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J Clin Microbiol. Oct 2008; 46(10): 3459–3464.
Published online Aug 27, 2008. doi:  10.1128/JCM.00973-08
PMCID: PMC2566105

Pyrazinamide Resistance among South African Multidrug-Resistant Mycobacterium tuberculosis Isolates[down-pointing small open triangle]

Abstract

Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Wayne's assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.

South Africa is ranked fourth among the 22 high-tuberculosis (TB)-burden countries designated by the World Health Organization (WHO), with an incidence rate of 940 cases per 100,000 citizens in 2006 (22). TB-human immunodeficiency virus (HIV)/AIDS coinfection rates are high, with more than 50% of adult TB patients also being HIV positive (22). Multidrug-resistant TB (MDR-TB) is further worsening South Africa's TB epidemic. National studies of MDR-TB conducted by the Medical Research Council (MRC) of South Africa in 2002 found that 1.6% of new TB cases, and 6.7% of cases in retreatment, had MDR-TB (i.e., resistance to isoniazid and rifampin) (22, 25). A total of 5,866 Mycobacterium tuberculosis isolates were collected during the survey. Information on previous treatment was available for 97.4% of the patients. Overall, 70.3% of the patients had no history of previous treatment, while 27.1% indicated that they had received treatment for TB before. Drug resistance to any of the four first-line drugs, isoniazid, rifampin, ethambutol, and streptomycin, was detected in 7.8% cultures from new TB patients and in 15.5% cultures from retreatment cases (21, 23). One hundred seventy-nine isolates were MDR M. tuberculosis isolates, of which 71 were available for this study (21).

According to the drug resistance survey conducted from 2001 to 2002 (21) by the MRC, South Africa, isolates with resistance to isoniazid were detected in 5.7% of new patients and in 11.8% of patients with a previous history of TB treatment. Rifampin resistance was found in 1.8% of new and 7.5% of retreatment cultures. Ethambutol resistance was very low at 0.8% in new patients and 2.4% in previously treated cases. Isolates with resistance to streptomycin were found in 4.3% of new and 8.1% of retreatment patients (21). Pyrazinamide is an important first-line drug used in the short-course treatment of TB in combination with isoniazid, rifampin, and ethambutol (11). Pyrazinamide is also one of the cornerstone drugs retained in the treatment of MDR-TB. Pyrazinamide can kill semidormant tubercle bacilli that persist in acidic inflammatory environments (7, 11) and are not affected by other anti-TB drugs. The intracellular sterilizing activity of pyrazinamide allows the treatment duration to be reduced from the earlier norm of 9 months to the current standard of 6 months, aiding compliance and decreasing the risk of developing MDR-TB. Routine drug susceptibility testing of pyrazinamide is complicated to perform, as the growth of the bacilli is impeded by the acidic conditions required for optimal drug activity (10). A recent study (9) describes a high (53.5%) frequency of pyrazinamide resistance among isolates from previously treated patients from the Western Cape, South Africa. However, no studies have addressed the countrywide frequency of pyrazinamide resistance among South African MDR M. tuberculosis isolates.

DNA sequencing studies of the pncA gene from pyrazinamide-resistant and pyrazinamide-susceptible isolates of M. tuberculosis have established an association between mutations in this gene and pyrazinamide resistance. Mutations in the pncA gene are known to be a major mechanism of pyrazinamide resistance (13, 17), although some studies have shown resistant isolates which have a wild-type gene, suggesting an alternative mechanism for pyrazinamide resistance (12, 16). Earlier observations by Butler and Kilburn (2), suggest that the resistance of M. tuberculosis isolates to pyrazinamide correlates with the absence of an amidase activity (pyrazinamidase). Since most pyrazinamide-resistant M. tuberculosis isolates have lost pyrazinamidase activity, enzyme activity assays are sometimes used to replace pyrazinamide susceptibility testing (12). Thus, the aim of this study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary TB patients through the use of phenotypic and genotypic methods. In vitro susceptibility to pyrazinamide by the Bactec 460 TB method was correlated with pyrazinamidase activity and the frequency of mutations in the pncA gene of M. tuberculosis isolates. Isolates with identical mutations were further analyzed by spoligotyping and variable-number tandem repeat of mycobacterial interspersed repetitive-unit (MIRU-VNTR) typing.

MATERIALS AND METHODS

Mycobacterial isolates.

A total of 5,866 M. tuberculosis isolates were collected during the national drug resistance survey conducted by the MRC, South Africa, from 2001 to 2002 (21). Of the 179 MDR M. tuberculosis isolates collected in the survey, 71 were tested for pyrazinamide susceptibility by the Bactec 460 TB method, Wayne's assay, and pncA gene sequencing. Of the 5,284 fully susceptible (for isoniazid, rifampin, ethambutol, and streptomycin) isolates that were collected during the survey, 59 were also studied for drug susceptibility to pyrazinamide by the above-mentioned methods. Isolates were excluded due to contamination, growth problems, and mislabeling. The characteristics of the 71 MDR M. tuberculosis isolates included in this study are provided in Table Table1.1. The identity of all isolates was confirmed by AccuProbe culture identification tests (Gen-Probe Inc., San Diego, CA) and spoligotyping. Isolates identified as mycobacteria other than M. tuberculosis were excluded from the study.

TABLE 1.
MDR M. tuberculosis isolates categorized by province, age, gender, HIV status, and treatment category

DNA extraction.

M. tuberculosis isolates were grown on Lowenstein-Jensen media at 37°C for 3 to 4 weeks. DNA from mycobacterial cells was extracted using a boiling technique (17). A 1-μl loopful of cells was suspended in 200 μl of TE buffer (10 mM Tris-Cl, 1 mM EDTA), pH 8.0, and heat-killed by incubation at 95°C for 15 to 20 min. The supernatant containing the extracted DNA was collected by centrifugation at 12,000 rpm for 7 min.

Drug susceptibility testing.

Pyrazinamide susceptibility testing was performed at the MRC Supranational Tuberculosis Reference Laboratory, in South Africa, by the radiometric Bactec 460 TB system (Becton Dickinson, Sparks, MD) at a pyrazinamide concentration of 100 μg/ml, as recommended by the manufacturer (18). A clinical Mycobacterium bovis isolate was included as a pyrazinamide-resistant control, and M. tuberculosis H37Rv (ATCC 27294) was included as a pyrazinamide-susceptible control. The recommended interpretive criteria for the ratio of the growth index (GI) of the drug-containing vial to the GI of the control vial were as follows: GI ratio of <9%, susceptible; 9 to 11%, borderline; and >11%, resistant.

Wayne's assay.

Pyrazinamidase activity was assayed according to Wayne's procedure (20). A heavy loopful of growth from an actively growing culture was inoculated onto the surface of Middlebrook 7H9 agar (Difco, Detroit, MI) containing 100 μg/ml pyrazinamide (Sigma-Aldrich, Steinheim, Germany) and 2 μg/ml sodium pyruvate (Merck, Darmstadt, Germany). The test tubes were incubated at 37°C for 7 days. After incubation, 1 ml of freshly prepared 1% ferrous ammonium sulfate [Fe(NH4)2(SO4)2 6H2O] (Fluka BioChemika, Buchs, Switzerland) solution was added to each tube, and the tubes were kept at 4°C for 4 h. A tube inoculated with M. bovis was included as a negative control and M. tuberculosis H37Rv (ATCC 27294) was used as a positive control. The pyrazinamidase activity assay was considered positive if a pink band was seen in the upper part of the butt.

Amplification and sequencing of the pncA gene.

The pncA gene was amplified from each isolate by using primers P1 and P6, as described previously (16). The expected size of the PCR product was 720 bp, which included the full length of the pncA gene (561 bp) as well as 104 bp of the upstream sequence and 55 bp of the downstream sequence.

PCR amplifications were carried out in a GeneAmp PCR system 9700 thermocycler (Perkin Elmer, Applied Biosystems, CA). Each reaction mixture (25 μl) contained 1.25 μl of the crude DNA extraction, 0.025 pmol of each PCR primer, and 12.5 μl HotStarTaq master mix (Qiagen, Germany). The reaction mixtures were subjected to 15 min at 95°C, followed by 30 cycles of 40 s at 94°C, 60 s at 55°C, and 40 s at 72°C, and terminated by 15 min at 72°C. Successful gene amplifications were confirmed by the Bioanalyzer DNA 1000 chip kit (Agilent Technologies).

PCR products from each strain were purified with the QIAquick PCR purification kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions. PCR products were subjected to a sequencing reaction using a BigDye Terminator cycle sequencing kit and primer P1. The amplification profile consisted of an initial 5 min for denaturation at 96°C, 30 cycles of 96°C for 30 s, 50°C for 15 s, and 60°C for 4 min, and an elongation time of 72°C for 15 min in an ABI 377 automatic DNA sequencer (Applied Biosystems Inc., Foster, CA). Nucleotide sequences were analyzed using CHROMAS software (Technelysium Ltd.).

Genotyping. (i) Spoligotyping.

Spoligotyping was performed to further characterize M. tuberculosis isolates with identical mutations in the pncA gene, according to an internationally standardized protocol (6) to detect the presence or absence of 43 variable spacers in the direct repeat (DR) region of M. tuberculosis. DNA was amplified by PCR using primer DRb and biotinylated DRa, complementary to either side of the DR unit. The resulting amplification products were then hybridized to a commercially available membrane (Isogen Bioscience BV, Bilthoven, The Netherlands), with covalently linked parallel rows of 43 synthetic oligonucleotides representing the unique spacer sequences between DR units in the DR region.

The conditions used for PCR were 3 min at 96°C, followed by 20 cycles of 1 min at 96°C, 1 min at 55°C, and 30 s at 72°C. A Microsoft Excel spreadsheet was used to analyze the spoligotyping results, and the spoligotyping image was converted into an octal digital format (3). The data were further analyzed by comparison with the fourth version of an international spoligotyping database (1).

(ii) MIRU-VNTR typing.

VNTR typing was used to further discriminate between strains that had identical pncA mutations and spoligotype patterns. As previously described (8), 12 primer sets were used to individually amplify regions with known tandem repeats.

Amplifications for all MIRU PCRs, except MIRU-24, were performed in a total volume of 10 μl containing 0.5 μl bacterial DNA, 0.25 μl (5 μM) of each PCR primer, 4 μl HotMasterMix (Eppendorf, Germany), and 5 μl double-distilled H2O. An initial denaturation of 2 min at 94°C was followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 1 min, and extension at 65°C for 30 s, followed by a final extension at 65°C for 15 min. Amplifications with the MIRU-24 primers were performed using 1 μl DNA, 0.5 μl of the MIRU-24 forward and reverse primers (10 μM), 6 μl HotStarTaq master mix (Qiagen, Germany), 2.5 μl Q-solution (Qiagen, Germany), and 1.5 μl double-distilled H2O. An initial denaturation of 15 min at 95°C was followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1.5 min, and extension at 72°C for 1.5 min, followed by a final extension at 72°C for 15 min.

Samples were visualized and analyzed with a Bioanalyzer DNA 1000 chip kit (Agilent Technologies). Amplicon sizes were used to estimate the number of complete repeats at each locus, as described by Frothingham and Meeker-O'Connell (5).

Analysis of strains with discrepant pyrazinamide susceptibility results.

MDR and fully susceptible M. tuberculosis isolates that had discordant results in any of the three methods applied (Bactec 460 TB system, Wayne's assay, and the sequencing method) were sent to a second laboratory (National Institute for Public Health, Oslo, Norway) for validation of susceptibility to pyrazinamide by retesting pyrazinamide susceptibility by the Bactec 460 TB system. The results from the arbiter laboratory were considered conclusive.

RESULTS

Pyrazinamide susceptibility testing by phenotypic methods.

Of the 71 MDR M. tuberculosis isolates tested by the three methods (Bactec 460 TB system, Wayne's assay, and sequencing of the pncA gene), 49 isolates had concordant results and 22 had discordant results in all three methods. Of the 49 MDR M. tuberculosis isolates with concordant results, 30 were resistant and 19 were susceptible to pyrazinamide by the Bactec 460 TB method. Of the 22 MDR M. tuberculosis isolates with discordant results in all three methods applied, 18 were resistant and 4 were sensitive to pyrazinamide by the Bactec 460 TB method.

The 22 MDR M. tuberculosis isolates with discordant results were retested for pyrazinamide susceptibility by the Bactec 460 TB system in a second laboratory (Norwegian Institute of Public Health, Oslo, Norway). Thus, 7 were found to be resistant (one isolate had borderline resistance), and 15 were sensitive to pyrazinamide. Therefore, on incorporation of retested results, 37 of 71 (52.1%) MDR M. tuberculosis isolates were resistant to pyrazinamide by the Bactec method (Table (Table22).

TABLE 2.
Results from pyrazinamide susceptibility testing of 71 MDR and 59 susceptible M. tuberculosis isolates by the Bactec 460 TB system, pncA gene sequencing, and pyrazinamidase activitya

Among the 59 isolates susceptible to rifampin, isoniazid, ethambutol, and streptomycin, the pyrazinamide susceptibility results of 54 isolates agreed in all three methods. Five isolates had discordant results in the methods applied and were retested for pyrazinamide susceptibility by the Bactec 460 TB system in a second laboratory. Of the 54 isolates with concordant results in all three methods, 51 were sensitive and 3 were resistant to pyrazinamide by the Bactec 460 TB method. Of the 5 isolates with discordant results, 3 were resistant (1 isolate showed borderline resistance) and 2 were sensitive to pyrazinamide on retesting in a second laboratory. On incorporation of retested Bactec results, 6 of 59 (10.2%) fully susceptible M. tuberculosis isolates were resistant to pyrazinamide (Table (Table22).

Of the 37 MDR-TB isolates found to be pyrazinamide resistant by the Bactec method (36 pyrazinamide-resistant and 1 borderline-resistant isolate), 32 isolates showed no pyrazinamidase activity, of which 30 isolates had mutations in the pncA gene (Table (Table2).2). Four of 6 fully susceptible isolates, which were pyrazinamide resistant by the Bactec method (5 pyrazinamide-resistant and 1 borderline-resistant isolate) showed no pyrazinamidase activity and had mutations in the pncA gene. Overall, using the Bactec 460 TB system as the reference method, 12 and 7 isolates had discordant results with Wayne's assay and sequencing of the pncA gene, respectively (Table (Table22).

Mutations in the pncA gene.

Twenty-five unique mutations out of a total of 44 different mutations were detected among 42 (36 MDR and 6 fully susceptible) M. tuberculosis isolates that had mutations in the pncA gene (Table (Table3).3). The majority of these were point mutations that resulted in amino acid substitutions. Point mutations included nucleotide substitution (37 of 44), deletions (3 of 44), and insertions (4 of 44) in the pncA gene (Table (Table3).3). Two of the isolates had synonymous (silent) nucleotide substitutions, confirmed by pyrazinamide sensitivity in the Bactec method and pyrazinamidase activity (Table (Table3).3). The most frequently encountered change was the A-to-G point mutation at position 35 (9 of 44). Isolates that shared identical mutations could be assigned to 10 different groups (Table (Table33).

TABLE 3.
Overview of 25 different mutations in the pncA gene of 42 (36 MDR and 6 fully susceptible) M. tuberculosis isolates from South Africab

Spoligotyping.

Twenty-eight M. tuberculosis isolates distributed in 10 different groups had identical mutations in the pncA gene and were characterized by spoligotyping (Table (Table3).3). On the basis of spoligotyping, 24 M. tuberculosis isolates were distributed in eight clusters (a cluster was defined as two or more isolates from different patients with identical spoligotype patterns and mutations in the pncA gene). Four of the 28 M. tuberculosis isolates had unique spoligotyping patterns. The eight clusters comprised isolates belonging to previously described shared types (ST): 18, 21, 34, 71, 92, 137, 719, and 926 (1).

The largest cluster was ST 137, comprising 9 isolates which had the same nucleotide substitution of A-to-G at position 35. Three isolates belonged to ST 926. The remaining clusters had two isolates each. To determine the relatedness of all isolates in clusters, additional molecular typing by MIRU-VNTR typing was performed.

MIRU-VNTR typing.

Twenty-four isolates distributed in eight clusters with identical mutations in the pncA gene and spoligotype patterns in each cluster were subjected to analysis of 12 MIRU loci. MIRU-VNTR typing had a greater resolving power than spoligotyping. Thus, no isolates were found to have the same MIRU-VNTR codes. However, isolates within each cluster displayed related MIRU-VNTR profiles.

Of the 12 MIRU-VNTR loci tested, MIRU-23 produced six alleles and was the most polymorphic. The least polymorphic loci were MIRU-16 and MIRU-24. At MIRU-16, tandem repeats were absent in 45.8% of the isolates. All isolates had one copy of the MIRU locus 24.

DISCUSSION

Global surveillance has shown that drug-resistant TB is widespread and is now a threat to TB control programs in many countries. Drug resistance complicates the treatment of TB cases, often leading to unsuccessful outcomes. In addition, MDR-TB causes high morbidity and mortality rates, particularly in immunocompromised populations. The treatment of drug-resistant and MDR-TB is expensive and has many problems. Therefore, strategies to detect, treat, and prevent drug resistance are urgently needed.

Treatment guidelines for MDR-TB patients in South Africa were implemented as national policy in 2001. Pyrazinamide is routinely used in first-line drug therapy for TB, but it is also one of the cornerstone drugs used in the treatment of MDR-TB and is included in the standardized MDR-TB treatment regimen in South Africa. However, due to technical difficulties, routine drug susceptibility testing for pyrazinamide is not performed, and consequently, little is known about the actual rates of pyrazinamide resistance in South Africa, despite similar data existing for other first-line anti-TB drugs.

A number of laboratory methods have been used to assess the susceptibility of M. tuberculosis to pyrazinamide. Conventional mycobacterial susceptibility testing methods, depending on growth of the organisms when exposed to drugs, are impeded by performance difficulties (requirement of a low pH, standardization of inoculum size and clumping effect) (10, 19). Other approaches such as Wayne's assay involve the detection of the presence or absence of pyrazinamidase activity as a marker of resistance, with resistant strains presumed to be lacking the enzyme (20). Mutations in the pncA gene encoding pyrazinamidase involved in the activation of pyrazinamide to the active form of pyrazinoic acid provide possible molecular markers for resistance determination (4).

Thus, in this study, we tested the susceptibility of 71 MDR and 59 fully susceptible M. tuberculosis isolates collected during the national drug resistance survey conducted by the MRC, South Africa, from 2001 to 2002 (21). Susceptibility to pyrazinamide was tested by the radiometric Bactec 460 TB system, Wayne's assay, and sequencing of the pncA gene. Thirty-seven of 71 (52.1%) MDR M. tuberculosis isolates were resistant to pyrazinamide by the Bactec 460 TB system. Similarly, there were 6 of 59 (10.2%) fully susceptible M. tuberculosis isolates that were resistant to pyrazinamide. Seven of 43 (16.3%) Bactec pyrazinamide-resistant M. tuberculosis isolates showed pyrazinamidase activity when tested by Wayne's assay. This suggests the participation of other M. tuberculosis genomic regions in conferring resistance to pyrazinamide and is supported by alternative mechanisms leading to pyrazinamide resistance: active efflux of bactericidal pyrazinoic acid from the organism (24) and defects in pyrazinamide uptake by the organism (15).

Thirty-nine of 43 (90.7%) Bactec pyrazinamide-resistant M. tuberculosis isolates showed pncA mutations in this study. The finding of pyrazinamide-resistant isolates that lack mutations in the pncA gene suggests that alternative mechanisms for drug resistance may exist. In addition, we found three isolates which were pyrazinamide susceptible but had mutations in the pncA gene, implying that mutations in this region do not affect the bacterium's susceptibility to pyrazinamide. It is generally considered that mutations leading to pyrazinamide resistance are scattered along the pncA gene (14). The mutations in the pncA gene, identified in this study, were found scattered outside the regions suggested to have some degree of clustering of mutations (Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12), as previously described (17). Our findings of diverse and scattered mutations along the pncA gene support similar observations in other studies (9, 14).

This study shows that the presence of mutations in the pncA gene correlates well with pyrazinamide resistance in the Bactec method results (for 39 of 42 isolates) and with a loss of pyrazinamidase activity (for 34 of 42 isolates). An A-to-G point mutation at position 35 in 21.4% of the isolates was the most common type of pncA mutation in the study. The isolates with identical mutations in the pncA gene could be differentiated from each other by a combination of the spoligotype patterns and analysis of 12 MIRU loci. Thus, all isolates containing mutations in the pncA gene were unique. In agreement with previous suggestions (9, 17), the detection of pncA mutations by direct sequencing of the pncA gene by PCR could provide a rapid method for the diagnosis of pyrazinamide-resistant M. tuberculosis, thereby contributing to more-effective management of TB patients and potentially limiting the spread of drug-resistant M. tuberculosis isolates.

Although, the strains studied may not be fully representative of all MDR M. tuberculosis strains from the national survey, the (alarmingly) high proportion (52.1%) of strains that were resistant to pyrazinamide highlights the need for further systematic testing for pyrazinamide during drug resistance surveys. Susceptibility tests for pyrazinamide are difficult to perform and need careful standardization. Furthermore, the clinical relevance of in vitro resistance to pyrazinamide also needs urgent investigation, as the drug is widely used for both drug-susceptible TB retreatment regimens and MDR-TB treatment. In conclusion, we show that a high proportion of the South African MDR-TB isolates tested are resistant to pyrazinamide, suggesting an evaluation of its clinical role in patients treated previously for TB as well as in MDR-TB treatment.

Acknowledgments

We thank Jeanette Brand (MRC, South Africa), Hilde Jacobsen (University of Bergen), and the staff at the Norwegian Institute of Public Health, Oslo, Norway, for their technical assistance.

This study was supported by Helse Vest, Norway, the University of Bergen, NORAD (scholarship to M.M.), and the Medical Research Council, South Africa.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 August 2008.

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