Microbiology & Immunology - Research Publications
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ItemComparative analysis of the complete genome of an epidemic hospital sequence type 203 clone of vancomycin-resistant Enterococcus faecium(BMC, 2013-09-01)BACKGROUND: In this report we have explored the genomic and microbiological basis for a sustained increase in bloodstream infections at a major Australian hospital caused by Enterococcus faecium multi-locus sequence type (ST) 203, an outbreak strain that has largely replaced a predecessor ST17 sequence type. RESULTS: To establish a ST203 reference sequence we fully assembled and annotated the genome of Aus0085, a 2009 vancomycin-resistant Enterococcus faecium (VREfm) bloodstream isolate, and the first example of a completed ST203 genome. Aus0085 has a 3.2 Mb genome, comprising a 2.9 Mb circular chromosome and six circular plasmids (2 kb-130 kb). Twelve percent of the 3222 coding sequences (CDS) in Aus0085 are not present in ST17 E. faecium Aus0004 and ST18 E. faecium TX16. Extending this comparison to an additional 12 ST17 and 14 ST203 E. faecium hospital isolate genomes revealed only six genomic regions spanning 41 kb that were present in all ST203 and absent from all ST17 genomes. The 40 CDS have predicted functions that include ion transport, riboflavin metabolism and two phosphotransferase systems. Comparison of the vancomycin resistance-conferring Tn1549 transposon between Aus0004 and Aus0085 revealed differences in transposon length and insertion site, and van locus sequence variation that correlated with a higher vancomycin MIC in Aus0085. Additional phenotype comparisons between ST17 and ST203 isolates showed that while there were no differences in biofilm-formation and killing of Galleria mellonella, ST203 isolates grew significantly faster and out-competed ST17 isolates in growth assays. CONCLUSIONS: Here we have fully assembled and annotated the first ST203 genome, and then characterized the genomic differences between ST17 and ST203 E. faecium. We also show that ST203 E. faecium are faster growing and can out-compete ST17 E. faecium. While a causal genetic basis for these phenotype differences is not provided here, this study revealed conserved genetic differences between the two clones, differences that can now be tested to explain the molecular basis for the success and emergence of ST203 E. faecium.
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ItemWhole Genome Comparisons Suggest Random Distribution of Mycobacterium ulcerans Genotypes in a Buruli Ulcer Endemic Region of Ghana(PUBLIC LIBRARY SCIENCE, 2015-03)Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.
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ItemSnapshot fecal survey of domestic animals in rural Ghana for Mycobacterium ulcerans(PEERJ INC, 2016-06-01)Identifying the source reservoirs of Mycobacterium ulcerans is key to understanding the mode of transmission of this pathogen and controlling the spread of Buruli ulcer (BU). In Australia, the native possum can harbor M. ulcerans in its gastrointestinal tract and shed high concentrations of the bacteria in its feces. To date, an analogous animal reservoir in Africa has not been identified. Here we tested the hypothesis that common domestic animals in BU endemic villages of Ghana are reservoir species analogous to the Australian possum. Using linear-transects at 10-meter intervals, we performed systematic fecal surveys across four BU endemic villages and one non-endemic village in the Asante Akim North District of Ghana. One hundred and eighty fecal specimens from a single survey event were collected and analyzed by qPCR for the M. ulcerans diagnostic DNA targets IS2404 and KR-B. Positive and negative controls performed as expected but all 180 test samples were negative. This structured snapshot survey suggests that common domestic animals living in and around humans do not shed M. ulcerans in their feces. We conclude that, unlike the Australian native possum, domestic animals in rural Ghana are unlikely to be major reservoirs of M. ulcerans.
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ItemInvestigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans(PUBLIC LIBRARY SCIENCE, 2014-09)BACKGROUND: The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127). CONCLUSION/SIGNIFICANCE: This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.
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ItemMycolactone Gene Expression Is Controlled by Strong SigA-Like Promoters with Utility in Studies of Mycobacterium ulcerans and Buruli Ulcer(PUBLIC LIBRARY SCIENCE, 2009-11)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.
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ItemThe Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis(PUBLIC LIBRARY SCIENCE, 2013-07-23)Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.
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ItemNo Preview AvailableComplete Genome Sequence of the Frog Pathogen Mycobacterium ulcerans Ecovar Liflandii(AMER SOC MICROBIOLOGY, 2013-02)In 2004, a previously undiscovered mycobacterium resembling Mycobacterium ulcerans (the agent of Buruli ulcer) was reported in an outbreak of a lethal mycobacteriosis in a laboratory colony of the African clawed frog Xenopus tropicalis. This mycobacterium makes mycolactone and is one of several strains of M. ulcerans-like mycolactone-producing mycobacteria recovered from ectotherms around the world. Here, we describe the complete 6,399,543-bp genome of this frog pathogen (previously unofficially named "Mycobacterium liflandii"), and we show that it has undergone an intermediate degree of reductive evolution between the M. ulcerans Agy99 strain and the fish pathogen Mycobacterium marinum M strain. Like M. ulcerans Agy99, it has the pMUM mycolactone plasmid, over 200 chromosomal copies of the insertion sequence IS2404, and a high proportion of pseudogenes. However, M. liflandii has a larger genome that is closer in length, sequence, and architecture to M. marinum M than to M. ulcerans Agy99, suggesting that the M. ulcerans Agy99 strain has undergone accelerated evolution. Scrutiny of the genes specifically lost suggests that M. liflandii is a tryptophan, tyrosine, and phenylalanine auxotroph. A once-extensive M. marinum-like secondary metabolome has also been diminished through reductive evolution. Our analysis shows that M. liflandii, like M. ulcerans Agy99, has the characteristics of a niche-adapted mycobacterium but also has several distinctive features in important metabolic pathways that suggest that it is responding to different environmental pressures, supporting earlier proposals that it could be considered an M. ulcerans ecotype, hence the name M. ulcerans ecovar Liflandii.