Pierre-Alain Fonteyne

Insects in the transmission of Mycobacterium ulcerans infection

Buruli ulcer, caused by Mycobacterium ulcerans, is a common disease of skin and bones, which usually occurs in the vicinity of rural tropical wetlands. Epidemiological studies have not established a natural reservoir or mode of transmission. We collected 30 plants from swamps in areas of Benin (Ouinhi and Zangnanado) and Ghana (Tontokrom) endemic for Buruli ulcer. We tested roots, stems, and leaves for M ulcerans by culture and nested PCR. , 3 We also studied five water bugs found on the roots of three of these plants. Of the 95 samples analysed, only the five insects were positive by PCR for M ulcerans. These insects were from the roots of plants belonging to the C y p e r u s, P a n i c u m, and E i c h h o r n i a genera. Two of them from the roots of the C y p e r u s a n d P a n i c u m were not identified but were probably water bugs of the family Naucoridae. Roots of E i c h h o r n i a sheltered three aquatic bugs, one belonging to the genus N a u c o r i s ( f a m i l y Naucoridae) and two to the genus D i p l o n y c h u s ( f a m i l y Belostomatidae). Only the insects were positive; not the roots, stems, or leaves. Three different genera of plants are involved, suggesting that the type of plant is not a factor, but it is the aquatic bugs in the roots that are of interest. These bugs are aggressive predators of other species of aquatic arthropods and molluscs. They fly from one nearby swamp or pond to another, and may bite human beings. Water bugs belonging to the generae N a u c o r i s and Diplonychus often bite villagers. A study of ten water bugs from the same areas showed that two were positive for acid-fast bacilli. Only occasional acid-fast bacilli were seen, suggesting that water bugs were mechanical vectors of M ulcerans. We propose a new hypothesis for a source of M ulcerans a n d one mode of its transmission to human beings. M ulcerans survives best under low oxygen tensions, such as exist in mud in the bottom of swamps. Some water-filtering organisms could concentrate M ulcerans and be ingested by waterdwelling predators, which are then a passive reservoir for M ulcerans. Bites of these insects or contamination of human skin by their faeces may explain why direct contact with water is not necessary to acquire Buruli ulcer. Trauma seems essential for the introduction of M ulcerans into the skin. T h e aetiological agent may be introduced directly by bites of these insects, or by trauma at skin sites contaminated by insect products containing M ulcerans. There may also be parallel modes of transmission of M ulcerans, such as by aerosols from the surface of swamps. If confirmed, we believe this would be the first implication of insects in the transmission of a mycobacterial disease.

Epidemiology of Human Sporotrichosis Investigated by Amplified Fragment Length Polymorphism

ABSTRACT Amplified fragment length polymorphism (AFLP) was used to analyze the genetic diversity of Peruvian strains of Sporothrix schenckii and to compare them to a panel of non-Peruvian strains. AFLP analysis suggests that the Peruvian strains can be divided into two homogeneous clusters with no reference to geographical origin or the clinical form of sporotrichosis. The strains from abroad present heterogeneous profiles, with the Bolivian strain and the Colombian strains related to one of the Peruvian population. Sequencing of internal transcribed spacer 2, used to examine the relationships over a longer distance, confirmed the division of Peruvian strains into two populations that can be identified on the basis of a single but specific sequence divergence. This paper introduces automated AFLP analysis as a valuable tool for further investigation of the epidemiology and ecology of S. schenckii.

Detection of Phospholipase C in Nontuberculous Mycobacteria and Its Possible Role in Hemolytic Activity

ABSTRACT Phospholipase C plays a key role in the pathogenesis of several bacterial infections, for example, those caused by Clostridium perfringens and Listeria monocytogenes. Previous studies have reported multiple copies of plc genes homologous to Pseudomonas aeruginosa plcH andplcN genes encoding the hemolytic and nonhemolytic phospholipase C enzymes in the genomes of Mycobacterium tuberculosis, M. marinum, M. bovis, and M. ulcerans. In this study we analyzed the possible relationship between phospholipase C and hemolytic activity in 21 strains of nontuberculous mycobacteria representing nine different species. Detection of phospholipase C enzymatic activity was carried out using thin-layer chromatography to detect diglycerides in the hydrolysates of radiolabeled phosphatidylcholine. DNA sequences of M. kansasii and M. marinum homologous to the genes encoding phospholipase C from M. tuberculosis and M. ulcerans were identified by DNA-DNA hybridization and sequencing. Finally, we developed a direct and simple assay to detect mycobacterial hemolytic activity. This assay is based on a modified blood agar medium that allows the growth and expression of hemolysis of slow-growing mycobacteria. Hemolytic activity was detected in M. avium, M. intracellulare, M. ulcerans, M. marinum, M. tuberculosis, andM. kansasii mycobacteria with phospholipase C activity, but not in M. fortuitum. No hemolytic activity was detected inM. smegmatis, M. gordonae, and M. vaccae. Whether or not phospholipase C enzyme plays a role in the pathogenesis of nontuberculous mycobacterial diseases needs further investigation.

Biochemical and Genetic Evidence for Phospholipase C Activity in Mycobacterium ulcerans

ABSTRACT This study reports the existence of phospholipase C and D enzymatic activities in Mycobacterium ulcerans cultures as determined by use of thin-layer chromatography to detect diglycerides in hydrolysates of radiolabeled phosphatidylcholine. M. ulcerans DNA sequences homologous to the genes encoding phospholipase C in Mycobacterium tuberculosis andPseudomonas aeruginosa were identified by sequence analysis and DNA-DNA hybridization. Whether or not the phospholipase C and D enzymes of M. ulcerans plays a role in the pathogenesis of the disease needs further investigation.