Persistent Association of Mycobacterium ulcerans with West African Predaceous Insects of the Family Belostomatidae

Abstract

A number of studies have suggested that Mycobacterium ulcerans, the etiological agent of Buruli ulcer, may be transmitted to humans by insect bites. M. ulcerans has been isolated from a predaceous aquatic insect, and PCR detection of M. ulcerans DNA in aquatic environments suggests that the organism is widely distributed within many invertebrate taxa and functional feeding groups. Thus, M. ulcerans may be concentrated through different trophic links. However, the specific environmental niche of M. ulcerans and route of transmission to humans remain a mystery. In this study, a biologically relevant infection model in which M. ulcerans-infected mosquito larvae were fed to a species of predaceous hemiptera (African Belostomatidae) was used to demonstrate the persistent colonization of M. ulcerans and subsequent transmission of bacteria to naïve prey. The association of M. ulcerans with specific anatomical compartments showed that M. ulcerans accumulates preferentially on the exoskeleton. In contrast, few organisms were found in dissected guts or salivary glands. No difference was found between the ability of wild-type M. ulcerans and an M. ulcerans isogenic mycolactonenegative mutant to colonize belostomatids. These data show that African belostomatids can successfully be colonized by M. ulcerans and support the trophic transfer of M. ulcerans within the environment.

Interaction of Mycobacterium ulcerans with Mosquito Species: Implications for Transmission and Trophic Relationships

“…The strains used in this study were WT M. ulcerans 1615 GFP (24) and an isogenic mycolactone-negative mutant, M. ulcerans 1615::TN118 GFP (24), and M. ulcerans V2 RFP, a fluorescently labeled clinical isolate from Australia (26). M. ulcerans 1615 GFP (ATCC 35840) is a well-characterized Malaysian human isolate with physical and biochemical properties very similar to those of the sequenced strain M. ulcerans Agy99 from Ghana (42).…”

“…This strain produces neither the core nor the side chain of mycolactone, is not cytotoxic, and is avirulent. M. ulcerans strains were fluorescently labeled by introduction of a green fluorescent protein (GFP) or a red derivative (RFP) using the phage-integrating vector psm5 as described previously (24,44). By using this method, the GFP/RFP gene is inserted into the chromosome of M. ulcerans in the phage attachment site (att) and has no effect on the virulence of the bacterium.…”

“…For bacterial culture, salivary glands were dissected and homogenized and 10-fold homogenate dilutions were plated on Middlebrook 7H9 (M7H9) medium supplemented with 10% oleic acid-albumin-dextrose enrichment (OADC; Difco) and incubated at 32°C. Large external insect morphological structures such as legs and other exoskeletal components could not be viewed easily by microscopy and were analyzed primarily by PCR for the presence of M. ulcerans as previously described (24).…”

“…A plasmid-encoded macrolide toxin, mycolactone, is the primary virulence determinant of M. ulcerans (8,41). Biting aquatic insects, such as several taxa in the Belostomatidae and Naucoridae families (Hemiptera), have been suggested as possible vectors of M. ulcerans in several laboratory experiments (16,19,20,24,31,32); however, there is little empirical evidence from field studies to support the contention that these biting insects vector M. ulcerans to humans (2). In Melbourne, Australia, recent epidemiological evidence suggests that mosquitoes may serve as vectors in the transmission of BU disease (10,11,12,34,35).…”

Buruli ulcer

“…30 Random transposon mutagenesis has also revealed other genes necessary for toxin production. 30,31 Pidot et al 28 have shown that BU patients and persons that have been exposed to M. ulcerans, but who remain disease free, can make antibodies against several of the PKS enzymatic domains. On the other hand, it remains to be demonstrated if antibodies can access the synthases in bacteria to prevent toxin production and if this can prevent BU.…”