Mycobacterium ulcerans in Mosquitoes Captured during Outbreak of Buruli Ulcer, Southeastern Australia

Mosquitoes positive for M. ulcerans were linked to outbreaks of Buruli ulcer in humans

Risk of Buruli Ulcer and Detection of Mycobacterium ulcerans in Mosquitoes in Southeastern Australia

Buruli ulcer (BU) is a destructive skin condition caused by infection with the environmental bacterium, Mycobacterium ulcerans. BU has been reported in more than 30 countries in Africa, the Americas, Asia and the Western Pacific. How people become infected with M. ulcerans is not completely understood, but numerous studies have explored the role of biting insects. In 2007, it was discovered that M. ulcerans could be detected in association with mosquitoes trapped in one town in southeastern Australia during a large outbreak of BU. In the present study we investigated whether there was a relationship between the incidence of BU in humans in several towns and the likelihood of detecting M. ulcerans in mosquitoes trapped in those locations. We found a strong association between the proportion of M. ulcerans-positive mosquitoes and the incidence of human disease. The results of this study strengthen the hypothesis that mosquitoes are involved in the transmission of M. ulcerans in southeastern Australia. This has implications for the development of strategies to control and prevent BU

Mycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer

Mycolactone A/B is a lipophilic macrocyclic polyketide that is the primary virulence factor produced by Mycobacterium ulcerans, a human pathogen and the causative agent of Buruli ulcer. In M. ulcerans strain Agy99 the mycolactone polyketide synthase (PKS) locus spans a 120 kb region of a 174 kb megaplasmid. Here we have identified promoter regions of this PKS locus using GFP reporter assays, in silico analysis, primer extension, and site-directed mutagenesis. Transcription of the large PKS genes mlsA1 (51 kb), mlsA2 (7 kb) and mlsB (42 kb) is driven by a novel and powerful SigA-like promoter sequence situated 533 bp upstream of both the mlsA1 and mlsB initiation codons, which is also functional in Escherichia coli, Mycobacterium smegmatis and Mycobacterium marinum. Promoter regions were also identified upstream of the putative mycolactone accessory genes mup045 and mup053. We transformed M. ulcerans with a GFP-reporter plasmid under the control of the mls promoter to produce a highly green-fluorescent bacterium. The strain remained virulent, producing both GFP and mycolactone and causing ulcerative disease in mice. Mosquitoes have been proposed as a potential vector of M. ulcerans so we utilized M. ulcerans-GFP in microcosm feeding experiments with captured mosquito larvae. M. ulcerans-GFP accumulated within the mouth and midgut of the insect over four instars, whereas the closely related, non-mycolactone-producing species M. marinum harbouring the same GFP reporter system did not. This is the first report to identify M. ulcerans toxin gene promoters, and we have used our findings to develop M. ulcerans-GFP, a strain in which fluorescence and toxin gene expression are linked, thus providing a tool for studying Buruli ulcer pathogenesis and potential transmission to humans

Correlation between detection of M. ulcerans in mosquitoes and incidence of BU, Bellarine Peninsula, 2005–09.

<p>The vertical axis shows the maximum likelihood estimate (MLE) of the proportion of M. ulcerans-positive mosquitoes or the average cumulative incidence of BU over five years by town (with 95% confidence intervals). There were no cases of BU at Breamlea or Wallington during the study period.</p

Detection of <i>M. ulcerans</i> in mosquitoes, Bellarine Peninsula, 2005–09.

a<p>Positive in at least two replicates for IS<i>2404</i> (± IS<i>2606</i> and KR).</p>b<p>Maximum likelihood estimate (MLE). MLE bias was corrected when ≥1 pool was positive; otherwise uncorrected.</p

BU incidence per 1,000 population by town, 2005–09.

<p>BU incidence per 1,000 population by town, 2005–09.</p

Sketch map of places and towns referred to in the text.

<p>Sketch map of places and towns referred to in the text.</p

Photo of Buruli ulcer lesion.

<p>Right elbow, 32 year old male with PCR and culture confirmed Buruli ulcer. The patient spent just 4 hours at Barwon Heads on 11 May 2008 and had no known contact with any other endemic areas. He first observed the ulcer in mid-October 2008.</p