Martha W. Rhodes

Evolution of Mycobacterium ulcerans and Other Mycolactone-Producing Mycobacteria from a Common Mycobacterium marinum Progenitor

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.

Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F.

ABSTRACT 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.

Mycobacteriosis in fishes: A review

Mycobacterium species have long been recognised as a significant source of morbidity and mortality in finfish aquaculture, as well as in wild finfishes. Mycobacteria infecting fishes also include zoonotic pathogens that can cause protracted illness, especially in immunocompromised individuals. Several basic aspects of mycobacterial pathobiology in aquatic animals remain poorly understood, although a number of important recent developments have been made, especially with respect to identification of novel Mycobacterium spp. infecting fishes and a new group of mycobacteria closely related to the human pathogen Mycobacterium ulcerans. This review will encompass important aspects of mycobacterial disease in fishes, discuss recent research including studies of mycobacteriosis in striped bass (Morone saxatilis) of Chesapeake Bay, USA, and suggest directions for future work.

Seasonal occurrence of mesophilic Aeromonas spp. as a function of biotype and water quality in temperate freshwater lakes

Abstract Densities of mesophilic aeromonads were compared in Virginia freshwater lakes with trophic classifications ranging from mesotrophic to hypereutrophic. Aeromonad concentrations were independent of trophic status and did not correlate statistically with water quality parameters used to measure trophic state (total phosphorus, chlorophyll a and Secchi depth). Bacterial indicators (fecal coliforms and Escherichia coli ) used to assess the public health safety of recreational waters correlated with mesophilic aeromonads only when sewage pollution occurred. Overall ( n = 101–107) there were small negative correlations of aeromonad densities with dissolved oxygen ( r = −0.30, P = r = −0.30, P = 0.05). During frequent sampling from April through October 1991, mean mesophilic aeromonad concentrations ranged from 10 3–4 cells 100 ml −1 with the lowest values measured during mid-summer. Over the course of twelve months, 273 aeromonad isolates were identified as belonging to the following biotypes: A. sobria (54%), A. hydrophila (29%), A. caviae (10%), and undetermined (7%). Seasonally, the frequency of isolation of A. sobria , considered the most pathogenic biotype, increased from 17 to 69% when water temperatures exceeded 20°C in a hypereutrophic lake. These temperatures typically occur during periods of peak recreational usage. Phenotypic characteristics associated with virulence were positive in 70% (hemolysis) and 42% (autoagglutination) of glycerol-stored strains ( n = 85), and in 15% (autoagglutination) of strains immediately processed ( n = 73). S-layer proteins associated with invasiveness were not found in 14 strains which autoagglutinated. To evaluate the public health significance of aeromonads in nutrient enriched lakes, our results demonstrate a need to couple investigations of mesophilic aeromonad ecology with virulence and biotype assessments.

Effects of sunlight and autochthonous microbiota onEscherichia coli survival in an estuarine environment

The effects of sunlight and the indigenous microbiota onEscherichia coli survival were examined with membrane diffusion chambers deployed in Chesapeake Bay shellfish growing waters. Chambers, fitted with an “upper” UV and visible light-transmitting copolymer film and “lower” semipermeable polycarbonate membrane, were deployed parallel to the water surface to maximize light exposure. Maximum values of a coefficient, kdens, describing changes in culturable cell densities after exposure to sunlight, were 1.7 h−1 and 0.7 h−1 in preliminary tank and in situ experiments, respectively. Mortality and sublethal stress, the latter measured with an electrochemical detection technique, were largest during the first 4 h of exposure. Owing to the light-attenuating properties of Chesapeake Bay water, light-induced cell mortality was significantly reduced at 0.25 m compared with surface exposed cells, and was undetected at 0.5–1.0 m except during seasons of maximal light penetration. Combined exposure to both sunlight and the autochthonous microbiota yielded significantly greater mortality than for either factor alone.

Use of Salmonella typhimurium WG49 to enumerate male-specific coliphages in an estuary and watershed subject to nonpoint pollution

The occurrence of male-specific RNA (FRNA) coliphages, proposed as indicators of enteric viruses, was determined in an estuary subject to nonpoint pollution that included fecal inputs from livestock. A host originally developed for detecting FRNA phages in sewage was applied to water and sediment samples. Phages were enumerated using the host Salmonella typhimurium WG49 containing an Escherichia coli plasmid coding for sex pili, and the female parent strain WG45. FRNA phages and fecal coliforms were enumerated in samples collected seasonally from an estuary and associated feeder streams and densities related to selected environmental parameters. Mean phage densities enumerated on WG49 ranged from < 1 to 50 100 ml−1 water and < 13 to 7200 100 g−1 dry sediment. Examination of 300 phages from estuarine and freshwater samples showed that ⩾ 99% were RNase-resistant, ⩾ 94% were lytic to the female parent salmonella strain (WG45), ⩽ 9% were lytic to male E. coli C3000, and none were lytic to female E. coli C. RNase resistant phages lytic to both salmonella strains were noncontractile flexible tailed phages and those lytic to male salmonella or E. coli hosts were filamentous phages. Electron micrographs of the only RNase-sensitive phage recovered that plaqued only male hosts showed cubic phage particles adsorbed to sex pili. Parallel enumerations of environmental samples on WG45 and WG49 yielded equal or greater phage densities on the former host. Purified phages from these samples were lytic to certain salmonella serovars recovered from the environment but did not cross react with fecal coliform or heterotrophic bacteria isolated from the environment. Although the WG49 host was inappropriate to estuarine and freshwater samples examined because of interference by somatic phages, WG45 and WG49 should be examined as hosts for enumerating salmonella phages. Similarly, the public health significance of somatic phages detected by these hosts should be determined. FRNA phages, with a single exception (1187 samples), were not detected in a condemned shellfish growing area subject to nonpoint pollution. This observation questions the application of FRNA phages as indicators of fecal contamination in waters impacted by diffuse fecal inputs.

Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay

ABSTRACT Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.

Characterization of photochromogenic Mycobacterium spp. from Chesapeake Bay striped bass Morone saxatilis.

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake Bay striped bass using phenotypic characterization and multilocus sequencing of 16S rRNA, hsp65 and rpoB genes. Genetic characterization reveals that these isolates are related to widely divergent portions of the mycobacterial phylogeny; however, some interesting trends are observed, such as a majority of isolates (10/17) belonging to the M. simiae-related grouping. Five additional isolates were assigned to the slow-growing mycobacteria (including 2 identified as M. marinum), while 2 are clearly shown to belong genetically to the fast-growing mycobacteria.

First report of Streptococcus parauberis in wild finfish from North America.

Streptococcosis is a common cause of pathology and mortality in fishes resulting in significant economic losses for the aquaculture industry. One etiologic agent of the disease, Streptococcus parauberis, has been associated with fish mortalities in Spain and Korea. Here we report the first identification of S. parauberis in wild finfish in Chesapeake Bay, USA. Gram-positive cocci were isolated from the spleens of striped bass, Morone saxatilis, and identified via species-specific primers and 16S rRNA gene sequencing. Biochemical characterization and antibiotic susceptibility tests were used to compare local isolates to isolates infecting aquacultured fishes and dairy cattle. This is also the first report of a plasmid in S. parauberis from any host.

Nested polymerase chain reaction assay for detection of Mycobacterium shottsii and M. pseudoshottsii in striped bass.

Wild striped bass Morone saxatilis in Chesapeake Bay are experiencing a high prevalence of mycobacteriosis, which produces granulomatous lesions of the skin and visceral organs. Culture-based studies have indicated that the newly described species Mycobacterium shottsii and M. pseudoshottsii are the dominant isolates from diseased fish. The classical fish pathogen M. marinum is also found, albeit at much lower frequencies. Both M. shottsii and M. pseudoshottsii are extremely slow-growing on standard selective media, and up to 12 months may be required for isolation and characterization. Epidemiological studies of mycobacteriosis in Chesapeake Bay would therefore benefit from rapid molecular assays with which to detect these species in fish. In this paper, we describe the development of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays capable of detecting M. shottsii, M. pseudoshottsii, and, in most instances, coinfections thereof in striped bass tissues. In addition, PCR-RFLP assays were designed to detect M. marinum and other as-yet-undescribed Mycobacterium spp. present in Chesapeake Bay striped bass. Comparison of these molecular assays with culture-based techniques using splenic tissue from wild striped bass yielded generally concordant results and demonstrated the applicability of these techniques to the study of wild fish.