Relationship between Pyrazinamide Resistance, Loss of Pyrazinamidase Activity, and Mutations in the
            <i>pncA</i>
            Locus in Multidrug-Resistant Clinical Isolates of
            <i>Mycobacterium tuberculosis</i>

Mestdagh, Marian; Fonteyne, Pierre-Alain; Realini, Laurence; Rossau, R.; Jannes, Geert; Mijs, Wouter; Smet, K. A. L. De; Portaels, Françoise; Eeckhout, E. Van den

doi:10.1128/aac.43.9.2317

Cited by 76 publications

(34 citation statements)

References 15 publications

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“…Several mutations, including missense, insertion, deletion and nonsense mutations, have been reported and located in both the putative promoter and coding regions of pncA [10]. PZA-resistant M. tuberculosis strains are usually correlated with defective PZase activity, but some PZA resistant strains have been reported to contain wild-type pncA and to maintain PZase activity [11-14], suggesting that other unknown resistance mechanisms could be responsible for the resistance phenotype.…”

Section: Introductionmentioning

confidence: 99%

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

et al. 2010

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BackgroundSusceptibility testing of pyrazinamide (PZA) against Mycobacterium tuberculosis is difficult to perform because the acidity of culture medium that is required for drug activity also inhibits the growth of bacteria. In Thailand, very limited information has been generated on PZA resistance, particularly among multidrug-resistant tuberculosis (MDR-TB) isolated from Thailand. Only two studies on PZA susceptibility among Thai M. tuberculosis strains have been reported; one used a pyrazinamidase assay, and the other used the BACTEC 460 TB for PZA susceptibility testing. In this study, we determined the percentage of strains possessing pyrazinamide resistance among pan-susceptible M. tuberculosis and MDR-TB isolates by using the pyrazinamidase assay, BACTEC MGIT 960 PZA method and pncA sequencing, and assessed the correlation in the data generated using these methods. The type and frequency of mutations in pncA were also determined.ResultsOverall, 150 M. tuberculosis isolates, consisting of 50 susceptible and 100 MDR-TB isolates, were tested for PZA susceptibility by BACTEC MGIT 960 PZA, the pyrazinamidase assay and pncA sequencing. The study indicated PZA resistance in 6% and 49% of susceptible and MDR-TB isolates, respectively. In comparison to the BACTEC MGIT 960 PZA, the PZase assay showed 65.4% sensitivity and 100% specificity, whereas pncA sequencing showed 75% sensitivity and 89.8% specificity. Twenty-four mutation types were found in this study, with the most frequent mutation (16%) being His71Asp. Of these mutations, eight have not been previously described. The Ile31Ser and Ile31Thr mutations were found both in PZA susceptible and resistant isolates, suggesting that mutation of this codon might not play a role on PZA resistance.ConclusionsOur findings suggest that phenotypic susceptibility testing is still essential for the detection of PZA resistance, especially for MDR-TB isolates. Some mutations were not associated with resistance and could lead to misinterpretation of the genotypic methods. This information could be helpful for clinicians in managing tuberculosis patients and frequencies, and the types of pncA mutations should offer baseline information on PZA resistance.

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Section: Introductionmentioning

confidence: 99%

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

et al. 2010

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“…However, some PZA-resistant strains without pncA mutations have been reported (48, 63–65). These could be due to false resistance as well as a small number of genuine PZA-resistant strains without pncA mutations.…”

Section: Mechanisms Of Pza Resistancementioning

confidence: 99%

Mechanisms of Pyrazinamide Action and Resistance

et al. 2014

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PZA is a unique anti-tuberculosis drug that plays a key role in shortening the TB therapy. PZA kills non-replicating persisters that other TB drugs fail to kill, and thus making it an essential drug for inclusion in any drug combinations for treating drug susceptible and drug-resistant TB such as MDR-TB. PZA acts differently from common antibiotics by inhibiting multiple targets such as energy production, trans-translation and perhaps pantothenate /coenzyme A required for persister survival. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase involved in conversion of the prodrug PZA to the active form POA. Mutations in the drug target RpsA are also found in some PZA-resistant strains. The recent finding that panD mutations are found in some PZA-resistant strains without pncA or rpsA mutations may suggest a third PZA resistance gene and a potential new target of PZA. Current phenotype based PZA susceptibility testing is not reliable due to false resistance, and sequencing of the pncA gene represents a more rapid, cost-effective and more reliable molecular test for PZA susceptibility testing and should be used for guiding improved treatment of MDR/XDR-TB. Finally, the story of PZA has important implications for not only TB therapy but also chemotherapy in general. PZA serves as a model prototype persister drug and hopefully a ‘tipping point’ that inspires new efforts at developing a new type of antibiotics or drugs that target non-replicating persisters for improved treatment of not only TB but also other persistent bacterial infections.

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“…An inability of the bacteria to release POA to the extracellular environment may be caused by low expression of PZAse or enzymatic malfunction due to pncA mutations 13, 14, 20–22 . However, some PZA-resistant isolates with pncA mutations retain PZAse activity 9, 21, 23, 24 , suggesting either inappropriate gene expression or an alternative mechanism such as an altered POA efflux pump.…”

Section: Introductionmentioning

confidence: 99%

Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance

et al. 2012

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Pyrazinamide is one of the most important drugs in the treatment of latent Mycobacterium tuberculosis infection. The emergence of strains resistant to pyrazinamide represents an important public health problem, as both first- and second-line treatment regimens include pyrazinamide. The accepted mechanism of action states that after the conversion of pyrazinamide into pyrazinoic acid by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. The pyrazinoic acid is protonated in the extracellular environment and then re-enters the mycobacterium, releasing the proton and causing a lethal disruption of the membrane. Although it has been shown that mutations causing significant loss of pyrazinamidase activity significantly contribute to pyrazinamide resistance, the mechanism of resistance is not completely understood. The pyrazinoic acid efflux rate may depend on multiple factors, including pyrazinamidase activity, intracellular pyrazinamidase concentration, and the efficiency of the efflux pump. Whilst the importance of the pyrazinoic acid efflux rate to the susceptibility to pyrazinamide is recognized, its quantitative effect remains unknown. Thirty-four M. tuberculosis clinical isolates and a Mycobacterium smegmatis strain (naturally resistant to PZA) were selected based on their susceptibility to pyrazinamide, as measured by Bactec 460TB and the Wayne method. For each isolate, the initial velocity at which pyrazinoic acid is released from the bacteria and the initial velocity at which pyrazinamide enters the bacteria were estimated. The data indicated that pyrazinoic acid efflux rates for pyrazinamide-susceptible M. tuberculosis strains fell within a specific range, and M. tuberculosis strains with a pyrazinoic acid efflux rate below this range appeared to be resistant. This finding contrasts with the high pyrazinoic acid efflux rate for Mycobacterium smegmatis, which is innately resistant to pyrazinamide: its pyrazinoic acid efflux rate was found to be 900 fold higher than the average efflux rate for M. tuberculosis strains. No significant variability was observed in the pyrazinamide flux rate. The pyrazinoic acid efflux rate explained 61% of the variability in Bactec pyrazinamide susceptibility, 24% of Wayne activity, and 51% of the Bactec 460TB growth index. In contrast, pyrazinamidase activity accounted for only 27% of the Bactec pyrazinamide susceptibility. This finding suggests that mechanisms other than pncA mutations (reduction of pyrazinamidase activity) are also implicated in pyrazinamide resistance, and that pyrazinoic acid efflux rate acts as a better proxy for pyrazinamide resistance than the presence of pncA mutations. This is relevant to the design of molecular diagnostics for pyrazinamide susceptibility, which currently rely on pncA gene mutation detection.

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Relationship between Pyrazinamide Resistance, Loss of Pyrazinamidase Activity, and Mutations in the pncA Locus in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis

Cited by 76 publications

References 15 publications

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital, Thailand

Mechanisms of Pyrazinamide Action and Resistance

Pyrazinoic acid efflux rate in Mycobacterium tuberculosis is a better proxy of pyrazinamide resistance

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