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Indian Journal of Medical Microbiology, Vol. 28, No. 1, January-March, 2010, pp. 26-29 Original Article Differentiation of clinical Mycobacterium tuberculosis complex isolates by their GyrB polymorphism NA Abass, *KM Suleiman, IM El Jalii Department of Preventive Medicine (ANA), College of Veterinary Medicine and Animal Production, Sudan University of Science and Technology. P.O. Box 204, Microbiology (SKM), Department of Preventive Medicine (EIM), Faculty of Veterinary Medicine, University of Khartoum, Postcode: 13314, Khartoum North- Sudan Correspondence Address: *Department of Preventive Medicine (ANA), College of Veterinary Medicine and Animal Production, Sudan University of Science and Technology. P.O. Box 204, Microbiology (SKM), Department of Preventive Medicine (EIM), Faculty of Veterinary Medicine, University of Khartoum, Postcode: 13314, Khartoum North- Sudan, kmsuleiman@yahoo.com Date of Submission: 01-Apr-2009 Code Number: mb10007 PMID: 20061759 DOI: 10.4103/0255-0857.58724 Abstract Purpose: To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical Mycobacterium tuberculosis complex isolates. Keywords: GyrB sequences, Mycobacterium tuberculosis complex, Mycobacteria other than tuberculosis Introduction 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 diagnosis and treatment. [1] The resurgence of tuberculosis and the appearance of multidrug-resistant strains of Mycobacterium tuberculosis have intensified the need for the increased use of rapid methods for isolating and identifying clinically encountered mycobacteria. [2] The closely related species, Mycobacterium tuberculosis, M. bovis, M. africanum, M. microti and M. canetti that form the M. tuberculosis complex (MTBC) are the causative agents of tuberculosis (TB) in human and animals. [3] Although the species are closely related genetically, they differ in their host, geographic range, certain phenotypes and pathogenicity. [4] M. tuberculosis and M. africanum are limited to humans, while M. bovis causes disease in a wide range of wild and domestic mammals, as well as in humans. M. microti has recently been reported to infect not only voles but also humans. [5] Extensive studies based on DNA similarity analysis reported 95-100% relatedness between the members of the complex. Sequencing of the 16S rRNA gene has shown that there are no sequence differences between the members of the complex. Furthermore, sequencing of the more variable internal transcribed spacers (ITSs) between 16S and 23S rRNA has led to the same conclusion, proving the existence of a close evolutionary relationship. DNA sequence analysis of the rpoB, katG, rpsL and gyrA genes have revealed a very strong identity among the M. tuberculosis complex. [6] Differentiation among the members is currently based mainly on the analysis of phenotypic characteristics such as colony morphology, growth rate, and biochemical tests such as niacin accumulation, nitrate reductase activity, resistance to thiophen-carboxylic acid hydrazide (TCH) and Pyrazinamide (PZA). [7],[8] These tests need sufficient bacterial growth, are time consuming and are not routinely performed by many laboratories. [9] Hence further methods allowing accurate and rapid species identification are urgently needed for clinical and epidemiological purposes. [10] Kasai, et al. [5] reported DNA sequence variations in the gyrB gene that may be useful for the species differentiation of the slowly growing mycobacteria and even for the differentiation of the members of the MTBC. The aim of this study was to differentiate clinical MTBC isolates among TB patients in Sudan using the gyrB- based PCR methods. Materials and Methods Mycobacterial isolates Seventy nine mycobacterial isolates from known TB patients (Khartoum, Sudan) and two reference strains (M. tuberculosis H37 Rv and M. bovis BCG) obtained from the National Tuberculosis Reference Laboratory, Khartoum, Sudan were investigated in this study. The isolates were cultivated on Loewenstein-Jensen medium. Preparation of mycobacterial DNA DNA extractions were performed as described by Roth et al., [11] with slight modifications. A loopful of colony from each isolate was suspended in 500 μl deionized distilled water in a 1.5 ml screw-capped microcentrifuge tube. The bacterial suspensions were vortexed and then boiled at 100°C for 20 min in a thermoblock (Biometra- Germany) to release DNA. The heat-treated samples were then centrifuged at 13000 rpm for 15 min. The supernatants containing the extracted DNA were transferred to sterile microcentrifuge tubes and stored at -20 o C until used for amplification. Amplification of gyrB gene (gyrB-PCR1) The target DNA for amplification was 1,020-bp fragment of the gyrB gene, which was used to identify members of the M. tuberculosis complex. The primers (Eurofins MWG, Operon, Germany) used were MTUB-f (5'-TCG GAC GCG TAT GCG ATA TC-3') and MTUB-r (5'-ACA TAC AGT TCG GAC TTG CG-3'). [9],[10] The 50-μl PCR mixture (Bioline, UK) contained 10 mM Tris-HCL (pH 8.3), 50 mM KCL, 1.5 mM MgCl2 , 200 μM of each deoxynucleoside triphosphate (dNTP), 20 pmol of each primer, 1U of Taq DNA polymerase and 2 μl of template DNA. [10] PCR amplifications were performed in an automated PROGENE thermal cycler by the protocol described by Niemann et al., [10] except the final extension for 10 minutes. A positive control containing chromosomal DNA of M. tuberculosis H37Rv (National Reference Laboratory) and a negative control without template DNA were included in each run. GyrB-based species specific PCR for M. tuberculosis complex (gyrB-PCR2) MTBC isolates confirmed with gyrB-PCR1 were analyzed further by species specific PCR using the specific primer set MTUB-756-Gf (5'-GAA GAC GGG GTC AAC GGT G) and MTUB-1450-Cr (5'-CCT TGT TCA CAA CGA CTT T CGC-3') (Eurofins MWG, Operon, Germany) for selective amplification of the gyrB fragments from M. tuberculosis. [5] . Three μl of the first amplification product were transferred to a PCR tube containing a reaction buffer, 1.25 U of Taq DNA polymerase, 0.1 mM of each deoxynucleoside triphosphate (dNTP) and 100 pmol of each primer. Deionized distilled water was added to each PCR tube to obtain a total volume of 20 μl. [5] The thermal cycling profiles were as follows: Five-minute incubation at 94°C, followed by 35 cycles at 95°C for 1 min and annealing-extension step at 72°C for 1.5 min. A final extension at 72°C for 10 min was carried out to ensure complete synthesis of the expected PCR products. Positive and negative control reaction in which PCR mixes were inoculated with M. tuberculosis H37Rv DNA and sterile distilled water without PCR product, respectively, were performed with each set of reactions. Electrophoresis Amplified products of the gyrB PCR1, gyrB PCR2 and DNA ladder were electrophorectically separated at a constant voltage of 80V (BIOMETRA) for 45 minutes in 1.5% agarose. Gels were visualized under UV transilluminator (BIOMETRA), photographed and stored as a soft copy (BIODOG ANALAYZ, BIOMETRA, Germany). Restriction fragment length polymorphism The DNA polymorphism in the 1020-bp gyrB fragment of confirmed M. tuberculosis by gyrB-PCR2 for 7 MTBC isolates were analyzed by restriction with RsaI as indicated by the manufacturer (Promega, USA). Samples for electrophoresis were prepared by adding 4 μl of the digest to 3 μl of the loading dye. [10] The 5 μl of DNA ladder (100 bp marker) and the mixture was separated in two per cent agarose gel by electrophoresis at a constant voltage of 90 v for 45 min. Results GyrB-PCR1 Seventy nine mycobacterial isolates were investigated by PCR to confirm their identity as members of the M. tuberculosis complex using the primer pair MTUB-f and MTUB-r specific for amplification of the 1,020-bp fragment of the gyrB gene. Seventy-seven (97.5%) of the isolates produced a 1,020-bp fragment of the gyrB gene and were identified as members of the M. tuberculosis complex whereas two (2.6%) of the isolates were negative and identified as MOTT. The presence of a single band of 1,020-bp was taken as a positive result, demonstrating that the isolate belonged to the M. tuberculosis complex [Figure - 1]. GyrB-PCR2 The 77 MTBC isolates confirmed with gyrB PCR1 were further differentiated by species-specific PCR using specific set of the primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrB fragments from M. tuberculosis. All tested isolates produced a band of 734-bp, which is specific for M. tuberculosis [Figure - 2]. GyrB PCR-RFLP To further confirmation of the differentiation system used, reference strains M. tuberculosis H37Rv and M. bovis BCG as well as seven clinical M. tuberculosis isolates confirmed by gyrB-PCR2 were analyzed by PCR-RFLP. All M. tuberculosis isolates showed the typical Rsa1 patterns (100, 360 and 560-bp) compared to reference strain, while 360 and 480-bp fragments were generated from M. bovis BCG [Figure - 3]. Discussion In this study, gyrB-based methods were performed to differentiate clinical mycobacterial isolates. Differentia tion of species in the clinical mycobacteriology laboratory is a difficult task because of the high degree of sequence conservation among members of the MTBC. [9] Seventy-seven (97%) of the 79 mycobacterial isolates analyzed by the gyrB-PCR1 produced specific bands (1020 bp fragment of the gyrB gene) and were identified as members of the M. tuberculosis complex. Two (2.6%) were negative and identified as MOTT, however, no further molecular characterization was performed to identify these two isolates to the species level. The use of MTUB-f and MTUB-r primers pair in this study allow the MTUB-specific amplification of a part of the gyrB and hence identification of MTBC isolates. [5],[10] The 77 isolates further examined by the gyrB-PCR2 produced specific bands that allowed the identification of the isolates as M. tuberculosis. This is in agreement with the results of Kasai et al., [5] who designed PCR primers that allowed the selective amplification of the gyrB fragment from each species of the M. tuberculosis complex. To evaluate the clinical applicability of the gyrB-PCR- RFLP, the target genes of two reference strains (M. tuberculosis H37Rv and M. bovis BCG) and seven clinical isolates were amplified and the products were digested by the endonuclease Rsa1. M. bovis BCG showed the typical M. bovis Rsa1 pattern (360-480-bp) described by Niemann et al. [10] All the M. tuberculosis strains showed the characteristic M. tuberculosis Rsa1 pattern (100, 360 and 560-bp). These results described that M. tuberculosis could be identified by their specific Rsa1 RFLP pattern (360 and 560). Production of (100-bp) band is significant because it was not considered previously by Niemann, [10] although Chimara et al., [9] who developed a new diagnostic algorithm of M. tuberculosis specific Rsa1 RFLP pattern (100, 385 and 560-bp), described this band. The gyrB PCR-RFLP using the restriction enzyme Rsa1 in this study is rapid and easy to-use to differentiate between M. tuberculosis (360/560-bp) and M. bovis (360/480-bp). Conclusion All the isolated mycobacteria from TB patients were confirmed to be M. tuberculosis using the gyrB-based PCR methods. These methods are highly applicable to clinical medicine because DNA sequencing is not required for rapid identification of these species. Acknowledgement The financial support of the German Academic Exchange Service (DAAD) is greatly acknowledged. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10007f2.jpg] [mb10007f1.jpg] [mb10007f3.jpg] |
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