Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus) in South Australia.

This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains.

Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range.

Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex.

The Mycobacterium avium complex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of the hsp65 gene has proven to efficiently classify isolates within the Mycobacterium genus but provides low resolution for distinguishing among members of the M. avium complex. Therefore, in this study, we have targeted the more variable 3' region of the hsp65 gene to determine whether it can effectively discriminate M. avium complex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified as M. avium complex. Sequences obtained indicate that M. intracellulare is genetically divergent from M. avium organisms, and distinct sequevars were obtained for M. avium subsets, including M. avium subsp. avium (bird type), M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains of M. avium subsp. paratuberculosis. A unique profile for M. avium subsp. silvaticum was not obtained. These results indicate that sequencing the 3' region of the hsp65 gene can simply and unambiguously distinguish species and subspecies of the M. avium complex.

Use of multilocus enzyme electrophoresis to examine genetic relationships amongst isolates of Mycobacterium intracellulare and related species.

As part of a larger study investigating diversity and distribution of Mycobacterium spp. in Australia, multilocus enzyme electrophoresis was used to assess genetic relationships at 17 enzyme loci amongst a collection of reference strains and isolates initially identified on biochemical and other grounds as M. intracellulare (70), "X' mycobacteria (10), M. scrofulaceum (7), M. avium (8) and M. avium subsp. paratuberculosis (2). Two of the isolates initially identified as M. intracellulare were shown to be quite distinct from the others. Both gave negative results in a species-specific DNA probe test, whilst one was positive by PCR. These results emphasize the uncertainties involved in identifying members of this group. The other M. intracellulare isolates formed a cohesive but diverse group, being divided into 48 electrophoretic types (ETs), with a mean genetic diversity of 0.38. Forty-three of these ETs contained only single isolates. There was no clear relationship between the serovar and ET designation. The index of association calculated for M. intracellulare was significantly different from zero, suggesting that it is a clonal species. PFGE was also applied to selected isolates from the ETs containing multiple isolates, and some of these could be differentiated further. The strains of M. scrofulaceum and "X' mycobacteria were distinct from M. intracellulare, but themselves were highly heterogeneous, with mean genetic diversities of 0.66 and 0.65, respectively. Each of these groups may represent more than one species. M. avium strains were distinct from the two M. avium subsp. paratuberculosis strains, as well as from the other mycobacteria studied.

AmpliBASE MT: a Mycobacterium tuberculosis diversity knowledgebase.

AmpliBASE MT is an online databank of high-resolution DNA fingerprints representing fluorescent amplified fragment length polymorphism (FAFLP) profiles or amplitypes developed for the Mycobacterium tuberculosis complex strains from 48 different countries. AmpliBASE MT is based on a relational database management system that is hyperlinked to visualize genotyping results in the form of DNA fingerprint images for individual strains. A flexible search system based on systematic comparisons of fragment sizes in base pairs allows inter-laboratory comparison of FAFLP profiles. Besides this, the database also displays previously published data on IS6110 profiles, spoligotypes, MIRU-VNTRs and large sequence polymorphisms along with the FAFLP records that will give the overall comparisons. Being the first of its kind, AmpliBASE MT is expected to be a very helpful tool in strengthening the concept of 'geographic genomics' and will be very helpful to molecular epidemiologists and those interested in diagnostic development for tuberculosis.

Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov.

A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined 'Mycobacterium canettii' and 'Mycobacterium caprae', on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this 'seal bacillus', although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis complex (the type strain is 6482(T)=ATCC BAA-688(T)=NCTC 13288(T)).

Global distribution of Mycobacterium tuberculosis spoligotypes.

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11708 patterns from as many clinical isolates originating from more than 90 countries. The 11708 spoligotypes were clustered into 813 shared types. A total of 1300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study.

The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.