reporter gene assay
(14), high-performance liquid chromatography mycolic acid
analysis (12), the E-test MIC method (35), and a
colorimetric method (36), have been proposed as
high-throughput assays for performing rapid susceptibility testing. The
results are available 3 to 14 days after initiation of testing
procedures.
We showed previously that susceptibility testing of M. tuberculosis (24) and other mycobacteria (4)
could be accomplished within 24 h after the mycobacteria were
incubated with antimycobacterial agents. The method is based on the
ability of mycobacteria to hydrolyze fluorescein diacetate (FDA) to
free fluorescein via nonspecific cellular esterases. Accumulation of
fluorescein in metabolically active mycobacterial cells can then be
easily detected by using a flow cytometer. By contrast, mycobacteria
that are killed or inhibited by antimycobacterial agents hydrolyze
significantly less FDA and therefore have less fluorescence.
Although the feasibility of using flow cytometry and FDA staining for
susceptibility testing of M. tuberculosis was demonstrated (24), the results were obtained with an attenuated strain
(H37Ra) of M. tuberculosis. In this report, we demonstrate
that flow cytometry and FDA staining can be used to detect the
susceptibility or resistance of clinical isolates of M. tuberculosis to ethambutol (EMB), isoniazid (INH), and rifampin
(RIF) 24 h after initiation of the testing procedure.
| |
MATERIALS AND METHODS |
Antimycobacterial agents.
EMB, INH, and RIF were obtained
from Sigma Chemical Co., St. Louis, Mo. Stock solutions of EMB and INH
were prepared at 10,000 µg/ml in distilled water and sterilized by
filtration with a 0.2-µm-pore-size filter before being dispensed in
1.0-ml aliquots and stored at 
70°C until used. RIF (10,000 µg/ml)
was prepared similarly, except that it was dissolved in dimethyl
sulfoxide (Sigma).
Mycobacteria and preparation.
Thirty-five clinical isolates
of M. tuberculosis with varied resistances to
antimycobacterial agents were obtained from the CDC. Each isolate was
grown from frozen stocks in 10.0 ml of 7H9 broth (Difco, Detroit,
Mich.) in a sterile 50.0-ml polypropylene screw-cap tube (Sarstedt,
Newton, N.C.) at 37°C in the presence of 5% CO2 until
the turbidity of the suspension was equivalent to a McFarland 1.0 standard. Approximately 5 to 14 days of incubation were required.
Agar proportion susceptibility testing.
The agar proportion
method, similar to that recommended by the National Committee for
Clinical Laboratory Standards (22), was used to determine
the percentage of M. tuberculosis organisms resistant to
each of the concentrations of antimycobacterial agents tested. Briefly,
appropriate concentrations of EMB, INH, and RIF were added to 7H10
medium tempered at 50 to 52°C to yield 5.0 µg of EMB/ml; 0.2, 1.0, and 5.0 µg of INH/ml; and 1.0 µg of RIF/ml. Subsequently, 5.0 ml of
medium containing each antimycobacterial agent was dispensed into
labeled quadrants of sterile petri plates. One quadrant was reserved
for 7H10 medium without any antituberculosis agent. After
solidification of the agar, the plates were inoculated with 0.1 ml of
10
2 and 104 dilutions of a McFarland 1.0 concentration of a suspension of each isolate of M. tuberculosis. The inoculated plates were then incubated at 37°C
in an atmosphere of 5% CO2 for 3 weeks. An isolate was
considered susceptible to an antimycobacterial agent if the number of
colonies that grew on the drug-containing plate was <1% of the number
of colonies that grew on the drug-free control. An isolate was
considered resistant if 1% or more grew on the drug-containing plate.
Flow cytometric susceptibility testing.
An aliquot (0.9 ml)
of each actively growing M. tuberculosis isolate was
transferred to a 2.0-ml screw-cap microtube (Sarstedt). The tubes were
then inoculated with 0.1 ml of a working dilution of INH at 50.0, 10.0, 2.0, or 0.2 µg/ml. Similarly, tubes containing suspensions of
M. tuberculosis isolates were inoculated with 0.1 ml of EMB
at 50.0 µg/ml or RIF at 10.0 µg/ml. Drug-free suspensions of
M. tuberculosis were also included as controls. The
suspensions were then incubated for 24 h at 37°C in the presence
of 5% CO2. After incubation, 0.2 ml of each assay
suspension was removed and placed in a sterile 2.0-ml screw-cap
microtube containing 0.2 ml of FDA (Sigma) prepared fresh daily at 500 ng/ml in phosphate-buffered saline at pH 7.4. The samples were then
incubated at 37°C for 30 min before being analyzed with a Bryte HS
flow cytometer with WinBryte software (Bio-Rad Laboratories, Hercules,
Calif.).
Initially, unstained viable M. tuberculosis cells were
detected and differentiated from non-M. tuberculosis
particles in 7H9 medium by forward and side angle light scatter.
Background events (particles) in the 7H9 medium and electronic noise
were eliminated by thresholding. Subsequently, viable M. tuberculosis cells incubated in the presence or absence of
antimycobacterial agents for 24 h were stained with FDA. For each
isolate at each concentration of antimycobacterial agent the flow
cytometer provided a histogram profile relating the number of M. tuberculosis organisms in each of 2,048 logarithmic channels of
increasing fluorescence intensity, a mean channel fluorescence value,
and a contour plot relating forward angle light scatter and intensity
of fluorescence. Two thousand events were acquired for each sample. In
addition, 1.5-µm-diameter fluorescent polystyrene beads (Bio-Rad
Laboratories) were used daily for calibration of the instrument.
Flow cytometric susceptibility index.
The susceptibility
index was determined by using the mean channel fluorescence value
obtained from histogram profiles (channels 0 through 2048) of the
population of FDA-stained M. tuberculosis cells in the
presence or absence of antimycobacterial agents. Subsequently, these
values were divided by 512, the number of channels per log decade. The
antilog was then determined for these values to obtain the relative
linear fluorescence value for each sample analyzed. Finally, for each
isolate the relative fluorescence value of each drug-containing sample
was divided by the relative fluorescence value of the drug-free control
to obtain the susceptibility index. An isolate of M. tuberculosis was considered susceptible to an antimycobacterial
agent if the susceptibility index was 0.75 or less. The calculation
eliminates the variability among isolates of M. tuberculosis
in their abilities to hydrolyze FDA in the absence of antimycobacterial
agents.
| |
RESULTS |
Detection of M. tuberculosis by flow cytometry.
Unstained viable M. tuberculosis organisms were readily
detected in 7H9 medium by flow cytometric analysis with forward angle light scatter and intensity of fluorescence displayed in a contour plot
profile of the acquired data (Fig. 1B). A
histogram profile of the unstained M. tuberculosis organisms
(events) showing the low intensity of fluorescence for each event
acquired was also obtained (Fig. 1A). When viable M. tuberculosis organisms were exposed to FDA (Fig. 1C and D), the
intensity of fluorescence increased in the population. The contour plot
profile (Fig. 1D) shows the intensity of fluorescence emitted by the
viable M. tuberculosis population after hydrolysis of FDA
relative to the degree of forward angle light scatter measured. The
histogram profile was used to determine that the mean channel
fluorescence of the population was 1,079 (Fig. 1C). By contrast, viable
M. tuberculosis organisms incubated for 24 h with 5.0 µg of INH/ml (Fig. 1E and F), showed a significant decrease in the
intensity of fluorescence displayed in the histogram after exposure to
FDA. The mean channel fluorescence of the population had decreased to
893 (Fig. 1E). In addition, the contour plot (Fig. 1F) obtained for the
M. tuberculosis organisms exposed to 5.0 µg of INH/ml was
considerably different from the contour plot of the drug-free control
(Fig. 1D). Furthermore, the susceptibility index value of 0.43 (Fig.
1E) demonstrated that the M. tuberculosis isolate was
affected by 5.0 µg of INH/ml.
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FIG. 1.
Intensity of fluorescence versus number of events (A, C,
and E) and forward angle light scatter (B, D, and F) displayed as
histogram or contour plot profiles, respectively, for M. tuberculosis organisms incubated for 24 h with (E and F) or
without (C and D) 5.0 µg of INH/ml and then exposed to FDA. Other
controls included M. tuberculosis organisms not incubated
with INH or exposed to FDA (A and B). The mean channel fluorescence
(MCF) values (the mean of the logarithmic intensity of fluorescence) of
M. tuberculosis organisms with or without incubation with
INH and exposed to FDA were used to calculate the susceptibility
indices (SI). An index value of less than 0.75 suggested that M. tuberculosis organisms hydrolyzed less FDA than the drug-free
control.
|
|
Susceptibility of clinical isolates of M. tuberculosis
to antimycobacterial agents.
Thirty-five clinical isolates of
M. tuberculosis with different susceptibilities to INH were
obtained from the CDC. Subsequently, the abilities of the isolates to
hydrolyze FDA after incubation for 24 h in various concentrations
of INH were determined by flow cytometry. Figure
2 shows the flow cytometric
susceptibility index values obtained for 5 of the 35 isolates with
different susceptibilities to various concentrations of INH. The flow
cytometric susceptibility index values decreased rapidly for four of
the five isolates. Isolates 497, 941, and 810 were susceptible to 0.02, 0.20, and 1.0 µg or more of INH/ml, respectively. Isolate 065 was
susceptible only to 5.0 µg of INH/ml, while isolate 024 was
completely resistant to all concentrations of INH tested. The
concentration of INH obtained by the susceptibility index for each of
the isolates correlated with the inhibitory concentration obtained by
the proportion method. Each of the isolates, except resistant isolate
024, had a susceptibility index value of 0.75 or less.
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FIG. 2.
Susceptibility index values of 5 of the 35 clinical
isolates of M. tuberculosis tested after incubation for
24 h with or without various concentrations of INH. Isolates 497 ( ), 941 ( ), 810 ( ), and 065 ( ) were susceptible to 0.02, 0.20, 1.0, or 5.0 µg or more of INH/ml, respectively. Isolate 024 (*) was resistant to all concentrations of INH tested.
|
|
We next determined the ability of the remaining 30 of the 35 clinical
isolates of
M. tuberculosis to hydrolyze FDA after
incubation
with various concentrations of INH for 24 h. The
susceptibility
results for each of the 35 isolates are listed in Table
1. The
inhibitory concentration of INH
obtained by the proportion method
was also obtained by flow cytometry
for all but four of the isolates.
By flow cytometry, isolate 322 was
resistant to 5.0 µg of INH/ml,
but it was susceptible by the
proportion method. Isolate 531 was
resistant to 0.2 and 1.0 µg of
INH/ml, but it was susceptible
by the proportion method. Similarly,
isolate 843 was resistant
to 1.0 µg of INH/ml, but it was susceptible
by the proportion
method. By contrast, isolate 863 was resistant to 0.2 µg of INH/ml
by the proportion method, but it was susceptible by the
flow cytometric
test. Agreement between the methods was 94, 94, and
97% at 0.2,
1.0, and 5.0 µg of INH/ml, respectively. Overall, the
agreement
was 95%.
In other studies, 26 isolates of
M. tuberculosis were tested
for susceptibility to EMB by the flow cytometric and proportion
methods
(Table
2). Agreement between the methods
was reached
for 24 of the 26 isolates. Isolates 204 and 813 were
resistant
to 5.0 µg of EMB/ml by flow cytometry, but they were
susceptible
by the proportion method. Agreement was 92%. When 24 isolates
of
M. tuberculosis were tested for susceptibility
to RIF by the
flow cytometric and proportion methods, four
discrepancies were
detected (Table
3).
Isolates 322, 509, and 843 were resistant
to 1.0 µg of RIF/ml by the
flow cytometric method but susceptible
by the proportion method. By
contrast, isolate 231 was resistant
by the proportion method but
susceptible by flow cytometry. The
overall agreement was 83%.
Reproducibility.
Table 4 shows
the reproducibility of the flow cytometric susceptibility test. Three
isolates (531, 126, and 072) with varied susceptibilities or
resistances to INH, EMB, and RIF were tested three times. Isolates
susceptible to INH, EMB, or RIF had susceptibility indexes ranging from
0.68 to 0.72, with an average standard deviation of 0.03. Similarly,
isolates resistant to concentrations of INH, EMB, or RIF had
susceptibility indexes ranging from 0.91 to 1.17. Their average
standard deviation was 0.04.
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TABLE 4.
Reproducibility of the flow cytometric susceptibility
test for three isolates of M. tuberculosis with varied
susceptibilities and resistances to INH, EMB, or RIF
|
|
| |
DISCUSSION |
The in vitro susceptibility testing of M. tuberculosis
is seriously limited by the time required to obtain results (5, 13, 15, 17, 22, 29, 32). 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 (9-11, 16, 19, 20, 25-27, 30, 31, 33). 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, 13, 21,
22) or formation of colonies (5, 21, 22) to assess the
effectiveness of an antimycobacterial agent. This may require weeks of
incubation (5, 21, 22). 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, 9, 24, 34). 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.
| |
ACKNOWLEDGMENTS |
We thank Bio-Rad Laboratories in cooperation with the Gundersen
Medical Foundation, Inc., LaCrosse, Wis., for support.
We greatly appreciate the support of Adolf L. Gundersen and Mark
Connelly along with Herbert M. Heili. We also thank Louise Kubista,
David Fett, Michelle Myrdal, and Daniel Muller for excellent advice and
assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Wisconsin State
Laboratory of Hygiene, University of Wisconsin, 465 Henry Mall,
Madison, WI 53706. Phone: (608) 262-3634. Fax: (608) 265-3451.
| |
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Journal of Clinical Microbiology, June 1998, p. 1568-1573, Vol. 36, No. 6
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
