This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Springer, B.
Right arrow Articles by Böttger, E. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Springer, B.
Right arrow Articles by Böttger, E. C.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 2008, p. 4064-4067, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01114-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Tuberculosis Drug Resistance in an Area of Low Endemicity in 2004 to 2006: Semiquantitative Drug Susceptibility Testing and Genotyping{triangledown}

Burkhard Springer,1,2,3 Romana C. Calligaris-Maibach,1,2 Claudia Ritter,1,2 and Erik C. Böttger1,2*

Institut für Medizinische Mikrobiologie, Universität Zürich, Zürich CH-8006, Switzerland,1 Nationales Zentrum für Mykobakterien, Zürich CH-8006, Switzerland,2 Institut für Medizinische Mikrobiologie und Hygiene, Österreichische Agentur für Gesundheit und Ernährungssicherheit, Graz A-8010, Austria3

Received 11 June 2008/ Returned for modification 11 September 2008/ Accepted 3 October 2008


arrow
ABSTRACT
 
We determined the quantitative levels and the genetic mechanisms of resistance in drug-resistant clinical isolates of Mycobacterium tuberculosis sampled over a period of 3 years (n = 45; 17 of the isolate were multidrug resistant). Our results led us to hypothesize that some strains categorized as resistant to isoniazid, ethambutol, or streptomycin by standard laboratory procedures of in vitro drug susceptibility testing may still respond to a treatment regimen that includes these agents.


arrow
TEXT
 
The treatment of multidrug-resistant (MDR) and extensively drug resistant (XDR) tuberculosis (TB) requires the use of second-line drugs. These drugs are less effective, more expensive, and more toxic than the first-line drugs. The rate of treatment success for drug-resistant TB, particularly MDR-TB or XDR-TB, is significantly lower than that for drug-susceptible TB (4, 11). In the laboratory, drug susceptibility testing of mycobacteria is substantially different from standard procedures in diagnostic bacteriology (3). Thus, rather than determination of the MICs, a single drug concentration, termed the critical concentration, is mostly used to categorize a clinical isolate as susceptible or resistant. These concentrations were introduced in the past to differentiate wild-type strains from strains with alterations in drug susceptibility, but they do not correspond to the drug concentrations present in serum or infected tissues (12, 17). With growing knowledge about the mechanisms that underlie drug resistance, it has become clear that drug resistance is multifaceted and that different mutations may lead to different levels of resistance. However, different levels of phenotypic resistance are not taken into account by using critical concentrations for in vitro drug susceptibility testing. This may be the reason why for more than one-third of cases of MDR-TB, standard short-course therapy has been found to be an effective treatment (1, 6).

In the study described here, we systematically evaluated quantitative resistance levels in drug-resistant clinical isolates of Mycobacterium tuberculosis sampled from 2004 to 2006; the TB case rates in Switzerland were 8.2/100,000 population in 2004, 7.6/100,000 in 2005, and 6.9/100,000 in 2006. The Bactec MGIT 960 system (Becton Dickinson Diagnostic Systems, Sparks, MD) was used for primary isolation and testing for susceptibility to first-line drugs, according to the manufacturer's instructions; the drug concentrations were 0.1 µg/ml for isoniazid, 1.0 µg/ml for rifampin (rifampicin), 5.0 µg/ml for ethambutol, and 100 µg/ml for pyrazinamide. From 2004 to 2006, a total of 45 clinical isolates of M. tuberculosis were categorized in the laboratory as resistant to one or more first-line drugs on the basis of standard critical concentration testing. The collection of strains investigated included all MDR strains sampled in Switzerland during that period, thus representing a nationwide survey for MDR-TB in a country with a low level of endemicity; MDR strains were submitted to the Nationales Zentrum für Mykobakterien as part of its function as a national reference center. By IS6110 profiling, the MDR strains were determined to represent independent, nonclonally related isolates. The panel of clinical isolates was retested for drug susceptibility at critical concentrations as well as at higher drug concentrations with the Bactec 460 TB system, as suggested by the manufacturer. The following drug concentrations were tested: isoniazid at 0.1 µg/ml, 0.4 µg/ml, 1.0 µg/ml, 3.0 µg/ml, and 10.0 µg/ml; rifampin at 1.0 µg/ml, 10.0 µg/ml, and 50.0 µg/ml; ethambutol at 2.5 µg/ml, 5.0 µg/ml, 12.5 µg/ml, and 50.0 µg/ml; streptomycin at 1.0 µg/ml, 10.0 µg/ml, and 50.0 µg/ml; ethionamide at 1.25 µg/ml and 12.5 µg/ml; amikacin at 1.0 µg/ml, 10.0 µg/ml, 50.0 µg/ml; and ofloxacin 2.0 µg/ml and 20.0 µg/ml. Interpretation was performed by using the standard interpretation procedure recommended by BD (resistant when the growth index for the drug-containing vial was greater than that for the drug-free control and susceptible when the growth index for the drug-containing vial was less than that for the drug-free control).

For molecular profiling, the GenoType MTBDRplus assay (Hain Lifescience, Nehren, Germany) was used (8, 16). The GenoType MTBDRplus assay is a reverse hybridization line probe assay designed for the rapid detection of isoniazid and rifampin resistance-conferring mutations. For the detection of mutational alterations associated with resistance to ethambutol or streptomycin, i.e., embB position 306 (10) and rpsL positions 42 and 87 (7), respectively, PCR-driven gene amplification and nucleic acid sequence determination were used.

Of 44 isolates found to be resistant to isoniazid at 0.1 µg/ml, 13 were found to be susceptible at 0.4 µg/ml (Table 1). According to the GenoType MTBDRplus assay, none of these 13 strains with low-level isoniazid resistance had a mutation in katG, but 11 of the 13 strains with low-level isoniazid resistance showed an inhA promoter mutation (–15C/T), leading to concomitant resistance to ethionamide (see Table 1 for details and Table 2 for a summary of the results). It has previously been shown that the overexpression of InhA leads to low or moderate levels of resistance to isoniazid and ethionamide (13). Thirty-one isolates were resistant to isoniazid at levels of 1 µg/ml or greater. The GenoType MTBDRplus assay detected the katG S315T mutation in 21 of these isolates. This particular mutation is the most frequent isoniazid resistance-conferring mutation in clinical strains (20). In our analysis, the isolates harboring the katG S315T mutation displayed resistance to various levels of isoniazid, ranging from 1 µg/ml to more than 10 µg/ml. One isolate showed a katG mutation, which led to the loss of hybridization of the katG wild-type probe but no binding to a specific mutated probe. Two isolates showed a katG and an inhA promoter mutation in parallel; in four isolates, a –15C/T inhA promoter mutation was found. All isolates harboring inhA promoter mutations were also resistant to ethionamide.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Quantitative resistance levels and genotypes of drug-resistant M. tuberculosis isolatesa


View this table:
[in this window]
[in a new window]

  		TABLE 2. Summary of genotypic resistance and quantitative resistance levels in clinical M. tuberculosis isolates categorized as resistant by critical concentration testinga

Nineteen isolates were found to be resistant to rifampin. All rifampin-resistant isolates exhibited a phenotype of high-level drug resistance (>50 µg/ml) and harbored well-known resistance mutations. The most frequent rpoB mutation in our collection of rifampin-resistant isolates was S531L (15 of 19 isolates), 2 isolates showed an H526Y mutation, and 1 isolate each had a H526D and a D516V mutation (Table 1). One isolate showed a rifampin-monoresistant phenotype and was susceptible to isoniazid, ethambutol, streptomycin, and pyrazinamide. Seventeen isolates showed simultaneous intermediate or high-level resistance to isoniazid and thus represented bona fide MDR strains. No XDR phenotype was present in our collection of MDR isolates, as the isolates remained susceptible to either the fluoroquinolones or the injectable second-line antibiotics (Table 1).

Given the small difference between the drug concentration used for in vitro drug susceptibility testing and the natural drug susceptibility of wild-type isolates of M. tuberculosis, testing for susceptibility to ethambutol is particularly problematic (14). Ethambutol resistance was mainly found in MDR strains. Fifteen isolates had MICs above 2.5 µg/ml, with 11/15 isolates displaying resistance to 5 µg/ml or greater. Two isolates showed a MIC of 12.5 µg/ml, but interestingly, no high-level resistance to ethambutol at 50 µg/ml was observed. In eight isolates, the previously described mutation at position 306 in embB was found by nucleic acid sequencing (10); these isolates displayed resistance to ethambutol at a concentration of at least 2.5 µg/ml.

Streptomycin, amikacin, and ofloxacin were tested only with selected clinical isolates, in particular, MDR strains. Two isolates showed ofloxacin resistance at 2.0 µg/ml but susceptibility at 20.0 µg/ml; both isolates were susceptible to amikacin (Table 1) and capreomycin (data not shown). One isolate was found to have high-level resistance to amikacin, with a MIC above 50.0 µg/ml; sequence analysis revealed the specific aminoglycoside resistance-conferring mutation A1408G in 16S rRNA (18). For streptomycin, there was a strict correlation between the genetic mechanism of resistance and the phenotypic resistance level conferred. While mutations in rpsL in general mediate high-level drug resistance, a significant part of clinical strains categorized as drug resistant exhibit a low-level-resistance phenotype and have no mutational target alteration in rpsL (15). Of 16 isolates tested, 13 showed resistance to streptomycin at 1.0 µg/ml. Five isolates showed a low- to intermediate-level-resistance phenotype and susceptibility to the drug at 10.0 µg/ml. All seven isolates displaying a high-level-resistance phenotype (resistant to 50.0 µg/ml) harbored the no-cost Lys->Arg alteration at position 42 of rpsL (3), indicating significant clinical selection pressure for this particular mutation (2, 19).

Quantitative drug susceptibility testing is essential for the recognition of residual drug activity against infecting M. tuberculosis strains. According to our data, no high-level ethambutol resistance exists. It thus remains to be determined whether an in vitro resistant phenotype implies the ineffectiveness of ethambutol in vivo, in particular as a component of combination therapy. In contrast, isolates displaying rifampin resistance were always high-level resistant, with MICs of >50 µg/ml. While promoter alterations in inhA result in low-level isoniazid resistance, they are associated with significant resistance to ethionamide. Some evidence indicates that isoniazid may be a valuable antibiotic, despite the presence of a low- or intermediate-level-resistance phenotype (5, 9). The distribution of isoniazid resistance-associated mutations in isoniazid-monoresistant isolates was different from that in multidrug-resistant isolates, with mutations in the inhA promoter region and, thus, low-level resistance being more common in monoresistant strains.

Taken together, our data indicate that some first-line drugs may be considered therapeutic treatment options, despite the presence of in vitro resistance at the critical concentration. Diagnostic mycobacteriology would benefit from standardized measures of quantitative drug susceptibility testing, in particular, for those drugs for which significant variations in phenotypic resistance levels are found in clinical isolates, e.g., isoniazid, ethambutol, and streptomycin.


arrow
ACKNOWLEDGMENTS
 
We thank P. Bosshard for data collection in the initial part of the project and P. Helbling for providing epidemiological information.

The study was supported in part by grants from the Bundesamt für Gesundheit, Bern, Switzerland, the Niedersächsischer Verein zur Bekämpfung der Tuberkulose Lungen- und Bronchialerkrankungen e.V., Hanover, Germany, and the University of Zurich.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Institut für Medizinische Mikrobiologie, Universität Zürich, Gloriastrasse 30/32, Zürich CH-8006, Switzerland. Phone: 41 44 634 26 60. Fax: 41 44 634 49 06. E-mail: boettger{at}immv.uzh.ch Back

{triangledown} Published ahead of print on 15 October 2008. Back


arrow
REFERENCES
 
    1
  1. Böttger, E. C. 2001. Drug-resistant tuberculosis. Lancet 357:1288-1289.[Medline]
    2. 2
  2. Böttger, E. C., B. Springer, M. Pletschette, and P. Sander. 1998. Fitness of antibiotic-resistant microorganisms and compensatory mutations. Nat. Med. 4:1343-1344.[CrossRef][Medline]
    3. 3
  3. Böttger, E. C., and B. Springer. 2008. Tuberculosis: drug resistance, fitness, and strategies for global control. Eur. J. Pediatr. 67:141-148.
    4. 4
  4. CDC. 2006. Emergence of Mycobacterium tuberculosis with extensive resistance to second-line drugs—worldwide, 2000-2004. MMWR Morb. Mortal. Wkly. Rep. 55:301-305.[Medline]
    5. 5
  5. Cynamon, M. H., Y. Zhang, T. Harpster, S. Cheng, and M. S. DeStefano. 1999. High-dose isoniazid therapy for isoniazid-resistant murine Mycobacterium tuberculosis infection. Antimicrob. Agents Chemother. 43:2922-2924.[Abstract/Free Full Text]
    6. 6
  6. Espinal, M. A., S. I. Kim, G. Suarez, K. M. Kam, A. G. Khomenko, G. B. Migliori, J. Baéz, A. Kochi, C. Dye, and M. C. Ravligione. 2000. Standard short-course chemotherapy for drug-resistant tuberculosis: treatment outcomes in 6 countries. JAMA 283:2537-2545.[Abstract/Free Full Text]
    7. 7
  7. Finken, M., P. Kirschner, A. Meier, A. Wrede, and E. C. Böttger. 1993. Molecular basis of streptomycin resistance in Mycobacterium tuberculosis: alterations of the ribosomal protein S12 gene and point mutations within a functional 16S ribosomal RNS pseudoknot. Mol. Microbiol. 9:1239-1246.[Medline]
    8. 8
  8. Hillemann, D., S. Rüsch-Gerdes, and E. Richter. 2007. Evaluation of the GenoType MTBDRplus assay for rifampin and isoniazid susceptibility testing of Mycobacterium tuberculosis strains and clinical specimens. J. Clin. Microbiol. 45:2635-2640.[Abstract/Free Full Text]
    9. 9
  9. Jayaram, R., R. K. Shandil, S. Gaonkar, P. Kaur, B. L. Suresh, B. N. Mahesh, R. Jayashree, V. Nandi, S. Bharath, E. Kantharaj, and V. Balasubramanian. 2004. Isoniazid pharmacokinetics-pharmacodynamics in an aerosol infection model of tuberculosis. Antimicrob. Agents Chemother. 48:2951-2957.[Abstract/Free Full Text]
    10. 10
  10. Johnson, R., A. M. Jordaan, L. Pretorius, E. Engelke, G. van der Spuy, M. Bosman, P. D. van Helden, R. Warren, and T. C. Victor. 2006. Ethambutol resistance testing by mutation detection. Int. J. Tuberc. Lung Dis. 10:68-73.[Medline]
    11. 11
  11. Kim, H. R., S. S. Hwang, H. J. Kim, S. M. Lee, C. G. Yoo, Y. W. Kim, S. K. Han, Y. S. Shim, and J. J. Yim. 2007. Impact of extensive drug resistance on treatment outcomes in non-HIV-infected patients with multidrug-resistant tuberculosis. Clin. Infect. Dis. 45:1290-1295.[CrossRef][Medline]
    12. 12
  12. Kim, S. J. 2005. Drug-susceptibility testing in tuberculosis: methods and reliability of results. Eur. Respir. J. 25:564-569.[Abstract/Free Full Text]
    13. 13
  13. Larsen, M. H., C. Vilchèze, L. Kremer, G. S. Basra, L. Parsons, M. Salfinger, L. Heifets, M. H. Hazbon, D. Alland, J. C. Sacchettini, and W. R. Jacobs, Jr. 2002. Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis. Mol. Microbiol. 46:453-466.[CrossRef][Medline]
    14. 14
  14. Madison, B., B. Robinson-Dunn, I. George, W. Gross, H. Lipman, B. Metchuck, A. Sloctsky, G. Washabange, G. Mazurek, and J. Riddershof. 2002. Multicenter evaluation of ethambutol susceptibility testing of Mycobacterium tuberculosis by agar proportion and radiometric methods. J. Clin. Microbiol. 40:3976-3979.[Abstract/Free Full Text]
    15. 15
  15. Meier, A., P. Sander, K. J. Schaper, M. Scholz, and E. C. Böttger. 1996. Correlation of molecular resistance mechanisms and phenotypic resistance levels in streptomycin-resistant Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 40:2452-2454.[Abstract]
    16. 16
  16. Miotto, P., F. Piana, D. M. Cirillo, and G. B. Migliori. 2008. Genotype MTBDRplus: a further step toward rapid identification of drug-resistant Mycobacterium tuberculosis. J. Clin. Microbiol. 46:393-394.[Free Full Text]
    17. 17
  17. Nuernberger, E., and J. Grosset. 2004. Pharmacokinetic and pharmacodynamic issues in the treatment of mycobacterial infections. Eur. J. Clin. Microbiol. Infect. Dis. 23:243-255.[CrossRef][Medline]
    18. 18
  18. Prammananan, T., P. Sander, B. A. Brown, K. Frischkorn, G. O. Onyi, Y. Zhang, E. C. Böttger, and R. J. Wallace, Jr. 1998. A single 16S ribosomal RNA substitution is responsible for resistance to amikacin and other 2-deoxystreptamine aminoglycosides in Mycobacterium abscessus and Mycobacterium chelonae. J. Infect. Dis. 177:1573-1581.[Medline]
    19. 19
  19. Sander, P., B. Springer, T. Prammananan, A. Sturmfels, M. Kappler, M. Pletschette, and E. C. Böttger. 2002. Fitness cost of chromosomal drug resistance-conferring mutations. Antimicrob. Agents Chemother. 46:1204-1211.[Abstract/Free Full Text]
    20. 20
  20. van Soolingen, D., P. E. de Haas, H. R. van Doorn, E. Kuijper, H. Rinder, and M. W. Borgdorff. 2000. Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in the Netherlands. J. Infect. Dis. 182:1788-1790.[CrossRef][Medline]

Journal of Clinical Microbiology, December 2008, p. 4064-4067, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.01114-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Springer, B., Lucke, K., Calligaris-Maibach, R., Ritter, C., Bottger, E. C. (2009). Quantitative Drug Susceptibility Testing of Mycobacterium tuberculosis by Use of MGIT 960 and EpiCenter Instrumentation. J. Clin. Microbiol. 47: 1773-1780 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Springer, B.
Right arrow Articles by Böttger, E. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Springer, B.
Right arrow Articles by Böttger, E. C.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»