Background
Multidrug resistant tuberculosis (MDR-TB) is a major concern hampering global tuberculosis control efforts [
1,
2]. According to new estimates from World Health Organization (WHO), there were around 0.48 million new cases of MDR-TB cases, and approximately 0.19 million deaths from MDR-TB worldwide in 2014 (WHO, 2015). Only behind India, China has the second burden of MDR-TB globally, with 52, 000 prevalent MDR-TB cases annually (WHO, 2015). A recent national survey of drug-resistant tuberculosis in China revealed that 5.7% of new TB cases and 25.6% of previously treated cases were MDR-TB, respectively, which were higher than the global average rates [
3]. Given its high rate of treatment failure, the epidemic of MDR-TB constitutes a serious public health problem in China [
3,
4].
Pyrazinamide (PZA) is one of cornerstone first-line anti-tuberculosis agents that is also commonly used as essential component in the therapeutic treatment of MDR-TB [
5,
6]. As the prodrug, PZA requires conversion into its active form pyrazinoic acid (POA) by the enzyme pyrazinamidase (PZase). PZase is encoded by the 561-nucleotide
pncA gene. Loss of PZase activity cause by genetic mutations in the
pncA gene is the main mechanism of resistance to PZA in
M. tuberculosis [
6]. Several recent literatures have reported that several PZA-resistant
M. tuberculosis isolates harbored the mutations in the promoter of
pncA gene, indicating these alternations may result in PZA resistance by influencing the expression of
pncA [
7‐
9]. In addition to
pncA, another gene named
rpsA, which encodes the 30S ribosomal protein S1, has been demonstrated to confer PZA resistance by structural alternation of POA binding [
10]. To date, the contribution of the
rpsA mutations conferring PZA resistance is controversial, which requires more experimental evidences to elucidate the role of
rpsA as a PZA resistance mechanism [
8].
Chongqing is the largest municipality in the southwestern China [
11]. Due to the underdeveloped setting, Chongqing is considered as a hotspot for both TB and MDR-TB in China [
12]. However, limited data is available for the prevalence and molecular characteristics of PZA resistance in
M. tuberculosis isolates, especially MDR-TB. In this study, our main goal was to investigate the prevalence of PZA resistance among MDR-TB isolates collected in Chongqing municipality. We also sought to analyze the mutant profiles of MDR-TB isolates conferring PZA resistance in this area.
Discussion
PZA plays a unique role in the treatment for MDR-TB in both first- and second-line regimens [
19,
20]. The prevalence of PZA among MDR-TB thus is a determining factor for initiation of PZA in the therapy regimens for these refractory patients [
21]. Here, our data demonstrated that 62.4% of MDR-TB exhibited resistance against PZA in Chongqing, which was similar to a recent literature from Beijing (57.7%) [
22], while higher than those from Zhejiang (43.1%) [
8], Shanghai (38.5%) [
23], United States (38.0%) [
19], and Thailand (49.0%) [
24]. The high prevalence of PZA resistance among MDR-TB patients from our report indicates that Chongqing is a hotspot of PZA resistance in China. In our study, more than 60% MDR patients received previous anti-TB therapy with PZA, which is significantly higher than the average national level (21.8%) [
3]. Hence, we speculate that the high proportion of PZA resistance may be contributed to the high rate of re-treated TB patients. The serious issue on PZA resistance highlights the diminished role of PZA in the treatment for MDR-TB in this setting with high MDR-TB burden. Prior to the use of PZA for treatment of MDR-TB cases, it is essential to perform in vitro susceptibility testing against PZA to formulate a suitable regimen [
21].
Another important finding from our observation was that we observed that there were high correlation between PZA resistance and several other drugs’ resistance, including OFLX, second-line injectable drugs, and PAS. Similar to our findings, a recent report from Alame-Emane and colleagues has revealed that PZA resistance in
M. tuberculosis arises after RIF and fluoroquinolone (FQ) resistance [
25]. Genetic mutations constitute the most important mechanism conferring drug resistance in
M. tuberculosis [
20]. Exposure to bacterial species to antimicrobial agents, including RIF, FQ and the aminoglycosides, induces the production of oxygen radicals, thereby conferring high frequency mutagenesis [
25‐
27]. Considering long duration of anti-TB treatment, we hypothesize that MDR bacteria will harbor more genetic mutations induced by prolonged exposure to these drugs, which may be responsible for the potential cross resistance between PZA and other drugs in our study.
In vitro susceptibility against PZA is essential for proper management of MDR-TB with regimen containing PZA [
21]. However, phenotypic DST for PZA is not routinely performed due to the requirement of harshly acidic environment [
17]. Molecular method based on detecting the mutations in
pncA and
rpsA serves as an alternative to predict the PZA susceptibility in
M. tuberculosis [
9]. In this study, our data demonstrated that genetic alternations in
pncA confer 88.0% of PZA resistance among MDR-TB in Chongqing. A number of studies have demonstrated a diverse prevalence of
pncA mutation among PZA resistant isolates in different regions, ranging from 45.7% in Brazil [
28], 70.6% in Iran [
29], 75.0% in Thailand [
24], 78.0% in Zhejiang [
8], 84.6% in Southern China [
9], and 94.1% in Sweden [
30]. Hence,
pncA mutations may differ from one geographic region to another. In addition, we found that
pncA mutations exhibited great diversity, and the most frequent mutant type in codon 142 only accounted for approximate 12% of PZA resistant isolates, which was also different from reports from other regions [
8,
9]. Given the diversity of
pncA mutations within more than 500-bp long segment, DNA sequencing of the entire
pncA is more effective for verification of PZA resistance rather than the routine methods by covering the mutant hotspots.
In addition, the third frequent mutation identified in this study was located at position −11 of the
pncA promoter region. In line with our observation, numerous literatures have observed this mutant type in PZA-resistant
M. tuberculosis isolates [
31,
32]. We found that this substitution at position −11 was associated with low level of
pncA expression, which was also consistent to the observation from Sheen et al. [
31]. The anti-TB activity of PZA depends on the transformation to POA by PZAse, which is encoded by
pncA gene. The loss of
pncA transcriptional level may result in the relative low PZAse activity, which is further associated with the phenotypic PZA resistance. Our results suggest that the promoter region of
pncA is recommended to be included in the sequence analysis of
pncA gene.
We acknowledge several limitations of this study. First, the small sample size is a major limitation of our report. And all the strains collected from one region also reduce their representativeness and thereby constrict the generalizability of findings. Further wider sampling will give more credence to this study. Second, although the primary data of this study suggest that loss of
pncA expression caused by promoter mutation confers PZA resistance in MDR-TB isolates, we could give no biochemical or transgenic substantiation of this statement. Therefore, there is an urgent need to confirm our findings with more experimental evidences in the future. Third, the high diversity of
pncA mutations in MTB-TB isolates from Chongqing underscores previous findings that there is no clear hotspot for
pncA mutations [
30], and several novel mutations in
pncA gene were found for first time among PZA-resistant isolates. Despite being highly correlated with the loss of PZA susceptibility, further experiments will be carried out to clarify the potential contributions of these mutations to PZA resistance. Nevertheless, our report firstly described the molecular characteristics of PZA resistance among MDR-TB isolates from Southern China, which provides important hints to diagnose PZA resistance and help guide therapy with PZA for MDR-TB patients in this region with high MDR-TB burden.