Novel pncA mutations in pyrazinamide-resistant isolates of Mycobacterium tuberculosis

Molecular Diagnosis Vol. 3 No. 4 1998

Novel pncA Mutations in Pyrazinamide-Resistant Isolates of Mycobacterium tuberculosis B R U C E S. SACHAIS,* I R V I N G N A C H A M K I N , ~ J U L I A N E K. MILLS,* D E B R A G. B. L E O N A R D * Philadelphia, Pennsylvania

The gene coding for pyrazinamidase (PZase) activity, pncA, in Mycobacterium tuberculosis' was recently cloned, and several mutations which correlate with in vitro resistance to pyrazinamide (PZA) have been identified. During the development of a clinical molecular assay for the detection of PZA resistance, two previously unreported mutations in isolates of PZA-resistant M. tuberculosis were identified. The assay that uses automated DNA sequencing is relatively rapid and allows the detection of any mutations present in the coding region of the pncA gene. The new mutations are both point mutations resulting in amino acid substitutions; 24lT~G results in FSOV, and 511G~C results in A171 R The identification of these mutations accentuates the utility of automated DNA sequencing in the clinical laboratory. Key words: drug resistance, PCR, DNA sequence.

More than 21,000 cases of Mycobacteriurn tuberculosis infection were reported in the United States

Scorpio and Zhang [7] cloned the pncA gene, which encodes the protein for PZase activity of M. tuberculosis. Further, they described five mutations in the pncA gene in PZA-resistant strains of M. tuberculosis: An additional 19 mutations in t h e pncA gene have been identified [5], bringing the total to 24 described pncA gene mutations (Fig. 1). These mutations account for approximately 70% of PZA-resistant M. tuberculosis" strains tested [5]. Because PZA is an important first-line therapy in the treatment of active tuberculosis [2], we developed a molecular assay for P Z A resistance that could be performed in parallel with other molecular drug resistance tests for isoniazid, rifampin, and streptomycin resistance [3]. During this development, we identified two previously undescribed mutations in PZA-resistant isolates.

in 1996 despite a 6.7% decrease from 1995 [1]. A combination of four antimycobacterial agents, including pyrazinamide (PZA), is commonly used to treat tuberculous infections [2]. Conventional microbiologic methods used to identify and perform drug susceptibility testing of M. tuberculosis' may take weeks to months, depending on the in vitro methods used, because of the slow growth rate of the organism. With the goal of providing rapid resuits, several laboratories have exploited molecular mechanisms of drug resistance in M. tuberculosis' to develop molecular assays, which can be routinely used in the clinical laboratory [3-6]. From the *Molecular Pathology Laboratory and iMicrobiology Laboratory, Department qf Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Methods

Reprint requests: Debra G.B. Leonard, MD, PhD, 7103 Founders, Department of Pathology and Laboratory Medicine, University of PennsylvaniaHealth System, 3400 Spruce Street, Philadelphia,PA 19104-4283. Copyright © 1998 by Churchill IJvingstone ® 1084-8592/98/0304-000758.00/0

From 1993 through June 1998, 147 M. tuberculosis isolates were identified in the clinical Microbiology Laboratory of the Hospital of the University

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Adelphia, N J) and sequenced in both the forward and reverse directions using pncA5' and pncA3', respectively, as primers. Sequencing fragments were analyzed using an ABI 377 DNA sequencer (Perkin-Elmer; Foster City, CA). Sequence analysis of all isolates allowed both strands from nucleotides 40 through 520 of the pncA gene to be compared with the wild-type pncA sequence.

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Nucleotide Position of Mutation

Fig. 1. Histogram of the location and frequency of pncA gene mutations reported in the literature [5,7] and in this report (*) for the gene region assessed by the sequence analysis assay.

of Pennsylvania. Four additional isolates for molecular P Z A resistance testing were sent to our laboratory by Dr Olarae Giger, from the Episcopal Hospital, in Philadelphia. Testing for in vitro P Z A resistance was performed by the National Jewish Center for Immunology and Respiratory Medicine (Denver, CO) (University of Pennsylvania isolates) or Smith Kline Clinical Laboratory (King of Prussia, PA) (Episcopal isolates) using the Bactec system at pH = 6.4 [8,9]. DNA was extracted from isolated colonies as previously described [3] and frozen at -70°C. Amplification of the coding region of the pncA gene (560 bp) was performed using the following primer set, modeled after primers previously described [5]: pncA5', 5 ' A T G C G G G C G T T G A T C A T C G T3' (nucleotides 1 to 20 of the pncA gene sequence) and pncA3', 5' C A G G A G C T G C A A A C C AACTCG3' (the reverse complement of nucleotides 560 to 540 of the pncA gene sequence). Amplification was performed using a Gene Amp 9600 Thermal Cycler (Perkin-Elmer; Norwalk, CT) with cycling conditions previously described [5] with the exception that 40 cycles of amplification were performed. The PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen; Chatsworth, CA) and cycle sequencing with Taq polymerase was performed using the AB! PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems; Foster City, CA) [3]. Each PCR product was purified using a Centri-sep spin column (Princeton Separations;

Of the 147 M. tuberculosis isolates identified in the University of Pennsylvania Microbiology Laboratory, only 12 were tested for in vitro susceptibility to PZA. Ten of these isolates were susceptible, and two were resistant to P Z A by in vitro testing. In addition, four clinical isolates from Episcopal Hospital were also tested for in vitro susceptibility to PZA and all were resistant.These 16 clinical isolates were used for P Z A susceptibility testing by sequence analysis. DNA, which had been extracted from these isolates at the time of initial culture and stored at -70°C, was analyzed for mutations in the pncA gene. A 560 base pair fragment of the pncA gene was amplified from all samples. Sequence analysis of 10/10 (100%) P Z A susceptible and 4/6 (67%) of the resistant samples showed a wild-type sequence. The two other PZA-resistant isolates had previously unreported mutations in the pncA gene, which would cause an amino acid substitution in the encoded protein. One novel mutation was found in isolate 93-179 (University of Pennsylvania isolate), shown to be P Z A resistant by in vitro testing. The pncA gene from this isolate contained a T to G mutation at base pair 241 on the forward strand (241T-~G), with the corresponding A to C mutation on the reverse strand. Repeat sequence analysis, including PCR amplification, confirmed the presence of this mutation. This point mutation results in the substitution of the phenylalanine at position 80 with a valine (F80V). Unique restriction sites for AatII and Ppu1253 I, as well as an additional site for Hin8 I, Hsp92 I, MaeII, Tail, and TtmI are created by this mutation. No restriction sites were lost. This isolate was also resistant to isoniazid, rifampin, streptomycin, and ethambutol by in vitro testing. The second novel mutation, 511G-~C, was identified in a second isolate, 98-25 (Episcopal isolate), which was also resistant to P Z A by in vitro testing

Novel pncA Mutations in M . t u b e r c u l o s i s

(Fig. 1). This gene mutation leads to a proline for alanine substitution at position 171 of the amino acid sequence (A171P). Unique restriction sites for Bscl07 I, Bsc4 I, BslI, B s t Z l l I, FauI, and MspA1 I are created by this mutation, whereas sites for BcaI, BceF I, BsoF I, BssF I, BstU I, HhaI, Hinpl I, M.SssI, Sell, and TauI are lost. This mutation is close to the 3' end of the coding region of the pncA gene.

Discussion The amidase activity encoded by pncA is required to convert P Z A to the active pyrazinoic acid [10]. Twenty-four mutations in the pncA gene that correlate with P Z A resistance have been reported [5,7]. These mutations are distributed along the entire length of the pncA gene (Fig. 1). The four most common mutations (at positions 70, 139, 254, and 388) account for approximately half of the resistant isolates reported in the literature (Fig. 1) [5,7]. All the previously reported mutations are located between nucleotides 56 and 422, except for one mutation at position 11. The mutation reported in this study at position 241 is within the region of previously reported mutations. However, the second mutation at position 511 is approximately 90 nucleotides 3' of the previously reported mutations. Sreevatsan et al. [5] made the observation that the majority of mutations associated with P Z A resistance are point mutations resulting in single amino acid substitutions, as is also true for rifampin and isoniazid drug resistance gene mutations. Both the new mutations identified in this study also result in a single amino acid substitution. Sreevatsan et al. [5] also observed the high frequency of proline amino acids introduced by the point mutations causing P Z A resistance. Whereas approximately 40% of the identified point mutations result in the introduction of a proline amino acid, approximately 68% of isolates with an identified point mutation in the pncA gene introduce a proline into the protein. One of the two mutations reported in this study also leads to a proline substitution. A proline substitution introduces a constraint on the secondary structure of a polypeptide chain and could compromise protein function. The other new mutation results in a substitution of valine for phenylalanine, which does not significantly alter the

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charge of the protein, but the side group of valine is smaller than that of phenylalanine and may alter the protein structure. No studies have evaluated the structure-function correlation for the PZase protein to allow analysis of the significance of these amino acid substitutions on protein function. To date, mutations in the pncA gene have not been identified in isolates that are susceptible to P Z A by in vitro methods [5,7]. None of the 10 susceptible isolates in this study had pncA gene mutations. However, four of the six PZA-resistant isolates tested had a wild-type sequence for the pncA gene. Such isolates have been previously reported in the study by Sreevatsan et al. [5], in which mutations in the pncA gene were identified in only 48 of 67 resistant isolates tested (72%). The significantly lower percentage of PZA-resistant isolates with identifiable pncA gene mutations in this study (33%) may be due to the few resistant isolates tested or to regional differences in susceptibility patterns for M. tuberculosis. Mutations may exist in the noncoding region of the gene, in the first or last 40 nucleotides of the gene coding sequence, or in other genes required for P Z A susceptibility, which are not assessed by this assay. However, only one noncoding region point mutation (at position - 11) has been reported in two isolates [5], and no mutations have been reported in the first or last 40 nucleotides of the gene [5,7]. It is also possible that these isolates were "false resistant," similar to those described by Scorpio and Zhang [7], as in vitro testing for P Z A resistance may be unreliable due to specific pH requirements and sensitivity to inoculum size [8,9]. PZase activity was not measured for these resistant isolates; therefore, a correlation between PZase activity and the reported pncA mutations is not possible. In summary, we have identified two novel mutations in the pncA gene that correlate with in vitro resistance to P Z A in M. tuberculosis. Results of our molecular susceptibility testing are available 48 to 72 hours after isolated colonies of M. tuberculosis are available, as compared with 2 to 3 weeks for in vitro testing. Drug susceptibility testing might also be possible using organisms grown in broth liquid culture; however, the processing methods for this approach were not addressed in the current study. Because mutations in this gene associated with resistance to P Z A are not localized to a specific gene region, it is important that clinical

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assays scan the entire gene sequence for mutations. Automated D N A sequencing represents a relatively rapid method for detection of novel mutations in the pncA coding sequence. Received June 1, 1998. Received in revised form June 11, 1998. Accepted August 27, 1998.

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gene and streptomycin resistance in single and multiple drug-resistant strains of Mycobacterium tuberculosis. Mol Microbiol 1993;10:521-527 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;41:636-640 Telenti A, Imboden R Marchesi F, Lowrie D, Cole S, Colston MJ, Matter L, Schopfer K, Bodmer T: Detection of rifampicin-resistance mutations in Mycobacteriurn tuberculosis. Lancet 1993;341:647650 Scorpio A, Zhang Y: Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculous drug pyrazinamide in tubercle bacillus. Nature Med 1996;2:662-667 Heifers LB: Drug susceptibility in the chemotherapy of mycobacterial infections. In pyrazinamide susceptibility. CDC Press, Boca Raton, 1991, pp. 109-115 Seddegi SH: Pyrazinamide (PZA) susceptibility testing. In Bactec 460TB system products and procedures manual. 1995, pp. VI-I-VI-7 Konno K, Feldmann FM, McDermott W: Pyrazinamide susceptibility and amidase activity of tubercle baculli. Am Rev Respir Dis 1967;95:461469