Whole Genome Analyses of Marine Fish Pathogenic Isolate,Mycobacterium sp. 012931

Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen.     

Comparative Genomic and Phylogenetic Approaches to Characterize the Role of Genetic Recombination in Mycobacterial Evolution

The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer (M. abscessus to Segniliparus and M. smegmatis to Streptomyces), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case (M. vanbaalenii to Streptomyces) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is relatively sparse, the acquisition of genes from non-mycobacterial lineages is a significant feature of mycobacterial evolution.     

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.     

Molecular Identification of Falciparum Malaria and Human Tuberculosis Co-Infections in Mummies from the Fayum Depression (Lower Egypt)

Due to the presence of the lake Quarun and to the particular nature of its irrigation system, it has been speculated that the Fayum, a large depression 80 kilometers south- west of modern Cairo, was exposed to the hazards of malaria in historic times. Similarly, it has been speculated that, in the same area, also human tuberculosis might have been far more widespread in the antiquity than in its recent past. If these hypotheses were confirmed, it would imply that frequent cases of co-infection between the two pathogens might have occurred in ancient populations. To substantiate those speculations, molecular analyses were carried out on sixteen mummified heads recovered from the necropolis of Abusir el Meleq (Fayum) dating from the 3^rd Intermediate Period (1064- 656 BC) to the Roman Period (30 BC- 300 AD). Soft tissue biopsies were used for DNA extractions and PCR amplifications using well-suited protocols. A partial 196-bp fragment of Plasmodium falciparum apical membrane antigen 1 gene and a 123-bp fragment of the Mycobacterium tuberculosis complex insertion sequence IS6110 were amplified and sequenced in six and five of the sixteen specimens, respectively. A 100% concordance rates between our sequences and those of P. falciparum and M. tuberculosis complex ones were obtained. Lastly, concomitant PCR amplification of P. falciparum and M. tuberculosis complex DNA specific fragments was obtained in four mummies, three of which are ¹⁴ C dated to the Late and Graeco-Roman Periods. Our data confirm that the hydrography of Fayum was extremely conducive to the spread of malaria. They also support the notion that the agricultural boom and dense crowding occurred in this region, especially under the Ptolemies, highly increased the probability for the manifestation and spread of tuberculosis. Here we extend back-wards to ca. 800 BC new evidence for malaria tropica and human tuberculosis co-occurrence in ancient Lower Egypt.     

Homology Modeling, Molecular Docking and DNA Binding Studies of Nucleotide Excision Repair UvrC Protein from M. tuberculosis

Mycobacterium tuberculosis is a Gram positive, acid-fast bacteria belonging to genus Mycobacterium, is the leading causative agent of most cases of tuberculosis. The pathogenicity of the bacteria is enhanced by its developed DNA repair mechanism which consists of machineries such as nucleotide excision repair. Nucleotide excision repair consists of excinuclease protein UvrABC endonuclease, multi-enzymatic complex which carries out repair of damaged DNA in sequential manner. UvrC protein is a part of this complex and thus helps to repair the damaged DNA of M. tuberculosis. Hence, structural bioinformatics study of UvrC protein from M. tuberculosis was carried out using homology modeling and molecular docking techniques. Assessment of the reliability of the homology model was carried out by predicting its secondary structure along with its model validation. The predicted structure was docked with the ATP and the interacting amino acid residues of UvrC protein with the ATP were found to be TRP539, PHE89, GLU536, ILE402 and ARG575. The binding of UvrC protein with the DNA showed two different domains. The residues from domain I of the protein VAL526, THR524 and LEU521 interact with the DNA whereas, amino acids interacting from the domain II of the UvrC protein included ARG597, GLU595, GLY594 and GLY592 residues. This predicted model could be useful to design new inhibitors of UvrC enzyme to prevent pathogenesis of Mycobacterium and so the tuberculosis.     

Complete Genome Sequence of Mycobacterium vaccae Type Strain ATCC 25954

Mycobacterium vaccae is a rapidly growing, nontuberculous Mycobacterium species that is generally not considered a human pathogen and is of major pharmaceutical interest as an immunotherapeutic agent. We report here the annotated genome sequence of the M. vaccae type strain, ATCC 25954.     

Sequence heterogeneity of an mpb70 gene analogue in Mycobacterium kansasii

The protein antigen MPB70 is a major component of culture supernatants of Mycobacterium bovis and is an active ingredient of bovine PPD used for skin-testing cattle for tuberculosis. We have shown that Mycobacterium kansasii possesses a similar gene that cross-reacts in a PCR test for M. bovis. Single strand conformational polymorphism analysis, and the DNA sequence of the PCR product, shows differences between M. kansasii strains, supporting the suggestion that M. kansasii is not a homogeneous species.     

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.     

Comparative Clinical Study of Different Multiplex Real Time PCR Strategies for the Simultaneous Differential Diagnosis between Extrapulmonary Tuberculosis and Focal Complications of Brucellosis

Background

Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis.

Methodology/Principal Findings

We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting bcsp31 and the IS711 sequence detecting all pathogenic species and biovars of Brucella genus, the IS6110 sequence detecting Mycobacterium genus, and the intergenic region senX3-regX3 specifically detecting Mycobacterium tuberculosis complex. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the Brucella genus and M. tuberculosis complex. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%).

Conclusions/Significance

In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of M. tuberculosis and Brucella spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.     

Distribution and Respiratory Activity of Mycobacteria in Household Water System of Healthy Volunteers in Japan

The primary infectious source of nontuberculous mycobacteria (NTM), which are known as opportunistic pathogens, appears to be environmental exposure, and it is important to reduce the frequency of exposure from environmental sources for preventing NTM infections. In order to achieve this, the distribution and respiratory activity of NTM in the environments must be clarified. In this study, we determined the abundance of mycobacteria and respiratory active mycobacteria in the household water system of healthy volunteers using quantitative PCR and a fluorescent staining method, because household water has been considered as one of the possible infectious sources. We chose healthy volunteer households in order to lessen the effect of possible residential contamination from an infected patient. We evaluated whether each sampling site (bathroom drain, kitchen drain, bath heater pipe and showerhead) have the potential to be the sources of NTM infections. Our results indicated that drains in the bathroom and kitchen sink are the niche for Mycobacterium spp. and M. avium cells were only detected in the bathtub inlet. Both physicochemical and biologic selective pressures may affect the preferred habitat of Mycobacterium spp. Regional differences also appear to exist as demonstrated by the presence (US) or absence (Japan) of Mycobacterium spp. on showerheads. Understanding of the country specific human activities and water usage will help to elucidate the infectious source and route of nontuberculous mycobacterial disease.     

Cost-effectiveness of blood agar for isolation of mycobacteria

Background

Mycobacterium species are grown using specific media that increase laboratory cost, thus hampering their diffusion in resource-limited countries. Preliminary data suggested that versatile blood agar may be also used for mycobacterial culture.

Methodology

We examined the growth of 41 different Mycobacterium species on 5% blood agar. Over a 24-month period we analysed isolation of mycobacteria after parallel inoculation of clinical specimens into both a reference automated system (BACTEC 9000 MB broth) and 5% blood agar slant tubes, after NaOH decontamination, and compared the cost of performing 1,000 analyses using these two techniques.

Conclusions

Mycobacterium reference species cultured on blood agar, with the exception of Mycobacterium ulcerans. Inoculation of 1,634 specimens yielded 95 Mycobacterium isolates. Blood agar performed significantly more efficiently than BACTEC 9000 MB broth (94 vs 88 isolates, P = 0.03). Decontamination of Candida albicans in 5 specimens by addition of amphotericin B in blood agar yielded one more M. tuberculosis isolate that could not be isolated in BACTEC broth. Uneven distribution of time to culture positivity for M. tuberculosis had a median (range) of 19±5 days using blood agar and 26±6 days using BACTEC 9000 MB broth. Cost for 1,000 analyses in France was estimated to be of 1,913 euros using the blood agar method and 8,990 euros using the BACTEC 9000 MB method. Blood agar should be regarded as a first-line medium for culturing Mycobacterium species. It saves time, is cost-effective, is more sensitive than, and at least as rapid as the automated method. This is of particular importance for resource-limited countries in which the prevalence of tuberculosis is high.     

An efficient alternative marker for specific identification of Mycobacterium tuberculosis

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers.     

IS1549 from Mycobacterium smegmatis Forms Long Direct Repeats upon Insertion

A new insertion element, IS1549, was identified serendipitously from Mycobacterium smegmatis LR222 during experiments using a vector designed to detect the excision of IS6110 from between the promoter region and open reading frame (ORF) of an aminoglycoside phosphotransferase gene. Six of the kanamycin-resistant isolates had a previously unidentified insertion element upstream of the ORF of the aph gene. The 1,634-bp sequence contained a single ORF of 504 amino acids with 85% G+C content in the third codon position. The putative protein sequence showed a distant relationship to the transposase of IS231, which is a member of the IS4 family of insertion elements. IS1549 contains 11-bp terminal inverted repeats and is characterized by the formation of unusually long and variable-length (71- to 246-bp) direct repeats of the target DNA during transposition. Southern blot analysis revealed that five copies of IS1549 are present in LR222, but not all M. smegmatis strains carry this element. Only strains with a 65-kDa antigen gene with a PCR-restriction fragment length polymorphism type identical to that of M. smegmatis 607 contain IS1549. None of 13 other species of Mycobacterium tested by PCR with two sets of primers specific for IS1549 were positive for the expected amplified product.     

Genotypic characteristics of a Mycobacterium sp. isolated from yellowtail Seriola quinqueradiata and striped jack Pseudocaranx dentex in Japan

In Japan, a Mycobacterium marinum‐like mycobacterium was isolated from the yellowtail, Seriola quinqueradiata. The species was identified as M. marinum by a commercial mycobacterial DNA‐DNA hybridization kit. Nevertheless, PCR restriction analysis of the DNA of its RNA polymerase β‐subunit gene definitively showed that this Mycobacterium sp. was M. ulcerans. PCR analysis revealed the genotypic characteristics of M. ulcerans in the Mycobacterium sp., only the mup053 gene sequence being absent, as has been found previously in other piscine mycobacteria such as M. marinum strains DL240490 and DL045 and M. pseudoshottsii. With one exception, this Mycobacterium sp. and M. pseudoshottsii had identical 16S rRNA gene sequences, which is also probably true of M. marinum strains DL240490 and DL045. Similarly, according to comparisons of the 16S rRNA gene, ITS region, and hsp65 gene sequences, this Mycobacterium sp. is more closely related to M. pseudoshottsii than to M. ulcerans or M. marinum. A PCR product of approximately 2000 bp was amplified from region of difference 9 in the Mycobacterium sp. The nucleotide sequence revealed insertion of IS2404, the sequence of which is 1366 bp long. The novel single nucleotide polymorphisms identified in this region distinguished this Mycobacterium sp. from M. marinum strain DL240490 and M. pseudoshottsii. The present findings raise the possibility that these species have a common ancestor. Further studies are required to improve our understanding of the relationship between their geographical origin and genetic diversity.     

Detection and molecular characterization of 9,000-year-old Mycobacterium tuberculosis from a Neolithic settlement in the Eastern Mediterranean

Background

Mycobacterium tuberculosis is the principal etiologic agent of human tuberculosis. It has no environmental reservoir and is believed to have co-evolved with its host over millennia. This is supported by skeletal evidence of the disease in early humans, and inferred from M. tuberculosis genomic analysis. Direct examination of ancient human remains for M. tuberculosis biomarkers should aid our understanding of the nature of prehistoric tuberculosis and the host/pathogen relationship.

Methodology/Principal Findings

We used conventional PCR to examine bone samples with typical tuberculosis lesions from a woman and infant, who were buried together in the now submerged site of Atlit-Yam in the Eastern Mediterranean, dating from 9250-8160 years ago. Rigorous precautions were taken to prevent contamination, and independent centers were used to confirm authenticity of findings. DNA from five M tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex.

Conclusions/Significance

Human tuberculosis was confirmed by morphological and molecular methods in a population living in one of the first villages with evidence of agriculture and animal domestication. The widespread use of animals was not a source of infection but may have supported a denser human population that facilitated transmission of the tubercle bacillus. The similarity of the M. tuberculosis genetic signature with those of today gives support to the theory of a long-term co-existence of host and pathogen.     

Nucleotide sequence of the Mycobacterium leprae katG region.

Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR. A 1.6-kb DNA fragment, which hybridized to an M. tuberculosis katG probe, was obtained from an M. leprae DNA template. Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M. leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment. Although the nucleotide sequence of the katG region of M. leprae was approximately 70% identical to that of the M. tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence. Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M. leprae katG region, and they seemed to be present in all seven M. leprae isolates tested. These results strongly suggest that M. leprae lacks a functional katG gene and catalase-peroxidase activity.     

Bacterial Classification of Fish-Pathogenic Mycobacterium Species by Multigene Phylogenetic Analyses and MALDI Biotyper Identification System

Mycobacterium marinum is difficult to distinguish from other species of Mycobacterium isolated from fish using biochemical methods. Here, we used genetic and proteomic analyses to distinguish three Mycobacterium strains: M. marinum strains MB2 and Europe were isolated from tropical and marine fish in Thailand and Europe, and Mycobacterium sp. 012931 strain was isolated from yellowtail in Japan. In phylogenetic trees based on gyrB, rpoB, and Ag85B genes, Mycobacterium sp. 012931 clustered with M. marinum strains MB2 and Europe, but in trees based on 16S rRNA, hsp65, and Ag85A genes Mycobacterium sp. 012931 did not cluster with the other strains. In proteomic analyses using a Bruker matrix-assisted laser desorption ionization Biotyper, the mass profile of Mycobacterium sp. 012931 differed from the mass profiles of the other two fish M. marinum strains. Therefore, Mycobacterium sp. 012931 is similar to M. marinum but is not the same, suggesting that it could be a subspecies of M. marinum.     

Improved Identification of Rapidly Growing Mycobacteria by a 16S–23S Internal Transcribed Spacer Region PCR and Capillary Gel Electrophoresis

The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S–23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S–23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.     

Rapid Global Expansion of the Fungal Disease Chytridiomycosis into Declining and Healthy Amphibian Populations

The fungal disease chytridiomycosis, caused by Batrachochytrium dendrobatidis, is enigmatic because it occurs globally in both declining and apparently healthy (non-declining) amphibian populations. This distribution has fueled debate concerning whether, in sites where it has recently been found, the pathogen was introduced or is endemic. In this study, we addressed the molecular population genetics of a global collection of fungal strains from both declining and healthy amphibian populations using DNA sequence variation from 17 nuclear loci and a large fragment from the mitochondrial genome. We found a low rate of DNA polymorphism, with only two sequence alleles detected at each locus, but a high diversity of diploid genotypes. Half of the loci displayed an excess of heterozygous genotypes, consistent with a primarily clonal mode of reproduction. Despite the absence of obvious sex, genotypic diversity was high (44 unique genotypes out of 59 strains). We provide evidence that the observed genotypic variation can be generated by loss of heterozygosity through mitotic recombination. One strain isolated from a bullfrog possessed as much allelic diversity as the entire global sample, suggesting the current epidemic can be traced back to the outbreak of a single clonal lineage. These data are consistent with the current chytridiomycosis epidemic resulting from a novel pathogen undergoing a rapid and recent range expansion. The widespread occurrence of the same lineage in both healthy and declining populations suggests that the outcome of the disease is contingent on environmental factors and host resistance.