No abstract

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis) A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 6C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mg ml "1 ), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15 T , has been deposited in the American Type Culture Collection as ATCC BAA-883 T and the National Collection of Type Cultures (UK) as NCTC 13318 T .

Mycobacterium ulcerans and Mycobacterium marinum are closely related pathogens which share an aquatic environment. The pathogenesis of these organisms in humans is limited by their inability to grow above 35°C. M. marinum causes systemic disease in fish but produces localized skin infections in humans. M. ulcerans causes Buruli ulcer, a severe human skin lesion. At the molecular level, M. ulcerans is distinguished from M. marinum by the presence of a virulence plasmid which encodes a macrolide toxin, mycolactone, as well as by hundreds of insertion sequences, particularly IS2404. There has been a global increase in reports of fish mycobacteriosis. An unusual clade of M. marinum has been reported from fish in the Red and Mediterranean Seas and a new mycobacterial species, Mycobacterium pseudoshottsii, has been cultured from fish in the Chesapeake Bay, United States. We have discovered that both groups of fish pathogens produce a unique mycolactone toxin, mycolactone F. Mycolactone F is the smallest mycolactone (molecular weight, 700) yet identified. The core lactone structure of mycolactone F is identical to that of M. ulcerans mycolactones, but a unique side chain structure is present. Mycolactone F produces apoptosis and necrosis on cultured cells but is less potent than M. ulcerans mycolactones. Both groups of fish pathogens contain IS2404. In contrast to M. ulcerans and conventional M. marinum, mycolactone F-producing mycobacteria are incapable of growth at above 30°C. This fact is likely to limit their virulence for humans. However, such isolates may provide a reservoir for horizontal transfer of the mycolactone plasmid in aquatic environments.Mycobacterium marinum is a globally distributed pathogen of marine and freshwater fish which also causes skin infections in humans (7, 9). M. marinum is phenotypically distinguished from other mycobacteria by its low optimal growth temperature, light-induced carotenoid production, and relatively rapid growth rate compared to other slow-growing Mycobacterium species. There is considerable heterogeneity among M. marinum isolates, and several subgroups have been described (28,(33)(34)(35).Mycobacteriosis was first diagnosed in fish from the Red Sea in 1990 (5). The infection was initially found in cultured sea bass (Dicentrarchus labrax) in Eilat and has since been found in over 20 different fish species and a hawksbill sea turtle. The Red Sea isolates differed phenotypically from other M. marinum strains by being scotochromogenic (having constitutive pigment production). Whereas most M. marinum strains form colonies on mycobacterial media within 8 days, initial growth was not obtained from these isolates for at least 2 weeks. Similar isolates have also been found in the Mediterranean Sea in Greece and Italy. Molecular characterization of the Israeli isolates from fish confirmed their identity as M. marinum, but analysis of the 16S rRNA gene showed that the isolates formed clades within the species (33, 34). Molecular comparison of the fish isolates with human isolates of...

Striped bass Morone saxatilis were infected intraperitoneally with approximately 10 5 Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21°C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from splenic tissue at all times p.i. Standard Ziehl-Neelsen staining, however, did not demonstrate acid-fast rods in most early inflammatory foci and granulomas. Large numbers of acid-fast rods were present in granulomas beginning at 8 wk p.i. Between 26 and 45 wk p.i., reactivation of disease was observed in some fish, with disintegration of granulomas, renewed inflammation, and elevated splenic bacterial densities approaching 10 9 colony-forming units g -1 . Infection with M. shottsii or M. gordonae did not produce severe pathology. Mild peritonitis was followed by granuloma formation in the mesenteries, but, with 1 exception, granulomas were not observed in the spleen or anterior kidney. M. shottsii and M. gordonae both established persistent infections in the spleen, but were present at densities at least 2 orders of magnitude less than M. marinum at all time points observed. Granulomas in the mesenteries of M. shottsii-and M. gordonaeinfected fish resolved over time, and no reactivation of disease was observed. KEY WORDS: Mycobacteriosis · Striped bass · Morone saxatilis · Mycobacterium marinum · Mycobacterium gordonae · Mycobacterium shottsii · Granuloma Resale or republication not permitted without written consent of the publisherDis Aquat Org 54: [105][106][107][108][109][110][111][112][113][114][115][116][117] 2003 respectively (Sakanari et al. 1983, Hedrick et al. 1987. Mycobacterium marinum was cultured from fish in the latter study, and has also been isolated from wild Pacific striped bass (Landsell et al. 1993). Recently, visceral and dermal lesions in striped bass from Chesapeake Bay and its tributaries were shown to be associated with mycobacterial infection (Vogelbein et al. 1999). An epizootiological study of striped bass from Virginia tributaries of Chesapeake Bay found up to 62.7% prevalence of mycobacteriosis, based on histological presence of characteristic granulomas. This suggests the disease has significant effects on wild stocks (Cardinal 2001).In addition to Mycobacterium marinum and several other Mycobacterium spp., 2 recently described species of mycobacteria, 'M. chesapeaki ' (Heckert et al. 2001) and M. shottsii (Rhodes et al. 2001(Rhodes et al. , 2003 in press), have been isolated from Chesapeake Bay striped bass. Mycobacteria were cultured from splenic tissue of approximately 76% of stripe...