pncA mutations in Mycobacterium tuberculosis clinical isolates from Korea

Background: Pyrazinamide (PZA) is among the first-line drugs to treat tuberculosis, and kills the semidormant mycobacteria only in a low-pH condition. And, the association between PZA resistance and pncA mutation has been studied. The purpose of this study was to compare PZA resistance to pyrazinamidase (PZase) activity and the genotype, to more understand the molecular basis of PZA resistance, and to expand the data of pncA mutations worldwide. Results: Of twenty-eight tested strains, six were susceptible to PZA and positive for PZase activity, and had no mutations in pncA genes. Twenty-one strains were all resistant to PZA and negative for PZase activity, and had mutations in the pncA gene, including 15 single point mutations, five insertions, and two deletions. One strain had no mutation in the pncA gene, even if it was resistant to PZA and negative for PZase activity. Three isolates had adenine to guanine point mutations of –11 upstream region, showing the most common type of pncA mutations with at least two different RFLP patterns. Conclusion: These data provide the more understanding of the molecular basis of PZA resistance. Furthermore, it was demonstrated that adenine to guanine point mutation of –11 upstream region is the most common type of pncA mutations, and that the results of pncA mutations should be carefully interpreted for the epidemiologic purposes. Background Pyrazinamide (PZA) is among the first-line drugs to treat tuberculosis, and kills the semidormant mycobacteria only in a low-pH condition. So, the biologic susceptibility test to PZA sometimes fails because of the poor growth of the mycobacteria in a low pH. Instead, pyrazinamidase (PZase) test, which was originally used for the differentiation of Mycobacterium tuberculosis from weakly niacin-positive strains of M. bovis, has been used to detect susceptible strains of M. tuberculosis, because the PZase converts PZA to pyrazinoic acid, an active form of the drug. pncA encodes the PZase, and mutations in pncA have been shown to be associated with biologic resistance or loss of PZase activity. The purpose of this study was to compare PZase activity to the genotype, to more understand the molecular basis of PZA resistance, and to expand the data of pncA mutations worldwide. Results and Discussion All five isolates collected from PNUH were revealed to be positive PZase activities in this study, and susceptible to PZA with minimal inhibitory concentrations (MIC) of less than 100 μg/mL, which are consistent with the initial routine tests. These isolates had pncA sequences identical with the sequences of pncA of M. tuberculosis. Among the twenty-three isolates collected from KIT, one was disclosed to be positive PZase activity and susceptible to PZA (MIC <100μg/mL), and had no pncA mutation in the entire open reading frame including the upstream region. So, this strain was demonstrated to be a PZAsusceptible strain. All of the remaining twenty-two isolates were negative for PZase activity and resistant to PZA (MIC >500μg/mL). Among them, twenty-one organisms (95.5%) had mutations in the pncA gene. Eleven organisms had twelve single point mutations, including two point mutations in one organism, which resulted in one silent mutation, one nonsense mutation, and 10 missense mutations. Of those, six mutations were newly found. Three isolates had upstream mutations at nucleotide –11, the single most common mutations, resulting in an A-to-G change. One had 3-bp insertion, resulting in a slipped-strand mispairing of PncA. Four had 1or 2-bp insertions, and two had 2or 234-bp deletions, all resulting in frameshift mutations in PncA (Table 1). The clustering tendency was appeared that 40.0% (6/15) of the single point mutations were located in the region between residues 132 to 142 of the PZase. Among the three strains of the promotor region mutations, two isolates showed closely related RFLP pattern. PZase test has been used for the differentiation of M. tuberculosis from M. bovis, M. avium complex from niacin-negative M. bovis, and M. marinum from M. kansasii [1], and also used for the detection of PZAresistant M. tuberculosis strains [2]. It has a shortcoming that if old colonies are used, false negative result of PZase activity might be obtained. It seems that in case of stationary or death phase of colonies, the enzyme activity is reduced not enough to get positive reaction. Actually, one of the twenty-three strains that had resulted in negative PZase reaction turned to be PZase-positive in this study. Twenty-one of 22 PZase-negative strains (95.5%) showed pncA mutations in this study. Other reports [35] also demonstrated that pncA mutations were found in 72 87% of biologically PZA-resistant strains, and in 97% of PZase-negative strains. Sreevatsan et al [5] suggested the possibility of another mechanisms of PZA resistance because no mutation in pncA or its upper promotor was found in 28% of biologically PZAresistant M. tuberculosis strains. In contrast, Hewlett et al [6] demonstrated the low reproducibility of biologic susceptibility to PZA, and Scorpio et al [3] proved false resistance to PZA in susceptible strains. So, if any, another resistance mechanism takes a minor part of PZA resistance, and more than 95% of PZAresistant M. tuberculosis strains are likely caused by pncA mutations. Twenty-two mutations included single nucleotide substitutions resulting silent, missense, or nonsense mutations, and deletions and insertions of as many as 234 nucleotides. Of those, six single point mutations of 7 strains were not described in the previous studies [3, 5, 7-14]. Therefore, we could agree that the mutations are arrayed along virtually the entire length of the gene [5], even though the clustering tendency was appeared that 40.0% (6/15) of the single point mutations were located in the region between residues 132 to 142 of the PZase. In addition, however, we think that the mutations in the promotor region, especially –11 upstream region, should be paid attention to. Even no strain showed this type of mutation in some studies, the results of the previous reports including the present study demonstrated that it is the most common type of pncA mutations [3, 5, 7, 9-11] (Table 2). Moreover, most of them were adenine to guanine substitutions. The consensus sequence TATAAT, known as the pribnow box or –10 site, is located approximately 10 base pairs upstream of the transcription start site of many bacterial genes. So, it seems that mutation of –11 upstream blocked binding of RNA polymerase to the promotor site, resulting no transcription in the correct place. However, further experiments such as in vitro mutagenesis should be performed to demonstrate the relationship between the promotor mutation and loss of PZase activity because of no consensus sequence in the pncA gene. pncA mutations were suggested to be a useful tool of epidemiologic investigations. Because of random mutations along the whole pncA gene, the same mutations among the unrelated isolates are rarely occurred theoretically. In Cheng et al’s report [11], twenty-one strains of the same mutations were demonstrated as highly related strains in strain typing. In the present study, the one strain of promotor mutation showed different RFLP pattern from the other two strains, indicating the different sources of infections. Conclusion These data provide the more understanding of the molecular basis of PZA resistance and expand the data of pncA mutations worldwide. Furthermore, it was demonstrated that adenine to guanine point mutation of – 11 upstream region is the most common type of pncA mutations. The results of pncA mutations should be carefully interpreted for the epidemiologic purposes. Materials and Methods Bacterial strains, PZA susceptibility and PZase activity: Twenty-eight clinical isolates of M. tuberculosis were included. Twenty-three PZase-negative clinical isolates were provided from Korean Institute of Tuberculosis (KIT), and these had originally been collected from various site of this country for the purpose of susceptibility tests. Five PZase-positive isolates were collected randomly among the clinical isolates grown at Pusan National University Hospital (PNUH). The type strain M. tuberculosis H37Rv was included as control. All isolates were grown in Lowenstein-Jensen medium at 37°C for 3 to 4 weeks. PZA susceptibility was tested by using Lowenstein-Jensen medium at pH 5.6 with 100 and 500 μg of PZA per mL. PZase assay was performed by the method described at Clinical Microbiology Procedure Handbook [1]. Genomic DNA preparation, PCR and DNA sequencing: DNA was extracted by using InstaGene matrix kit (Bio-Rad Laboratories Inc., Hercules, CA). A 720-bp segment including the entire open reading frame of pncA gene was amplified by using the conditions and the set of primers P1 and P6 [3]. The PCR products were cut from the gel and purified with the QiaAmp PCR purification kit (QIAGEN GmbH, Germany), according to the manufacturer’s instructions. The gel-purified PCR products were quantitated and adjusted to 200-μmol concentration, and used for direct sequencing by using the ABI 377 automatic DNA sequencer (Applied Biosystems Inc., Foster, CA) with each 4 pM of above-mentioned primers. The RFLP analysis was performed for the three clinical isolates showing the same pncA mutations by an internationally standardized method [15], and the hybridized membrane was detected by the colorimetric method using the Roche digoxigenin detection kit (F. Hoffmann-La Roche Ltd., Switzerland). The 245-bp mycobacterial IS probe was amplified by PCR with a DIG DNA labeling kit (F. Hoffmann-La Roche) using INS-1 and INS-2 primers. References 1. Isenberg HD: Mycobacteriology: Identification tests for mycobacteria. In Clinical Microbiology Procedure Handbook. Edited by Isenberg HD,vol. 1. Washington DC: ASM Press, 1995, 3.12.17. 2. Konno K, Feldmann FM, McDermott W: Pyrazinamide susceptibility and amidase activity of tubercle bacilli. Am Rev Respir Dis 1967, 95:461-9. 3. Scorpio A, Lindholm Levy P, Heifets L, Gilman R, Siddiqi S, Cynamon M, Zhang Y: Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother 1997, 41:540-3. 4. Hirano K, Takahashi M, Kazumi Y, Fukasawa Y, Abe C: Mutation in pncA is a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis. Tuber Lung Dis 1997, 78:117-22. 5. Sreevatsan S, Pan X, Zhang Y, Kreiswirth BN, Musser JM: Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms. Antimicrob Agents Chemother 1997, 41:636-40. 6. Hewlett D, Jr., Horn DL, Alfalla C: Drug-resistant tuberculosis: inconsistent results of pyrazinamide susceptibility testing. JAMA 1995, 273:916-7. 7. Marttila HJ, Marjamaki M, Vyshnevskaya E, Vyshnevskiy BI, Otten TF, Vasilyef AV, Viljanen MK: pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from northwestern Russia. Antimicrob Agents Chemother 1999, 43:1764-6. 8. Lemaitre N, Sougakoff W, Truffot-Pernot C, Jarlier V: Characterization of new mutations in pyrazinamide-resistant strains of Mycobacterium tuberculosis and identification of conserved regions important for the catalytic activity of the pyrazinamidase PncA. Antimicrob Agents Chemother 1999, 43:1761-3. 9. Morlock GP, Crawford JT, Butler WR, Brim SE, Sikes D, Mazurek GH, Woodley CL, Cooksey RC: Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2000, 44:2291-5. 10. Mestdagh M, Fonteyne PA, Realini L, Rossau R, Jannes G, Mijs W, De Smet KA, Portaels F, Van den Eeckhout E: Relationship between pyrazinamide resistance, loss of pyrazinamidase activity, and mutations in the pncA locus in multidrug-resistant clinical isolates of Mycobacterium tuberculosis. Antimicrob Agents Chemother 1999, 43:2317-9. 11. Cheng SJ, Thibert L, Sanchez T, Heifets L, Zhang Y: pncA mutations as a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis: spread of a monoresistant strain in Quebec, Canada. Antimicrob Agents Chemother 2000, 44:528-32. 12. Scorpio A, Zhang Y: Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nat Med 1996, 2:662-7. 13. Brown TJ, Tansel O, French GL: Simultaneous identification and typing of multi-drugresistant Mycobacterium tuberculosis isolates by analysis of pncA and rpoB. J Med Microbiol 2000, 49:651-6. 14. Escalante P, Ramaswamy S, Sanabria H, Soini H, Pan X, Valiente-Castillo O, Musser JM: Genotypic characterization of drug-resistant Mycobacterium tuberculosis isolates from Peru. Tuber Lung Dis 1998, 79:111-8. 15. van Embden JD, Cave MD, Crawford JT, Dale JW, Eisenach KD, Gicquel B, Hermans P, Martin C, McAdam R, Shinnick TM, et al.: Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J Clin Microbiol 1993, 31:406-9. Table 1. pncA nucleotide and amino acid changes in PZase negative M. tuberculosis clinical isolates from Korea Mutation Site Nucleotide Changes Amino acid changes No. of isolates -11 A to G Mutation in promotor 3 23 TCG insertion slipped-strand mispairing 1 41 G41A Missense (Cys14 →Tyr) 1 56 234-bp deletion Frameshift 1 172 T172C Missense (Phe58 →Leu) 1 180 C180T Silent (Gly60 →Gly) 1 190 T190G Missense (Tyr64 →Asp) 1 212 A212G Missense (His71 →Arg) 1 227 C227T Missense (Thr76 →Ile) 1 317 CT insertion Frameshift 1 382 AG insertion Frameshift 1 393 GT insertion Frameshift 1 393 T insertion Frameshift 1 395 G395T Missense (Gly132 →Val) 2 403 A403C Missense (Thr135 →Pro) 1 407 A407G Missense (Asp136 →Gly) 1 421 C421T Nonsense (Gln141 →Termination) 1 425 C425T Missense (Thr142 →Met) 1 513 GC deletion Frameshift 1 a Previously described mutation [5, 7, 10, 11]. b Previously described mutations [14]. c Previously described mutations [9]. d Previously described mutations [13]. e Previously described mutations [3, 10]. Table 2. Type and frequency of mutations described in the pncA promotor region Mutation Site Nucleotide Changes Frequency Reference -16∼-11 AACGTA to GGCAGTT 1/37 [9] -12 T to G 1/31 [7] -11 A to C 1/8 (mutants made in vitro) [3] -11 A to G 2/48 [5] -11 A to G 5/31 [7] -11 A to G 1/17 [10] -11 A to G 1/33 [11] -11 A to G 3/22 The present study -7 T to C 1/37 [9] a Among 53 isolates, 21 were closely related strains showing the same pncA mutation, which were considered as one strain. 1 2 3 4 5 M 8 kb 7 6 4.8 3.5 2.7 1.9 1.5 1.15 1 FIG. 1. RFLP analysis patterns of three strains containing promotor mutations. M, molecular markers; 1-3, tested strains containing –11 upstream adenine to guanine substitutions; 4, M. tuberculosis H37Rv; 5, clinical isolate as a control strain. Pre-publication history 2 April 2001 First round of reviews pncA mutations in Mycobacterium tuberculosis clinical isolates from Korea Soon Kew Park [[email protected]] Jung You Lee [[email protected]] Chulhun Ludgerus Chang [[email protected]] Min Kee Lee [[email protected]] Han Chul Son [[email protected]] Cheol Min Kim [[email protected]] Hyun Jung Jang [[email protected]] Hee Kyung Park [[email protected]] Seok Hoon Jeong [[email protected]] Ying Zhang Comments The paper by Park et al. reports characterization of PZA-resistant TB strains in terms of pncA mutations and Pzase activity. The paper adds new information to the profile of pncA mutations in PZA-resistant TB strains. However, the paper is not well written and contains numerous language problems. The authors should have someone who is conversant in English correct the language problems. Major points 1. The claim that –11 mutation is the most common type of pncA mutation has to be substantiated by comparing with other frequently occurring mutations (at nucleotide level) such as those located between residues 132-142 of PncA. Thus, Table 2, which summarizes the –11 mutations in different studies, should also contain information on comparison with other mutations occurring in region 132-142, in order to make a valid claim. 2. Do the two strains with the same Gly132->Val mutation have the same IS6110 fingerprint? Minor points 1. In the Abstract, line 5, “more” should be change to “better”, the same applies to other part of the text; Line 5, “data” should be changed to “profile”. Line 10, “even if” should be changed to “even though”. 2. In the Background, line 3 and 7 and elsewhere in the text, “biologic” should be deleted. References are missing in this section. Please add references. 3. The 132-142 mutation clustering has been reported by Scorpio et al. and Lemaitre et al.. So please cite the references when discussing this point. 4. “was appeared” in various part of the text is incorrect use of language and should be modified. “resulting” should be changed to “resulting in” in the Results section. 5. On p4, line 25, “because of no consensus ....”does not make any sense and should be deleted. Level of interest A paper whose findings are important to those with closely related research interests Advice on publication Unable to decide on acceptance or rejection until I see revised version Quality of written English Not acceptable for publication unless it is rewritten Competing interests None declared. Have you in the past five years received reimbursements, fees, funding, or salary from an organisation that may in any way gain or lose financially from the publication of this paper? No. Do you hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this paper? No. Do you have any other financial competing interests? No. Are there any non-financial competing interests you would like to declare in relation to this paper? No. Open peer review Submission of this report to BMC is taken as confirmation that you are happy for your signed report to be posted on the BMC website as part of the pre-publication history of this paper. Elfriede Van den Eeckhout Comments The English needs correction. Some more references and more detailed protocols are needed . Overall, the conclusion drawn by the authors is acceptable. Abstract line 2: kills the semidormant mycobacteria:refes to an in vivo situation line 3: only in a low-pH condition refers to an in vitro situation Both conditionsshould not be mixed . line 3: drop “And,” line 11: the most common type of pncA mutations in this study line 13: These data help to better understand line 15: interpreted for epidemiologic purposes (drop “the”) Background line 2&3: same remark as line 3 in abstract line 3: in vitro susceptibility testing rather than biologic ... line 6: the PZase converts the prodrug PZA to pyrazinoic acid, the active form Results and discussion line 12: to be PZase-positive line 13: is consistent line 14: M. tuberculosis H37Rv (+ reference to study by Cole et al., please) line 15: found to be PZase-positive line 16: and accordingly, had no ... line 17: drop small sentence line 24-25: adapt this sentence line 2 following page: reduced below the limits to get positive reaction. This explains why... before turned out line 5: of PZA-resistant strains (drop “biologically”) line 9-10: if any other resistance mechanism exists, it only takes ... line 11: add “in this study” line 14: change “agree” into “confirm” line 18: adapt sentence!! line 23: resulting into inhibition of transcription line 1-2 following page: one of the strains with the promoter mutation showed a RFLP pattern different from those of the other two, suggesting different ... Conclusion line 8: interpreted for epidemiologic purposes (drop “the”) Materials and methods line 13: various sites line 14: susceptibility testing line 15: a PZA-susceptible and thus PZase-positive control line 17: please give reference of mentioned protocol line 18: give, in short, the PZase-testing protocol line 25-26: was performed by an internationally standardized method for the three... Table 1 It would be better to mention the two point mutation, one of which silent, occurring in the same isolate, together in the table; Also in the results section it should be more clearly indicated that the 12 point mutation mentioned is in fact a silent mutation within an isolate that also harbours another “real” point mutation. Table 2 You consider the 21 closely related strains showing the same mutation, among the 53 from reference 11, as one strain, on a total of 33. To be consequent, you should then also reduce your number from three to two on a total from 21 instead of 22, because also two of you three strains were closely related. Level of interest A paper whose findings are important to those with closely related research interests Advice on publication Unable to decide on acceptance or rejection untill I see revised version Quality of written English Not acceptable for publication unless it is rewritten (according to the given comments) Competing interests None declared Pre-publication history 8 May 2001 Response to referees’ comments pncA mutations in Mycobacterium tuberculosis clinical isolates from Korea Soon Kew Park [[email protected]] Jung You Lee [[email protected]] Chulhun Ludgerus Chang [[email protected]] Min Kee Lee [[email protected]] Han Chul Son [[email protected]] Cheol Min Kim [[email protected]] Hyun Jung Jang [[email protected]] Hee Kyung Park [[email protected]] Seok Hoon Jeong [[email protected]] ([email protected]) ([email protected]) ([email protected]@hyowon.cc.pusan.ac.kr) ([email protected]) Pre-publication history 21 May 2001 Second round of reviews pncA mutations in Mycobacterium tuberculosis clinical isolates from Korea Soon Kew Park [[email protected]] Jung You Lee [[email protected]] Chulhun Ludgerus Chang [[email protected]] Min Kee Lee [[email protected]] Han Chul Son [[email protected]] Cheol Min Kim [[email protected]] Hyun Jung Jang [[email protected]] Hee Kyung Park [[email protected]] Seok Hoon Jeong [[email protected]] Review on revised manuscript by Ying Zhang I have looked at the revised version of the manuscript. It is much improved. One point that was raised earlier regarding the reference of the pncA sequence on p3, the 4th line of Results and Discussion, should be addressed more carefully. The authors should cite the original pncA sequence with GenBank accession number U59967 at least. So I suggest that the authors cite U59967 and if they want also the H37Rv GenBank accession number AL021899. If they can fix this reference, I will recommend acceptance for publication. Please make sure that they do this correction before acceptance. Review on revised manuscript by Elfriede Van den Eeckhout Overall, most original criticisms have been answered. The use of English language is much better than in the original manuscript. I have noticed that Table 2 got a new content. I advise publication of the revised manuscript. Anyway still some minor remarks: *Background: line 2: kills the semidormant mycobacteria: refers to an in vivo situation line 3: only in a low-pH condition refers to an in vitro situation Both conditions should not be mixed. Answer) I am sorry, but I think that both descriptions are about in vitro conditions. Heifets et al (cited in the manuscript) described below; The number of viable tubercle bacilli remained almost unchanged over 3 to 4 weeks of cultivation in a liquid medium at pH 4.8 to 5.0. We qualified this state as semidormant rather than dormant, assuming that the relatively stable number of viable cells was a reflection of the simultaneous existence of cells in various states of dormancy, multiplication, and dying. OK, by adding the Heifets' reference, I see that you are talking about in vitro culturing of mycobacteria anyway. In the original manuscript, you did not mention this reference, so it appeared as if you stated that bacilli in the semidormant state also those within the patient's lesions are killed by PZA only at a low pH. There is still no agreement about the need of a low pH for PZA to be effective within the patient. line 3: in vitro susceptibility testing rather than biologic ... Answer) It was revised. You didn't revise it correctly: In vitro susceptibility testing sometimes fails.... *Results and discussion: line 12 and line 15: to be PZase-positive Answer) The phrase is hyphenated only when it is used as an adjective. Thus, “PZase-positive strains” BUT “the strains were PZase positive.” The absence of the hyphen was not the problem. The problem was that you wrote "the isolates ... were revealed to be positive PZase activities" (line 12) and "to be positive PZase activity" (line 15). This is incorrect use of the English language. It should read "the isolates were PZase-positive". line 2 following page: reduced below the limits to get positive reaction. This explains why... before turned out Ans) It was revised. You didn't revise it correctly. This explains why one of the 23 strains that had resulted in a negative PZase reaction before, turned out to be PZase positive in this study. line 11: add “in this study” Ans) The description means that more than 95% of resistance could be caused by pncA mutations in general. So, I think the above phrase may not be necessary. I do not agree. The results from the different studies give percentages varying between 72 and 97%, as you are citing yourself. Why then put 95% three phrases further in the text? ... suggesting different sources of infection. ... were collected in 1990 and 1997, suggesting that strains with the same mutations came from different sources. *Materials and Methods: .... collected randomly among (drop "from") Löwenstein