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Journal of Clinical Microbiology, April 2000, p. 1498-1501, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
New Agar Medium for Testing Susceptibility of
Mycobacterium tuberculosis to Pyrazinamide
Leonid
Heifets* and
Tracy
Sanchez
National Jewish Medical and Research Center,
Denver, Colorado
Received 3 September 1999/Returned for modification 27 December
1999/Accepted 25 January 2000
 |
ABSTRACT |
A new agar medium to perform pyrazinamide (PZA) susceptibility
testing with Mycobacterium tuberculosis has been developed. This medium has an acidic pH of 6.0 instead of the usual for agar media, pH 6.8, to provide optimal conditions for PZA activity, and it
also differs from conventional Middlebrook 7H10/7H11 agar in that
animal serum (fetal or calf bovine or fetal equine serum) is used
instead of oleic acid-albumin-dextrose-catalase to support good growth
of M. tuberculosis at the low pH of 6.0. A critical concentration of 900 or 1,200 µg of PZA/ml in this medium made it
possible to differentiate between PZA-susceptible and PZA-resistant clinical isolates. This agar medium has the following advantages compared to a liquid medium: it allows determination of the actual proportion of PZA-resistant bacteria in the isolate and it is simple
and inexpensive. In addition, it has the potential of being used for a
direct susceptibility test with PZA, but this approach will require
further confirmation. Further studies to develop critical
concentrations of other drugs for this low-pH medium, as well as to
investigate the possibility of cultivation in regular (non-CO2) incubators, are in progress.
| |
INTRODUCTION |
Pyrazinamide (PZA) is one of the
first-line drugs in the standard treatment regimen currently used for
tuberculosis patients. It is recommended in the United States
(12), and it is desirable for any country with high
prevalence of drug resistance (3), that a drug
susceptibility test for pretreatment isolates from new tuberculosis
patients be performed, at least with the first-line drugs. A variety of
techniques can be used for testing antituberculosis drugs other than
PZA, including the agar proportion method in its direct and indirect
versions. The only PZA test approved by the Food and Drug
Administration in the United States is the radiometric method using the
special PZA liquid medium at pH 6.0 in the BACTEC-460 system
(10). This method is quite expensive and may not be
affordable for many laboratories, especially in developing countries.
Unlike the test with other drugs by the agar proportion method, the
test in a liquid medium does not provide any information on the actual proportion of the resistant bacteria in the patient's isolate and it
cannot be used as a direct test. The previous suggestion of using the
agar proportion method for a test with PZA (1, 2) did not
lead to this test finding its way into the clinical laboratory
practices, because of very poor growth of Mycobacterium tuberculosis isolates at pH 5.5 (11).
The aims of this study were (i) to develop an acidic agar medium that
would satisfy the requirement for PZA inhibitory activity and provide,
at the same time, good growth of M. tuberculosis and (ii) to
evaluate this medium with PZA-susceptible and PZA-resistant M. tuberculosis laboratory strains and clinical isolates.
| |
MATERIALS AND METHODS |
Antimicrobial agent.
PZA was purchased from Sigma Chemical
Co. (St. Louis, Mo.). The necessary solutions were made in distilled
water. Three solutions were made to have the final concentrations of
300, 900, and 1,200 µg/ml in the agar medium (see below).
Culture medium preparation.
The commercially available
Middlebrook 7H10 agar base (BBL, Becton Dickinson and Co.,
Cockeysville, Md.) was dissolved in deionized water at 14.4 g per
660 ml. Then 4.7 g of monopotassium phosphate
(KH2PO4) was added to acidify the medium. In
addition, 0.72 g of casein hydrolysate and 4.0 ml of glycerol were
added. After being autoclaved at 121°C for 12 min, the medium was
split into four sterile flasks, 160 ml each. The flasks were placed into the water bath to cool down to 54°C. After that, 20 ml of the
sterile animal serum (fetal or calf bovine serum [FBS and CBS] or
fetal equine serum [FES]; Sigma Chemical Co.) mixed with 20 ml of the
PZA solution (or distilled water for the control) was added to a total
volume of 200 ml per flask. The final concentration of the serum was
10%, and the final pH was 6.15 ± 0.1. For the purpose of growth
comparison, similar media were made with oleic acid-albumin-dextrose-catalase (OADC) or albumin-dextrose-catalase (ADC) instead of the animal serum.
The media were poured into the 100- by 15-mm four-segment plastic
dishes, one segment for the drug-free medium and the three remaining
segments for the agar containing three PZA concentrations. After
completion of the quality controls for sterility and ability to support
growth, the plates were stored at 4°C, protected from light, for a
period not longer than 8 weeks.
Drug susceptibility test.
A culture of M. tuberculosis, cultivated in 7H9 broth at 37°C for a period of 4 to 7 days, was adjusted, using the same medium, to the optical density
of McFarland standard no. 1. Two dilutions of this suspension,
10
2 and 104, were used as an inoculum, 0.1 ml per segment, to inoculate two plates. The plates were sealed in
individual polyethylene CO2-permeable bags (XPEDX, Denver,
Colo.) and incubated right side up (agar down) at 37°C in the
presence of 5 to 7% CO2 for a period of 21 days.
Afterwards, the plates were removed from the incubator and placed on
the bench upside down (agar up) at room temperature for at least 4 h (or overnight) to eliminate the condensate. The plates were examined
without opening the polyethylene bags using a dissecting microscope.
The colonies on each segment were counted, and the numbers of colonies
on drug-containing segments were compared with that on the drug-free control.
Strains.
Quality control (QC) strains were M. tuberculosis H37Rv, susceptible to all
antituberculosis drugs (ATCC 27294), and M. tuberculosis ATCC 35828, monoresistant to PZA. Other laboratory strains included three susceptible to all drugs, Erdman, Atencio, and 9719, as well as
two PZA-resistant mutants developed by us from susceptible strains
(H37Rv and 9719) by selection in the presence of 1,200 µg
of PZA/ml on agar plates at pH 6.0. In addition to the laboratory strains, 53 clinical isolates were included in this study. Twenty-four of these strains were isolated from newly diagnosed patients and were
reported by our clinical laboratory as susceptible to PZA. Our clinical
laboratory has identified 29 clinical isolates as resistant to PZA
based on the conventional test in the BACTEC broth at pH 6.0. All 53 isolates, along with two QC strains (susceptible H37Rv and
PZA-resistant ATCC 35828), were retested in this study by the BACTEC
radiometric method using three PZA concentrations, 100, 300, and 900 µg/ml, to determine the MIC, as described previously (4).
| |
RESULTS |
Effect of ADC, OADC, and animal sera on growth at low pH.
It
was previously speculated that oleic acid may inhibit bacterial growth
at low pHs, and therefore it was suggested that ADC be used instead of
OADC for 7H10/7H11 agar media to obviate the growth inhibition at the
acidic pH of 5.5 (1, 2). We have compared the growth rates
of three strains (H37Rv, Erdman, and Atencio) on pH 6.0 agar medium supplemented with 10% of OADC, ADC, FES, or FBS, all
obtained from Sigma. For this purpose, we prepared two sets of agar
plates (to have duplicates) containing each of the supplements, one
having standard pH 6.8 and one with pH 6.0. These plates were
inoculated simultaneously with 0.5 ml of the bacterial suspension
adjusted to the optical density of McFarland standard no. 1 and than
diluted 106 to have approximately 100 to 200 CFU per plate.
The results of experiments with three laboratory strains are shown in
Table
1. These preliminary data showed no
significant
difference in levels of recovery of
M. tuberculosis on the pH
6.8 agar in the presence of different
supplements. At pH 6.0,
the recovery of growth (number of CFU per
plate) on media supplemented
with FBS or FES was equal to, and
sometimes even greater than,
that on the standard OADC-containing
medium and no less than that
on the media with pH 6.8. At the same
time, at pH 6.0 growth on
the media supplemented with ADC was partially
suppressed compared
to growth on the media supplemented with either
OADC or FBS. In
addition, the size and appearance of colonies at the
3-week reading
on the pH 6.0 medium with FBS were no different from
those at
pH 6.8, whereas a reduction in size on the pH 6.0 media
supplemented
with OADC or ADC was observed.
PZA susceptibility test results with PZA-resistant mutants.
The agar proportion method should provide the opportunity for
determining the actual proportion of resistant bacteria in the population. To investigate the applicability of this option to the PZA
susceptibility test, we conducted experiments with artificially prepared mixtures containing various proportions of PZA-resistant bacteria with the original susceptible strains. For this purpose, we
developed PZA-resistant mutants by selection from two pansusceptible strains (H37Rv and 9719) on agar plates containing 1,200 µg of PZA/ml. Mixtures contained 10, 25, or 50% PZA-resistant
bacteria. These mixtures were tested along with the original
susceptible strains and their resistant mutants. All five cultures were
tested by two methods, one using BACTEC PZA broth at 100, 300, and 900 µg/ml and the other using agar plates at 300, 900, and 1,200 µg/ml.
The broth-determined MICs of PZA for two susceptible strains
(H
37Rv and 9719) were
100 µg/ml in the BACTEC pH 6.0 broth medium.
While the growth of one of these strains (9719) was
completely
inhibited by all drug concentrations incorporated in the
agar
medium, a substantial proportion (35.9%) of another strain
(H
37Rv)
was not inhibited by 300 µg/ml in agar (Tables
2 and
3).
Growth
of both PZA-resistant mutants (bottom rows in Tables
2 and
3)
was not inhibited by any of the drug concentrations used for either
medium; the mutants showed full resistance to all concentrations
used
in the BACTEC broth (MIC > 900 µg/ml). Suspensions prepared
with the intention of having 10, 25, or 50% PZA-resistant bacteria
in
the mixtures showed proportions of resistant bacteria grown
on the agar
plates approximating those in the prepared mixtures.
This correlation
is indicative of the ability to indicate the
proportion of the
PZA-resistant bacteria in a specimen, even if
such proportion is as low
as 10%.
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TABLE 2.
Evaluation of two PZA susceptibility testing methods
using mixtures of the original susceptible strain H37Rv
and its PZA-resistant mutant
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TABLE 3.
Evaluation of two PZA susceptibility testing methods
using mixtures of the original susceptible strain 9719 with its
PZA-resistant mutant
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|
PZA susceptibility test with clinical isolates.
Tables
4 and 5
include data obtained with 53 clinical isolates and 2 QC strains, a
total of 55 cultures tested in this series of experiments. The results
of the test in agar medium supplemented with CBS were compared with
those of the BACTEC method using different PZA concentrations. Tables 4
and 5 analyze the results for 900 and 1,200 µg/ml of PZA in agar
medium versus 300 or 900 µg/ml in the BACTEC medium. This analysis
indicated 100% agreement for 25 PZA-susceptible strains (including
H37Rv strain) tested with PZA at either 900 or 1,200 µg/ml incorporated in the agar medium versus 300 µg/ml in the
BACTEC medium (Table 4). From a total of 30 strains (29 clinical
isolates and 1 QC strain [ATCC 35828]) identified as resistant to PZA
at 300 µg/ml by the BACTEC method, resistance to PZA in agar medium
was observed for 29 strains (including the QC strain) with a
concentration of 900 µg/ml (96.7%) and for 27 strains (including the
QC strain) with 1,200 µg/ml (90%).
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TABLE 4.
Comparison of the PZA susceptibility testing of
M. tuberculosis strains on agar plates versus in
the BACTEC broth with PZA at 300 µg/ml
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TABLE 5.
Comparison of the PZA susceptibility testing of
M. tuberculosis strains on agar plates versus in
the BACTEC broth with PZA at 900 µg/ml
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|
When the breakpoint of 900 µg/ml was used in the BACTEC system (Table
5), the agreement in results for susceptible strains
(including
H
37Rv) was 92.9% (26 of 28 strains) for PZA at 900
µg/ml
in agar medium and 96.4% (27 of 28 strains) for PZA at 1,200
µg/ml
in
agar.
In addition, we have compared two cultivation conditions for the PZA
test in the low-pH agar medium with 1,200 µg of the drug/ml:
in the
CO
2 incubator (in an atmosphere containing about 7%
CO
2)
and in a regular incubator without CO
2. No
difference between
these two conditions has been found, either in
regard to the ability
of the low-pH agar medium to support growth of
the tested strains
or in the results of the PZA test. These preliminary
data indicated
the need for further investigation of the possibility of
using
this low-pH agar medium for cultivation and drug susceptibility
testing of
M. tuberculosis cultures in regular incubators
without
CO
2.
| |
DISCUSSION |
The most reliable among all currently available methods
for a test of the susceptibility of M. tuberculosis to PZA
is the radiometric BACTEC technique (10), especially when
three PZA concentrations are used to determine the MIC (4).
There were reports that this method may give false-positive and
false-negative results when a single concentration, 100 µg/ml, is
used (4-6). Nevertheless, the reliability of this method
(with 300 µg/ml) has been confirmed by detection, using DNA
sequencing, of mutations specific for PZA resistance in the
pyrazinamidase gene, pncA, in strains identified by the
BACTEC technique as PZA resistant but not in susceptible strains
(8, 9). Therefore, the BACTEC PZA test has been used in this
study as a "gold standard" for evaluation of the PZA susceptibility
test in the new agar medium. The BACTEC method, though reliable, has
certain disadvantages. One of them is that it can be used only as an
indirect method, which requires initial isolation of a pure culture;
and it cannot be used as a direct test with acid-fast bacillus-positive
specimens. The total turnaround time of this method is about 4 weeks.
Among other problems associated with the BACTEC technology are high cost, the need for disposal of a substantial volume of
14C-radiolabeled culture vials (which is not permitted in
many countries), and the fact that most of the tuberculosis
laboratories in the world do not use, and cannot afford to use, the
BACTEC system. In addition, the BACTEC method does not provide
information on the actual proportion of the PZA-resistant bacteria in
the patient's isolate. While a number of alternative methods for
susceptibility testing with other drugs are widely available, the
BACTEC method is considered by many investigators as the only reliable
technique for a test with PZA.
Previously, there were attempts to develop an agar-based PZA
susceptibility test, and one of them involved using an ADC supplement instead of OADC in a pH 5.5 agar medium (1, 2). This method did not find its way into clinical laboratories because of insufficient growth of M. tuberculosis isolates on this medium.
We have detected some suppression of growth of M. tuberculosis on the acidic agar medium (pH 6.0) supplemented with
either ADC or OADC. We found, on the other hand, that the acidic pH did not have such a negative effect when the medium was supplemented with
an animal serum (FBS, CBS, or FES). Moreover, the growth of M. tuberculosis on the agar medium supplemented with the animal serum
at pH 6.0 was even better than that on the conventional 7H11 agar, at
pH 6.0 or 6.8, supplemented with OADC and especially with ADC.
Previously we have shown a correlation between the pH of the medium and
the PZA concentration necessary to inhibit growth of M. tuberculosis. For example, 50 µg/ml was required to inhibit growth at pH 5.5 in a liquid medium, but the same effect could have
been achieved with 300 to 400 µg/ml at pH 6.0 (7).
In the present study we found that the growth of 25 PZA-susceptible
M. tuberculosis strains was inhibited in a pH 6.0 agar medium (supplemented with an animal serum) with a concentration of 900 or 1,200 µg of PZA/ml but that good growth occurred in the drug-free
controls. At the same time, 29 out of 30 PZA-resistant strains produced
sufficient growth in the presence of PZA at 900 µg/ml in this medium.
The predictive value of the agar method by comparison with the
conventional BACTEC method (100 and 300 µg/ml) was 100% for
susceptible strains and 96.7% for resistant strains. We have also
demonstrated that a test by the proportion method using the pH 6.0 agar
medium can provide clear information on the proportion of PZA-resistant
bacteria in the inoculum.
Based on the results presented in this report, we are proposing a new
agar medium, different from the Middlebrook 7H11 agar in pH (6.0 instead of 6.8) and in the growth supplement (animal serum instead of
OADC). This medium can be used for susceptibility testing of the
M. tuberculosis isolates with PZA by an agar proportion method. The best results were obtained when CBS (10%) and 900 µg of
PZA/ml (as a critical concentration) were incorporated into this
medium. Further studies with a larger number of strains are needed to
make a final choice between 900 and 1,200 µg/ml of PZA as the
critical concentration. One of the advantages of the agar proportion
test with PZA on this medium over the conventional PZA test in the
BACTEC system is that it is less expensive. Materials to prepare a
biplate containing PZA and drug-free agar cost about 56 cents. The
total direct cost, including labor, is about $1.00. The agar medium can
be prepared in-house, while the supplies alone for the BACTEC PZA test
cost about $12, according to the catalog, and have to be purchased from
the manufacturer.
A susceptibility test with all first-line antituberculosis drugs in the
United States is now recommended for all new patients, but this
requirement cannot be implemented for PZA, one of the first-line drugs,
in laboratories that are not equipped with the BACTEC-460 system.
Introduction of the new medium described in this report will allow
performance of the PZA susceptibility test in any laboratory that is
capable of performing the agar proportion susceptibility test with
other drugs.
One of the important advantages of the agar proportion test with PZA is
that it has a potential of being used not only as an indirect test with
previously isolated cultures but also as a direct test with raw
specimens. Such an approach may shorten the total turnaround time to
only 3 weeks, a time that is well known from experience in applying the
direct test with other drugs to acid-fast bacillus-positive sputum
specimens (4). The validity of a direct PZA test by the agar
proportion method requires additional studies. Whether the direct or
indirect PZA susceptibility test is done by the agar proportion method,
only this technology can provide information on the actual proportion
of resistant bacteria in the patient's isolate. Other promising
directions for studies with the low-pH (pH 6.0) agar medium,
supplemented with animal sera, include the possibility of cultivation
in a regular (without CO2) incubator and evaluation of
these conditions for testing the susceptibility to drugs other than PZA.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 1400 Jackson
St., Denver, CO 80206. Phone: (303) 398-1384. Fax: (303)
398-1953. E-mail: heifetsl{at}njc.org.
| |
REFERENCES |
| 1.
|
Butler, W. R., and J. O. Kilburn.
1982.
Improved method for testing susceptibility of Mycobacterium tuberculosis to pyrazinamide.
J. Clin. Microbiol.
16:1106-1109[Abstract/Free Full Text].
|
| 2.
|
Butler, W. R., and J. O. Kilburn.
1983.
Susceptibility of Mycobacterium tuberculosis to pyrazinamide and its relationship to pyrazinamidase activity.
Antimicrob. Agents Chemother.
24:600-601[Abstract/Free Full Text].
|
| 3.
|
Heifets, L., and J. Cangelosi.
1999.
Drug susceptibility testing of Mycobacterium tuberculosis: a neglected problem at the turn of the century.
Int. J. Tuberc. Lung Dis.
3:564-581[Medline].
|
| 4.
|
Heifets, L. B.
1991.
Drug susceptibility tests in the management of chemotherapy of tuberculosis, p. 89-122.
In
L. B. Heifets (ed.), Drug susceptibility in the chemotherapy of mycobacterial infections. CRC Press, Boca Raton, Fla.
|
| 5.
|
Hewlett, D.,
D. Horn, and C. Alfalla.
1995.
Drug-resistant tuberculosis: inconsistent results of pyrazinamide susceptibility testing.
JAMA
273:916-917[Abstract/Free Full Text]. (Letter.)
|
| 6.
|
Miller, M. A.,
L. Thibert,
F. Desjardins,
S. H. Siddiqi, and A. Dascal.
1996.
Growth inhibition of Mycobacterium tuberculosis by polyoxyethylene stearate present in the BACTEC pyrazinamide susceptibility test.
J. Clin. Microbiol.
34:84-86[Abstract].
|
| 7.
|
Salfinger, M., and L. Heifets.
1988.
Determination of pyrazinamide MICs for Mycobacterium tuberculosis at different pHs by the radiometric method.
Antimicrob. Agents Chemother.
32:1002-1004[Abstract/Free Full Text].
|
| 8.
|
Scorpio, A.,
P. Lindholm-Levy,
L. Heifets,
R. Gilman,
S. Siddiqi,
M. Cynamon, and Y. Zhang.
1997.
Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.
Antimicrob. Agents Chemother.
41:540-543[Abstract].
|
| 9.
|
Scorpio, A., and A. Zhang.
1996.
Mutations of pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus.
Nat. Med.
2:662-667[CrossRef][Medline].
|
| 10.
|
Siddiqi, S.
1992.
Radiometric (BACTEC) tests for slowly growing mycobacteria, p. 1-25.
In
H. Isenberg (ed.), Clinical microbiology procedures handbook, vol. 5.14. American Society for Microbiology, Washington, D.C.
|
| 11.
|
Stottmeier, K. D.,
R. E. Beam, and G. P. Kubica.
1967.
Determination of drug susceptibility of mycobacteria to pyrazinamide in 7H10 agar.
Am. Rev. Respir. Dis.
96:1072-1075[Medline].
|
| 12.
|
Tenover, F. C.,
J. T. Crawford,
R. E. Heubner,
L. J. Geiter,
C. R. Horsburgh, and R. C. Good.
1993.
The resurgence of tuberculosis: is your laboratory ready?
J. Clin. Microbiol.
31:767-770[Free Full Text].
|
Journal of Clinical Microbiology, April 2000, p. 1498-1501, Vol. 38, No. 4
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
