A Murine Model of Mycobacterium kansasii Infection Reproducing Necrotic Lung Pathology Reveals Considerable Heterogeneity in Virulence of Clinical Isolates.

Among non-tuberculous mycobacteria, Mycobacterium kansasii is one of the most pathogenic, able to cause pulmonary disease indistinguishable from tuberculosis in immunocompetent susceptible adults. The lack of animal models that reproduce human-like lung disease, associated with the necrotic lung pathology, impairs studies of M. kansasii virulence and pathogenicity. In this study, we examined the ability of the C57BL/6 mice, intratracheally infected with highly virulent M. kansasii strains, to produce a chronic infection and necrotic lung pathology. As a first approach, we evaluated ten M. kansasii strains isolated from Brazilian patients with pulmonary disease and the reference strain M. kansasii ATCC 12478 for virulence-associated features in macrophages infected in vitro; five of these strains differing in virulence were selected for in vivo analysis. Highly virulent isolates induced progressive lung disease in mice, forming large encapsulated caseous granulomas in later stages (120-150 days post-infection), while the low-virulent strain was cleared from the lungs by day 40. Two strains demonstrated increased virulence, causing premature death in the infected animals. These data demonstrate that C57BL/6 mice are an excellent candidate to investigate the virulence of M. kansasii isolates. We observed considerable heterogeneity in the virulence profile of these strains, in which the presence of highly virulent strains allowed us to establish a clinically relevant animal model. Comparing public genomic data between Brazilian isolates and isolates from other geographic regions worldwide demonstrated that at least some of the highly pathogenic strains isolated in Brazil display remarkable genomic similarities with the ATCC strain 12478 isolated in the United States 70 years ago (less than 100 SNPs of difference), as well as with some recent European clinical isolates. These data suggest that few pathogenic clones have been widely spread within M. kansasii population around the world.

Whole-Genome Sequences of Mycobacterium abscessus subsp. massiliense Isolates from Brazil.

The Mycobacterium abscessus complex comprises multidrug-resistant, opportunistic, and rapidly growing pathogens responsible for severe infections. Here, we report the genome composition of four Mycobacterium abscessus subsp. massiliense isolates from three sources: two from the lung of a cystic fibrosis patient, one from a mammary cyst, and one from a gutter system.

Exploring the "Latin American Mediterranean" family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela.

BACKGROUND: The Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available. RESULTS: Genotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23. CONCLUSIONS: Our results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

Strain classification of Mycobacterium tuberculosis isolates in Brazil based on genotypes obtained by spoligotyping, mycobacterial interspersed repetitive unit typing and the presence of large sequence and single nucleotide polymorphism.

Rio de Janeiro is endemic for tuberculosis (TB) and presents the second largest prevalence of the disease in Brazil. Here, we present the bacterial population structure of 218 isolates of Mycobacterium tuberculosis, derived from 186 patients that were diagnosed between January 2008 and December 2009. Genotypes were generated by means of spoligotyping, 24 MIRU-VNTR typing and presence of fbpC103, RDRio and RD174. The results confirmed earlier data that predominant genotypes in Rio de Janeiro are those of the Euro American Lineages (99%). However, we observed differences between the classification by spoligotyping when comparing to that of 24 MIRU-VNTR typing, being respectively 43.6% vs. 62.4% of LAM, 34.9% vs. 9.6% of T and 18.3% vs. 21.5% of Haarlem. Among isolates classified as LAM by MIRU typing, 28.0% did not present the characteristic spoligotype profile with absence of spacers 21 to 24 and 32 to 36 and we designated these conveniently as "LAM-like", 79.3% of these presenting the LAM-specific SNP fbpC103. The frequency of RDRio and RD174 in the LAM strains, as defined both by spoligotyping and 24 MIRU-VNTR loci, were respectively 11% and 15.4%, demonstrating that RD174 is not always a marker for LAM/RDRio strains. We conclude that, although spoligotyping alone is a tool for classification of strains of the Euro-American lineage, when combined with MIRU-VNTRs, SNPs and RD typing, it leads to a much better understanding of the bacterial population structure and phylogenetic relationships among strains of M. tuberculosis in regions with high incidence of TB.

Múltiplas estirpes de isolados de Mycobacteriumbovis identificados por tipagem molecular em bovinos abatidos em matadouros-frigoríficos / Multiple strains of Mycobacterium bovis isolates identified by molecular typing of bovine animals slaughtered in slaughterhouse

O objetivo do presente trabalho foi utilizar métodos bacteriológicos e moleculares para a identificação do Mycobacteriumbovis em lesões observadas em carcaças de bovinos durante a inspeção postmortem de rotina em matadouros-frigoríficos com serviço de inspeção oficial. Foi acompanhado o abate e a inspeção de 825.394 bovinos, sadios ao exame ante mortem pelo serviço de inspeção oficial em dez matadouros-frigoríficos do estado da Bahia. Carcaça de 180 bovinos apresentaram lesões sugestivas de tuberculose e por outras linfadenites. No isolamento bacteriano, 25 amostras apresentaram crescimento disgônico de colônias de coloração creme-amareladas em meio de cultura Stonebrink-Leslie. Desses isolados, 14 foram identificados como M. bovis PCR multiplex e pela técnica do spoligotyping foram discriminados oito diferentes espoligotipos do M. bovis, sendo sete descritos na literatura e um novo spoligotipo sem descrição anterior. O espoligotipo majoritário foi o SB0121, com cinco amostras, sendo descrito no Brasil e em outros países, seguidos por dois clusters, SB295 e SB1055, com dois isolados cada. O espoligotipo SB1145 e SB1648 foram referidos apenas no Brasil e Dinamarca, respectivamente. O espoligotipo SB140 já foi encontrado no Brasil, Argentina, Uruguai e Paraguai. Estes resultados demonstram que os espoligotipos obtidos são compartilhados, até o momento, entre estados brasileiros e entre países da América Latina e Europa. Sendo assim, a discriminação molecular de isolados de M. bovis através do Spoligotyping constitui-se numa ferramenta para estudos epidemiológicos da tuberculose bovina no Estado da Bahia.

The aim of this study was to use bacteriological and molecular methods to identify Mycobacteriumbovis in lesions observed in cattle carcasses during routine post-mortem inspection in slaughterhouses with official inspection service. It was accompanied the slaughter and inspection of 825,394 cattle, healthy ante mortem examination by the official inspection service in ten slaughterhouses in the state of Bahia. Carcasses of 180 cattle presented lesions suggestive of tuberculosis and other lymphadenitis. In bacterial isolation, 25 samples showed dysgonic growth of colonies of creamy-yellow in medium-Stonebrink Leslie. From these isolates, 14 were identified as M. bovis and the multiplex PCR technique spoligotyping was discriminated against eight different spoligotypes of M. bovis, seven previously described in the literature and a new spoligotypes without former description. The major spoligotypes was SB0121, with five samples which has been described in Brazil and other countries, followed by two clusters, SB295 and SB1055, with two isolates each. The SB1145 and SB1648 spoligotypes were reported only in Brazil and Denmark, respectively. The spoligotypes SB140 has been found in Brazil, Argentina, Uruguay and Paraguay. These results demonstrate that the spoligotypes obtained are shared, so far, among Brazilian states and among Latin America and Europe. Thus, molecular discrimination of isolates of M. bovis by Spoligotyping constitutes a tool for epidemiological studies of bovine tuberculosis in the state of Bahia.

Distinct genotypic profiles of the two major clades of Mycobacterium africanum.

BACKGROUND: Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis (TB) and a member of the M. tuberculosis complex (MTC). Additional MTC species that cause TB in humans and other mammals include Mycobacterium africanum and Mycobacterium bovis. One result of studies interrogating recently identified MTC phylogenetic markers has been the recognition of at least two distinct lineages of M. africanum, known as West African-1 and West African-2. METHODS: We screened a blinded non-random set of MTC strains isolated from TB patients in Ghana (n = 47) for known chromosomal region-of-difference (RD) loci and single nucleotide polymorphisms (SNPs). A MTC PCR-typing panel, single-target standard PCR, multi-primer PCR, PCR-restriction fragment analysis, and sequence analysis of amplified products were among the methods utilized for the comparative evaluation of targets and identification systems. The MTC distributions of novel SNPs were characterized in the both the Ghana collection and two other diverse collections of MTC strains (n = 175 in total). RESULTS: The utility of various polymorphisms as species-, lineage-, and sublineage-defining phylogenetic markers for M. africanum was determined. Novel SNPs were also identified and found to be specific to either M. africanum West African-1 (Rv1332(523); n = 32) or M. africanum West African-2 (nat(751); n = 27). In the final analysis, a strain identification approach that combined multi-primer PCR targeting of the RD loci RD9, RD10, and RD702 was the most simple, straight-forward, and definitive means of distinguishing the two clades of M. africanum from one another and from other MTC species. CONCLUSION: With this study, we have organized a series of consistent phylogenetically-relevant markers for each of the distinct MTC lineages that share the M. africanum designation. A differential distribution of each M. africanum clade in Western Africa is described.

Single nucleotide polymorphisms (SNPs) at -238 and -308 positions in the TNFalpha promoter: clinical and bacteriological evaluation in leprosy.

Tumor necrosis factor alpha (TNFalpha) plays a key role in orchestrating the complex events involved in inflammation and immune response. The presence of single nucleotide polymorphisms (SNPs) within the promoter region of the TNFa gene has been associated with a number of diseases. The aim of this study was to investigate the distribution of polymorphisms at positions -238 (G/A) and -308 (G/A) at the TNFalpha promoter, and its association to the outcome of different clinical forms of leprosy. Furthermore, the bacteriological index (BI) was evaluated among genotyped multibacillary (MB) patients in order to investigate the possible influence of each polymorphism on the bacterial load. This study included a total of 631 leprosy patients being 401 MB and 230 paucibacillary (PB), that was further separated according to its ethnicity (Afro- and Euro-Brazilians). The combination of SNPs in haplotypes generated three different arrangements: TNFG-G, TNFG-A and TNFA-G. In spite of the marked differences observed in the frequency of the haplotypes along the ethnic groups, no statistical differences were observed in haplotype frequencies between MB and PB patients. The BI analyses showed a lower bacteriological index among the -308 carriers, while the BI of the -238 carriers was higher. Although no significance has been achieved in this analysis regarding the influence of the polymorphisms to the development of the clinical outcome, it seems that in a different stage (among the MB patients) the polymorphisms could contribute to the degree of severity observed.