Aquatic snails, passive hosts of Mycobacterium ulcerans.

Accumulative indirect evidence of the epidemiology of Mycobacterium ulcerans infections causing chronic skin ulcers (i.e., Buruli ulcer disease) suggests that the development of this pathogen and its transmission to humans are related predominantly to aquatic environments. We report that snails could transitorily harbor M. ulcerans without offering favorable conditions for its growth and replication. A novel intermediate link in the transmission chain of M. ulcerans becomes likely with predator aquatic insects in addition to phytophage insects. Water bugs, such as Naucoris cimicoides, a potential vector of M. ulcerans, were shown to be infected specifically by this bacterium after feeding on snails experimentally exposed to M. ulcerans.

Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbor these bacteria in the environment.

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.

Susceptibility of Mycobacterium ulcerans to a combination of amikacin/rifampicin.

The effectiveness of rifampicin (RIF), amikacin (AMK) and their combination were estimated in the treatment of mice experimentally infected by Mycobacterium ulcerans and the risk of relapse after the treatment was evaluated. After 7 weeks of treatment with RIF or with the combination of AMK/RIF and 8 weeks with AMK alone, no viable bacilli were found in the infected tissues and these remained uninfected during the following 6 months. Among the mice treated with AMK alone, three mice relapsed, but the minimal inhibitory concentration of AMK for these isolates remained unchanged. With RIF alone, two mice relapsed and the minimal inhibitory concentration of these isolated strains was higher. However, with all the mice treated with both RIF and AMK, no relapse was observed.

Isolation of three Mycobacterium ulcerans strains resistant to rifampin after experimental chemotherapy of mice

By use of a murine model for Buruli ulcer, Mycobacterium ulcerans was found to be susceptible to rifampin, with the MIC being 0.5 to 1 micro g/ml. Three mutants were isolated after rifampin monotherapy. Two were resistant to rifampin at 8 micro g/ml, and one was resistant to rifampin at 32 micro g/ml. The mutants harbored Ser416Phe mutations and His420Tyr mutations in the rpoB gene, and these mutations have also been found to be responsible for rifampin resistance in the leprosy and tubercle bacilli. The results indicate that while rifampin may be active against M. ulcerans, it should never be used as monotherapy in humans.

Isolation of three Mycobacterium ulcerans strains resistant to rifampin after experimental chemotherapy of mice.

By use of a murine model for Buruli ulcer, Mycobacterium ulcerans was found to be susceptible to rifampin, with the MIC being 0.5 to 1 micro g/ml. Three mutants were isolated after rifampin monotherapy. Two were resistant to rifampin at 8 micro g/ml, and one was resistant to rifampin at 32 micro g/ml. The mutants harbored Ser416Phe mutations and His420Tyr mutations in the rpoB gene, and these mutations have also been found to be responsible for rifampin resistance in the leprosy and tubercle bacilli. The results indicate that while rifampin may be active against M. ulcerans, it should never be used as monotherapy in humans.

Aquatic insects as a vector for Mycobacterium ulcerans.

Mycobacterium ulcerans is an emerging environmental pathogen which causes chronic skin ulcers (i.e., Buruli ulcer) in otherwise healthy humans living in tropical countries, particularly those in Africa. In spite of epidemiological and PCR data linking M. ulcerans to water, the mode of transmission of this organism remains elusive. To determine the role of aquatic insects in the transmission of M. ulcerans, we have set up an experimental model with aquariums that mimic aquatic microenvironments. We report that M. ulcerans may be transmitted to laboratory mice by the bite of aquatic bugs (Naucoridae) that are infected with this organism. In addition, M. ulcerans appears to be localized exclusively within salivary glands of these insects, where it can both survive and multiply without causing any observable damage in the insect tissues. Subsequently, we isolated M. ulcerans from wild aquatic insects collected from a zone in the Daloa region of Ivory Coast where Buruli ulcer is endemic. Taken together, these results point to aquatic insects as a possible vector of M. ulcerans.