Aquatic Plants Stimulate the Growth of and Biofilm Formation by Mycobacterium ulcerans in Axenic Culture and Harbor These Bacteria in the Environment

Abstract

Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans. Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown. We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M. ulcerans and promoted biofilm formation. Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M. ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic. Genotype analysis showed that plant-associated M. ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens. These observations implicate aquatic plants as a reservoir of M. ulcerans and add a new potential link in the chain of transmission of M. ulcerans to humans.Mycobacterium ulcerans is an emerging environmental pathogen (2). It is the etiologic agent of Buruli ulcer, a necrotic skin disease highly prevalent in many countries throughout west Africa and one of the most common mycobacterial diseases in humans after tuberculosis and leprosy (2). M. ulcerans is the only mycobacterium known to produce a toxin, a polyketide-derived macrolide called mycolactone (10, 11). The infection begins with a painless nodule or papule that spreads over the surrounding tissue. Ischemic and necrotized tissue disappears and is replaced by centralized ulceration of the limb. At present, the only effective treatment consists of excision of the lesions, and this is often followed by extensive skin grafting (6).Until very recently M. ulcerans had never been isolated in culture from the environment. Indirect evidence from epidemiological studies suggests that M. ulcerans is an environmental mycobacterium present in swampy areas. Humans are thought to be infected through minor wounds or skin abrasions via contact with mycobacterium-containing water (33). In a previous study we demonstrated that aquatic insects are a possible mode of transmission of M. ulcerans to humans. We showed that, in an experimental mouse model, aquatic insects (Naucoridae) were able to transmit an infection by biting, thereby inoculating bacilli that had accumulated in the salivary glands of the insects (16). We were then able to isolate in pure culture M. ulcerans from the salivary glands of Naucoridae captured in a region of endemicity in the Ivory Coast. However it seems unlikely that these insects are the only environmental source of M. ulcerans. In another recent study, data gathered using an M. ulcerans-specific PCR to survey environments of endemicity in southeastern Australia identified aquatic plants as a possible reservoir of this pathogen (30).In this report, we test the hypothesis that M. ulcerans is associated with aquatic plants by studying the effects of crude aquatic plant extracts on the growth of M. ulcer...

Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer

“…In an aquatic environment, M. ulcerans might obtain its energy and carbon from degradation of plant saccharides, as these stimulate growth in vitro (Marsollier et al 2004b). Little is known, however, about its metabolism in insect tissues, although it may be significant that both M. marinum and M. ulcerans encode four potential chitinases/transglycosidases (BuruList/ mast/P4.27.html), one of which is attached to a PE domain and thus is expected to be in the cell envelope.…”

“…M. ulcerans is associated with aquatic vegetation such as algae (Marsollier et al 2004b); snails and other organisms that feed on algae may serve as passive hosts (Marsollier et al 2004a). One report has shown that M. ulcerans can multiply in the salivary glands of carnivorous water bugs, such as Naucoris cimicoides (Marsollier et al 2002).…”

Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease

“…When more discriminatory methods based on detection of variable number tandem repeats were used, many IS2404-positive environmental samples were reported to lack M. ulcerans (18). In light of these recent fi ndings, the relative frequency or abundance of M. ulcerans among aquatic invertebrates or other environmental reservoirs, remains tenuous, and thus, the role of aquatic insect vectors is uncertain.A series of laboratory experiments provided initial evidence for biting hemipteran vectors of M. ulcerans (19)(20)(21)(22)(23). Marsollier et al (9,24) demonstrated that a South American isolate of M. ulcerans could survive and multiply within the salivary glands of aquatic bugs indigenous to France (Naucoridae: Naucoris cimicoides).…”

Punaises aquatiques et transmission deMycobacterium ulcerans

“…Une étude histologique a montré une localisation prépondérante sinon exclusive du bacille dans les glandes salivaires des punaises, et ce, paradoxalement, sans aucun dommage du parenchyme tissulaire malgré la capacité du bacille à produire une toxine cytotoxique [8,9]. Par ailleurs, nous avons rapporté que des plantes aquatiques servaient de support à la formation de biofilms [10,11] et pouvaient être à l'origine de la contamination transitoire de proies phytophages (mollusques) des punaises. Un travail de terrain réalisé dans des zones endémi-ques au Bénin a permis de confirmer la validité des données sur l'écologie et la transmission de M. ulcerans des végétaux aquatiques à l'homme via des punaises d'eau [8].…”