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Antimicrobial Agents and Chemotherapy, September 1999, p. 2317-2319, Vol. 43, No. 9
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Relationship between Pyrazinamide Resistance, Loss
of Pyrazinamidase Activity, and Mutations in the pncA Locus
in Multidrug-Resistant Clinical Isolates of Mycobacterium
tuberculosis
M.
Mestdagh,1
P. A.
Fonteyne,2
L.
Realini,2
R.
Rossau,3
G.
Jannes,3
W.
Mijs,3
K. A. L.
De
Smet,3
F.
Portaels,2 and
E.
Van
den Eeckhout1,*
Laboratory for Pharmaceutical Biotechnology,
FFW, University of Ghent, 9000 Ghent,1
Unit of Mycobacteriology, Institute of Tropical Medicine, 2000 Antwerp,2 and Innogenetics n.v., 9052 Ghent,3 Belgium
Received 25 March 1999/Returned for modification 16 June
1999/Accepted 13 July 1999
 |
ABSTRACT |
Sixty-two Mycobacterium tuberculosis isolates were
tested for pyrazinamidase activity, and their pyrazinamide
susceptibility was determined by the radiometric method. Sequencing of
pncA genes in the 23 resistant strains revealed mutations
in 16 pyrazinamidase-negative strains, 11 of which had not been
previously described. Six isolates containing wild-type
pncA might possess alternative resistance mechanisms.
| |
TEXT |
Pyrazinamide (PZA), one of the
first-line drugs for tuberculosis treatment, is assumed to be a
prodrug, converted by the bacterial enzyme pyrazinamidase (PZase) to
the toxic pyrazinoic acid (POA), the target of which remains unknown
(7). Since most PZA-resistant Mycobacterium
tuberculosis strains have lost PZase activity, enzyme activity
assays are sometimes used to replace PZA susceptibility testing, which
is problematic because of growth problems at the acid pH (±5.5)
required for in vitro activity of PZA (2, 4). Single
mutations in the pncA gene encoding this PZase are
considered the major PZA resistance mechanism in M. tuberculosis (5, 11, 12, 15). Our objective was to gain
further insight into PZA resistance mechanisms and their
molecular-biological background by comparing different methods for
detection of PZA resistance in M. tuberculosis.
Fifty-four multidrug-resistant M. tuberculosis strains were
isolated from clinical specimens collected in Bangladesh, Azerbaijan, Siberia, Belgium, Abkhazia, Rwanda, Congo-Brazzaville, Kazakhstan, and
Canada, and eight isolates were obtained from the Scottish Mycobacteria
Reference Laboratory (SMRL). M. tuberculosis H37Ra (NCTC
7417) was included as a PZA-susceptible control strain. One
Mycobacterium bovis isolate from Belgium and one from Egypt from the collection of the Institute of Tropical Medicine were used as
PZA-resistant control organisms. M. tuberculosis strains were grown to confluence on Löwenstein-Jensen medium, and
M. bovis strains were grown on Stonebrink medium.
Isolates were tested for PZA susceptibility by the BACTEC radiometric
method (3, 10, 13) at 100 µg of PZA per ml according to
the manufacturer's instructions (14).
The PZase activity of all 65 isolates was assayed, following the
modified version of the Wayne (17) test, as described by Jakschik (6). For each isolate, one test tube and one
control tube were prepared. The test medium contained 6.5 g of
Dubos broth base (Difco Laboratories, Detroit, Mich.), 1.0 g of
PZA (Janssen Chimica, Geel, Belgium), 2.0 g of sodium pyruvate
(Janssen Chimica), and 15.0 g of agar (Difco Laboratories) in 1 liter of water. In the control medium, PZA was omitted. Freshly
prepared autoclaved media were divided into 5-ml aliquots in glass
tubes and were allowed to cool in the upright position. Next, 0.2 to
0.3 ml of sterilized water was aseptically added to both test and
control tubes. Tubes were inoculated with a loopful of a freshly grown culture of each isolate by stabbing the agar two to three times onto
the bottom. After incubation at 37°C for 4 days, 1.0 ml of a freshly
prepared 1% ferro(II) ammonia sulfate (Acros Chimica, Geel, Belgium)
solution was added to each tube, and the tubes were kept at 4°C for
4 h. A PZase activity assay was considered positive when a dark
red to brownish ring appeared on the agar-water interface.
Genomic DNA was extracted as described previously (16).
pncA genes were amplified by PCR (9) with primers
P1 and P6 (12) to obtain DNA fragments of about 720 bp. The
cycling parameters were 94°C for 1 min, 56°C for 1 min, and 72°C
for 1 min; 40 cycles were performed. These PCR products were used to
perform cycle sequencing by using fluorescence-labelled
dideoxynucleotide terminators with the PRISM Ready Reaction Dye Deoxy
Terminator Cycle Sequencing kit (Applied Biosystems, Foster City,
Calif.) and primers P1, P3, P4, and P6 (12) according to the
manufacturer's instructions. Sequence data were generated with an
automated DNA sequencer, model 373A (Applied Biosystems).
Thirty-nine isolates were susceptible to PZA; they all displayed PZase
activity. The pncA sequence of these isolates was not determined, because earlier investigations (11, 12, 15) of
sequences in 75 PZA-susceptible M. tuberculosis strains did not reveal any nucleotide change. Twenty-three isolates were resistant to PZA. For these strains, sequencing of the pncA open
reading frame and an 85-bp upstream, putative regulatory region was
performed. The results obtained are summarized in Table
1. In 16 PZA-resistant isolates, the lack
of PZase activity was correlated with mutations leading to amino acid
substitutions (10 of 16), large deletions (1 of 16), frameshifts (4 of
16) by single nucleotide insertion or deletion, or promoter mutations
(1 of 16). In four PZA-resistant isolates lacking PZase activity,
wild-type pncA sequence was present. Three PZA-resistant
isolates displayed PZase activity: the wild-type pncA
sequence was present in two of them, while the other contained a
nucleotide change leading to an amino acid substitution (isolate 98-32 [Ala to Val at codon 171]). For strains with discrepant results
between susceptibility testing and the PZase assay (PZA resistant, but
PZase positive) or between the PZase assay and pncA
sequencing (PZase negative, but pncA wild type), the results obtained were confirmed in a second test round.
View this table:
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TABLE 1.
PZase activity, changes in pncA nucleotide
sequence, and corresponding amino acid changes of the 23 PZA-resistant
M. tuberculosis isolates in this study
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|
Detection of PZase activity, as described by Wayne (17) and
modified by Jakschik (6), is an easy and very rapid
technique suited for any mycobacterial laboratory. Considering BACTEC
PZA susceptibility determination as a reference, PZase negativity was
correlated with PZA resistance in all 20 strains in this study. However, for only 39 of the 42 PZase-positive strains, a
PZA-susceptible pattern was obtained on BACTEC. This confirms the
findings of Butler and Kilburn (1) and Miller et al.
(8) that PZA-resistant isolates are not always PZase
negative. Our conclusion is that PZase test results should only be
interpreted in one direction
a negative result indicating PZA
resistancewhereas care should be taken in interpreting positive test results.
Mutations were present in 17 (74%) of the 23 PZA-resistant isolates as
defined by the BACTEC radiometric method. The six strains lacking
pncA mutations are divided into two groups by the PZase test: one group of two strains was PZase positive, and a second group
of four strains was PZase negative. Resistance to PZA in the first
group may be due to mutations in the POA drug target, which has not yet
been found. The second group could have undergone downregulation of the
pncA gene, directed on the translation phase or on
regulation of transcription, as an alternative to inactivation of the
gene product by point mutations in the open reading frame. Two of these
strains had a completely identical restriction fragment length
polymorphism pattern (97-56 and 97-58 [data not shown]). Additional
research on these strains should help to elucidate the mechanism of
action of the antituberculous agent POA.
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ACKNOWLEDGMENTS |
This work was supported by a grant of the Institute for Enhancement
of the Scientific-Technological Research in the Industry (IWT) of the
Flemish Government and by grant no. G.0368.98 of the Funding for
Scientific Research in Belgium.
Clinical specimens from Bangladesh were obtained thanks to A. Van Deun,
and eight M. tuberculosis isolates were obtained from the
Scottish Mycobacteria Reference Laboratory thanks to John Norton (Murex
Biotech Limited). We thank the team of M. Van Montagu (University of
Ghent) for preparation of the primers.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory for
Pharmaceutical Biotechnology, FFW, University of Ghent, Harelbekestraat 72, 9000 Ghent, Belgium. Phone: 32-9.264.80.52. Fax: 32-9.220.66.88. E-mail: Elfriede.VandenEeckhout{at}rug.ac.be.
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Antimicrobial Agents and Chemotherapy, September 1999, p. 2317-2319, Vol. 43, No. 9
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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