Abstract
Testing for susceptibility to pyrazinamide (PZA) and analysis of the pncA gene sequences of 423 Mycobacterium tuberculosis complex isolates have revealed a unique silent nucleotide substitution that enables the rapid identification of “M. canettii” (proposed name). Moreover, the lack of a defined mutation within the pncA gene strongly suggests that an alternative mechanism is responsible for PZA resistance. Our results indicate that DNA sequencing of the pncA gene has the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing of the M. tuberculosis complex.
The Mycobacterium tuberculosis complex (MTB complex) consists of the closely related organisms M. tuberculosis, M. africanum, M. bovis, M. bovis BCG, M. caprae, M. microti, M. pinnipedii, the dassie bacillus, and “M. canettii” (proposed name) (3, 5, 6, 26). Although the members of the complex may differ in their epidemiologies, host spectra, geographic ranges, pathogenicities, and antituberculosis drug susceptibilities and although they display different phenotypic characteristics by conventional biochemical tests, they show high degrees of genetic homogeneity (10, 25).
Conventional phenotypic methods for the identification of the members of the MTB complex are laborious and time-consuming, and they require a large biomass (13). Thus, molecular methods (such as DNA sequencing of the oxyR, pncA, gyrB, or hsp65 gene; analysis of spacers between direct repeats in the direct repeat region; and deletion analysis of the regions of difference [RD]) provide a more rapid and accurate approach to the differentiation of the members of the MTB complex (8, 9, 16, 23, 24).
The findings of a recent report that examined seven genes (katG, gyrB, gyrA, rpoB, hsp65, sodA, and the 16S rRNA gene) indicate that M. canettii represents the most ancient phylogenetic lineage of the tubercle bacilli (10). Until recently, little attention was paid to M. canettii, a rare member of the complex that infects only humans and that shows a geographical restriction to Africa; however, it has been found in patients as they travel to other parts of the world, leading to the need for the identification of this organism outside of the African continent (7, 19, 27).
M. canettii is characterized by eugonic growth, with smooth, white, and glossy colonies on solid medium. Its biochemical characteristics indicate that it is unable to produce niacin but that it is capable of nitrate reduction and has positive urease, Tween 80 hydrolysis, and catalase activities (at 22°C, although not at 68°C). Genetically, it can be distinguished from other members of the MTB complex by an unusual composition of the direct repeat cluster when it is assessed by spoligotyping. Finally, it is naturally resistant to thiophene-2-carboxylic acid hydrazide and pyrazinamide (PZA) (9, 19, 27). PZA (a nicotinamide analog) is a prodrug which is converted to its active form, pyrazinoic acid (POA), by the mycobacterial enzyme pyrazinamidase (PZase) (18, 28). It has been observed that PZA-resistant M. tuberculosis isolates usually lose their PZase activity (14). When the gene encoding PZase (pncA) from PZA-resistant clinical isolates was sequenced, it was found that 72 to 97% of all isolates tested contained a mutation in the structural gene or in the putative promoter region, both of which were predicted to impair PZase activity (23, 28). In addition, M. bovis and M. bovis BCG, which are naturally resistant to PZA, encode a single substitution of PZase, His57Asp, which distinguishes them from other members of the MTB complex (22).
The aim of the present study was to determine whether the natural resistance to PZA of M. canettii could be correlated with a similar pncA mutation and, if so, whether the mutation could be used to distinguish M. canettii from other members of the MTB complex. On the basis of the accumulated findings, we also aimed our study to examine the use of pncA sequencing for the rapid detection of PZA resistance.
A total of nine M. canettii isolates were studied: M. canettii CIPT140010059 from the Institut Pasteur, Paris, France; M. canettii 910563 from the Institut Pasteur; M. canettii 217/94, isolated at the Swiss Reference Laboratory for Mycobacteria, Zurich, Switzerland (19); M. canettii So93 (smooth colony type), isolated at the National Institute of Public Health and Environment, Bilthoven, The Netherlands (27); M. canettii So93R, the rough colony type subculture of a single colony of So93; M. canettii Percy 25, Percy 65, and Percy 229, isolated at the Percy Military Hospital, Clamart, France (7); and M. canettii AFB 0600720 identified at the Wadsworth Center, Albany, NY. M. canettii isolates 217/94 (kindly provided by Gaby Pfyffer); So93 and So93R (kindly provided by Dick van Soolingen); and Percy 25, 65, and 229 (kindly provided by Michel Fabre) were maintained and cultured at the Institut Pasteur de Guadeloupe.
Identification of all strains as M. canettii was confirmed by a PCR-based deletion analysis with two panels of RDs (RD1, RD9, and RD10 and RD4, RD12, and TbD1) and by PCR-restriction analysis of the hsp65 gene, as described earlier (9, 17; L. M. Parsons, J. Dormandy, A. Clobridge, J. R. Driscoll, M. Oxtoby, H. W. Taber, and M. Salfinger, Abstr. National TB Controllers Workshop, 2004). Routine drug susceptibility testing was performed with all strains by use of the radiometric BACTEC 460TB system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) and by the agar proportion method (12, 13); M. canettii AFB 0600720 was tested in the BACTEC 460TB system at the Wadsworth Center. All other M. canettii isolates were tested by the agar proportion method at the Unité de la Tuberculose et des Mycobactéries, Institut Pasteur de Guadeloupe, as described earlier (9). M. canettii Percy 25, 65, and 229 were also tested with the BACTEC MGIT 960 system (Becton Dickinson Diagnostic Instrument Systems) (M. Fabre, personal communication).
Using two primers, pncA-P1 (5′-GCT-GGT-CAT-GTT-CGC-GAT-CG-3′) and pncA-P6 5′-GCT-TTG-CGG-CGA-GCG-CTC-CA-3′), which flanked the entire pncA gene and its upstream promoter, we generated a 700-bp product from each isolate, as described previously (22, 23). The same primers were used for DNA sequencing of both strands with an automated 3700 DNA sequencer (Applied Biosystems, Foster City, CA). The resulting sequences were compared with the wild-type pncA sequence from M. tuberculosis H37Rv for the detection of mutations associated with PZA resistance. The DNA sequencing was carried out by the Molecular Genetics Core Facility at the Wadsworth Center.
The mutations of the pncA gene that were identified in the nine M. canettii strains are summarized in Table 1. All strains were resistant to PZA. Analysis of the DNA sequence of pncA revealed an A138G (Ala46Ala) silent nucleotide substitution in all nine M. canettii strains. In an independent study just published, the same substitution was described as being present in five M. canettii strains by Huard et al. (11). Strain CIPT140010059 in our study carried an additional T387C (Asp129Asp) silent substitution, while strain Percy 65 carried an additional 10 nucleotide substitutions, 4 of which were silent and 6 of which introduced amino acid changes in PZase (Table 1).
TABLE 1.
Nucleotide polymorphisms in the pncA gene of the 11 M. canettii isolates studied
| Strains (origin) | Nucleotide (amino acid) changes in pncAa |
|---|---|
| M. canettii CIPT140010059 (Institute Pasteur, Paris, France) | A138G (Ala46Ala), T387C (Asp129Asp) |
| M. canettii 910563 (Institute Pasteur, Paris, France) | A138G (Ala46Ala) |
| M. canettii 217/94 (Swiss Reference Laboratory for Mycobacteria, | |
| Zurich, Switzerland) | A138G (Ala46Ala) |
| M. canettii So93 (National Institute of Public Health and | |
| Environment, Bilthoven, The Netherlands) | A138G (Ala46Ala) |
| M. canettii So93R (the rough colony type subculture of M. canettii | |
| So93; National Institute of Public Health and Environment, | |
| Bilthoven, The Netherlands) | A138G (Ala46Ala) |
| M. canettii Percy 25 (Percy Military Hospital, Clamart, France) | A138G (Ala46Ala) |
| M. canettii Percy 229 (Percy Military Hospital, Clamart, France) | A138G (Ala46Ala) |
| M. canettii Percy 65 (Percy Military Hospital, Clamart, France) | A138G (Ala46Ala), C42T (Cys14Cys), G261C (Thr87Thr), C349T (Leu117Leu), T387C (Asp129Asp), A110C (Glu37Ala), G187A (Asp63Asn), G273C (Glu91Asp), T451A (Leu151Met), T506C, G507A (Val169Ala), G522C (Glu174Asp) |
| M. canettii AFB0600000720 (Wadsworth Center, Albany, NY) | A138G (Ala46Ala) |
Boldface indicates an M. canettii-specific silent substitution.
In a second phase of the study, we analyzed the pncA gene by DNA sequencing of a large number (n = 412) of MTB complex isolates (294 M. tuberculosis, 45 M. bovis, 43 M. bovis BCG, 24 M. africanum, 4 M. microti, and 2 M. caprae isolates) to confirm that the A138G substitution identified in the M. canettii strains is unique to this member of the MTB complex. Susceptibility testing of these strains was performed with the BACTEC 460TB system. Identification of the strains was determined by PCR-based deletion analysis, as described above. Of the 412 strains, 240 (58.3%) were susceptible and 172 (41.7%) were resistant to 100 μg/ml PZA in the BACTEC 460TB system.
Analysis of the pncA gene sequence of this large number of clinical isolates showed that none of the changes in the M. canettii strains were present in other members of the MTB complex (Table 2). DNA sequencing confirmed a wild-type pncA sequence for 195 (81.2%) of the 240 non-M. canettii strains that were susceptible to PZA (Table 2). Three (1.3%) PZA-susceptible M. tuberculosis strains carried silent substitutions which differed from those in M. canettii (Tables 1 and 2). A further 42 (17.5%) M. tuberculosis strains that were susceptible in the BACTEC 460TB system carried the same pncA mutation (Thr47Ala). However, it is noteworthy that these M. tuberculosis strains, which were resistant to the same drugs and which shared a characteristic spoligotype pattern, all belonged to the Beijing/W family (Table 2). They are therefore derived from a single clone of a strain that was originally identified during nosocomial outbreaks in New York City in the early 1990s (2). Further analysis of these Beijing/W strains revealed that the mutation observed in pncA resulted in elevated PZA MICs compared to those for PZA-susceptible strains but in lower MICs compared to those for PZA-resistant strains, as determined by more rigorous assays (data not shown) (6a).
TABLE 2.
Pyrazinamide susceptibility testing with the BACTEC 460TB system and pncA DNA sequencing analysis results for 412 non-M. canettii isolates of the Mycobacterium tuberculosis complexa
| No. of strains | Identification | BACTEC 460TB result | pncA mutation | Nucleotide change |
|---|---|---|---|---|
| 166 | MTB | S | No mutation | NA |
| 42 | MTB | S | Thr47Ala | A139G |
| 2 | MTB | S | Ser65Ser* | C195T |
| 1 | MTB | S | Ala20Ala* | G60A |
| 24 | AFR | S | No mutation | NA |
| 4 | MICR | S | No mutation | NA |
| 1 | CAP | S | No mutation | NA |
| 14 | MTB | R | Frameshift mutation | Deletion of nucleotide 70 (G) |
| 13 | MTB | R | Leu85Pro | T254C |
| 6 | MTB | R | NA | A→−11 upstream |
| 3 | MTB | R | Ser104Arg | C312G |
| 3 | MTB | R | Leu172Pro | T515C |
| 2 | MTB | R | Gln10Pro | A39C |
| 2 | MTB | R | Cys14Arg and Ser65Ser* | T40C and C195G |
| 2 | MTB | R | His51Gln | C153A |
| 2 | MTB | R | Leu116Arg | T347G |
| 2 | MTB | R | Trp119Cys | G357T |
| 2 | MTB | R | Thr142Pro and Ser65Ser* | A424C and C195T |
| 2 | MTB | R | Frameshift mutation | Nucleotide insertion (G) between codons 77 and 78 |
| 1 | MTB | R | NA | Deletion of start codon and −5 nucleotides upstream |
| 1 | MTB | R | Val21Gly | T62G |
| 1 | MTB | R | Leu35Arg and His82Asp | T104 and C244G |
| 1 | MTB | R | Ala46Val | C138T |
| 1 | MTB | R | Frameshift mutation | Deletion of nucleotide 146 (A) |
| 1 | MTB | R | His51Arg | A152G |
| 1 | MTB | R | Ser67Pro | T199C |
| 1 | MTB | R | Trp68Arg | T202C |
| 1 | MTB | R | His71Arg | A212G |
| 1 | MTB | R | Gly78Cys | G232T |
| 1 | MTB | R | Tyr103Asp | T307G |
| 1 | MTB | R | Gly108Arg | G322C |
| 1 | MTB | R | Frameshift mutation | Nucleotide insertion (G) between codons 104 and 105 |
| 1 | MTB | R | Frameshift mutation | Deletion of codons 128, 129, 130 |
| 1 | MTB | R | Val128Gly | T383G |
| 1 | MTB | R | Frameshift mutation | Nucleotide insertion (GG) between codons 130 and 131 |
| 1 | MTB | R | Cys138Tyr | G413A |
| 1 | MTB | R | Val139Leu | G415C |
| 1 | MTB | R | Val139Ala | T416C |
| 1 | MTB | R | Gln141Stop | C421T |
| 1 | MTB | R | Thr142Met | C425T |
| 1 | MTB | R | Frameshift mutation | Nucleotide insertion (CT) between codons 155 and 156 |
| 1 | MTB | R | Leu156Pro | T467C |
| 1 | MTB | R | Gly162Ser | G485A |
| 1 | MTB | R | Gly162Asp | G486A |
| 1 | MTB | R | Ser164Pro and Glu173Glu* | T490C and G519A |
| 1 | MTB | R | Ala171Val | C512T |
| 1 | MTB | R | Frameshift mutation | Nucleotide insertion (CG) at codon 177 |
| 1 | MTB | R | Val180Phe | G538T |
| 1 | MTB | R | Frameshift mutation | Deletion of four nucleotides (542, 543, 544, 545) |
| 45 | BOV | R | His57Asp | C169G |
| 43 | BCG | R | His57Asp | C169G |
| 1 | CAP | R† | No mutation | NA |
Abbreviations and symbols: MTB, M. tuberculosis; BOV, M. bovis; AFR, M. africanum; CAP, M. caprae; MICR, M. microti; S, susceptible; R, resistant; NA, not applicable; *, silent mutation; †, a live culture could not be retrieved to confirm the accuracy of the discrepant result (M. caprae strains are naturally PZA susceptible).
PZA resistance-associated mutations were detected in 171 (99.94%) of the 172 PZA-resistant strains. The single M. caprae strain that exhibited resistance to PZA in the BACTEC 460TB system did not encode any mutations in the sequenced region (Table 2). Unfortunately, a live culture could not be retrieved to confirm the validity of this discrepant result (M. caprae strains are naturally PZA susceptible) (1). All of the 45 M. bovis isolates and the 43 M. bovis BCG isolates encoded the distinguishing His57Asp mutation, consistent with previous results (Table 2) (22).
Testing of the members of the MTB complex for their susceptibilities to PZA has been complicated by the fact that this drug is active only at acidic pH; a significant number of M. tuberculosis isolates would not grow at pH 5.5 in conventional solid media (4, 20). Subsequently, a more accurate, broth-based radiometric test system (the BACTEC 460TB system) with pH 6.0 medium was described, and this has become the current recommended assay (21). However, in a study that examined 428 MTB complex strains, at least 4 (0.8%) grew in the presence of 100 μg/ml PZA in the BACTEC 460TB system and yet were PZase positive, suggesting either that for some strains the radiometric assay is inaccurate or that resistance is mediated by another mechanism (15). Our results indicate that sequence analysis of pncA is also an accurate predictor of PZA resistance (99.9%). This capability, combined with the rapid turnaround time of the sequence analysis, makes it an attractive new reference assay for PZA susceptibility testing.
Most importantly, our work with the M. canettii isolates suggests that their natural resistance to PZA is not pncA based. Rather, another mechanism of PZA resistance must be operative, such as PZA uptake, pncA regulation, or POA efflux, a conclusion that is in agreement with the observations of others (15, 28). Further investigations are warranted to determine the mechanism of PZA resistance in M. canettii. Moreover, the sequence analysis identified a single nucleotide polymorphism (A138G) as a positive indicator of M. canettii, allowing this organism to be easily distinguished from other members of the MTB complex.
The natural reservoir, host range, and mode of transmission of M. canettii are unknown. Many clinical laboratories do not differentiate among the members of the MTB complex. Thus, it is very likely that the geographic distribution and prevalence of M. canettii, as well as those of other non-M. tuberculosis members of the MTB complex, are underestimated. The results of the present study establish that analysis of the pncA gene by DNA sequencing can rapidly and accurately identify M. canettii and can differentiate it from other members of the MTB complex.
In conclusion, besides the potential to shorten the turnaround time and increase the accuracy of PZA susceptibility testing, the broader use of DNA sequencing analysis of pncA will help to accumulate additional valuable information on the epidemiology, transmission, and pathogenesis of M. bovis, M. bovis BCG, and M. canettii infections.
Acknowledgments
We thank Keith Derbyshire for critical review of the manuscript; Anne Clobridge, Susan Larsen, Jayanti Sekhar, and Andrea Doney for excellent technical assistance in performing the susceptibility tests; and Gaby Pfyffer, Dick van Soolingen, and Michel Fabre for providing some of the M. canettii strains.
Footnotes
Published ahead of print on 29 November 2006.
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