Pyrosequencing assay for rapid identification of Mycobacterium tuberculosis complex species
© Drancourt et al; licensee BioMed Central Ltd. 2011
Received: 25 May 2011
Accepted: 19 October 2011
Published: 19 October 2011
Identification of the Mycobacterium tuberculosis complex organisms to the species level is important for diagnostic, therapeutic and epidemiologic perspectives. Indeed, isolates are routinely identified as belonging to the M. tuberculosis complex without further discrimination in agreement with the high genomic similarity of the M. tuberculosis complex members and the resulting complex available identification tools.
We herein develop a pyrosequencing assay analyzing polymorphisms within glpK, pykA and gyrB genes to identify members of the M. tuberculosis complex at the species level. The assay was evaluated with 22 M. tuberculosis, 21 M. bovis, 3 M. caprae, 3 M. microti, 2 M. bovis BCG, 2 M. pinnipedii, 1 M. canettii and 1 M. africanum type I isolates. The resulted pyrograms were consistent with conventional DNA sequencing data and successfully identified all isolates. Additionally, 127 clinical M. tuberculosis complex isolates were analyzed and were unambiguously identified as M. tuberculosis.
We proposed a pyrosequencing-based scheme for the rapid identification of M. tuberculosis complex isolates at the species level. The assay is robust, specific, rapid and can be easily introduced in the routine activity.
The Mycobacterium tuberculosis complex (MTC) includes M. tuberculosis, M. bovis and BCG-derived clones, M. africanum, M. canettii, M. microti, M. caprae and M. pinnipedii. Recently, an eight member named Mycobacterium mungi was identified in banded mongooses (Mungos mungo) in Botswana . While there is variable host-specificity among the different MTC members, every species, but M. mungi, has been implicated in human tuberculosis with M. tuberculosis being the most common pathogen [3–6]. The exact contribution of each species in human disease may be underestimated due to the limited capacity of laboratories to identify MTC isolates at the species level in routine practice. Indeed, phenotypic methods are time-consuming and the results are difficult to interpret. Molecular assays are hampered by the high genetic similarity reflected by the complete conservation of the 16S rRNA and rpoB genes of the MTC members [7, 8]. This situation indeed, led to propose that the various MTC species would be more accurately described as ecotypes . Despite the high degree of nucleotide sequence homology, some genomic markers such as pncA, mpt40, hupB, gyrB and wbbl1 genes in addition to the regions of difference (RD) have been successfully used for the differentiation of MTC members through direct analysis such as in spoligotyping or after enzymatic digestion of PCR products [10–16]. Nevertheless, most of these techniques allow only partial discrimination between the MTC species and are too fastidious to be implemented in the routine practice of microbiology laboratories. The Genotype MTBC (Hain Lifescience, GmbH, Nehren, Germany) is a commercial DNA-strip based assay identifying MTC species through the detection of gyrB gene single nucleotide polymorphisms (SNPs) and RD1 . The test is easy to use, but failed to identify M. canettii and M. pinnipeddii.
Pyrosequencing is a real-time non-electrophoretic DNA sequencing technique that can be easily adapted for routine use in the clinical microbiology laboratory. This technique was successfully used for the identification of non-tuberculous mycobacteria, identification of the Beijing family of M. tuberculosis and detection of rifampin-resistance among MTC members [18–20]. We here developed a pyrosequencing assay for the rapid identification of the MTC species based on polymorphisms within the glpK, pykA and gyrB genes.
A set of 8 reference MTC isolates and 47 M. africanum type 2 and human and animal clinical isolates representative of all MTC species (excluding M. mungi and M. africanum type II) after molecular identification as previously described  was used in this study [15, 16, 20]. Maria Laura Boschiroli (ANSES, Maisons-Alfort, France) graciously provided DNA extracts of MTC organisms isolated from different animal species (cattle, badger, cat, dog, sea lion, wild boar, wild deer, marmoset and chimpanzee). In addition, 127 clinical MTC organisms identified using ITS-real time PCR were analyzed (Additional file 1). These strains were isolated at the microbiology laboratory of Farhat Hached Hospital, Sousse, Tunisia and the mycobacteria reference laboratory of the Institut Hospitalier Universitaire POLMIT, Marseille, France. This study involves mycobacteria isolated from animals and from patients as a part of the routine diagnostic activity of laboratories and does not involve animals or patients themselves. No informed consent was obtained from individuals in agreement with French law, as the study concerns only microbiota and not the individuals themselves.
glpK and pykA gene analyses
Sequencing and pyrosequencing primers used in this study.
Primer sequence (5' - 3')
Product size (bp)
After analysis of the glpK and pykA gene sequences, MTC species-specific polymorphic regions were targeted for pyrosequencing . To allow discrimination between all MTC members, single nucleotide polymorphisms (SNPs) within the gyrB gene were also used . Specific primers were designed using the PSQ assay (Qiagen) (Table1). In a first step, simplex PCR reactions were performed as described above with 1-min of elongation instead of 2.5 min. PCR products were the n subjected to simplex pyrosequencing analysis using the PyroMark Q96 ID System (Qiagen) as previously described . SNP analyses were performed using the SNP program of the PyroMark Q96 ID software. Sequencing analysis was realized using the sequencing program with a 6(GATC) dispension order. Secondly, we performed multiplex PCR and pyrosequencing reactions with the same conditions as for simplex analysis. The choice of the gene fragments to be simultaneously analyzed was dictated by the PyroMark Q96 ID software so that polymorphic regions did not overlap. For multiplex SNP analysis, two sequencing primers were hybridized either to a single or two PCR amplified loci.
A set of clinical isolates of non tuberculous mycobacteria (Mycobacterium smegmatis, Mycobacterium immunogenum, Mycobacterium bolletii, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium massillense, Mycobacterium avium and Mycobacterium intracellulare) and other bacteria (Escherichia coli, Klebsiella pneumoniae, Enterococcus feacalis, Salmonella enterica, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Citrobacter freundii and Stenotrophomonas maltophilia) were tested on the pyrosequencing analysis to assess the specificity of the assay. Isolates were identified by sequencing the 16S rRNA or the rpoB gene and/or using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Bruker Daltonics, Wissembourg, France) .
Results and discussion
The pykA (encoding for the pyruvate kinase) and glpK (encoding for the glycerol kinase) genes were previously analyzed to disclose the genetic basis of difference in the glycerol metabolism between M. tuberculosis and M. bovis in comparison with other MTC species [23, 24]. The authors reported a specific gene structure for M. tuberculosis, M. bovis and M. bovis BCG but this particularity has not been further used for identifying MTC organisms. In our study, we extended this previous observation to all MTC species and a larger collection of clinical isolates in the perspective of MTC member's identification.
Our proposed scheme allows in one step the specific identification of M. tuberculosis, the leading cause of human tuberculosis worldwide and M. africanum type I, an important cause of human tuberculosis in west Africa [6, 25]. M. bovis, an important zoonotic tuberculosis agent and M. bovis BCG a safe vaccine exceptionally responsible for disseminated disease in immuno-compromised vaccinated neonates and bladder cancer-patients, could be unambiguously identified in a second reaction after excluding M. tuberculosis and M. africanum type I [26, 27]. The remaining MTC species are less common in human disease and their identification using specific genomic marker described here could be directed by epidemiologic data.
The ability of rapid and accurate identification of MTC members has an important impact both for public health and veterinary facilities. Although a few patients infected with M. bovis have been treated by antibiotic regimens incorporating the pyrazinamide, the natural resistance of these species to pyrazinamide makes pyrazinamide generally not recommended for treating such patients . Moreover, it can help epidemiologists and health care professionals to measure the relative contribution of each MTC species in human and animal disease, to identify specific MTC-specie outbreak and to rapidly intercept the impact of the zoonotic transmission particularly in case of M. bovis infections so that appropriate control measure could be undertaken.
Pyrosequencing analysis of the glpK, pykA and gyrB genes provides a robust and easy tool for the rapid identification of all MTC species that can be easily introduced in the routine laboratory activity, just requiring basics skills in PCR. The assay allows the simultaneous analyses of up to 96 isolates within 10 to 20 min after a 4-hour DNA extraction and PCR amplification rounds comprising of 3 different PCRs, and 7 different pyrosequencing steps which can be run in parallel. Moreover, pyrosequencing has several advantages compared to other molecular methods: it determines the exact sequence thereby providing the same accuracy as conventional sequencing methods, the technique dispenses with the need for labeled nucleotides, labeled primers and electrophoresis.
The authors would like to thank Maria Laura BOSCHIROLI for generously providing DNA from different animal Mycobacterium tuberculosis complex isolates. IBK is financially supported by the Oeuvre Antituberculeuse des Bouches du Rhône, Marseille, France.
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