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Phenotypic and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Ningbo, China



Detection of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis (TB) patients is critical, especially in dealing with multidrug-resistant Mycobacterium tuberculosis (MDR-TB) case. Up to date, PZA drug susceptibility testing (DST) has not been regularly performed in China. The prevalence and molecular characteristics of PZA resistance in M.tuberculosis isolates, especially MDR-TB have not been studied in Ningbo, China. This study aimed to analyze the phenotypic and molecular characterization of PZA resistance among MDR-TB isolates in Ningbo.


A total of 110 MDR-TB isolates were collected from the TB patients who were recorded at local TB dispensaries in Ningbo. All clinical isolates were examined by drug susceptibility testing and genotyping. DNA sequencing was used to detect mutations in the pncA gene associated with PZA resistance.


The prevalence of PZA resistance among MDR-TB strains in Ningbo was 59.1%. With regard to the history and the outcome of treatments among MDR-TB cases, the percentages of re-treated MDR-TB patients in the PZA-resistant group and of successful patients in PZA-susceptible group were significantly higher than the ones in the PZA-susceptible group and in the PZA-resistant group, respectively (P = 0.027, P = 0.020). The results showed that the resistance of streptomycin (67.7% vs 46.7%, P = 0.027), ethambutol (56.9% vs 33.3%, P = 0.015), ofloxacin (43.1% vs 11.1%, P = 0.000), levofloxacin (43.1% vs 11.1%, P = 0.000), pre-XDR (pre-Xtensively Drug Resistance) (38.5% vs 15.6%, P = 0.009), were more frequently adverted among PZA-resistant isolates compared with PZA-susceptible isolates. In addition, 110 MDR-TB was composed of 87 (PZA resistant, 78.5%) Beijing strains and 23 (PZA resistant, 21.5%) non-Beijing strains. Fifty-four out of 65 (83.1%) PZA-resistant MDR strains harbored a mutation located in the pncA gene and the majority (90.7%) were point mutations. Compared with the phenotypic characterization, DNA sequencing of pncA has sensitivity and specificity of 83.1 and 95.6%.


The mutations within pncA gene was the primary mechanism of PZA resistance among MDR-TB and DNA sequencing of pncA gene could provide a rapid detection evidence in PZA drug resistance of MDR-TB in Ningbo.

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Multidrug-resistant tuberculosis (MDR-TB) which is resistant to isoniazid and rifampin continues to be a great public health threat [1, 2]. World Health Organization (WHO) reported that about 0.48 million new cases of rifampicin-resistant TB, which 78% had multidrug-resistant TB, worldwide in 2018 were reported [3]. China has been the second burden in MDR-TB in the world with 66 thousands prevalent MDR-TB cases annually [3]. One recent report based on drug-resistant TB national survey in China showed that 7.1% of new TB cases and 21% of previously treated cases were MDR-TB [4]. The estimated incidence of TB was 0.04% residents in 2019 in Ningbo and the epidemic of MDR-TB has been considered one of a serious public health concerns in China because of its treatment failure [4, 5].

WHO recommends a treatment regimen including pyrazinamide (PZA) as cornerstone first-line anti-tuberculosis agent to the majority of MDR-TB patients [6, 7]. PZA as a pro-drug becomes toxic to M.tuberculosis under acidic conditions (PH = 5.5) requires conversion into its active form pyrazinoic acid with the enzyme pyrazinamidase, which was encoded by the pncA gene [8, 9]. The main genetic mechanism of pyrazinamide resistance lies in the mutations within the 561-nucleotide pncA gene [10, 11]. Because of its uniquely bactericidal effect, detection of PZA resistance in TB patients is critical, especially in dealing with MDR-TB case.

Up to date, PZA drug susceptibility testing (DST) has not been regularly investigated in China, thus, limited data has been updated reporting PZA resistance among MDR-TB isolates in the mainland of China [12, 13]. The prevalence and molecular characteristics of PZA resistance in M.tuberculosis isolates, especially MDR-TB have not been studied in Ningbo, China. The objective was to investigate the prevalence of PZA resistance among MDR-TB isolates in Ningbo and to analyze the characteristics of mutated pncA gene conferring PZA resistance.


Study design

The first study of drug-resistant TB in the entire Ningbo from 2015 to 2017 by Ningbo Center for Disease Control and Prevention (CDC). A total of 1325 participants registering and diagnosed with TB at local TB dispensaries, were recruited in the present study and all the medical and related information were collected by well-trained nurses in the local hospitals. The isolates collected from TB patients were cultured on Lowenstein-Jensen (L-J) medium for 4–8 weeks and the culture with growing colonies were delivered to the Ningbo Tuberculosis Control Institute for further drug susceptibility testing. New cases were defined as patients previously receiving none or less than one month of anti-TB treatment. Re-treated cases were defined as patients previously receiving more than one month of anti-TB treatment. One hundred and ten consecutive MDR-TB patients out of 1325 patients with full medical and microbiological information were assessed for study eligibility.

Drug susceptibility testing

Tests for susceptibility to four first-line anti-TB drugs and six second-line drugs were performed with the proportional method recommended by WHO [14]. The concentrations of drugs in L-J medium were as follows: isoniazid (INH), 0.2 μg /mL; rifampicin (RIF), 40 μg /mL; ethambutol (EMB), 2 μg /mL; streptomycin (SM), 4 μg /mL; ofloxacin (OFLX), 2 μg /mL; levofloxacin (LVX), 2 μg /mL; kanamycin (KAN), 30 μg /mL; amikacin (AMK), 30 μg /mL; capromycin (CAP), 40 μg /mL; protionamide (PTO), 40 μg /mL and p-aminosalicylic acid (PAS), 1 μg /mL [14]. The PZA drug susceptibility testing was performed with a Bactec MGIT 960 system and critical concentration was 100 μg /mL [15]. Quality control was performed during DST using the H37RV reference strains.


Drug-resistant Mycobacterium tuberculosis types

MDR-TB was defined as those resistant to both isoniazid and rifampicin. Pre-XDR TB was defined as MDR-TB additionally resistant to either quinolone family or second-line anti-TB injectable drugs. XDR-TB was defined as MDR-TB resistant to any member of the quinolone family and at least one of the remaining second-line anti-TB injectable drugs [3].

Treatment outcomes

All patients were following the standard treatment outcomes. Standard WHO outcome definitions were used to detect MDR TB including cure, treatment completion, treatment failure, causes of death, default, and transferring out [16, 17]. Successful outcomes and poor outcomes were defined as cure or treatment completion failure or death, respectively. These were considered as known outcomes, whereas unknown outcomes included default, transferring, or continuing treatment.

DNA extraction and sequencing

The crude DNA was extracted from freshly harvested bacteria [18]. The cultured bacteria was extracted from the surface of L-J medium were suspended in 500 μL Tris-EDTA (TE) buffer and heated in a 95 °C water bath for 30 min. The genomic DNA was used as template for amplification. The pncA gene was amplified with the following primers: pncA-F 5′-GTCGGTCATGTTCGCGATCG-3′ and pncA-R 5′- GCTTTGCGGGCGAGCGCTCCA-3′ [19]. The 50 μL PCR mixture was prepared as follows: 25 μL 2 × GoldStar MasterMix (CWBio, Beijing, China), 5 μL of DNA template, and 0.2 μM of each primer set. The amplifications of pncA was performed using the following conditions: 5 min of denaturation at 94 °C followed by 35 cycles (in which each cycle consisted of 94 °C for 1 min, 58 °C for 1 min, and 1 min of extension at 72 °C) and a final extension of 72 °C for 5 min. PCR products were carried out at Personalbio company (Shanghai, China). Gene polymorphisms were aligned with pncA of reference strain H37RV (ATCC) using DNAstar MegAlign (version 7.1) software.


Members of the strains of Beijing family were identified by the RD105 multiplex PCR [20].

Statistical analysis

The percentages between PZA-resistant and PZA-susceptible MDR strains resistant to SM and others were compared and analyzed by Crosstabs and Chi square test. The sensitivity and specificity of different methods were examined by Wilson score confidence interval method. Results were considered statistically significant at a two-tailed level of 0.05. Statistical analysis were conducted using SPSS 21.0 (SPSS., USA).


Demographic characteristics and drug susceptibility profiles

Totally, 110 (8.3%) out of 1325 clinical isolates were identified as MDR-TB, including 29.1% pre-XDR and 6.4% XDR. Around 70.0% strains were isolated from male patients (Table 1). The average age of the 110 MDR-TB patients was 46.4 years (range 19–85 years). Around 40.9% isolates were from new cases. Among 110 MDR-TB clinical isolates, 59.1, 47.3, 30.0, 30.0, 8.2, 7.3, 3.6, 1.8 and 5.5% were resistant to SM, EMB, OFLX, LVX, KAN, AMK, CAP, PTO and PAS, respectively.

Table 1 Risk factor associate with pyrazinamide resistance among 110 MDR isolates

With regard to the treatment history and treatment outcomes among MDR-TB cases, the percentage of re-treated MDR-TB patients in the PZA-resistant group was significantly higher than those in the PZA-susceptible group (P = 0.027), and the percentage of treatment successful patients in PZA-susceptible group was significantly higher than in the PZA-resistant group(P = 0.020).

The resistance phenotypic of other drugs between PZA-resistant and PZA-susceptible groups were further examined (Table 1). The resistance of streptomycin-(P = 0.027), ethambutol-(P = 0.015), ofloxacin-(P<0.001), levofloxacin-(P<0.001) and pre-XDR-(P = 0.009) were more frequently detected among PZA-resistant groups compared with PZA-susceptible groups.

Mutations in the pncA gene

Totally, 50.9% MDR-TB isolates were observed a mutation located in the pncA gene, including 91.1% of single nucleotide substitutions and 8.9% of frame-shift mutation.

Great mutant diversity in pncA gene was observed and 40 different mutant types conferred PZA resistance among MDR strains in Ningbo (Table 2). Two PZA-susceptible isolates harbored a genetic mutation in pncA gene, including 1 strains in codon 8 and 1 strains in codon 76. Considering the phenotypic PZA susceptibility as a gold standard, detection of mutation in pncA gene exhibited a sensitivity of 83.1% and a specificity of 95.6% (Table 3).

Table 2 Mutations of PncA gene among MDR-TB isolates
Table 3 Performance of PncA mutations for predicting PZA susceptibility


PZA as an important first-line anti-tuberculosis drug plays a crucial role in the therapeutic treatment of MDR-TB [21, 22]. Considering the unique effect of PZA, the detection of PZA among MDR-TB is a significant factor for initiation of PZA in the therapy regimens for these refractory patients [23, 24]. This study showed that the PZA resistance rate among MDR-TB in Ningbo was 59.1%, which higher than those in Zhejiang Province (43.1%) [25], Shanghai (38.5%) [26], Thailand (49.0%) [27], United states (38.0%) [28], and similar to a recent result from Beijing (57.7%) [29].

About 60% MDR patients received previous anti-TB therapy with PZA in our study, which is significantly higher than the national level (21.8%) [4]. Our results demonstrated that the high frequency of PZA resistance may contribute to the high rate of re-treated TB patients. Therefore, we proposed to diminish the role of PZA in the treatment for MDR-TB in Ningbo. It was necessary to formulate a suitable regimen by detecting PZA resistance before using of PZA for treatment of MDR-TB cases.

Bacterial species induces the production of oxygen radicals which result in high frequency mutagenesis by exposing to antimicrobial agents, including RIF, FQ and the aminoglycosides [30, 31]. A recent report from Alame-Emane and colleagues reveled that PZA resistance in M.tuberculosis arised after RIF and fluoroquinolone (FQ) resistance [32]. In line with our findings, one recent result from our observation was that there were high correlation between PZA resistance and several other drugs resistance, including SM, EMB, OFLX and LVX. Regarding long duration of anti-TB treatment, MDR bacteria existed more genetic mutations which may be responsible for the potential cross resistance between PZA and other drugs in our study. It is necessary to perform PZA susceptibility testing for proper management of MDR-TB with regimen containing PZA. However, traditional PZA drug susceptibility testing is not routinely performed due to the requirement of harshly acidic environment which many isolates of M.tuberculosis failed to grow [33]. As an alternative testing to predict the PZA susceptibility in M.tuberculosis, Molecular method based on detecting the mutation in pncA is essential [34, 35].

Previous study showed that genetic mutations constituted the most import mechanism conferring drug resistance in M.tuberculosis [36]. The result from our study suggested that genetic alternations in pncA confer 83.1% of PZA resistance among MDR-TB in Ningbo. Previous studies demonstrated a diverse prevalence of pncA mutation among PZA resistance isolates in different regions, ranging from 45.7% in Brazil [37], 70.6% in Iran [38], 75.0% in Thailand [27], 78.0% in Zhejiang [25], 84.6% in Southern China [39], and 94.1% in Sweden [40]. Additionally, pncA mutations exhibited great diversity in our study, although pncA mutations were not statistically significant between 2 genotypes - Beijing family and non-Beijing family. Hence, DNA sequencing of the entire pncA was more effective for detection of PZA resistance rather than the routine methods by covering the mutant hot-sports. Moreover, other reports suggested that many MDR-TB and XDR-TB outbreaks were caused by strains of the Beijing family which had an increased tendency to develop drug resistance [41, 42]. However, the data from our study showed that there was no significant difference between PZA-resistant and PZA-susceptible group in genotype. The genetic background capable of accumulating resistance was not observed in our study. The effect of PZA resistance during chemotherapy on treatment outcomes are still lack of evidence [43, 44]. Our subjects were compared with successful and poor treatment outcomes in terms of resistance to PZA in this study. According to our observations, treatment outcomes were significantly better with PZA susceptible MDR patients, suggest the need for PZA resistance test to optimize treatment.

To our knowledge, this study was the first investigation on phenotypic and molecular characterization of PZA resistance among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Ningbo. Nonetheless, some limitations of this study need to be considered. First, the small sample size cannot be representative for TB patients in the whole Ningbo. Second, only MDR-TB isolates were detected in our study. Therefore, further experiments need to be analyzed the contributions of pncA mutations to PZA resistance in non-MDR isolates. However, this study provides critical evidence to diagnose PZA resistance and help guide the treatment with PZA for MDR-TB patients in this region with high MDR-TB burden.


The sequencing of the pncA gene in our study provided rapid and reliable information against PZA susceptibility for MDR-TB isolates in Ningbo. The PZA-resistant isolates in MDR-TB were likely to have concomitant resistance to streptomycin, ethambutol, ofloxacin, levofloxacin and pre-XDR. DNA sequencing of the entire pncA was more effective to predict PZA resistance rather than the routine methods by covering the mutant hotspots because of its high degree of diversity in pncA gene. The high prevalence of PZA resistance among MDR suggested that we should adjust PZA in a timely and accurate manner in a treatment regimen for MDR-TB in this setting with TB burden. Future study should aim to clarify the potential contributions of other gene mutations to PZA resistance caused by various medication treatments.

Availability of data and materials

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World Health Organization






















Pre-extensively extensive drug resistance


Extensive drug resistance


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We sincerely thank all the participants in this project.


This research was supported by Natural Science Foundation of Ningbo (grant:2019A610385) and Ningbo Health Branding Subject Fund (PPXK2018–10). The funder had no role in study design, data collection, analysis, interpretation of data and writing the manuscript.

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YC participated in the design of the study, performed the analysis and interpretation of the data, drafted and revised the manuscript. DB, XL and TC participated in the analysis and interpretation of the data. TH and YL participated in the collection of data, revised the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Tianfeng He or Yi Lin.

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Che, Y., Bo, D., Lin, X. et al. Phenotypic and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Ningbo, China. BMC Infect Dis 21, 605 (2021).

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