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Antimicrobial Agents and Chemotherapy, April 2000, p. 1075-1077, Vol. 44, No. 4
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Mutations in the Beginning of the rpoB Gene Can Induce Resistance to Rifamycins in both Helicobacter pylori and Mycobacterium tuberculosis

Markus Heep,^* Ulrich Rieger, Daniela Beck, and Norbert Lehn

Institute for Medical Microbiology, Regensburg, Germany

Received 26 July 1999/Returned for modification 23 September 1999/Accepted 10 January 2000

	ABSTRACT

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A clinical isolate of Helicobacter pylori that developed resistance to rifabutin during therapy carried an rpoB gene that retained a wild-type cluster region sequence but had acquired a novel codon 149 (V149F) mutation. In transformation experiments, the mutation was shown to confer high-level rifabutin resistance. The equivalent mutation (V176F) was present in several resistant isolates of Mycobacterium tuberculosis.

	TEXT

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Rifabutin and other derivatives of rifampin are inhibitory against Helicobacter pylori at very low concentrations in vitro (1, 4). Triple therapy including rifabutin, amoxicillin, and a proton pump inhibitor has been effective in the eradication of H. pylori after failure of other therapies and in spite of resistance to other antibiotics (10). Resistance to rifampin and rifabutin is caused by amino acid exchanges in the  subunit of the DNA-directed RNA polymerase (RpoB). Mutations at codons 146, 507 to 533, 563 to 572, and 687 of the rpoB gene in Escherichia coli (11) or at codons 507 to 533 (cluster region) in Mycobacterium tuberculosis (8, 9) have been shown to induce resistance.

All resistant mutants of H. pylori ATCC 43504 selected in vitro in a previous investigation (4) showed mutations in the cluster region encompassing codons 525 to 545 and codon 586.

Here we describe a clinical isolate of H. pylori that developed resistance during therapy. The patient was treated with lanzoprazole and rifabutin, but amoxicillin was discontinued due to intolerance. Isolates of H. pylori before (DR62a) and after (DR62n) treatment were available for evaluation. Culture, storage, E-test, agar dilution assay, PCR, and sequencing of the cluster region were performed as described recently (4). Table 1 shows the primers for the amplification and evaluation of large and small segments of the respective rpoB regions. Sequencing was performed in both directions (Perkin-Elmer Applied Biosystems).

View this table: [in this window] [in a new window]   TABLE 1.   PCR primers for amplification and sequencing of the respective H. pylori and M. tuberculosis rpoB segments

DR62n was resistant to rifampin (E-test, MIC of >256 µg/ml) and rifabutin but, in contrast to previous findings (4), showed no difference from the published wild-type sequence (14) in the cluster region. The MICs of rifabutin for H. pylori DR62a and DR62n were 0.002 and 8 µg/ml, respectively (Table 2). Both strains yielded homologous patterns when typed by arbitrarily primed PCR (2; unpublished work). Amplification and sequencing of DR62n rpoB (rifabutin resistant) from bp 387 to bp 916 revealed a codon exchange, GTCTTC (V149F). DR62a (rifabutin susceptible) showed wild-type sequence in both the V149 region and the cluster region.

View this table: [in this window] [in a new window]   TABLE 2.   MICs (agar dilution) of rifampin and rifabutin for H. pylori with wild-type rpoB sequence (DR62a and 2802A) and for H. pylori harboring a novel mutation, V149F (DR62n and 2802A-R)a

In order to confirm that the amino acid mutation V149F is responsible for high-level resistance, large fragments of the rpoB gene (bp 54 to 916 harboring codon 149 and bp 1271 to 2106 harboring the cluster region) from DR62n and DR62a were amplified and sequenced. The amino acid mutation V149F was the only difference between DR62n and DR62a or the published wild-type sequence (14). All fragments were transformed into a competent, rifabutin-susceptible strain, H. pylori 2802A. Transformation of H. pylori 2802A was done, using a dense suspension of a fresh culture adjusted to a McFarland standard of >4 in 1 ml at an optical density value at 550 nm (OD₅₅₀) of 10, diluted to an OD₅₅₀ value of 0.2 in brucella broth with 8% fetal calf serum. One milliter of a suspension with an OD₅₅₀ value of 0.2 (in a 24-well plate) was incubated at 36°C with 3 to 5% CO₂ for 6 h. At 0, 2, and 4 h, 3 to 10 µl of a PCR product (1 to 3 µg of DNA) was added. Cells were cultured on Wilkins-Chalgren agar containing rifabutin (0.03 µg/ml) under microaerophilic conditions for 3 to 5 days. Single colonies were picked and grown on selective agar for MIC determination, storage, DNA amplification, and sequencing.

Repeated transformation of H. pylori 2802A with PCR products harboring the V149F mutation (bp 54 to 916 from DR62n rpoB) generated 10⁵ to 10⁶ CFU/well (2802A-R) showing the same level of resistance as seen in the donor strain (Table 2). Transformation with PCR products of the wild-type 5' region (bp 54 to 916) or cluster region (bp 1371 to 2106) of H. pylori rpoB generated fewer than five resistant colonies per well.

Positive controls for the cluster region (e.g., Q527R) were amplified from the DNA of rifabutin-resistant H. pylori ATCC 43504 variants (4) and generated 10⁵ to 10⁶ CFU/well (data not shown).

After we had the confirmation of a newly described mutation outside the cluster region conferring high-level resistance in H. pylori, we wondered whether this arrangement might also occur in M. tuberculosis. PCR-based systems for the detection of mutations in the cluster region are of outstanding importance for the determination of rifampin susceptibility of slow-growing organisms like M. tuberculosis but sometimes fail to match with a resistant phenotype. Reported failure rates range from 3 to 6% (3, 5, 7, 9, 13, 16, 17). DNA samples of six M. tuberculosis isolates from Japan for which MICs of rifampin were elevated but with wild-type sequence in the rpoB cluster region were provided by the authors of reference 17.

Primers for PCR and sequencing are shown in Table 1. Three out of six samples showed the mutation GTCTTC (V176F), consistent with our findings in DR62n. The amino acid changes detected are shown in Table 2 and mapped in Fig. 1. There is no report in the literature so far of a mutation in this region in mycobacteria.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Alignment of the homologous amino acid sequences of the N-terminal region of RpoB of different species. The figure shows mapping of codons associated with the rifamycin resistance phenotype (boxes).

Rudi Rossau et al. (Innogenetics) recently presented data at a meeting (R. Rossau, W. Mus, G. Jannes, K. de Smet, H. van Heuverswijn, H. Traore, and F. Portaels, 20th Annu. Congr. Eur. Soc. Mycobacteriol., abstr. OC27, p. 44, 1999) which are in strong agreement with ours and emphasize the importance of our findings. They investigated 203 resistant M. tuberculosis isolates, finding discrepant results in four isolates. Three out of four samples showed the mutation V176F, induced by the same base exchange. Their collection contained no isolates from Japan. However, no comparable mutation in eight resistant M. tuberculosis strains with the wild-type cluster region was found by Telenti et al. (8; A. Telenti, personal communication; no MIC given).

The amino acid mutation V146F conferring high-level resistance in E. coli was first observed by Lisitsyn et al. in 1984 (6) and verified by Severinov et al. (12) by site-directed mutagenesis (Fig. 1). Recently, three mutations associated with resistance were described for a Rickettsia typhi isolate (15). Mutations were observed at codons 151 (F151L), 201, and 271. Codon 151 is located next to the region which has been shown to confer resistance and is possibly the one responsible for the resistant phenotype. Out of six M. tuberculosis isolates, three with the higher level of resistance to rifampin (8 to 32 µg/ml) carried the V176F mutation, while the low-level resistance (1 to 4 µg/ml) remains unexplained.

Mutations in the 5' region of the rpoB gene inducing rifamycin resistance in H. pylori and possibly in M. tuberculosis have not been sufficiently examined. Resistance can be caused by a single base exchange outside the cluster region. This should be considered in future developments of molecular biology-based testing for mycobacteria.

The high homology of this region on the amino acid level (Fig. 1) indicates that there might be mutations conferring resistance in other species like Legionella pneumophila or Staphylococcus aureus as well.

Nucleotide sequence accession numbers. The relevant sequence fragments of M. tuberculosis strain 84 and H. pylori DR62n have been deposited in the GenBank database under accession no. AF 177294 and AF 177295, respectively.

	ACKNOWLEDGMENTS

The DNA samples of resistant M. tuberculosis isolates were kindly provided by Shigeru Kohno, Nagasaki University, Nagasaki, Japan. H. Bock, Frankfurt, Germany, provided the biopsy specimens from the patient before and after H. pylori eradication therapy. We thank Regine Birngruber for excellent technical assistance.

	FOOTNOTES

^* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie, Uniklinik Regensburg, D-93053, Regensburg, Germany. Phone: 49 941 944-6414. Fax: 49 941 944-6402. E-mail: Markus.Heep{at}klinik.uni-regensburg.de.

	REFERENCES

Top Abstract Text References

1.	Akada, J. K., M. Shirai, K. Fujii, K. Okita, and T. Nakazawa. 1999. In vitro anti-Helicobacter pylori activities of new rifamycin derivatives, KRM-1648 and KRM-1657. Antimicrob. Agents Chemother. 43:1072-1076[Abstract/Free Full Text].
2.	Berg, D. E., L.-G. Janaki, T. I. Engin, S. Kalpana, and N. S. Akopyants. 1997. H. pylori DNA fingerprinting using the arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD) method. Methods Mol. Med. 8:117-132.
3.	Cooksey, R. C., G. P. Morlock, S. Glickman, and J. T. Crawford. 1997. Evaluation of a line probe assay kit for characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from New York City. J. Clin. Microbiol. 35:1281-1283[Abstract].
4.	Heep, M., D. Beck, E. Bayerdörffer, and N. Lehn. 1999. Rifampin and rifabutin resistance mechanism in Helicobacter pylori. Antimicrob. Agents Chemother. 43:1497-1499[Abstract/Free Full Text].
5.	Kapur, V., L. L. Li, S. Iordanescu, M. R. Hamrick, A. Wanger, B. N. Kreiswirth, and J. M. Musser. 1994. Characterization by automated DNA sequencing of mutations in the gene (rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas. J. Clin. Microbiol. 32:1095-1098[Abstract/Free Full Text].
6.	Lisitsyn, N. A., E. D. Sverdlov, E. P. Moiseyeva, O. N. Danilevskaya, and V. G. Nikiforov. 1984. Mutation to rifampicin resistance at the beginning of the RNA polymerase beta subunit gene in Escherichia coli. Mol. Gen. Genet. 196:173-174[CrossRef][Medline].
7.	Matsiota-Bernard, P., G. Vrioni, and E. Marinis. 1998. Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Greece. J. Clin. Microbiol. 36:20-23[Abstract/Free Full Text].
8.	Musser, J. M. 1995. Antimicrobial agent resistance in mycobacteria: molecular genetic insights. Clin. Microbiol. Rev. 8:496-514[Abstract].
9.	Ohno, H., H. Koga, S. Kohno, T. Tashiro, and K. Hara. 1996. Relationship between rifampin MICs for and rpoB mutations of Mycobacterium tuberculosis strains isolated in Japan. Antimicrob. Agents Chemother. 40:1053-1056[Abstract].
10.	Perri, F., V. Festa, and A. Andriulli. 1998. Treatment of antibiotic-resistant Helicobacter pylori infection. N. Engl. J. Med. 339:53[Free Full Text].
11.	Severinov, K., M. Soushko, A. Goldfarb, and V. Nikiforov. 1993. Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase. J. Biol. Chem. 268:14820-14825[Abstract/Free Full Text].
12.	Severinov, K., M. Soushko, A. Goldfarb, and V. Nikiforov. 1994. RifR mutations in the beginning of the Escherichia coli rpoB gene. Mol. Gen. Genet. 244:120-126[Medline].
13.	Telenti, A., P. Imboden, F. Marchesi, D. Lowrie, S. Cole, M. J. Colston, L. Matter, K. Schopfer, and T. Bodmer. 1993. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341:647-650[CrossRef][Medline].
14.	Tomb, J. F., O. White, A. R. Kerlavage, R. A. Clayton, G. G. Sutton, R. D. Fleischmann, K. A. Ketchum, H. P. Klenk, S. Gill, B. A. Dougherty, K. Nelson, J. Quackenbush, L. Zhou, E. F. Kirkness, S. Peterson, B. Loftus, D. Richardson, R. Dodson, H. G. Khalak, A. Glodek, K. McKenney, L. M. Fitzegerald, N. Lee, M. D. Adams, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539-547[CrossRef][Medline].
15.	Troyer, J. M., S. Radulovic, S. G. Andersson, and A. F. Azad. 1998. Detection of point mutations in rpoB gene of rifampin-resistant Rickettsia typhi. Antimicrob. Agents Chemother. 42:1845-1846[Abstract/Free Full Text].
16.	Williams, D. L., L. Spring, L. Collins, L. P. Miller, L. B. Heifets, P. R. Gangadharam, and T. P. Gillis. 1998. Contribution of rpoB mutations to development of rifamycin cross-resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 42:1853-1857[Abstract/Free Full Text].
17.	Yang, B., H. Koga, H. Ohno, K. Ogawa, M. Fukuda, Y. Hirakata, S. Maesaki, K. Tomono, T. Tashiro, and S. Kohno. 1998. Relationship between antimycobacterial activities of rifampicin, rifabutin and KRM-1648 and rpoB mutations of Mycobacterium tuberculosis. J. Antimicrob. Chemother. 42:621-628[Abstract/Free Full Text].

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Antimicrobial Agents and Chemotherapy, April 2000, p. 1075-1077, Vol. 44, No. 4
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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