PAGES: 745-748 DOI: Short communication
Mycobacterium tuberculosis Complex Differentiation Using gyrB-Restriction Fragment Length Polymorphism Analysis

Erica Chimara +, Lucilaine Ferrazoli, Sylvia Cardoso Leão*

Setor de Micobactérias, Seção de Bacteriologia, Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, 01246-902 São Paulo, SP, Brasil
*Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, SP, Brasil


Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species M. tuberculosis, M. bovis subsp. bovis, M. bovisBCG, M. africanum, M. pinnipedii, and "M. canetti" were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, São Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis  BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovisBCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.

Tuberculosis is one of the leading causes of death due to infectious agents. Ninety-five per cent of cases occur in the developing world, where few resources are available for diagnostic and treatment (ATS 2000). Members of Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in humans and animals and, despite genetic close relationship, they differ in epidemiology, pathogenicity, geographic range, host preference, and in importance for tuberculosis disease in humans. MTBC includes M. tuberculosisM. bovis subsp. bovisM. bovis subsp. capraeM. bovis BCG vaccine strain, M. africanum (subtypes I and II)M. microti, and "M. canetti". The last member of this list has still not been officially recognized on the list of Bacterial Names with Standing in Nomenclature [http://www.bacterio.]. A novel member, M. pinnipedii, proposed by Cousins et al. (2003), has been well-characterized as a specie of M. tuberculosis complex. Genetically, all members of this complex are extremely similar, having 99.9% similarity at the nucleotide level and identical 16SrRNA sequences (Brosch et al. 2002).

Early diagnosis is essential for tuberculosis control. The high degree of sequence conservation among members of MTBC makes differentiation of species in the clinical mycobacteriology laboratory a difficult task. Routine differentiation is still based on phenotypic characteristics, such as oxygen preference, niacin accumulation, nitrate reductase activity, colony morphology, and resistance to two compounds, 2-thiophen-carboxylic acid hydrazide (TCH) and pyrazinamide (PZA) (Collins et al. 1997). These tests need sufficient bacterial growth, are time-consuming and are not performed by many laboratories routinely. Species belonging to MTBC cannot be differentiated by small subunit rRNA (16SrRNA) or internal transcribed spacer (ITS) sequencing. Molecular techniques were designed to rapidly differentiate MTBC species, as Spoligotyping (Kamerbeek et al. 1997), methods to detect mutations in pncA and oxyR genes (Scorpio et al. 1996, Sreevatsan et al. 1996), mtp40-PCR (Del Portillo et al. 1991, Liébana et al. 1996), and PCR-amplification of regions of difference (RD) (Parsons et al. 2002, Huard et al. 2003), among others. There are some limitations for the use of these techniques. pncA,oxyR, and mtp40 are useful for differentiation of M. tuberculosis and M. bovis, but not for other MTBC members. Spoligotyping requires analysis of several isolates in the same experiment and RD analysis requires several amplification steps.

The gyrB gene encodes the B subunit of DNA gyrase (topoisomerase II), an enzyme universally distributed and essential for bacterial replication. Kasai et al. (2000) obtained gyrB nucleotide sequences from different strains of M. tuberculosis, M. bovis, M. africanum, and M. mi-croti. A two-step method for differentiation of these species was developed, based on gyrB. In the first step, a 1020 bp fragment of the gene is amplified with specific primers, which do not generate amplicons from other species of mycobacteria. In the second step, the amplicon is digested with restriction enzymes. Analysis of restriction fragment length polymorphisms (RFLP) allows distinction of M. tuberculosis, M. bovis, M. africanum, and M. microti. Niemann et al. (2000) extended these observations and proposed a diagnostic algorithm of gyrB-RFLP patterns to differentiate M. tuberculosis/M. africanum II from M. africanum I, M. bovis subsp. bovisM. bovis subsp. caprae and M. microti.

In order to implement gyrB-RFLP in the Reference Laboratory at Instituto Adolfo Lutz, in São Paulo, Brazil, we analyzed gyrB-RFLP patterns from reference strains, including M. tuberculosis H37Rv (ATCC 27294), M. bovis (AN5), and M. bovis BCG (Pasteur), "M. canetti" isolate 140010059 and M. africanum isolate 140010068 from the National Reference Laboratory for Mycobacteria at Institut Pasteur (Paris, France) culture collection, kindly provided by Dr Véronique Vincent, and M. pinnipedii isolate 1458, kindly provided by Amelia Bernardelli (Senasa, Buenos Aires, Argentina). Amplifications were performed using purified DNA or bacteria lysed by freezing and thawing, using the protocol described by Kasai et al. (2000), with modifications. Mycobacteria DNA was added to PCR mix containing 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 1U of Taq DNA polymerase (Invitrogen), 0.2 mM of each deoxynucleoside triphosphate, and 100 pmol of each primer MTUBf (5'TCGGACGCGTATGCGATATC) and MTUBr (5'ACATACAGTTCGGACTTGCG). After initial denaturation for 10 min at 95ºC, 35 amplification cycles were completed, each consisting of 1 min at 94ºC, 1 min at 65ºC and 1,5 min at 72ºC. A final extension of 10 min at 72ºC was applied. The amplification product was analyzed by restriction with RsaI, TaqI and SacII. Restriction digests were separated in 2% agarose gels together with 50 bp DNA ladder (Invitrogen), applied in a separate lane. Gels were stained with ethidium bromide and photographed on UV transiluminator. RFLPs in the 1020-bp gyrB fragments were analyzed using GelCompar II v. 2.5 program (AppliedMaths, Sint-Martens-Latem, Belgium) (Fig. 1).

After analysis of the results we observed that some bands showed different sizes from those described by Niemann et al. (2000). Also, small bands, not considered by Kasai et al. (2000) and Niemann et al. (2000), were clearly visible and helped in species differentiation (Fig. 1). Thus, we developed a new table based on band sizes calculated using GelCompar and confirmed by analysis of sequences deposited by Kasai et al. (2000) in DNA DataBank of Japan ( (Fig. 2). This new table describes for the first time the gyrB-RFLP patterns of "M. canetti", indistinguishable from M. tuberculosis, and of M. pinnipedii,indistinguishable from M. africanum I. This last result confirms findings described by Cousins et al. (2003), demonstrating that seal isolates were genetically closer to M. tuberculosis and M. africanum than to M. bovis.

To evaluate the clinical applicability of gyrB-RFLP, we analyzed 311 MTBC isolates cultured from patients living in different cities of the state of São Paulo. A set of 307 clinical MTBC isolates, were consecutively received at the Mycobacteriology Laboratory at Instituto Adolfo Lutz (IAL) for susceptibility and identification tests from Jan 9 2003 to Mar 7 2003. From these isolates, 83 (27.1%) came from cities of the state of São Paulo and 224 (72.9%) from the Metropolitan Region of Great São Paulo. They were cultured from sputum (268 samples), bronchial washes (8), lymph nodes (9), pleural fluids (4), urine (4), blood (4), cerebrospinal fluids (4), gastric washes (3), abscesses from unknown sites (2), and skin biopsy (1). For differentiation of MTBC, isolates were examined using biochemical and drug susceptibility tests. Biochemical tests include nitrate reduction, niacin accumulation test (BBL Taxo TB niacin test, Becton Dickinson, US) and growth in the presence of TCH (5 µg/ml), p-nitrobenzoic acid (PNB) (0.5 mg/ml) and cycloserine (0.02 mg/ml). Drug susceptibility was determined by the resistance ratio method in Löwenstein Jensen (LJ) medium containing isoniazid (INH), rifampicin (RF), ethambutol (EMB) and streptomycin (SM) (Collins et al. 1997). Pyrazinamide (PZA) susceptibility was tested by detection of pyrazinamidase activity (Collins et al. 1997). At Instituto Adolfo Lutz, drug susceptibility test is performed under specific criteria. All isolates cultured from cases of HIV positive, multidrug resistance tuberculosis, relapses, default and treatment failure are tested to INH, RF, and PZA. If some resistance occurs, the isolates are further tested to EMB and SM. Isolates not matching any of these criteria were investigated exclusively by biochemical tests. According to the IAL criteria, 198 isolates were subjected to susceptibility tests and 109 isolates to biochemical tests for identification. Out of 307 isolates, 306 (99.7%) were identified as M. tuberculosis and one as M. bovis BCG. Two clinical isolates of M. bovis and two of M. bovis BCG belonging to IAL strain collection were also included for analysis of gyrB patterns.

gyrB-RFLP revealed 306 isolates with the profile common to M. tuberculosisM. africanum II, and "M. canetti" and five with the pattern common to M. bovis subsp. bovis and M. bovis BCG. Lack of restriction with SacII excluded the presence of M. bovis subsp. caprae (data not shown).

Despite evidences that the prevalence of M. africanum II and "M. canetti" in non-African populations is extremely low, data presented here cannot confirm that these species are not present among the analyzed isolates, first because M. tuberculosis, "M. canetti", and M. africanum subtype II are indistinguishable by gyrB-RFLP, and second because this study was not specifically designed to verify this point. M. bovis and M. bovisBCG were also not differentiated by gyrB-RFLP. Both are sensitive to the usual antituberculosis drugs, isoniazid and rifampin, but resistant to pyrazinamide. Therefore, from the point of view of treatment, simple differentiation of M. tuberculosis from M. bovis, achieved by gyrB-RFLP, is satisfactory.

No association between gyrB genotype and drug resistance was found, as expected. Among 198 isolates subjected to susceptibility tests, 165 (83.3%) were sensitive to INH, RF, PZA, and 33 (16.7%) were resistant at least to one drug (data not shown). All 198 isolates presented indistinguishable gyrB-RFLP pattern common toM. tuberculosis, "M. canetti", and M. africanum II.

In conclusion, gyrB-RFLP can differentiate MTBC species with easy-to perform PCR, enzymatic restriction and gel electrophoresis. Consequently, it holds many advantages for the clinical laboratory, in terms of improvement in accuracy and savings in time and cost. It has the potential to reduce time required for identification when compared to conventional methods. The test can be simplified by the use of only two enzymes, RsaI and TaqI, which are sufficiently discriminatory. The third enzyme, SacII could be reserved for situations in which differentiation of M. bovis subsp. bovis and M. bovis subsp. caprae may be required.

Application of gyrB-RFLP for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II and "M canetti", M. africanum subtype I from M. pinnipedii, and M. bovis from M. bovis BCG. For this purpose, other strategies have to be pursued.



To Maria Alice da Silva Telles, Sueli Yoko Mizuka Ueki, Carmen Maria Giampaglia, Maria Conceição Martins, and Jorge Luiz Mello Sampaio for suggestions and technical help.



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