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Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China

Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant... Background: Pyrazinamide (PZA) plays a unique role in the treatment for multidrug-resistant tuberculosis (MDR-TB) in both first- and second-line regimens. The aim of this study was to investigate the prevalence and molecular characterization of PZA resistance among MDR-TB isolates collected in Chongqing municipality. Methods: A total of 133 MDR-TB isolates were collected from the smear-positive tuberculosis patients who were registered at local TB dispensaries of Chongqing. PZA susceptibility testing was determined with a Bactec MGIT 960 system. In addition, the genes conferring for PZA resistance were screened by DNA sequencing. Results: Of these 133 MDR-TB isolates, 83 (62.4%) were determined as PZA-resistant by MGIT 960. In addition, streptomycin- (83.1% vs. 56.0%, P < 0.01), ofloxacin- (51.8% vs. 18.0%, P < 0.01), kanamycin- (22.9% vs. 2.0%, P <0.01), amikacin- (18.1% vs. 2.0%, P = 0.01), capromycin-resistance (12.0% vs. 2.0%, P =0.05), were morefrequently observed among PZA-resistant isolates compared with PZA-susceptible isolates. Sequence analysis revealed that 73 out of 83 (88. 0%) MDR strains harbored a mutation located in the pncA gene, including 55 (75.3%, 55/73) of single nucleotide substitutions and 18 (24.7%, 18/73) of frameshift mutation, while no genetic mutation associated with PZA resistance was found in the rpsA gene. The pncA expression of strains harboring substitution from A to G at position −11 in the promoter region of pncA was significantly lower than that of H37Rv (P <0.01). Conclusions: In conclusion, our data have demonstrated that the analysis of the pncA gene rather than rpsA gene provides rapid and accurate information regarding PZA susceptibility for MDR-TB isolates in Chongqing. In addition, loss of pncA expression caused by promoter mutation confers PZA resistance in MDR-TB isolates. Background Only behind India, China has the second burden of MDR- Multidrug resistant tuberculosis (MDR-TB) is a major con- TB globally, with 52, 000 prevalent MDR-TB cases annu- cern hampering global tuberculosis control efforts [1, 2]. ally (WHO, 2015). A recent national survey of drug- According to new estimates from World Health resistant tuberculosis in China revealed that 5.7% of new Organization (WHO), there were around 0.48 million new TB cases and 25.6% of previously treated cases were MDR- cases of MDR-TB cases, and approximately 0.19 million TB, respectively, which were higher than the global average deaths from MDR-TB worldwide in 2014 (WHO, 2015). rates [3]. Given its high rate of treatment failure, the epi- demic of MDR-TB constitutes a serious public health problem in China [3, 4]. * Correspondence: [email protected]; [email protected] Equal contributors Pyrazinamide (PZA) is one of cornerstone first-line Clinical Laboratory, Chongqing Tuberculosis Control Institute, No. 71, anti-tuberculosis agents that is also commonly used as Longteng Street, Jiulongpo District, Chongqing 400050, People’s Republic of essential component in the therapeutic treatment of China National Center for Tuberculosis Control and Prevention, Chinese Center for MDR-TB [5, 6]. As the prodrug, PZA requires conver- Disease Control and Prevention, No. 155, Chang Bai Road, Changping sion into its active form pyrazinoic acid (POA) by the District, Beijing 102206, People’s Republic of China enzyme pyrazinamidase (PZase). PZase is encoded by Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pang et al. BMC Infectious Diseases (2017) 17:711 Page 2 of 8 the 561-nucleotide pncA gene. Loss of PZase activity testing was determined with a Bactec MGIT 960 system cause by genetic mutations in the pncA gene is the main according to the manufacturer’sinstructions. Thecritical mechanism of resistance to PZA in M. tuberculosis [6]. concentration of PZA in the liquid medium was 100 μg/ Several recent literatures have reported that several ml [8]. The MDR-TB strains were defined as those resist- PZA-resistant M. tuberculosis isolates harbored the mu- ant to both isoniazid and rifampicin. In addition, XDR-TB tations in the promoter of pncA gene, indicating these was defined as MDR-TB resistant to any member of the alternations may result in PZA resistance by influencing quinolone family and at least one of the remaining the expression of pncA [7–9]. In addition to pncA,an- second-line anti-TB injectable drugs [15]. other gene named rpsA, which encodes the 30S riboso- mal protein S1, has been demonstrated to confer PZA DNA extraction and amplification resistance by structural alternation of POA binding [10]. Genomic DNA was extracted from freshly cultured bac- To date, the contribution of the rpsA mutations confer- teria as previously described [16]. Briefly, the harvested ring PZA resistance is controversial, which requires bacteria from the surface of L-J medium were suspended more experimental evidences to elucidate the role of in 500 μl Tris-EDTA (TE) buffer and heated in a 95 °C rpsA as a PZA resistance mechanism [8]. water bath for 30 min. The crude DNA was used as tem- Chongqing is the largest municipality in the south- plate for amplification. The fragments of pncA and rpsA western China [11]. Due to the underdeveloped setting, were amplified with the following primers: pncA-F 5′- Chongqing is considered as a hotspot for both TB and AACAGTTCATCCCGGTTC-3′ and pncA-R 5′- MDR-TB in China [12]. However, limited data is avail- GCGTCATGGACCCTATATC-3′; rpsA-F 5′-CGGAG- able for the prevalence and molecular characteristics of CAACCCAACAATA-3′ and rpsA-R 5′-GTGGACAG- PZA resistance in M. tuberculosis isolates, especially CAACGACTTC-3′, respectively [17]. The 50 μL PCR MDR-TB. In this study, our main goal was to investigate mixture was prepared as follows: 25 μl 2 × GoldStar Mas- the prevalence of PZA resistance among MDR-TB terMix (CWBio, Beijing, China), 5 μL of DNA template, isolates collected in Chongqing municipality. We also and 0.2 μM of each primer set. PCR parameters for ampli- sought to analyze the mutant profiles of MDR-TB fication were 5 min at 94 °C followed by 35 cycles of 94 °C isolates conferring PZA resistance in this area. for 1 min, 58 °C for 1 min, 72 °C for 1 min and a final extension of 72 °C for 5 min. PCR products were sent to Methods Tsingke company for sequencing. All sequence data were Patient enrollment aligned with pncA and rpsA of reference strain H37Rv All smear-positive tuberculosis patients who were regis- (ATCC) using BioEdit (version 7.1.3.0) software. tered at local TB dispensaries between November 2014 and February 2016 were enrolled in this study. Informa- Quantitative reverse transcription PCR (QRT-PCR) tion was obtained from patient’s medical record. Two Bacteria were harvested from L-J medium after inocula- sputum samples were obtained from each smear-positive tion for 4 weeks. The total RNA was isolated following a patient for culturing in the county-level laboratories. standard Trizol RNA extraction protocol supplied by Invi- After 4–8 weeks of incubation, cultures with growing trogen (Invitrogen, Life Technologies, USA) [18]. Followed colonies on Löwenstein-Jensen (L-J) medium were sent by treatment with DNaseI (Invitrogen, Life Technologies, to the Chongqing Tuberculosis Control Institute for USA), the reverse transcription was carried out using further drug susceptibility testing. Re-treated cases were SuperScriptIII RT kit (Invitrogen, Life Technologies, defined as patients having previously received more than USA). The relative expression level of pncA gene was one month of anti-TB treatment. detected by QRT-PCR in a 20 μl system containing 10 μL of 2 × UltraSYBR Mixture (CWBio, Beijing, China), 2 μL Drug susceptibility testing of cDNA template, and 0.2 μM of each primer set (pncA- Drug susceptibility testing (DST) was performed with the QF 5′-GAAGCGGCGGACTACCATC-3′ and pncA-QR proportional method recommended by WHO [3, 13]. The 5′-AGTGGCGTGCCGTTCTCG-3′). PolyA was set as concentrations of drugs in L-J medium were as follows: the internal control in respective PCR experiments [18]. isoniazid (INH), 0.2 μg/ml; rifampicin (RIF), 40 μg/ml; ethambutol (EMB), 2 μg/ml; streptomycin (SM), 4 μg/ml; Data analysis ofloxacin (OFLX), 2 μg/ml; kanamycin (KAN), 30 μg/ml; Chi square test was used to evaluate the associations amikacin (AMK), 30 μg/ml; capromycin (CAP), 40 μg/ml; among multiple categorical variables, and the statistical re- and protionamide (PTO), 40 μg/ml and p-aminosalicylic sults were summarized with odds ratios (ORs) with 95% acid (PAS) 1 μg/ml [14]. A strain was declared resistant to confidence intervals (CIs). All calculations were performed an antimicrobial agent when the growth rate exceeded 1% in SPSS 13.0 (SPSS Inc., USA). Differences with a P value compared with the control. In addition, PZA susceptibility less than 0.05 were declared statistically significant. Pang et al. BMC Infectious Diseases (2017) 17:711 Page 3 of 8 Results revealed no statistically significant difference between Demographic characteristics and drug susceptibility profiles the PZA-resistant and PZA-susceptible group in gender A total of 133 (10.8%) of 1236 clinical isolates were iden- and age (P > 0.05). tified as MDR-TB, including 38 (28.6%) pre-XDR and 17 We further analyzed the resistance profiles of other (12.8%) XDR. Overall, 80 (60.2%) strains were isolated drugs among PZA-resistant and PZA-susceptible M. tu- from male patients and 53 (39.8%) from female patients. berculosis strains. Statistical analysis revealed that SM- The average age of the patients was 46.6 years (range (83.1% vs. 56.0%, P < 0.01), OFLX- (51.8% vs. 18.0%, 19–76 years). In addition, 36.8% of isolates were from P < 0.01), KAN- (22.9% vs. 2.0%, P < 0.01), AMK- new cases, and 63.2% from re-treated cases. Out of the (18.1% vs. 2.0%, P = 0.01), CAP-resistance (12.0% vs. 133 MDR isolates tested, 97 (72.9%) were resistant to 2.0%, P = 0.05), pre-XDR (36.1% vs. 16.0%, P < 0.02) and SM, 40 (30.1%) to EMB, 52 (39.1%) to OFLX, 20 (15.0%) XDR (19.3% vs. 2.0%, P < 0.01) were more frequently ob- to KAN, 16 (12.0%) to AMK, 11 (8.3%) to CAP and 15 served among PZA-resistant isolates compared with (11.3%) to PAS (Table 1). PZA-susceptible isolates, while no difference was identi- fied between EMB-resistant and EMB-susceptible strains Factors associated with PZA resistance (31.3% vs. 28.0%, P = 0.69) (Table 1). As shown in Table 1, 83 (62.4%) isolates were deter- mined as PZA-resistant by MGIT, while the other 50 Mutations in the pncA and rpsA gene (37.6%) were susceptible to PZA. In regard to the distri- A total of 73 out of 83 (88.0%) MDR strains harbored a bution of MDR-TB cases treatment history, the percent- mutation located in the pncA gene, including 55 (75.3%, age of re-treated MDR-TB patients in the PZA-resistant 55/73) of single nucleotide substitutions and 18 (24.7%, group was significantly higher than in the PZA- 18/73) of frameshift mutation. As summarized in Table 2, susceptible group (odd ratio (OR) [95% confidence inter- we observed great mutant diversity in pncA gene, and val (CI): 3.26[1.55–6.82], P < 0.01). In contrast, our data there were 48 different mutant types conferring PZA Table 1 Risk factor associate with PZA resistance among 133 MDR isolates Characteristics No. (%) of isolates No. (%) of isolates OR P value (n = 133) R S (95% CI) PZA PZA (n = 83) (n = 50) Sex Male 80 (60.2) 47 (56.6) 33 (66.0) 0.67 (0.33–1.39) 0.29 Female 53 (39.8) 36 (43.4) 17 (34.0) 1.0 (Ref.) – Age group < 30 31 (23.3) 18 (21.7) 13 (26.0) 1.0 (Ref.) – 30–59 80 (60.2) 55 (66.3) 25 (50.0) 1.59 (0.68–3.74) 0.29 ≥ 60 22 (16.5) 10 (12.0) 12 (24.0) 0.60 (0.20–1.81) 0.36 Treatment history New case 49 (36.8) 22 (26.5) 27 (54.0) 1.00 (Ref.) – Re-treated 84 (63.2) 61 (73.5) 23 (46.0) 3.26 (1.55–6.82) <0.01 Resistance to: SM 97 (72.9) 69 (83.1) 28 (56.0) 3.87 (1.74–8.63) <0.01 EMB 40 (30.1) 26 (31.3) 14 (28.0) 1.17 (0.54–2.54) 0.69 OFLX 52 (39.1) 43 (51.8) 9 (18.0) 4.89 (2.11–11.35) <0.01 KAN 20 (15.0) 19 (22.9) 1 (2.0) 14.58 (1.88–112.44) <0.01 AMK 16 (12.0) 15 (18.1) 1 (2.0) 10.81 (1.38–84.58) 0.01 CAP 11 (8.3) 10 (12.0) 1 (2.0) 6.71 (0.83–54.12) 0.05 PAS 15 (11.3) 13 (15.7) 2 (4.0) 4.46 (0.96–20.65) 0.04 Pre-XDR 38 (28.6) 30 (36.1) 8 (16.0) 2.97 (1.23–7.16) 0.02 XDR 17 (12.8) 16 (19.3) 1 (2.0) 11.70 (1.50–91.22) <0.01 MDR is defined as Mycobacterium tuberculosis strain resistant to at least isoniazid and rifampin Pre-XDR is defined as MDR strain additionally resistant to either ofloxacin or kanamycin, but not both XDR is defined as Mycobacterium tuberculosis strain resistant to isoniazid, rifampin, ofloxacin and kanamycin Pang et al. BMC Infectious Diseases (2017) 17:711 Page 4 of 8 Table 2 Mutations of PZA-resistant MDR-TB isolates within pncA Locus Position of nucleotide Nucleotide substitution Position of amino acid Amino acid substitution No. of isolates pncA −11 TAT → TGT −4 Tyr → Cys 4 2 ATG → ACG 1 Met → Thr 1 20 GTC → GGC 7 Val → Gly 2 24 GAC → GAG 8 Asp → Glu 1 28 CAG → TAG 10 Gln → Stop 1 35 GAC → GCC 12 Asp → Ala 2 37 TTC → GTC 13 Phe → Val 1 40 TGC → CGC 14 Cys → Arg 2 40 TGC → CGC 14 Cys → Arg 1 94 TTC → GTC 32 Phe → Val 1 123 TAC → TAG 41 Tyr → Stop 1 146 GAC → GCC 49 Asp → Gly 1 146 GAC → GCC 49 Asp → Ala 2 151 CAC → TAC 51 His → Tyr 1 151 CAC → CGC 51 His → Arg 1 152 CAC → CCC 51 His → Pro 1 170 CAC → CGC 57 His → Arg 1 185 CCG → CTG 62 Pro → Leu 5 206 CCG → CTG 69 Pro → Leu 1 213 CAT → CAG 71 His → Gln 1 226 ACT → CCT 76 Thr → Pro 3 232 GGC → AGC 78 Gly → Asp 1 245 CAT → CGT 82 His → Arg 1 286 AAG → CAG 96 Lys → Gln 1 307 TAC → CAC 103 Tyr → His 1 309 TAC → TAG 103 Tyr → Stop 1 319 GAA → AAA 107 Glu → Lys 1 395 GGT → GAT 132 Gly → Asp 9 425 ACG → ATG 142 Thr → Met 1 437 GCG → GTG 146 Ala → Val 1 464 GTG → GGG 155 Val → Gly 1 488 GTG → GCG 163 Val → Ala 1 515 CTG → CCG 172 Leu → Pro 2 52 insertion of GC 1 130 insertion of C 1 136 deletion of G 2 139 insertion of CA 1 232 insertion of C 1 243 insertion of T 1 288 insertion of A 1 341 deletion of ACGCC 1 342 deletion of GCCAC 2 376 deletion of GATGAGGTC 1 392 insertion of G 1 Pang et al. BMC Infectious Diseases (2017) 17:711 Page 5 of 8 Table 2 Mutations of PZA-resistant MDR-TB isolates within pncA (Continued) Locus Position of nucleotide Nucleotide substitution Position of amino acid Amino acid substitution No. of isolates 392 insertion of GG 1 393 insertion of GGT 1 408 insertion of CA 1 408 insertion of A 2 Total 73 resistance among MDR strains in Chongqing. The most prevalence of PZA among MDR-TB thus is a determin- prevalent mutation associated with PZA resistance was ing factor for initiation of PZA in the therapy regimens found in codon 132 of pncA (12.3%, 9/73), resulting in for these refractory patients [21]. Here, our data demon- the amino aicd substitution of Gly to Asp. As the second strated that 62.4% of MDR-TB exhibited resistance affected codon, five isolates had a mutation in codon 62 against PZA in Chongqing, which was similar to a recent (6.8%, 5/73). Notably, the third frequent mutation was literature from Beijing (57.7%) [22], while higher than the nucleotide substitution from A to G at position −11 those from Zhejiang (43.1%) [8], Shanghai (38.5%) [23], in the promoter region of pncA, accounting for 5.4% of United States (38.0%) [19], and Thailand (49.0%) [24]. mutant isolates. We also found that 4 PZA-susceptible The high prevalence of PZA resistance among MDR-TB isolates carried a genetic mutation in pncA, including 2 patients from our report indicates that Chongqing is a strains in codon 62, one in codon 67 and one in codon hotspot of PZA resistance in China. In our study, more 154 (Table 3). In addition, no genetic mutation associ- than 60% MDR patients received previous anti-TB ther- ated with PZA resistance was found in the rpsA gene in apy with PZA, which is significantly higher than the this study. average national level (21.8%) [3]. Hence, we speculate We analyzed the performance of pncA mutations for that the high proportion of PZA resistance may be con- predicting PZA susceptibility. When setting the pheno- tributed to the high rate of re-treated TB patients. The typic PZA susceptibility as a gold standard, we found serious issue on PZA resistance highlights the dimin- that detection of mutations in pncA gene exhibited a ished role of PZA in the treatment for MDR-TB in this sensitivity of 88.0% (95% CI, 80.9%–95.0%) and a specifi- setting with high MDR-TB burden. Prior to the use of city of 92.0% (95% CI, 84.5%–99.5%) (Table 4). PZA for treatment of MDR-TB cases, it is essential to perform in vitro susceptibility testing against PZA to for- Loss of pncA expression due to promoter mutation mulate a suitable regimen [21]. We further explore whether the substitution at position Another important finding from our observation was −11 affected the expression level of pncA. As shown in that we observed that there were high correlation be- Fig. 1, compared with reference strain H37Rv, the rela- tween PZA resistance and several other drugs’ resist- tive expression level of pncA in MDR87, MDR88, ance, including OFLX, second-line injectable drugs, and MDR114 and MDR126 carried the substitution at pos- PAS. Similar to our findings, a recent report from ition −11 were 0.25 fold, 0.19 fold, 0.36 fold and 0.22 Alame-Emane and colleagues has revealed that PZA re- fold, respectively. Statistical analysis revealed that the sistance in M. tuberculosis arises after RIF and fluoro- pncA expression of strains harboring promoter mutation quinolone (FQ) resistance [25]. Genetic mutations at position −11 was significantly lower than that of constitute the most important mechanism conferring H37Rv (P < 0.01). drug resistance in M. tuberculosis [20]. Exposure to bac- terial species to antimicrobial agents, including RIF, FQ Discussion and the aminoglycosides, induces the production of oxy- PZA plays a unique role in the treatment for MDR-TB gen radicals, thereby conferring high frequency muta- in both first- and second-line regimens [19, 20]. The genesis [25–27]. Considering long duration of anti-TB Table 3 Mutations in PZA-susceptible MDR-TB isolates within pncA gene Locus Position of nucleotide Nucleotide substitution Position of amino acid Amino acid substitution No. of isolates pncA 184 CCG → ACG 62 Pro → Thr 1 185 CCG → CAG 62 Pro → Gln 1 200 TCG → TGG 67 Ser → Trp 1 461 AGG → AAG 154 Arg → Lys 1 Total 4 Pang et al. BMC Infectious Diseases (2017) 17:711 Page 6 of 8 Table 4 Performance of pncA mutations for predicting PZA susceptibility Mutation PZA susceptibility Total Sensitivity Specificity PPV NPV in pncA (95% CI, %) (95% CI, %) (95% CI, %) (95% CI, %) RS Yes 73 4 77 88.0 92.0 94.8 82.1 (80.9–95.0) (84.5–99.5) (89.8–99.8) (72.1–92.2) No 10 46 56 Total 83 50 133 R, resistant, S susceptible, PPV positive predictive value, NPV negative predictive value, CI confidence interval treatment, we hypothesize that MDR bacteria will harbor than the routine methods by covering the mutant more genetic mutations induced by prolonged exposure hotspots. to these drugs, which may be responsible for the poten- In addition, the third frequent mutation identified in tial cross resistance between PZA and other drugs in this study was located at position −11 of the pncA our study. promoter region. In line with our observation, numerous In vitro susceptibility against PZA is essential for literatures have observed this mutant type in PZA- proper management of MDR-TB with regimen contain- resistant M. tuberculosis isolates [31, 32]. We found that ing PZA [21]. However, phenotypic DST for PZA is not this substitution at position −11 was associated with low routinely performed due to the requirement of harshly level of pncA expression, which was also consistent to acidic environment [17]. Molecular method based on the observation from Sheen et al. [31]. The anti-TB ac- detecting the mutations in pncA and rpsA serves as an tivity of PZA depends on the transformation to POA by alternative to predict the PZA susceptibility in M. tuber- PZAse, which is encoded by pncA gene. The loss of culosis [9]. In this study, our data demonstrated that pncA transcriptional level may result in the relative low genetic alternations in pncA confer 88.0% of PZA resist- PZAse activity, which is further associated with the ance among MDR-TB in Chongqing. A number of stud- phenotypic PZA resistance. Our results suggest that the ies have demonstrated a diverse prevalence of pncA promoter region of pncA is recommended to be in- mutation among PZA resistant isolates in different re- cluded in the sequence analysis of pncA gene. gions, ranging from 45.7% in Brazil [28], 70.6% in Iran We acknowledge several limitations of this study. First, [29], 75.0% in Thailand [24], 78.0% in Zhejiang [8], the small sample size is a major limitation of our report. 84.6% in Southern China [9], and 94.1% in Sweden [30]. And all the strains collected from one region also reduce Hence, pncA mutations may differ from one geographic their representativeness and thereby constrict the region to another. In addition, we found that pncA mu- generalizability of findings. Further wider sampling will tations exhibited great diversity, and the most frequent give more credence to this study. Second, although the mutant type in codon 142 only accounted for approxi- primary data of this study suggest that loss of pncA mate 12% of PZA resistant isolates, which was also dif- expression caused by promoter mutation confers PZA ferent from reports from other regions [8, 9]. Given the resistance in MDR-TB isolates, we could give no bio- diversity of pncA mutations within more than 500-bp chemical or transgenic substantiation of this statement. long segment, DNA sequencing of the entire pncA is Therefore, there is an urgent need to confirm our find- more effective for verification of PZA resistance rather ings with more experimental evidences in the future. Fig. 1 Relative expression level of pncA gene in 4 M. tuberculosis isolates with mutation in the position −11 of promoter region Pang et al. BMC Infectious Diseases (2017) 17:711 Page 7 of 8 Third, the high diversity of pncA mutations in MTB-TB Competing interests The authors declare that they have no competing interests. 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 Publisher’sNote for first time among PZA-resistant isolates. Despite be- Springer Nature remains neutral with regard to jurisdictional claims in ing highly correlated with the loss of PZA susceptibility, published maps and institutional affiliations. further experiments will be carried out to clarify the po- Author details tential contributions of these mutations to PZA resist- 1 National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on ance. Nevertheless, our report firstly described the Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China. molecular characteristics of PZA resistance among Clinical Laboratory, Chongqing Tuberculosis Control Institute, No. 71, MDR-TB isolates from Southern China, which provides Longteng Street, Jiulongpo District, Chongqing 400050, People’s Republic of important hints to diagnose PZA resistance and help China. National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Chang Bai Road, guide therapy with PZA for MDR-TB patients in this re- Changping District, Beijing 102206, People’s Republic of China. gion with high MDR-TB burden. Received: 26 February 2017 Accepted: 22 September 2017 Conclusion In conclusion, our data have demonstrated that the ana- lysis of the pncA gene rather than rpsA gene provides References 1. Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, Jensen rapid and accurate information regarding PZA suscepti- P, Bayona J. Multidrug-resistant and extensively drug-resistant tuberculosis: a bility for MDR-TB isolates in Chongqing. In addition, threat to global control of tuberculosis. Lancet. 2010;375(9728):1830–43. loss of pncA expression caused by promoter mutation 2. Zhang Z, Pang Y, Wang Y, Liu C, Zhao Y. Beijing genotype of Mycobacterium tuberculosis is significantly associated with linezolid confers PZA resistance in MDR-TB isolates. Considering resistance in multidrug-resistant and extensively drug-resistant tuberculosis the high degree of diversity of pncA gene mutations, in China. Int J Antimicrob Agents. 2014;43(3):231–5. DNA sequencing of the entire pncA is more effective for 3. Zhao Y, Xu S, Wang L, Chin DP, Wang S, Jiang G, Xia H, Zhou Y, Li Q, Ou X, et al. National survey of drug-resistant tuberculosis in China. N Engl J Med. verification of PZA resistance rather than the routine 2012;366(23):2161–70. methods by covering the mutant hotspots. The high 4. Orenstein EW, Basu S, Shah NS, Andrews JR, Friedland GH, Moll AP, Gandhi prevalence of PZA resistance among MDR highlights NR, Galvani AP. Treatment outcomes among patients with multidrug- resistant tuberculosis: systematic review and meta-analysis. Lancet Infect Dis. the diminished role of PZA in the treatment for MDR- 2009;9(3):153–61. TB in this setting with high TB burden. 5. Verdugo D, Fallows D, Ahuja S, Schluger N, Kreiswirth B, Mathema B. Epidemiologic Correlates of Pyrazinamide-Resistant Mycobacterium Abbreviations tuberculosis in New York City. Antimicrob Agents Chemother. 2015;59(10): AMK: Amikacin; CAP: Capromycin; CIs: Confidence intervals; DST: Drug 6140–50. susceptibility testing; EMB: Ethambutol; INH: Isoniazid; KAN: Kanamycin; MDR- 6. Zhang Y, Mitchison D. The curious characteristics of pyrazinamide: a review. TB: Multidrug-resistant tuberculosis; OFLX: Ofloxacin; ORs: Odds ratios; PAS: P- Int J Tuberc Lung Dis. 2003;7(1):6–21. aminosalicylic acid; POA: Pyrazinoci acid; PTO: Protionamide; 7. Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/ PZA: Pyrazinamide; PZase: Pyrazinamidase; RIF: Rifampicin; SM: Streptomycin nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat Med. 1996;2(6):662–7. Acknowledgements 8. Xia Q, Zhao LL, Li F, Fan YM, Chen YY, Wu BB, Liu ZW, Pan AZ, Zhu M. We would like to thank the staffs from the National Tuberculosis Reference Phenotypic and genotypic characterization of pyrazinamide resistance Laboratory for their technical support. among multidrug-resistant Mycobacterium tuberculosis isolates in Zhejiang. China Antimicrob Agents Chemother. 2015;59(3):1690–5. Funding 9. Tan Y, Hu Z, Zhang T, Cai X, Kuang H, Liu Y, Chen J, Yang F, Zhang K, Tan S, This study was supported by the Major State Basic Research Development et al. Role of pncA and rpsA gene sequencing in detection of pyrazinamide Program of China (2014CB744403). resistance in Mycobacterium tuberculosis isolates from southern China. J Clin Microbiol. 2014;52(1):291–7. Availability of data and materials 10. Shi W, Zhang X, Jiang X, Yuan H, Lee JS, Barry CE 3rd, Wang H, Zhang W, The datasets used and/or analysed during the current study available from Zhang Y. Pyrazinamide inhibits trans-translation in Mycobacterium the corresponding author on reasonable request. tuberculosis. Science. 2011;333(6049):1630–2. 11. Zhang D, An J, Wang J, Hu C, Wang Z, Zhang R, Wang Y, Pang Y. Molecular Authors’ contributions typing and drug susceptibility of Mycobacterium tuberculosis isolates from Conceived and designed the experiments: YP, DZ, JL, YL. Performed the Chongqing Municipality, China. Infect Genet Evol. 2013;13:310–6. experiments: YP, DZ, HZ, JS. YH, JL. Analyzed the data: YP, DZ, JL, YL. Wrote 12. Zhang D, An J, Wang Y, Pang Y. Genetic diversity of multidrug-resistant the paper: YP, DZ, JL, YL. All authors contributed to the final manuscript. All tuberculosis in a resource-limited region of China. Int J Infect Dis. authors read and approved the final manuscript. 2014;29:7–11. 13. World Health Organization: Global tuberculosis report 2015. Geneva: World Ethics approval and consent to participate Health Organization; 2015. This study was approved by the Ethics Committee of the Chongqing 14. World Health Organization. Anti-tuberculosis drug resistance in the world. Tuberculosis Control Institute. Patients enrolled in this study provided the Geneva: World Health Organization; 2008. signed Informed Consent form. 15. Pang Y, Zhou Y, Zhao B, Liu G, Jiang G, Xia H, Song Y, Shang Y, Wang S, Zhao YL. Spoligotyping and drug resistance analysis of Mycobacterium Consent for publication tuberculosis strains from national survey in China. PLoS One. 2012;7(3): Not applicable. e32976. Pang et al. BMC Infectious Diseases (2017) 17:711 Page 8 of 8 16. Zhang Z, Lu J, Wang Y, Pang Y, Zhao Y. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother. 2014;58(1):364–9. 17. Pang Y, Wang Z, Zheng H, Song Y, Wang Y, Zhao Y. Pyrazinamide resistance determined by liquid culture at low pH better correlates with genetic mutations in MDR tuberculosis isolates. J Microbiol Methods. 2015;119:142–4. 18. Pang Y, Lu J, Wang Y, Song Y, Wang S, Zhao Y. Study of the rifampin monoresistance mechanism in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2013;57(2):893–900. 19. Kurbatova EV, Cavanaugh JS, Dalton T, Click ES, Cegielski JP. Epidemiology of pyrazinamide-resistant tuberculosis in the United States, 1999-2009. Clin Infect Dis. 2013;57(8):1081–93. 20. Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int J Tuberc Lung Dis. 2015;19(11):1276–89. 21. Zhang Y, Chiu Chang K, Leung CC, Wai Yew W, Gicquel B, Fallows D, Kaplan G, Chaisson RE, Zhang W. ‘Z(S)-MDR-TB’ versus ‘Z(R)-MDR-TB’: improving treatment of MDR-TB by identifying pyrazinamide susceptibility. Emerg Microbes Infect. 2012;1(7):e5. 22. Gu Y, Yu X, Jiang G, Wang X, Ma Y, Li Y, Huang H. Pyrazinamide resistance among multidrug-resistant tuberculosis clinical isolates in a national referral center of China and its correlations with pncA, rpsA, and panD gene mutations. Diagn Microbiol Infect Dis. 2016;84(3):207–11. 23. Xu P, Wu J, Yang C, Luo T, Shen X, Zhang Y, Nsofor CA, Zhu G, Gicquel B, Gao Q. Prevalence and transmission of pyrazinamide resistant Mycobacterium tuberculosis in China. Tuberculosis (Edinb). 2016;98:56–61. 24. Jonmalung J, Prammananan T, Leechawengwongs M, Chaiprasert A. Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital. Thailand BMC Microbiol. 2010;10:223. 25. Alame-Emane AK, Xu P, Pierre-Audigier C, Cadet-Daniel V, Shen X, Sraouia M, Siawaya JF, Takiff H, Gao Q, Gicquel B. Pyrazinamide resistance in Mycobacterium tuberculosis arises after rifampicin and fluoroquinolone resistance. Int J Tuberc Lung Dis. 2015;19(6):679–84. 26. Ysern P, Clerch B, Castano M, Gibert I, Barbe J, Llagostera M. Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones. Mutagenesis. 1990;5(1):63–6. 27. Baharoglu Z, Mazel D. Vibrio cholerae triggers SOS and mutagenesis in response to a wide range of antibiotics: a route towards multiresistance. Antimicrob Agents Chemother. 2011;55(5):2438–41. 28. Bhuju S, Fonseca Lde S, Marsico AG, de Oliveira Vieira GB, Sobral LF, Stehr M, Singh M, Saad MH. Mycobacterium tuberculosis isolates from Rio de Janeiro reveal unusually low correlation between pyrazinamide resistance and mutations in the pncA gene. Infect Genet Evol. 2013;19:1–6. 29. Doustdar F, Khosravi AD, Farnia P. Mycobacterium tuberculosis genotypic diversity in pyrazinamide-resistant isolates of Iran. Microb Drug Resist. 2009; 15(4):251–6. 30. Jureen P, Werngren J, Toro JC, Hoffner S. Pyrazinamide resistance and pncA gene mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2008;52(5):1852–4. 31. Sheen P, Lozano K, Gilman RH, Valencia HJ, Loli S, Fuentes P, Grandjean L, Zimic M. pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis. Tuberculosis (Edinb). 2013;93(5): 515–22. 32. Portugal I, Barreiro L, Moniz-Pereira J, Brum L. pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates in Portugal. 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Prevalence and molecular characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis isolates from Southern China

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Medicine & Public Health; Infectious Diseases; Parasitology; Medical Microbiology; Tropical Medicine; Internal Medicine
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Abstract

Background: Pyrazinamide (PZA) plays a unique role in the treatment for multidrug-resistant tuberculosis (MDR-TB) in both first- and second-line regimens. The aim of this study was to investigate the prevalence and molecular characterization of PZA resistance among MDR-TB isolates collected in Chongqing municipality. Methods: A total of 133 MDR-TB isolates were collected from the smear-positive tuberculosis patients who were registered at local TB dispensaries of Chongqing. PZA susceptibility testing was determined with a Bactec MGIT 960 system. In addition, the genes conferring for PZA resistance were screened by DNA sequencing. Results: Of these 133 MDR-TB isolates, 83 (62.4%) were determined as PZA-resistant by MGIT 960. In addition, streptomycin- (83.1% vs. 56.0%, P < 0.01), ofloxacin- (51.8% vs. 18.0%, P < 0.01), kanamycin- (22.9% vs. 2.0%, P <0.01), amikacin- (18.1% vs. 2.0%, P = 0.01), capromycin-resistance (12.0% vs. 2.0%, P =0.05), were morefrequently observed among PZA-resistant isolates compared with PZA-susceptible isolates. Sequence analysis revealed that 73 out of 83 (88. 0%) MDR strains harbored a mutation located in the pncA gene, including 55 (75.3%, 55/73) of single nucleotide substitutions and 18 (24.7%, 18/73) of frameshift mutation, while no genetic mutation associated with PZA resistance was found in the rpsA gene. The pncA expression of strains harboring substitution from A to G at position −11 in the promoter region of pncA was significantly lower than that of H37Rv (P <0.01). Conclusions: In conclusion, our data have demonstrated that the analysis of the pncA gene rather than rpsA gene provides rapid and accurate information regarding PZA susceptibility for MDR-TB isolates in Chongqing. In addition, loss of pncA expression caused by promoter mutation confers PZA resistance in MDR-TB isolates. Background Only behind India, China has the second burden of MDR- Multidrug resistant tuberculosis (MDR-TB) is a major con- TB globally, with 52, 000 prevalent MDR-TB cases annu- cern hampering global tuberculosis control efforts [1, 2]. ally (WHO, 2015). A recent national survey of drug- According to new estimates from World Health resistant tuberculosis in China revealed that 5.7% of new Organization (WHO), there were around 0.48 million new TB cases and 25.6% of previously treated cases were MDR- cases of MDR-TB cases, and approximately 0.19 million TB, respectively, which were higher than the global average deaths from MDR-TB worldwide in 2014 (WHO, 2015). rates [3]. Given its high rate of treatment failure, the epi- demic of MDR-TB constitutes a serious public health problem in China [3, 4]. * Correspondence: [email protected]; [email protected] Equal contributors Pyrazinamide (PZA) is one of cornerstone first-line Clinical Laboratory, Chongqing Tuberculosis Control Institute, No. 71, anti-tuberculosis agents that is also commonly used as Longteng Street, Jiulongpo District, Chongqing 400050, People’s Republic of essential component in the therapeutic treatment of China National Center for Tuberculosis Control and Prevention, Chinese Center for MDR-TB [5, 6]. As the prodrug, PZA requires conver- Disease Control and Prevention, No. 155, Chang Bai Road, Changping sion into its active form pyrazinoic acid (POA) by the District, Beijing 102206, People’s Republic of China enzyme pyrazinamidase (PZase). PZase is encoded by Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pang et al. BMC Infectious Diseases (2017) 17:711 Page 2 of 8 the 561-nucleotide pncA gene. Loss of PZase activity testing was determined with a Bactec MGIT 960 system cause by genetic mutations in the pncA gene is the main according to the manufacturer’sinstructions. Thecritical mechanism of resistance to PZA in M. tuberculosis [6]. concentration of PZA in the liquid medium was 100 μg/ Several recent literatures have reported that several ml [8]. The MDR-TB strains were defined as those resist- PZA-resistant M. tuberculosis isolates harbored the mu- ant to both isoniazid and rifampicin. In addition, XDR-TB tations in the promoter of pncA gene, indicating these was defined as MDR-TB resistant to any member of the alternations may result in PZA resistance by influencing quinolone family and at least one of the remaining the expression of pncA [7–9]. In addition to pncA,an- second-line anti-TB injectable drugs [15]. other gene named rpsA, which encodes the 30S riboso- mal protein S1, has been demonstrated to confer PZA DNA extraction and amplification resistance by structural alternation of POA binding [10]. Genomic DNA was extracted from freshly cultured bac- To date, the contribution of the rpsA mutations confer- teria as previously described [16]. Briefly, the harvested ring PZA resistance is controversial, which requires bacteria from the surface of L-J medium were suspended more experimental evidences to elucidate the role of in 500 μl Tris-EDTA (TE) buffer and heated in a 95 °C rpsA as a PZA resistance mechanism [8]. water bath for 30 min. The crude DNA was used as tem- Chongqing is the largest municipality in the south- plate for amplification. The fragments of pncA and rpsA western China [11]. Due to the underdeveloped setting, were amplified with the following primers: pncA-F 5′- Chongqing is considered as a hotspot for both TB and AACAGTTCATCCCGGTTC-3′ and pncA-R 5′- MDR-TB in China [12]. However, limited data is avail- GCGTCATGGACCCTATATC-3′; rpsA-F 5′-CGGAG- able for the prevalence and molecular characteristics of CAACCCAACAATA-3′ and rpsA-R 5′-GTGGACAG- PZA resistance in M. tuberculosis isolates, especially CAACGACTTC-3′, respectively [17]. The 50 μL PCR MDR-TB. In this study, our main goal was to investigate mixture was prepared as follows: 25 μl 2 × GoldStar Mas- the prevalence of PZA resistance among MDR-TB terMix (CWBio, Beijing, China), 5 μL of DNA template, isolates collected in Chongqing municipality. We also and 0.2 μM of each primer set. PCR parameters for ampli- sought to analyze the mutant profiles of MDR-TB fication were 5 min at 94 °C followed by 35 cycles of 94 °C isolates conferring PZA resistance in this area. for 1 min, 58 °C for 1 min, 72 °C for 1 min and a final extension of 72 °C for 5 min. PCR products were sent to Methods Tsingke company for sequencing. All sequence data were Patient enrollment aligned with pncA and rpsA of reference strain H37Rv All smear-positive tuberculosis patients who were regis- (ATCC) using BioEdit (version 7.1.3.0) software. tered at local TB dispensaries between November 2014 and February 2016 were enrolled in this study. Informa- Quantitative reverse transcription PCR (QRT-PCR) tion was obtained from patient’s medical record. Two Bacteria were harvested from L-J medium after inocula- sputum samples were obtained from each smear-positive tion for 4 weeks. The total RNA was isolated following a patient for culturing in the county-level laboratories. standard Trizol RNA extraction protocol supplied by Invi- After 4–8 weeks of incubation, cultures with growing trogen (Invitrogen, Life Technologies, USA) [18]. Followed colonies on Löwenstein-Jensen (L-J) medium were sent by treatment with DNaseI (Invitrogen, Life Technologies, to the Chongqing Tuberculosis Control Institute for USA), the reverse transcription was carried out using further drug susceptibility testing. Re-treated cases were SuperScriptIII RT kit (Invitrogen, Life Technologies, defined as patients having previously received more than USA). The relative expression level of pncA gene was one month of anti-TB treatment. detected by QRT-PCR in a 20 μl system containing 10 μL of 2 × UltraSYBR Mixture (CWBio, Beijing, China), 2 μL Drug susceptibility testing of cDNA template, and 0.2 μM of each primer set (pncA- Drug susceptibility testing (DST) was performed with the QF 5′-GAAGCGGCGGACTACCATC-3′ and pncA-QR proportional method recommended by WHO [3, 13]. The 5′-AGTGGCGTGCCGTTCTCG-3′). PolyA was set as concentrations of drugs in L-J medium were as follows: the internal control in respective PCR experiments [18]. isoniazid (INH), 0.2 μg/ml; rifampicin (RIF), 40 μg/ml; ethambutol (EMB), 2 μg/ml; streptomycin (SM), 4 μg/ml; Data analysis ofloxacin (OFLX), 2 μg/ml; kanamycin (KAN), 30 μg/ml; Chi square test was used to evaluate the associations amikacin (AMK), 30 μg/ml; capromycin (CAP), 40 μg/ml; among multiple categorical variables, and the statistical re- and protionamide (PTO), 40 μg/ml and p-aminosalicylic sults were summarized with odds ratios (ORs) with 95% acid (PAS) 1 μg/ml [14]. A strain was declared resistant to confidence intervals (CIs). All calculations were performed an antimicrobial agent when the growth rate exceeded 1% in SPSS 13.0 (SPSS Inc., USA). Differences with a P value compared with the control. In addition, PZA susceptibility less than 0.05 were declared statistically significant. Pang et al. BMC Infectious Diseases (2017) 17:711 Page 3 of 8 Results revealed no statistically significant difference between Demographic characteristics and drug susceptibility profiles the PZA-resistant and PZA-susceptible group in gender A total of 133 (10.8%) of 1236 clinical isolates were iden- and age (P > 0.05). tified as MDR-TB, including 38 (28.6%) pre-XDR and 17 We further analyzed the resistance profiles of other (12.8%) XDR. Overall, 80 (60.2%) strains were isolated drugs among PZA-resistant and PZA-susceptible M. tu- from male patients and 53 (39.8%) from female patients. berculosis strains. Statistical analysis revealed that SM- The average age of the patients was 46.6 years (range (83.1% vs. 56.0%, P < 0.01), OFLX- (51.8% vs. 18.0%, 19–76 years). In addition, 36.8% of isolates were from P < 0.01), KAN- (22.9% vs. 2.0%, P < 0.01), AMK- new cases, and 63.2% from re-treated cases. Out of the (18.1% vs. 2.0%, P = 0.01), CAP-resistance (12.0% vs. 133 MDR isolates tested, 97 (72.9%) were resistant to 2.0%, P = 0.05), pre-XDR (36.1% vs. 16.0%, P < 0.02) and SM, 40 (30.1%) to EMB, 52 (39.1%) to OFLX, 20 (15.0%) XDR (19.3% vs. 2.0%, P < 0.01) were more frequently ob- to KAN, 16 (12.0%) to AMK, 11 (8.3%) to CAP and 15 served among PZA-resistant isolates compared with (11.3%) to PAS (Table 1). PZA-susceptible isolates, while no difference was identi- fied between EMB-resistant and EMB-susceptible strains Factors associated with PZA resistance (31.3% vs. 28.0%, P = 0.69) (Table 1). As shown in Table 1, 83 (62.4%) isolates were deter- mined as PZA-resistant by MGIT, while the other 50 Mutations in the pncA and rpsA gene (37.6%) were susceptible to PZA. In regard to the distri- A total of 73 out of 83 (88.0%) MDR strains harbored a bution of MDR-TB cases treatment history, the percent- mutation located in the pncA gene, including 55 (75.3%, age of re-treated MDR-TB patients in the PZA-resistant 55/73) of single nucleotide substitutions and 18 (24.7%, group was significantly higher than in the PZA- 18/73) of frameshift mutation. As summarized in Table 2, susceptible group (odd ratio (OR) [95% confidence inter- we observed great mutant diversity in pncA gene, and val (CI): 3.26[1.55–6.82], P < 0.01). In contrast, our data there were 48 different mutant types conferring PZA Table 1 Risk factor associate with PZA resistance among 133 MDR isolates Characteristics No. (%) of isolates No. (%) of isolates OR P value (n = 133) R S (95% CI) PZA PZA (n = 83) (n = 50) Sex Male 80 (60.2) 47 (56.6) 33 (66.0) 0.67 (0.33–1.39) 0.29 Female 53 (39.8) 36 (43.4) 17 (34.0) 1.0 (Ref.) – Age group < 30 31 (23.3) 18 (21.7) 13 (26.0) 1.0 (Ref.) – 30–59 80 (60.2) 55 (66.3) 25 (50.0) 1.59 (0.68–3.74) 0.29 ≥ 60 22 (16.5) 10 (12.0) 12 (24.0) 0.60 (0.20–1.81) 0.36 Treatment history New case 49 (36.8) 22 (26.5) 27 (54.0) 1.00 (Ref.) – Re-treated 84 (63.2) 61 (73.5) 23 (46.0) 3.26 (1.55–6.82) <0.01 Resistance to: SM 97 (72.9) 69 (83.1) 28 (56.0) 3.87 (1.74–8.63) <0.01 EMB 40 (30.1) 26 (31.3) 14 (28.0) 1.17 (0.54–2.54) 0.69 OFLX 52 (39.1) 43 (51.8) 9 (18.0) 4.89 (2.11–11.35) <0.01 KAN 20 (15.0) 19 (22.9) 1 (2.0) 14.58 (1.88–112.44) <0.01 AMK 16 (12.0) 15 (18.1) 1 (2.0) 10.81 (1.38–84.58) 0.01 CAP 11 (8.3) 10 (12.0) 1 (2.0) 6.71 (0.83–54.12) 0.05 PAS 15 (11.3) 13 (15.7) 2 (4.0) 4.46 (0.96–20.65) 0.04 Pre-XDR 38 (28.6) 30 (36.1) 8 (16.0) 2.97 (1.23–7.16) 0.02 XDR 17 (12.8) 16 (19.3) 1 (2.0) 11.70 (1.50–91.22) <0.01 MDR is defined as Mycobacterium tuberculosis strain resistant to at least isoniazid and rifampin Pre-XDR is defined as MDR strain additionally resistant to either ofloxacin or kanamycin, but not both XDR is defined as Mycobacterium tuberculosis strain resistant to isoniazid, rifampin, ofloxacin and kanamycin Pang et al. BMC Infectious Diseases (2017) 17:711 Page 4 of 8 Table 2 Mutations of PZA-resistant MDR-TB isolates within pncA Locus Position of nucleotide Nucleotide substitution Position of amino acid Amino acid substitution No. of isolates pncA −11 TAT → TGT −4 Tyr → Cys 4 2 ATG → ACG 1 Met → Thr 1 20 GTC → GGC 7 Val → Gly 2 24 GAC → GAG 8 Asp → Glu 1 28 CAG → TAG 10 Gln → Stop 1 35 GAC → GCC 12 Asp → Ala 2 37 TTC → GTC 13 Phe → Val 1 40 TGC → CGC 14 Cys → Arg 2 40 TGC → CGC 14 Cys → Arg 1 94 TTC → GTC 32 Phe → Val 1 123 TAC → TAG 41 Tyr → Stop 1 146 GAC → GCC 49 Asp → Gly 1 146 GAC → GCC 49 Asp → Ala 2 151 CAC → TAC 51 His → Tyr 1 151 CAC → CGC 51 His → Arg 1 152 CAC → CCC 51 His → Pro 1 170 CAC → CGC 57 His → Arg 1 185 CCG → CTG 62 Pro → Leu 5 206 CCG → CTG 69 Pro → Leu 1 213 CAT → CAG 71 His → Gln 1 226 ACT → CCT 76 Thr → Pro 3 232 GGC → AGC 78 Gly → Asp 1 245 CAT → CGT 82 His → Arg 1 286 AAG → CAG 96 Lys → Gln 1 307 TAC → CAC 103 Tyr → His 1 309 TAC → TAG 103 Tyr → Stop 1 319 GAA → AAA 107 Glu → Lys 1 395 GGT → GAT 132 Gly → Asp 9 425 ACG → ATG 142 Thr → Met 1 437 GCG → GTG 146 Ala → Val 1 464 GTG → GGG 155 Val → Gly 1 488 GTG → GCG 163 Val → Ala 1 515 CTG → CCG 172 Leu → Pro 2 52 insertion of GC 1 130 insertion of C 1 136 deletion of G 2 139 insertion of CA 1 232 insertion of C 1 243 insertion of T 1 288 insertion of A 1 341 deletion of ACGCC 1 342 deletion of GCCAC 2 376 deletion of GATGAGGTC 1 392 insertion of G 1 Pang et al. BMC Infectious Diseases (2017) 17:711 Page 5 of 8 Table 2 Mutations of PZA-resistant MDR-TB isolates within pncA (Continued) Locus Position of nucleotide Nucleotide substitution Position of amino acid Amino acid substitution No. of isolates 392 insertion of GG 1 393 insertion of GGT 1 408 insertion of CA 1 408 insertion of A 2 Total 73 resistance among MDR strains in Chongqing. The most prevalence of PZA among MDR-TB thus is a determin- prevalent mutation associated with PZA resistance was ing factor for initiation of PZA in the therapy regimens found in codon 132 of pncA (12.3%, 9/73), resulting in for these refractory patients [21]. Here, our data demon- the amino aicd substitution of Gly to Asp. As the second strated that 62.4% of MDR-TB exhibited resistance affected codon, five isolates had a mutation in codon 62 against PZA in Chongqing, which was similar to a recent (6.8%, 5/73). Notably, the third frequent mutation was literature from Beijing (57.7%) [22], while higher than the nucleotide substitution from A to G at position −11 those from Zhejiang (43.1%) [8], Shanghai (38.5%) [23], in the promoter region of pncA, accounting for 5.4% of United States (38.0%) [19], and Thailand (49.0%) [24]. mutant isolates. We also found that 4 PZA-susceptible The high prevalence of PZA resistance among MDR-TB isolates carried a genetic mutation in pncA, including 2 patients from our report indicates that Chongqing is a strains in codon 62, one in codon 67 and one in codon hotspot of PZA resistance in China. In our study, more 154 (Table 3). In addition, no genetic mutation associ- than 60% MDR patients received previous anti-TB ther- ated with PZA resistance was found in the rpsA gene in apy with PZA, which is significantly higher than the this study. average national level (21.8%) [3]. Hence, we speculate We analyzed the performance of pncA mutations for that the high proportion of PZA resistance may be con- predicting PZA susceptibility. When setting the pheno- tributed to the high rate of re-treated TB patients. The typic PZA susceptibility as a gold standard, we found serious issue on PZA resistance highlights the dimin- that detection of mutations in pncA gene exhibited a ished role of PZA in the treatment for MDR-TB in this sensitivity of 88.0% (95% CI, 80.9%–95.0%) and a specifi- setting with high MDR-TB burden. Prior to the use of city of 92.0% (95% CI, 84.5%–99.5%) (Table 4). PZA for treatment of MDR-TB cases, it is essential to perform in vitro susceptibility testing against PZA to for- Loss of pncA expression due to promoter mutation mulate a suitable regimen [21]. We further explore whether the substitution at position Another important finding from our observation was −11 affected the expression level of pncA. As shown in that we observed that there were high correlation be- Fig. 1, compared with reference strain H37Rv, the rela- tween PZA resistance and several other drugs’ resist- tive expression level of pncA in MDR87, MDR88, ance, including OFLX, second-line injectable drugs, and MDR114 and MDR126 carried the substitution at pos- PAS. Similar to our findings, a recent report from ition −11 were 0.25 fold, 0.19 fold, 0.36 fold and 0.22 Alame-Emane and colleagues has revealed that PZA re- fold, respectively. Statistical analysis revealed that the sistance in M. tuberculosis arises after RIF and fluoro- pncA expression of strains harboring promoter mutation quinolone (FQ) resistance [25]. Genetic mutations at position −11 was significantly lower than that of constitute the most important mechanism conferring H37Rv (P < 0.01). drug resistance in M. tuberculosis [20]. Exposure to bac- terial species to antimicrobial agents, including RIF, FQ Discussion and the aminoglycosides, induces the production of oxy- PZA plays a unique role in the treatment for MDR-TB gen radicals, thereby conferring high frequency muta- in both first- and second-line regimens [19, 20]. The genesis [25–27]. Considering long duration of anti-TB Table 3 Mutations in PZA-susceptible MDR-TB isolates within pncA gene Locus Position of nucleotide Nucleotide substitution Position of amino acid Amino acid substitution No. of isolates pncA 184 CCG → ACG 62 Pro → Thr 1 185 CCG → CAG 62 Pro → Gln 1 200 TCG → TGG 67 Ser → Trp 1 461 AGG → AAG 154 Arg → Lys 1 Total 4 Pang et al. BMC Infectious Diseases (2017) 17:711 Page 6 of 8 Table 4 Performance of pncA mutations for predicting PZA susceptibility Mutation PZA susceptibility Total Sensitivity Specificity PPV NPV in pncA (95% CI, %) (95% CI, %) (95% CI, %) (95% CI, %) RS Yes 73 4 77 88.0 92.0 94.8 82.1 (80.9–95.0) (84.5–99.5) (89.8–99.8) (72.1–92.2) No 10 46 56 Total 83 50 133 R, resistant, S susceptible, PPV positive predictive value, NPV negative predictive value, CI confidence interval treatment, we hypothesize that MDR bacteria will harbor than the routine methods by covering the mutant more genetic mutations induced by prolonged exposure hotspots. to these drugs, which may be responsible for the poten- In addition, the third frequent mutation identified in tial cross resistance between PZA and other drugs in this study was located at position −11 of the pncA our study. promoter region. In line with our observation, numerous In vitro susceptibility against PZA is essential for literatures have observed this mutant type in PZA- proper management of MDR-TB with regimen contain- resistant M. tuberculosis isolates [31, 32]. We found that ing PZA [21]. However, phenotypic DST for PZA is not this substitution at position −11 was associated with low routinely performed due to the requirement of harshly level of pncA expression, which was also consistent to acidic environment [17]. Molecular method based on the observation from Sheen et al. [31]. The anti-TB ac- detecting the mutations in pncA and rpsA serves as an tivity of PZA depends on the transformation to POA by alternative to predict the PZA susceptibility in M. tuber- PZAse, which is encoded by pncA gene. The loss of culosis [9]. In this study, our data demonstrated that pncA transcriptional level may result in the relative low genetic alternations in pncA confer 88.0% of PZA resist- PZAse activity, which is further associated with the ance among MDR-TB in Chongqing. A number of stud- phenotypic PZA resistance. Our results suggest that the ies have demonstrated a diverse prevalence of pncA promoter region of pncA is recommended to be in- mutation among PZA resistant isolates in different re- cluded in the sequence analysis of pncA gene. gions, ranging from 45.7% in Brazil [28], 70.6% in Iran We acknowledge several limitations of this study. First, [29], 75.0% in Thailand [24], 78.0% in Zhejiang [8], the small sample size is a major limitation of our report. 84.6% in Southern China [9], and 94.1% in Sweden [30]. And all the strains collected from one region also reduce Hence, pncA mutations may differ from one geographic their representativeness and thereby constrict the region to another. In addition, we found that pncA mu- generalizability of findings. Further wider sampling will tations exhibited great diversity, and the most frequent give more credence to this study. Second, although the mutant type in codon 142 only accounted for approxi- primary data of this study suggest that loss of pncA mate 12% of PZA resistant isolates, which was also dif- expression caused by promoter mutation confers PZA ferent from reports from other regions [8, 9]. Given the resistance in MDR-TB isolates, we could give no bio- diversity of pncA mutations within more than 500-bp chemical or transgenic substantiation of this statement. long segment, DNA sequencing of the entire pncA is Therefore, there is an urgent need to confirm our find- more effective for verification of PZA resistance rather ings with more experimental evidences in the future. Fig. 1 Relative expression level of pncA gene in 4 M. tuberculosis isolates with mutation in the position −11 of promoter region Pang et al. BMC Infectious Diseases (2017) 17:711 Page 7 of 8 Third, the high diversity of pncA mutations in MTB-TB Competing interests The authors declare that they have no competing interests. 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 Publisher’sNote for first time among PZA-resistant isolates. Despite be- Springer Nature remains neutral with regard to jurisdictional claims in ing highly correlated with the loss of PZA susceptibility, published maps and institutional affiliations. further experiments will be carried out to clarify the po- Author details tential contributions of these mutations to PZA resist- 1 National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on ance. Nevertheless, our report firstly described the Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China. molecular characteristics of PZA resistance among Clinical Laboratory, Chongqing Tuberculosis Control Institute, No. 71, MDR-TB isolates from Southern China, which provides Longteng Street, Jiulongpo District, Chongqing 400050, People’s Republic of important hints to diagnose PZA resistance and help China. National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Chang Bai Road, guide therapy with PZA for MDR-TB patients in this re- Changping District, Beijing 102206, People’s Republic of China. gion with high MDR-TB burden. Received: 26 February 2017 Accepted: 22 September 2017 Conclusion In conclusion, our data have demonstrated that the ana- lysis of the pncA gene rather than rpsA gene provides References 1. Gandhi NR, Nunn P, Dheda K, Schaaf HS, Zignol M, van Soolingen D, Jensen rapid and accurate information regarding PZA suscepti- P, Bayona J. Multidrug-resistant and extensively drug-resistant tuberculosis: a bility for MDR-TB isolates in Chongqing. In addition, threat to global control of tuberculosis. Lancet. 2010;375(9728):1830–43. loss of pncA expression caused by promoter mutation 2. Zhang Z, Pang Y, Wang Y, Liu C, Zhao Y. Beijing genotype of Mycobacterium tuberculosis is significantly associated with linezolid confers PZA resistance in MDR-TB isolates. Considering resistance in multidrug-resistant and extensively drug-resistant tuberculosis the high degree of diversity of pncA gene mutations, in China. Int J Antimicrob Agents. 2014;43(3):231–5. DNA sequencing of the entire pncA is more effective for 3. Zhao Y, Xu S, Wang L, Chin DP, Wang S, Jiang G, Xia H, Zhou Y, Li Q, Ou X, et al. National survey of drug-resistant tuberculosis in China. N Engl J Med. verification of PZA resistance rather than the routine 2012;366(23):2161–70. methods by covering the mutant hotspots. The high 4. Orenstein EW, Basu S, Shah NS, Andrews JR, Friedland GH, Moll AP, Gandhi prevalence of PZA resistance among MDR highlights NR, Galvani AP. Treatment outcomes among patients with multidrug- resistant tuberculosis: systematic review and meta-analysis. Lancet Infect Dis. the diminished role of PZA in the treatment for MDR- 2009;9(3):153–61. TB in this setting with high TB burden. 5. Verdugo D, Fallows D, Ahuja S, Schluger N, Kreiswirth B, Mathema B. Epidemiologic Correlates of Pyrazinamide-Resistant Mycobacterium Abbreviations tuberculosis in New York City. Antimicrob Agents Chemother. 2015;59(10): AMK: Amikacin; CAP: Capromycin; CIs: Confidence intervals; DST: Drug 6140–50. susceptibility testing; EMB: Ethambutol; INH: Isoniazid; KAN: Kanamycin; MDR- 6. Zhang Y, Mitchison D. The curious characteristics of pyrazinamide: a review. TB: Multidrug-resistant tuberculosis; OFLX: Ofloxacin; ORs: Odds ratios; PAS: P- Int J Tuberc Lung Dis. 2003;7(1):6–21. aminosalicylic acid; POA: Pyrazinoci acid; PTO: Protionamide; 7. Scorpio A, Zhang Y. Mutations in pncA, a gene encoding pyrazinamidase/ PZA: Pyrazinamide; PZase: Pyrazinamidase; RIF: Rifampicin; SM: Streptomycin nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat Med. 1996;2(6):662–7. Acknowledgements 8. Xia Q, Zhao LL, Li F, Fan YM, Chen YY, Wu BB, Liu ZW, Pan AZ, Zhu M. We would like to thank the staffs from the National Tuberculosis Reference Phenotypic and genotypic characterization of pyrazinamide resistance Laboratory for their technical support. among multidrug-resistant Mycobacterium tuberculosis isolates in Zhejiang. China Antimicrob Agents Chemother. 2015;59(3):1690–5. Funding 9. Tan Y, Hu Z, Zhang T, Cai X, Kuang H, Liu Y, Chen J, Yang F, Zhang K, Tan S, This study was supported by the Major State Basic Research Development et al. Role of pncA and rpsA gene sequencing in detection of pyrazinamide Program of China (2014CB744403). resistance in Mycobacterium tuberculosis isolates from southern China. J Clin Microbiol. 2014;52(1):291–7. Availability of data and materials 10. Shi W, Zhang X, Jiang X, Yuan H, Lee JS, Barry CE 3rd, Wang H, Zhang W, The datasets used and/or analysed during the current study available from Zhang Y. Pyrazinamide inhibits trans-translation in Mycobacterium the corresponding author on reasonable request. tuberculosis. Science. 2011;333(6049):1630–2. 11. Zhang D, An J, Wang J, Hu C, Wang Z, Zhang R, Wang Y, Pang Y. Molecular Authors’ contributions typing and drug susceptibility of Mycobacterium tuberculosis isolates from Conceived and designed the experiments: YP, DZ, JL, YL. Performed the Chongqing Municipality, China. Infect Genet Evol. 2013;13:310–6. experiments: YP, DZ, HZ, JS. YH, JL. Analyzed the data: YP, DZ, JL, YL. Wrote 12. Zhang D, An J, Wang Y, Pang Y. Genetic diversity of multidrug-resistant the paper: YP, DZ, JL, YL. All authors contributed to the final manuscript. All tuberculosis in a resource-limited region of China. Int J Infect Dis. authors read and approved the final manuscript. 2014;29:7–11. 13. World Health Organization: Global tuberculosis report 2015. Geneva: World Ethics approval and consent to participate Health Organization; 2015. This study was approved by the Ethics Committee of the Chongqing 14. World Health Organization. Anti-tuberculosis drug resistance in the world. Tuberculosis Control Institute. Patients enrolled in this study provided the Geneva: World Health Organization; 2008. signed Informed Consent form. 15. Pang Y, Zhou Y, Zhao B, Liu G, Jiang G, Xia H, Song Y, Shang Y, Wang S, Zhao YL. Spoligotyping and drug resistance analysis of Mycobacterium Consent for publication tuberculosis strains from national survey in China. PLoS One. 2012;7(3): Not applicable. e32976. Pang et al. BMC Infectious Diseases (2017) 17:711 Page 8 of 8 16. Zhang Z, Lu J, Wang Y, Pang Y, Zhao Y. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother. 2014;58(1):364–9. 17. Pang Y, Wang Z, Zheng H, Song Y, Wang Y, Zhao Y. Pyrazinamide resistance determined by liquid culture at low pH better correlates with genetic mutations in MDR tuberculosis isolates. J Microbiol Methods. 2015;119:142–4. 18. Pang Y, Lu J, Wang Y, Song Y, Wang S, Zhao Y. Study of the rifampin monoresistance mechanism in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2013;57(2):893–900. 19. Kurbatova EV, Cavanaugh JS, Dalton T, Click ES, Cegielski JP. Epidemiology of pyrazinamide-resistant tuberculosis in the United States, 1999-2009. Clin Infect Dis. 2013;57(8):1081–93. 20. Zhang Y, Yew WW. Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015. Int J Tuberc Lung Dis. 2015;19(11):1276–89. 21. Zhang Y, Chiu Chang K, Leung CC, Wai Yew W, Gicquel B, Fallows D, Kaplan G, Chaisson RE, Zhang W. ‘Z(S)-MDR-TB’ versus ‘Z(R)-MDR-TB’: improving treatment of MDR-TB by identifying pyrazinamide susceptibility. Emerg Microbes Infect. 2012;1(7):e5. 22. Gu Y, Yu X, Jiang G, Wang X, Ma Y, Li Y, Huang H. Pyrazinamide resistance among multidrug-resistant tuberculosis clinical isolates in a national referral center of China and its correlations with pncA, rpsA, and panD gene mutations. Diagn Microbiol Infect Dis. 2016;84(3):207–11. 23. Xu P, Wu J, Yang C, Luo T, Shen X, Zhang Y, Nsofor CA, Zhu G, Gicquel B, Gao Q. Prevalence and transmission of pyrazinamide resistant Mycobacterium tuberculosis in China. Tuberculosis (Edinb). 2016;98:56–61. 24. Jonmalung J, Prammananan T, Leechawengwongs M, Chaiprasert A. Surveillance of pyrazinamide susceptibility among multidrug-resistant Mycobacterium tuberculosis isolates from Siriraj Hospital. Thailand BMC Microbiol. 2010;10:223. 25. Alame-Emane AK, Xu P, Pierre-Audigier C, Cadet-Daniel V, Shen X, Sraouia M, Siawaya JF, Takiff H, Gao Q, Gicquel B. Pyrazinamide resistance in Mycobacterium tuberculosis arises after rifampicin and fluoroquinolone resistance. Int J Tuberc Lung Dis. 2015;19(6):679–84. 26. Ysern P, Clerch B, Castano M, Gibert I, Barbe J, Llagostera M. Induction of SOS genes in Escherichia coli and mutagenesis in Salmonella typhimurium by fluoroquinolones. Mutagenesis. 1990;5(1):63–6. 27. Baharoglu Z, Mazel D. Vibrio cholerae triggers SOS and mutagenesis in response to a wide range of antibiotics: a route towards multiresistance. Antimicrob Agents Chemother. 2011;55(5):2438–41. 28. Bhuju S, Fonseca Lde S, Marsico AG, de Oliveira Vieira GB, Sobral LF, Stehr M, Singh M, Saad MH. Mycobacterium tuberculosis isolates from Rio de Janeiro reveal unusually low correlation between pyrazinamide resistance and mutations in the pncA gene. Infect Genet Evol. 2013;19:1–6. 29. Doustdar F, Khosravi AD, Farnia P. Mycobacterium tuberculosis genotypic diversity in pyrazinamide-resistant isolates of Iran. Microb Drug Resist. 2009; 15(4):251–6. 30. Jureen P, Werngren J, Toro JC, Hoffner S. Pyrazinamide resistance and pncA gene mutations in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2008;52(5):1852–4. 31. Sheen P, Lozano K, Gilman RH, Valencia HJ, Loli S, Fuentes P, Grandjean L, Zimic M. pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis. Tuberculosis (Edinb). 2013;93(5): 515–22. 32. Portugal I, Barreiro L, Moniz-Pereira J, Brum L. pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates in Portugal. 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BMC Infectious Diseases – Springer Journals

Published: Nov 6, 2017

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