A Single-Step Sequencing Method for the Identification of Mycobacterium tuberculosis Complex Species

The Mycobacterium tuberculosis complex (MTC) comprises several closely related species responsible for strictly human and zoonotic tuberculosis. Some of the species are restricted to Africa and were responsible for the high prevalence of tuberculosis. However, their identification at species level is difficult and expansive. Accurate species identification of all members is warranted in order to distinguish between strict human and zoonotic tuberculosis, to trace source exposure during epidemiological studies, and for the appropriate treatment of patients. In this paper, the Exact Tandem Repeat D (ETR-D) intergenic region was investigated in order to distinguish MTC species. The ETR-D sequencing unambiguously identified MTC species type strain except M. pinnipedii and M. microti, and the results agreed with phenotypic and molecular identification. This finding offers a new tool for the rapid and accurate identification of MTC species in a single sequencing reaction, replacing the current time-consuming polyphasic approach. Its use could assist public health interventions and aid in the control of zoonotic transmission in African countries, and could be of particular interest with the current emergence of multidrug-resistant and extended-resistance isolates

Isolamento de micobactérias não-tuberculosas em São José do Rio Preto entre 1996 e 2005 Nontuberculous mycobacteria isolated in São José do Rio Preto, Brazil between 1996 and 2005

OBJETIVO: Estudar a ocorrência de micobactérias não-tuberculosas e a variabilidade das espécies isoladas na região atendida pelo Instituto Adolfo Lutz-Regional de São José do Rio Preto-no período entre 1996 e 2005, assim como mostrar a importância do diagnóstico laboratorial. MÉTODOS: A partir de amostras pulmonares e extrapulmonares, foi realizado o isolamento de micobactérias, e estas foram identificadas por métodos fenotípicos e pelo método molecular polymerase chain reaction-restriction enzyme analysis. RESULTADOS: Foram isoladas 317 cepas de micobactérias não-tuberculosas: complexo Mycobacterium avium, 182 (57,4%); M. gordonae, 33 (10,4%); M. fortuitum, 25(7,9%); M. chelonae, 8 (2,5%); complexo M. terrae, 8 (2,5%); M. kansasii, 7 (2,2%); e espécies menos freqüentes, 54 (17%). No período, foram caracterizados 72 casos (33,3%) de micobacterioses, de acordo com os critérios bacteriológicos estabelecidos pela American Thoracic Society (2007).Desses, complexo M. avium foi responsável por 56 casos, sendo que 29 (51,8%) foram caracterizados como doença disseminada. M. fortuitum foi responsável por 6 casos; M. gordonae, 3; M. chelonae, 2; M. abscessus, 1; M. kansasii, 1; M. intracellulare, 1; M. malmoense, 1; e Mycobacterium ssp., 1. CONCLUSÕES: Os resultados obtidos mostraram a importância do diagnóstico bacteriológico das micobacterioses, pois a identificação das espécies possibilita a introdução de um tratamento adequado precocemente.<br>OBJECTIVE: To study the incidence of nontuberculous mycobacteria and the range of species isolated between 1996 and 2005 at a regional branch of the Adolfo Lutz Institute-located in the city of São José do Rio Preto, Brazil-and to show the importance of laboratory testing. METHODS: Mycobacteria were isolated from pulmonary and extrapulmonary specimens and identified through phenotyping and molecular methods (polymerase chain reaction-restriction enzyme analysis). RESULTS: We isolated 317 nontuberculous mycobacterium strains: Mycobacterium avium complex, 182 (57.4%); M. gordonae, 33 (10.4%); M. fortuitum, 25 (7.9%); M. chelonae, 8 (2.5%); M. terrae complex, 8(2.5%); M.kansasii, 7 (2.2%); and less frequent species, 54 (17%). During this period, 72 cases (33.3%) were characterized as mycobacteriosis, according to bacteriological criteria established by the American Thoracic Society in 2007. Of those 72 cases, 56 were attributed to M.avium complex. Of those 56, 29 (51.8%) were characterized as disseminated disease. Six cases were attributed to M. fortuitum, 3 to M. gordonae, 2 to M. chelonae, 1 to M. abscessus, 1 to M. kansasii, 1 to M. intracellulare, 1 to M. malmoense and 1 to Mycobacterium ssp. CONCLUSIONS: These results show the importance of the bacteriological diagnosis, since identification of the species enables early and appropriate treatment

Mycobacterium franklinii sp. nov., a species closely related to members of the Mycobacterium chelonae-Mycobacterium abscessus group

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to 'Mycobacterium franklinii' DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA-DNA hybridization demonstrated that 'M. franklinii DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524(T) (=ATCC BAA-2149(T)) is the type strain

Genomic epidemiology of a national outbreak of post-surgical Mycobacterium abscessus wound infections in Brazil

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of Mycobacterium abscessus and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche