Mycobacterium tuberculosis rpoB gene DNA sequencing: implications for detection of rifamycin resistance

J Antimicrob Chemother 1999; 44: 294–295

Vitali Sintchenko*, Peter J. Jelfs, William K. Chew and Gwendolyn L. Gilbert

Centre for Infectious Diseases and Microbiology Laboratory Services, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, New South Wales, Australia

Sir,

There is a growing demand from clinicians for rapid molecular diagnostic tests for multidrug-resistant Mycobacterium tuberculosis (MDRTB) detection. In their recent paper, Yang et al. 1 have drawn attention to the contribution of different insertion, deletion and missense mutations within a hypervariable region of rpoB, the gene encoding the ß-subunit of the DNA-dependent RNA polymerase of M. tuberculosis, in the development of resistance to rifamycins. They confirmed the observation of Bodmer et al. 2 that the activity of rifabutin in rifampicin-resistant isolates depends on both the mutation position and the type of amino acid change in the rpoB gene. High-frequency mutations in codons 531, 526 and 516 may serve as predictors of resistance to rifampicin, which itself is a useful surrogate marker for MDRTB. 3 The authors 1 have further extended the pool of low-frequency genetic alterations in this region associated with in-vitro resistance to rifampicin and rifabutin.2 ,4 ,5 However, the clinical implications and the diagnostic utility of their detection remain controversial.

To illustrate this point we now report a previously uncharacterized mutation in the rpoB gene of a multidrug-resistant clinical isolate of M. tuberculosis from the sputum of a 31-year-old man who had recently migrated to Australia from Pakistan. He had a long history of chest infections and diabetes mellitus. His pulmonary tuberculosis failed to respond clinically and bacteriologically to fully supervised standard first-line antituberculous treatment and subsequent therapy with ethambutol, pyrazinamide,capreomycin, amikacin and ciprofloxacin. Clinical recovery and sputum culture conversion occurred only after radical surgical lobectomy.

Susceptibility testing on the isolate was performed by the BACTEC 460 (Becton Dickinson, Towson, MD, USA) radiometric method. The isolate was resistant to rifampicin (MIC >2 mg/L), isoniazid and streptomycin, and sensitive to rifabutin (MIC 0.25 mg/L). The commercially available line probe assay (INNO-LiPA Rif.TB; Innogenetics NV, Haven, Belgium), which was performed according to the manufacturer's instructions, showed a wild-type pattern suggesting in-vitro sensitivity to rifampicin. To resolve this contradiction the rifampicin resistance-determining 69 bp region of the rpoB gene was amplified as described by Ohno et al. 5 except that 1U of AmpliTaq DNA polymerase (Perkin–Elmer, Norwalk, CT, USA) per reaction mixture was used. The cycling protocol was: initial denaturation, 95°C for 5 min following by 30 cycles of 1 min at 95°C, 1 min at 55°C and 1 min at 72°C, with a final cycle of 10 min at 72°C. The presence of a 260-bp product in 2% agarose gel indicated a successful amplification of mycobacterium DNA. Amplified DNA was directly sequenced on an automated sequencer with fluorescence-labelled dideoxynucleotide terminators (Model 373, ABI Prism, Perkin– Elmer, Foster City, CA, USA) with IP1 and IP2 primers. 5 DNA sequence analysis revealed a single-point nucleotide substitution affecting the codon of Gln517 (Escherichia coli rpoB codon numbering system 4) (CAG->A) (GenBank accession number AF112973). Neither insertion nor deletion mutations were found.

This case provides evidence to support the opinion that rapid molecular susceptibility testing of M. tuberculosis must be confirmed by conventional rifampicin susceptibility testing. At the same time, it also demonstrates the role of DNA sequence analysis for the detection of new genetic alterations. As the activity of rifamycins in M. tuberculosis largely depends on both the mutation position and the type of substitution in the rpoB gene, this region of clinical isolates with rifampicin-resistant phenotype should be sequenced to identify resistant alleles. Other clusters of mutations in the rpoBgene or other target sites can be associated with resistance to rifampicin and rifabutin. 1 ,5 ,6 ,7

In addition, there are many opportunities for different kinds of mutations that are not critical for the binding activity of RNA polymerase. We believe that the recent successful sequencing of the entire genome of M. tuberculosis will facilitate our understanding of its drug resistance.

Detection of low-frequency M. tuberculosis mutants that remain susceptible to rifabutin is important, considering the limited management options for MDRTB infection. Our analysis of 22 MDRTB isolates in Australia in 1996–1998 revealed that four (18%) rifampicin-resistant MDRTB isolates were rifabutin-sensitive. All of them carried low-frequency rpoB gene alterations in codons 516 and 522.8

Several PCR-based methods including heteroduplex formation, single strand conformation polymorphism, direct DNA sequencing and LiPA can be used for rapid detection of rifamycin resistance in clinical isolates. 7 INNO-LiPA can detect only high-frequency mutations 3 and most other PCR-based methods detect only the presence of a mutation, not the actual substitution in the rpoB gene. This limits our ability to estimate the level of resistance to rifamycins and may lead to false-positive and false-negative reports. Nevertheless, there is a need to quantify antibiotic resistance in order to manage and control MDRTB.

In conclusion, we suggest that PCR analysis should be supported by DNA sequencing of relevant MDRTB target genes. Cumulative data concerning the relationship between the phenotype, clinical response, and type and position of mutations are recommended to facilitate the rapid response required to limit the extent and severity of MDRTB transmission and infection.

Notes

* Corresponding author. Tel: +61-2-9845-6255; Fax: +61-2-9893-8659; E-mail: vitalis{at}icpmr.wsahs.nsw.gov.au Back

References

1 . Yang, B., Koga, H., Ohno, H., Ogawa, K., Fukuda, M., Hirakata, Y. et al. (1998). Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 42, 621–8.[Abstract]

2 . Bodmer, T., Zurcher, G., Imboden, P. & Telenti, A. (1995). Mutation position and type of substitution in the ß-subunit of the RNA polymerase influence in-vitro activity of rifamycins in rifampicin-resistant Mycobacterium tuberculosis. Journal of Antimicrobial Chemotherapy 35, 345–8.[ISI][Medline]

3 . Rossau, R., Traore, H., Beenhouwer, H., Mijs, W., Jannes, G., de Rijk, P. et al. (1997). Evaluation of the INNO-LiPA Rif.TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin. Antimicrobial Agents and Chemotherapy 41, 2093–8.[Abstract]

4 . Telenti, A., Imboden, P., Marchesi, F., Lowrie, D., Cole, S., Colston, M. J. et al. (1993). Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341, 647–50.[ISI][Medline]

5 . Ohno, H., Koga, H., Kohno, S., Tashiro, T. & Hara, K. (1996). Relationship between rifampin MIC for rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrobial Agents and Chemotherapy 40, 1053–6.[Abstract]

6 . Williams, D. L., Spring, L., Collins, L., Miller, L. P., Gangadharam, P. R. J. & Gillis, T. P. (1998). Contribution of rpoB mutations to development of rifampicin cross-resistance in Mycobacterium tuberculosis.Antimicrobial Agents and Chemotherapy 42, 1853–7.[Abstract/Free Full Text]

7 . Drobniewski, F. A. & Wilson, S. M. (1998). The rapid diagnosis of isoniazid and rifampicin resistance in Mycobacterium tuberculosis—a molecular story. Journal of Medical Microbiology 47, 189–96.[Abstract]

8 . Sintchenko, V., Chew, W. K., Jelfs, P. J. & Gilbert, G. L. (1999). Mutations in rpoB gene and rifabutin susceptibility of multidrug-resistant Mycobacterium tuberculosis strains isolated in Australia. Pathology 31, in press.





This Article
Extract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Disclaimer
Request Permissions
Google Scholar
Articles by Sintchenko, V.
Articles by Gilbert, G. L.
PubMed
PubMed Citation
Articles by Sintchenko, V.
Articles by Gilbert, G. L.