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.

Genetic diversity and population structure of Mycobacterium marinum: new insights into host and environmental specificities.

Mycobacterium marinum causes a systemic tuberculosis-like disease in fish and skin infections in humans that can spread to deeper structures, resulting in tenosynovitis, arthritis, and osteomyelitis. However, little information is available concerning (i) the intraspecific genetic diversity of M. marinum isolated from humans and animals; (ii) M. marinum genotype circulation in the different ecosystems, and (iii) the link between M. marinum genetic diversity and hosts (humans and fish). Here, we conducted a genetic study on 89 M. marinum isolates from humans (n = 68) and fish (n = 21) by using mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing. The results show that the M. marinum population is genetically structured not only according to the host but also according to the ecosystem as well as to tissue tropism in humans. This suggests the existence of different genetic pools in the function of the biological and ecological compartments. Moreover, the presence of only certain M. marinum genotypes in humans suggests a different zoonotic potential of the M. marinum genotypes. Considering that the infection is linked to aquarium activity, a significant genetic difference was also detected when the human tissue tropism of M. marinum was taken into consideration, with a higher genetic polymorphism in strains isolated from patients with cutaneous forms than from individuals with deeper-structure infection. It appears that only few genotypes can produce deeper infections in humans, suggesting that the immune system might play a filtering role.

Mycobacterium marinum infections in humans and tracing of its possible environmental sources.

The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M. marinum presence in the aquarists' fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n = 20), aquarium animals (n = 8), and an aquarium environment (n = 10). Isolate identification was carried out using 16S rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M. marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium peregrinum . The presence of M. marinum was proven in the aquarium environments of two patients. Although M. marinum is described as being present in water, it was detected only in fish.

Discriminatory potential of a novel set of Variable Number of Tandem Repeats for genotyping Mycobacterium marinum.

Mycobacterium marinum is a free-living bacterium that infects fish and amphibians, but is also an opportunistic pathogen in humans. Although in previous studies, the bacterium has shown a small number of Variable Number of Tandem Repeat (VNTR) polymorphic loci, the discriminatory power of the known VNTR loci is unlikely to be sufficient to distinguish such a globally ubiquitous waterborne microorganism, which has a presumably high genetic variance. The object of this study was to test M. marinum isolates from east China for strain diversity using previously described loci as well as novel VNTR loci, and to identify more discriminating VNTR loci that could enrich the genotyping tools for M. marinum. We found that 7 of the 12 tested VNTR loci, of which 5 were known loci and 2 loci were newly identified, showed good discriminatory power for the 14 M. marinum clinical isolates (Hunter-Gaston Index [HGI] value=0.990). The pathogenicity of isolates representing different VNTR clusters was also studied in a zebrafish (Danio rerio) infection model; in this model, 6 isolates representing 4 VNTR clusters induced chronic infections, whereas the American strain ATCC BAA-535 caused acute and lethal infections.

Evaluation of BacT/Alert 3D automated unit for detection of nontuberculous mycobacteria requiring incubation at 30 degrees C for optimal growth.

The reliability of the BacT/Alert 3D unit for automated detection of nontuberculous mycobacteria (NTM) that grow optimally at 30 °C was assessed. This system reliably maintained a temperature of 30 °C and detected 50% of the clinical NTM strains (5 Mycobacterium marinum and 3 Mycobacterium gordonae strains) faster than 37 °C culture.

Regulation of the 18 kDa heat shock protein in Mycobacterium ulcerans: an alpha-crystallin orthologue that promotes biofilm formation.

Mycobacterium ulcerans is the causative agent of the debilitating skin disease Buruli ulcer, which is most prevalent in Western and Central Africa. M. ulcerans shares >98% DNA sequence identity with Mycobacterium marinum, however, M. marinum produces granulomatous, but not ulcerative, lesions in humans and animals. Here we report the differential expression of a small heat shock protein (Hsp18) between strains of M. ulcerans (Hsp18(+) ) and M. marinum (Hsp18(-) ) and describe the molecular basis for this difference. We show by gene deletion and GFP reporter assays in M. marinum that a divergently transcribed gene called hspR_2, immediately upstream of hsp18, encodes a MerR-like regulatory protein that represses hsp18 transcription while promoting its own expression. Naturally occurring mutations within a 70 bp segment of the 144 bp hspR_2-hsp18 intergenic region among M. ulcerans strains inhibit hspR_2 transcription and explain the Hsp18(+) phenotype. We also propose a biological role for Hsp18, as we show that this protein significantly enhances bacterial attachment or aggregation during biofilm formation. This study has uncovered a new member of the MerR family of transcriptional regulators and suggests that upregulation of hsp18 expression was an important pathoadaptive response in the evolution of M. ulcerans from a M. marinum-like ancestor.

Extrapulmonary infections associated with nontuberculous mycobacteria in immunocompetent persons.

Over the past several years, the prevalence of human disease caused by nontuberculous mycobacteria (NTM) has increased. Whether the increase in cases is real or whether more cases are being recognized remains unclear. Despite a considerable increase in knowledge about NTM infections, they still represent a diagnostic and therapeutic challenge for several reasons: 1) pathogenic isolates may be indistinguishable from contaminant or saprophytic isolates; 2) timely and reliable identification of isolates may depend on proper communication between clinicians and laboratory staff; 3) lack of standardized susceptibility testing makes adoption of tailored therapies unrealistic; and 4) lack of treatment guidelines exposes patients to toxic drugs and disappointing outcomes. Laboratory research and multicenter controlled trials are needed to improve diagnosis and treatment of these infections.

Region of difference 1 in nontuberculous Mycobacterium species adds a phylogenetic and taxonomical character.

The esat-6 and cfp-10 genes are essential for virulence in Mycobacterium tuberculosis. Among nontuberculous mycobacteria, we found these genes only in M. kansasii, M. szulgai, M. marinum, and M. riyadhense, with unique sequences. This adds a phylogenetic and taxonomical characteristic and may represent a virulence factor for nontuberculous mycobacteria.

Fish gambler's tenosynovitis caused by Mycobacterium marinum: environmental investigation of a fishing pond in Southern Taiwan.

We describe a patient with Mycobacterium marinum tenosynovitis associated with a fish spine injury acquired at a gambling fishing pond in southern Taiwan and identify the source of the infection. M. marinum was isolated from fishing ponds and underground water and wastewater at the site. The isolates shared the same pulsed-field gel electrophoresis pattern as the patient. M. marinum was not detected in 54 samples obtained from 27 fish. Mycobacterium gordonae was isolated from 24 samples collected from the fish. Mycobacterium abscessus was isolated from 3 fish samples (Lateolabrax japonicus 1 and Sciaenops ocellatus 2). M. abscessus and M. gordonae were isolated from all water samples. This investigation provides strong evidence that the predisposing factor for the M. marinum infection was with a fish spine injury acquired at a gambling fishing pond. The source of the infection was the contaminated pond water.

The evolving story of Mycobacterium tuberculosis clade members detected in fish.

Advances in molecular analyses have permitted documentation of an increasing spectrum of mycobacteria infecting fish. Although some of these mycobacteria are not closely related, several species belong to the Mycobacterium tuberculosis clade. One member of the clade, M. marinum, is well known as an agent of piscine mycobacteriosis. Three other clade species, M. shottsii, M. pseudoshottsii and M. 'chesapeaki', have recently been identified as predominant disease agents in a widespread, continuing epizootic in wild striped bass of the Chesapeake Bay. A fifth clade member, M. ulcerans, has recently been indirectly detected in wild, African cichlid fish. As M. ulcerans is the third most common human mycobacterial infection worldwide, even such indirect evidence of M. ulcerans in fish must be more thoroughly investigated. Complicating the differentiation of these clade members is the growing recognition of intraspecies and interspecies variation in phenotypes, genes and virulence. Thus, researchers must be aware of the variety of piscine isolates within the M. tuberculosis clade. This review summarizes the methods of detection and differentiation for this important group of mycobacteria.

Mycobacterium marinum, a further infectious agent associated with sarcoidosis: the polyetiology hypothesis.

A 39-y-old male had a diagnosis of sarcoidosis and corticosteroid therapy was started. Surprisingly, following his discharge from hospital, Mycobacterium marinum was isolated in 1 of 3 sputum samples taken 7 weeks earlier on admission. After this, Mycobacterium marinum-DNA was identified in the stored lung biopsies by the PCR-RFLP of the hsp65 gene.

Identification of Mycobacterium marinum macrophage infection mutants.

Mycobacterium marinum is an important pathogen of humans, amphibians and fish. Most pathogenic mycobacteria, including M. marinum, infect, survive and replicate primarily intracellularly within macrophages. We constructed a transposon mutant library in M. marinum using Tn5367 delivered by phage transduction in the shuttle phasmid phAE94. We screened 529 clones from the transposon library directly in macrophage infection assays. All clones were screened for their ability to initially infect macrophages as well as survive and replicate intracellularly. We identified 19 mutants that fit within three classes: class I) defective for growth in association with macrophages (42%), class II) defective for macrophage infection (21%) and class III) defective for infection of and growth in association with macrophages (37%). Although 14 of the macrophage infection mutants (Mim) carry insertions in genes that have not been previously identified, five are associated with virulence of mycobacteria in animal models. These observations confirm the utility of mutant screens directly in association with macrophages to identify new virulence determinants in mycobacteria. We complemented four of the Mim mutants with their M. tuberculosis homologue, demonstrating that secondary mutations are not responsible for the observed defect in macrophage infection. The genes we identified provide insight into the molecular mechanisms of macrophage infection by M. marinum.

Multilocus variable-number tandem repeat typing of Mycobacterium ulcerans.

The apparent genetic homogeneity of Mycobacterium ulcerans contributes to the poorly understood epidemiology of M. ulcerans infection. Here, we report the identification of variable number tandem repeat (VNTR) sequences as novel polymorphic elements in the genome of this species. A total of 19 potential VNTR loci identified in the closely related M. marinum genome sequence were screened in a collection of 23 M. ulcerans isolates, one Mycobacterium species referred to here as an intermediate species, and five M. marinum strains. Nine of the 19 loci were polymorphic in the three species (including the intermediate species) and revealed eight M. ulcerans and five M. marinum genotypes. The results from the VNTR analysis corroborated the genetic relationships of M. ulcerans isolates from various geographical origins, as defined by independent molecular markers. Although these results further highlight the extremely high clonal homogeneity within certain geographic regions, we report for the first time the discrimination of the two South American strains from Surinam and French Guyana. These findings support the potential of a VNTR-based genotyping method for strain discrimination within M. ulcerans and M. marinum.

A case of granulomatous skin lesions caused by Mycobacterium marinum in the Campania region.

The diagnosis of cutaneous Mycobacterium marinum infection is frequently presumptive, as detection by conventional methods is difficult. We describe a patient with granulomatous skin lesions on the right dorsal hand and forearm. Histological examinations were presumptive for mycobacterium lesions. We identified Mycobacterium marinum directly in the patient's lesional skin biopsy combining polymerase chain reaction (PCR) amplification using Mycobacterium genus-specific primers, and subsequent restriction enzyme analysis enabling identification to the species level. The symptoms were no longer present after specific therapy, thereby confirming the initial diagnosis.

Mycobacterium marinum infections in fish and humans in Israel.

Israeli Mycobacterium marinum isolates from humans and fish were compared by direct sequencing of the 16S rRNA and hsp65 genes, restriction mapping, and amplified fragment length polymorphism analysis. Significant molecular differences separated all clinical isolates from the piscine isolates, ruling out the local aquaculture industry as the source of human infections.

Genotyping Mycobacterium ulcerans and Mycobacterium marinum by using mycobacterial interspersed repetitive units.

A novel category of variable tandem repeats (VNTR) called mycobacterial interspersed repetitive units (MIRUs) has been identified for Mycobacterium ulcerans (n = 39), M. marinum (n = 27), and one related organism. Fifteen MIRU loci were identified in the genome of M. marinum and were used to genotype M. ulcerans, M. marinum, and an M. marinum-like organism that is considered a possible missing link between M. marinum and M. ulcerans. Seven MIRU loci were polymorphic, and locus-specific PCRs for four of these loci differentiated seven M. ulcerans genotypes, four M. marinum genotypes, and a unique genotype for the missing link organism. The seven M. ulcerans genotypes were related to six different geographic origins of isolates. All isolates from West and Central Africa, including old and recent isolates, belonged to the same genotype, emphasizing the great spatiotemporal homogeneity among African isolates. Unlike the M. ulcerans genotypes, the four M. marinum genotypes could not be clearly related to the geographic origins of the isolates. According to MIRU-VNTR typing, all M. ulcerans and M. marinum isolates of American origin were closely related, suggesting a common American ancestor for these two pathogenic species on the American continents. MIRU typing has significant potential value for discriminating between reoccurrence and reinfection for M. ulcerans disease.

Mycobacterium marinum strains can be divided into two distinct types based on genetic diversity and virulence.

Mycobacterium marinum causes a systemic tuberculosis-like disease in a large number of poikilothermic animals and is used as a model for mycobacterial pathogenesis. In the present study, we infected zebra fish (Danio rerio) with different strains of M. marinum to determine the variation in pathogenicity. Depending on the M. marinum isolate, the fish developed an acute or chronic disease. Acute disease was characterized by uncontrolled growth of the pathogen and death of all animals within 16 days, whereas chronic disease was characterized by granuloma formation in different organs and survival of the animals for at least 4 to 8 weeks. Genetic analysis of the isolates by amplified fragment length polymorphism showed that M. marinum strains could be divided in two clusters. Cluster I contained predominantly strains isolated from humans with fish tank granuloma, whereas the majority of the cluster II strains were isolated from poikilothermic species. Acute disease progression was noted only with strains belonging to cluster I, whereas all chronic-disease-causing isolates belonged to cluster II. This difference in virulence was also observed in vitro: cluster I isolate Mma20 was able to infect and survive more efficiently in the human macrophage THP-1 and the carp leukocyte CLC cell lines than was the cluster II isolate Mma11. We conclude that strain characteristics play an important role in the pathogenicity of M. marinum. In addition, the correlation between genetic variation and host origin suggests that cluster I isolates are more pathogenic for humans.

The pathogen Mycobacterium marinum, a faster growing close relative of Mycobacterium tuberculosis, has a single rRNA operon per genome.

Although Mycobacterium marinum and Mycobacterium tuberculosis are very closely related they differ significantly in their growth rates. The Type strain of M. marinum and one clinical isolate were investigated and, like M. tuberculosis, were found to have a single rRNA (rrn) operon per genome located downstream from murA gene and controlled by two promoters. No sequence differences were found that account for the difference in the growth rates of the two species. We infer that M. tuberculosis has the capacity to synthesize rRNA much faster than it actually does; and propose that the high number of insertion sequences in this species attenuate growth rate to lower values.

Strain variation in Mycobacterium marinum fish isolates.

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.

Strain variation in Mediterranean and Red Sea Mycobacterium marinum isolates.

Four different PCR fingerprinting techniques were tested to distinguish possible strain variations in fourteen Mycobacterium marinum isolates, thirteen from Mediterranean and Red Sea fishes and one from a patient in Sardinia, Italy. PCR ribotyping and ERIC (enterobacterial repetitive consensus sequences)-PCR were found to be non-discriminative, whereas IS (insertion sequences)-PCR and GTG (GTG sequences repeats)-PCR could distinguish the clinical isolate from the piscine isolates, two Italian piscine isolates from all other isolates, but not the Greek isolates from the Israeli isolates. Our results indicate that GTG-PCR and IS-PCR have superior discriminative properties and are thus useful molecular tools for epidemiological studies of M. marinum.