Publications by authors named "Pamela L C Small"

25 Publications

  • Page 1 of 1

Functional Diversity as a New Framework for Understanding the Ecology of an Emerging Generalist Pathogen.

Ecohealth 2016 09 29;13(3):570-581. Epub 2016 Jun 29.

Bournemouth University, School of Applied Sciences, Dorset, BH12 5BB, UK.

Emerging infectious disease outbreaks are increasingly suspected to be a consequence of human pressures exerted on natural ecosystems. Previously, host taxonomic communities have been used as indicators of infectious disease emergence, and the loss of their diversity has been implicated as a driver of increased presence. The mechanistic details in how such pathogen-host systems function, however, may not always be explained by taxonomic variation or loss. Here we used machine learning and methods based on Gower's dissimilarity to quantify metrics of invertebrate functional diversity, in addition to functional groups and their taxonomic diversity at sites endemic and non-endemic for the model generalist pathogen Mycobacterium ulcerans, the causative agent of Buruli ulcer. Changes in these metrics allowed the rapid categorisation of the ecological niche of the mycobacterium's hosts and the ability to relate specific host traits to its presence in aquatic ecosystems. We found that taxonomic diversity of hosts and overall functional diversity loss and evenness had no bearing on the mycobacterium's presence, or whether the site was in an endemic area. These findings, however, provide strong evidence that generalist environmentally persistent bacteria such as M. ulcerans can be associated with specific functional traits rather than taxonomic groups of organisms, increasing our understanding of emerging disease ecology and origin.
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http://dx.doi.org/10.1007/s10393-016-1140-xDOI Listing
September 2016

Comparative Sigma Factor-mRNA Levels in Mycobacterium marinum under Stress Conditions and during Host Infection.

PLoS One 2015 7;10(10):e0139823. Epub 2015 Oct 7.

Department of Cell and Molecular Biology, Uppsala University Biomedical Centre, Uppsala, Sweden.

We have used RNASeq and qRT-PCR to study mRNA levels for all σ-factors in different Mycobacterium marinum strains under various growth and stress conditions. We also studied their levels in M. marinum from infected fish and mosquito larvae. The annotated σ-factors were expressed and transcripts varied in relation to growth and stress conditions. Some were highly abundant such as sigA, sigB, sigC, sigD, sigE and sigH while others were not. The σ-factor mRNA profiles were similar after heat stress, during infection of fish and mosquito larvae. The similarity also applies to some of the known heat shock genes such as the α-crystallin gene. Therefore, it seems probable that the physiological state of M. marinum is similar when exposed to these different conditions. Moreover, the mosquito larvae data suggest that this is the state that the fish encounter when infected, at least with respect to σ-factor mRNA levels. Comparative genomic analysis of σ-factor gene localizations in three M. marinum strains and Mycobacterium tuberculosis H37Rv revealed chromosomal rearrangements that changed the localization of especially sigA, sigB, sigD, sigE, sigF and sigJ after the divergence of these two species. This may explain the variation in species-specific expression upon exposure to different growth conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139823PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596819PMC
June 2016

Mycobacterium ulcerans fails to infect through skin abrasions in a guinea pig infection model: implications for transmission.

PLoS Negl Trop Dis 2014 Apr 10;8(4):e2770. Epub 2014 Apr 10.

University of Tennessee, Knoxville, Tennessee, United States of America.

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.
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http://dx.doi.org/10.1371/journal.pntd.0002770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983084PMC
April 2014

Identifying the Achilles' heel of multi-host pathogens: The concept of keystone "host" species illustrated by transmission.

Environ Res Lett 2013 ;8(4):045009

UMR MIVEGEC (IRD 224-CNRS 5290-UM1-UM2), Montpellier, France ; French School of Public Health, Interdisciplinary research center on biodiversity, climate change and infectious diseases, Montpellier, France.

Pathogens that use multiple host species are an increasing public health issue due to their complex transmission, which makes them difficult to mitigate. Here, we explore the possibility of using networks of ecological interactions among potential host species to identify the particular disease-source species to target to break down transmission of such pathogens. We fit a mathematical model on prevalence data of in western Africa and we show that removing the most abundant taxa for this category of pathogen is not an optimal strategy to decrease the transmission of the mycobacterium within aquatic ecosystems. On the contrary, we reveal that the removal of some taxa, especially Oligochaeta worms, can clearly reduce rates of pathogen transmission and should be considered as a keystone organism for its transmission because it leads to a substantial reduction in pathogen prevalence regardless of the network topology. Besides its potential application for the understanding of ecology, we discuss about how networks of species interactions can modulate transmission of multi-host pathogens.
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http://dx.doi.org/10.1088/1748-9326/8/4/045009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3925833PMC
January 2013

Aquatic macroinvertebrate assemblages of Ghana, West Africa: understanding the ecology of a neglected tropical disease.

Ecohealth 2014 Jun 4;11(2):168-83. Epub 2013 Dec 4.

Department of Entomology, Michigan State University, East Lansing, MI, 48824, USA,

Buruli ulcer (BU) is an emerging, but neglected tropical disease, where there has been a reported association with disturbed aquatic habitats and proposed aquatic macroinvertebrate vectors such as biting Hemiptera. An initial step in understanding the potential role of macroinvertebrates in the ecology of BU is to better understand the entire community, not just one or two taxa, in relation to the pathogen, Mycobacterium ulcerans, at a large spatial scale. For the first time at a country-wide scale this research documents that M. ulcerans was frequently detected from environmental samples taken from BU endemic regions, but was not present in 30 waterbodies of a non-endemic region. There were significant differences in macroinvertebrate community structure and identified potential indicator taxa in relation to pathogen presence. These results suggest that specific macroinvertebrate taxa or functional metrics may potentially be used as aquatic biological indicators of M. ulcerans. Developing ecological indicators of this pathogen is a first step for understanding the disease ecology of BU and should assist future studies of transmission.
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http://dx.doi.org/10.1007/s10393-013-0886-7DOI Listing
June 2014

Mycobacterium ulcerans causes minimal pathogenesis and colonization in medaka (Oryzias latipes): an experimental fish model of disease transmission.

Microbes Infect 2012 Aug 14;14(9):719-29. Epub 2012 Mar 14.

Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.

Mycobacterium ulcerans causes Buruli ulcer in humans, a progressive ulcerative epidermal lesion due to the mycolactone toxin produced by the bacterium. Molecular analysis of M. ulcerans reveals it is closely related to Mycobacterium marinum, a pathogen of both fish and man. Molecular evidence from diagnostic PCR assays for the insertion sequence IS2404 suggests an association of M. ulcerans with fish. However, fish infections by M. ulcerans have not been well documented and IS2404 has been found in other mycobacteria. We have thus, employed two experimental approaches to test for M. ulcerans in fish. We show here for the first time that M. ulcerans with or without the toxin does not mount acute or chronic infections in Japanese Medaka "Oryzias latipes" even at high doses. Moreover, M. ulcerans-infected medaka do not exhibit any visible signs of infection nor disease and the bacteria do not appear to replicate over time. In contrast, similar high doses of the wild-type M. marinum or a mycolactone-producing M. marinum "DL" strain are able to mount an acute disease with mortality in medaka. Although these results would suggest that M. ulcerans does not mount infections in fish we have evidence that CLC macrophages from goldfish are susceptible to mycolactones.
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http://dx.doi.org/10.1016/j.micinf.2012.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389220PMC
August 2012

Detection of Mycobacterium ulcerans in the environment predicts prevalence of Buruli ulcer in Benin.

PLoS Negl Trop Dis 2012 Jan 31;6(1):e1506. Epub 2012 Jan 31.

University of Tennessee, Knoxville, Tennessee, USA.

Background: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). In West Africa there is an association between BU and residence in low-lying rural villages where aquatic sources are plentiful. Infection occurs through unknown environmental exposure; human-to-human infection is rare. Molecular evidence for M. ulcerans in environmental samples is well documented, but the association of M. ulcerans in the environment with Buruli ulcer has not been studied in West Africa in an area with accurate case data.

Methodology/principal Finding: Environmental samples were collected from twenty-five villages in three communes of Benin. Sites sampled included 12 BU endemic villages within the Ouheme and Couffo River drainages and 13 villages near the Mono River and along the coast or ridge where BU has never been identified. Triplicate water filtrand samples from major water sources and samples from three dominant aquatic plant species were collected. Detection of M. ulcerans was based on quantitative polymerase chain reaction. Results show a significant association between M. ulcerans in environmental samples and Buruli ulcer cases in a village (p = 0.0001). A "dose response" was observed in that increasing numbers of M. ulceran- positive environmental samples were associated with increasing prevalence of BU cases (R(2) = 0.586).

Conclusions/significance: This study provides the first spatial data on the overlap of M. ulcerans in the environment and BU cases in Benin where case data are based on active surveillance. The study also provides the first evidence on M. ulcerans in well-defined non-endemic sites. Most environmental pathogens are more broadly distributed in the environment than in human populations. The congruence of M. ulcerans in the environment and human infection raises the possibility that humans play a role in the ecology of M. ulcerans. Methods developed could be useful for identifying new areas where humans may be at high risk for BU.
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http://dx.doi.org/10.1371/journal.pntd.0001506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3269429PMC
January 2012

Ecology and transmission of Buruli ulcer disease: a systematic review.

PLoS Negl Trop Dis 2010 Dec 14;4(12):e911. Epub 2010 Dec 14.

Department of Entomology, Michigan State University, East Lansing, Michigan, USA.

Buruli ulcer is a neglected emerging disease that has recently been reported in some countries as the second most frequent mycobacterial disease in humans after tuberculosis. Cases have been reported from at least 32 countries in Africa (mainly west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. Large lesions often result in scarring, contractual deformities, amputations, and disabilities, and in Africa, most cases of the disease occur in children between the ages of 4-15 years. This environmental mycobacterium, Mycobacterium ulcerans, is found in communities associated with rivers, swamps, wetlands, and human-linked changes in the aquatic environment, particularly those created as a result of environmental disturbance such as deforestation, dam construction, and agriculture. Buruli ulcer disease is often referred to as the "mysterious disease" because the mode of transmission remains unclear, although several hypotheses have been proposed. The above review reveals that various routes of transmission may occur, varying amongst epidemiological setting and geographic region, and that there may be some role for living agents as reservoirs and as vectors of M. ulcerans, in particular aquatic insects, adult mosquitoes or other biting arthropods. We discuss traditional and non-traditional methods for indicting the roles of living agents as biologically significant reservoirs and/or vectors of pathogens, and suggest an intellectual framework for establishing criteria for transmission. The application of these criteria to the transmission of M. ulcerans presents a significant challenge.
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http://dx.doi.org/10.1371/journal.pntd.0000911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3001905PMC
December 2010

A Mycobacterium ulcerans toxin, mycolactone, induces apoptosis in primary human keratinocytes and in HaCaT cells.

Microbes Infect 2010 Dec 25;12(14-15):1258-63. Epub 2010 Aug 25.

Department of Clinical and Experimental Medicine, University of Piemonte Orientale A. Avogadro, Via Solaroli 17, 28100 Novara, Italy.

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.
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http://dx.doi.org/10.1016/j.micinf.2010.08.005DOI Listing
December 2010

Interaction of Mycobacterium ulcerans with mosquito species: implications for transmission and trophic relationships.

Appl Environ Microbiol 2010 Sep 30;76(18):6215-22. Epub 2010 Jul 30.

Department of Biology, Millersville University, Millersville, Pennsylvania 17551, USA.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans. Here we show that Aedes aegypti, A. albopictus, Ochlerotatus triseriatus, and Culex restuans larvae readily ingest wild-type M. ulcerans, isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis), and tertiary (Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.
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http://dx.doi.org/10.1128/AEM.00340-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937476PMC
September 2010

Heterogeneity in the stereochemistry of mycolactones isolated from M. marinum: toxins produced by fresh vs. saltwater fish pathogens.

Chem Commun (Camb) 2009 Dec 26(47):7402-4. Epub 2009 Oct 26.

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.

A novel mycolactone has been identified from Mycobacterium marinum infecting freshwater fish.
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http://dx.doi.org/10.1039/b917014fDOI Listing
December 2009

Mycolactone inhibits monocyte cytokine production by a posttranscriptional mechanism.

J Immunol 2009 Feb;182(4):2194-202

Cytokine and Signal Transduction Laboratory, Kennedy Institute of Rheumatology Division, London, United Kingdom.

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1beta, IL-6, IL-10, and IFN-gamma-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-kappaB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.
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http://dx.doi.org/10.4049/jimmunol.0802294DOI Listing
February 2009

Limited repair and structural damages displayed by skeletal muscles loaded with mycolactone.

Microbes Infect 2009 Feb 16;11(2):238-44. Epub 2008 Dec 16.

Centre de Recherche du Centre Hospitalier de l'Université Laval, 2705 boulevard Laurier, bureau RC-9500, Québec (Qc) G1V 4G2, Canada.

Mycolactone produced by Mycobacterium ulcerans is the toxin responsible for most of the pathology in Buruli ulcer, the cutaneous signature of a complex disease. Although mycolactone cytopathicity is well described in various in vitro and in vivo models, the effect of this molecule on mammalian skeletal muscles has not been addressed. This is particularly surprising since muscle damage is characteristic of severe Buruli ulcer. We have thus investigated the impact of mycolactone on the mouse soleus muscle during degenerative and regenerative phases. Mice were intramuscularly injected with 300 microg of mycolactone and soleus muscles assessed histologically, biochemically and functionally at 7 and 42 days post-injection. Our results show that mycolactone induces local acute and chronic inflammatory responses which are respectively associated with a 65% and 68% decrease in maximal isometric force production (P(0)) relative to sham injections. In addition, muscle stiffness and total hydroxyproline content rose by 46% and 134% at day 42 relative to sham injections indicating an extensive fibrotic process in injured soleus muscles. Histological observations demonstrate significant muscle necrosis and atrophy with limited signs of regeneration. Together, our data indicate that mycolactone not only induces muscle damage but also prevents muscle regeneration to occur. These results may help to explain why patients with Buruli ulcer, experience muscle weakness and contracture.
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http://dx.doi.org/10.1016/j.micinf.2008.11.016DOI Listing
February 2009

A Landscape-based model for predicting Mycobacterium ulcerans infection (Buruli Ulcer disease) presence in Benin, West Africa.

Ecohealth 2008 Mar 8;5(1):69-79. Epub 2008 Feb 8.

Quantitative Fisheries Center, Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA.

Mycobacterium ulcerans infection (Buruli ulcer [BU] disease) is an emerging tropical disease that causes severe morbidity in many communities, especially those in close proximity to aquatic environments. Research and control efforts are severely hampered by the paucity of data regarding the ecology of this disease; for example, the vectors and modes of transmission remain unknown. It is hypothesized that BU presence is associated with altered landscapes that perturb aquatic ecosystems; however, this has yet to be quantified over large spatial scales. We quantified relationships between land use/land cover (LULC) characteristics surrounding individual villages and BU presence in Benin, West Africa. We also examined the effects of other village-level characteristics which we hypothesized to affect BU presence, such as village distance to the nearest river. We found that as the percent urban land use in a 50-km buffer surrounding a village increased, the probability of BU presence decreased. Conversely, as the percent agricultural land use in a 20-km buffer surrounding a village increased, the probability of BU presence increased. Landscape-based models had predictive ability when predicting BU presence using validation data sets from Benin and Ghana, West Africa. Our analyses suggest that relatively small amounts of urbanization are associated with a decrease in the probability of BU presence, and we hypothesize that this is due to the increased availability of pumped water in urban environments. Our models provide an initial approach to predicting the probability of BU presence over large spatial scales in Benin and Ghana, using readily available land use data.
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http://dx.doi.org/10.1007/s10393-007-0148-7DOI Listing
March 2008

Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis.

Genome Res 2008 May 10;18(5):729-41. Epub 2008 Apr 10.

Department of Microbiology, Monash University, Clayton 3800, Australia.

Mycobacterium marinum, a ubiquitous pathogen of fish and amphibia, is a near relative of Mycobacterium tuberculosis, the etiologic agent of tuberculosis in humans. The genome of the M strain of M. marinum comprises a 6,636,827-bp circular chromosome with 5424 CDS, 10 prophages, and a 23-kb mercury-resistance plasmid. Prominent features are the very large number of genes (57) encoding polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and the most extensive repertoire yet reported of the mycobacteria-restricted PE and PPE proteins, and related-ESX secretion systems. Some of the NRPS genes comprise a novel family and seem to have been acquired horizontally. M. marinum is used widely as a model organism to study M. tuberculosis pathogenesis, and genome comparisons confirmed the close genetic relationship between these two species, as they share 3000 orthologs with an average amino acid identity of 85%. Comparisons with the more distantly related Mycobacterium avium subspecies paratuberculosis and Mycobacterium smegmatis reveal how an ancestral generalist mycobacterium evolved into M. tuberculosis and M. marinum. M. tuberculosis has undergone genome downsizing and extensive lateral gene transfer to become a specialized pathogen of humans and other primates without retaining an environmental niche. M. marinum has maintained a large genome so as to retain the capacity for environmental survival while becoming a broad host range pathogen that produces disease strikingly similar to M. tuberculosis. The work described herein provides a foundation for using M. marinum to better understand the determinants of pathogenesis of tuberculosis.
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http://dx.doi.org/10.1101/gr.075069.107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2336800PMC
May 2008

Distribution of Mycobacterium ulcerans in buruli ulcer endemic and non-endemic aquatic sites in Ghana.

PLoS Negl Trop Dis 2008 Mar 26;2(3):e205. Epub 2008 Mar 26.

Department of Microbiology, University of Tennessee, Knoxville, Tennessee, USA.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer.
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http://dx.doi.org/10.1371/journal.pntd.0000205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268743PMC
March 2008

First cultivation and characterization of Mycobacterium ulcerans from the environment.

PLoS Negl Trop Dis 2008 Mar 26;2(3):e178. Epub 2008 Mar 26.

Institute of Tropical Medicine, Antwerpen, Belgium.

Background: Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.

Methodology/principal Findings: One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5' end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3' end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.

Conclusion: Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.
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http://dx.doi.org/10.1371/journal.pntd.0000178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2268003PMC
March 2008

Mycolactone is responsible for the painlessness of Mycobacterium ulcerans infection (buruli ulcer) in a murine study.

Infect Immun 2008 May 3;76(5):2002-7. Epub 2008 Mar 3.

Department of Human Pathology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.

Buruli ulcer is a chronic skin disease caused by Mycobacterium ulcerans, which produces a toxic lipid mycolactone. Despite the extensive necrosis and tissue damage, the lesions are painless. This absence of pain prevents patients from seeking early treatment and, as a result, many patients experience severe sequelae, including limb amputation. We have reported that mice inoculated with M. ulcerans show loss of pain sensation and nerve degeneration. However, the molecules responsible for the nerve damage have not been identified. In order to clarify whether mycolactone alone can induce nerve damage, mycolactone A/B was injected to footpads of BALB/c mice. A total of 100 microg of mycolactone induced footpad swelling, redness, and erosion. The von Frey sensory test showed hyperesthesia on day 7, recovery on day 21, and hypoesthesia on day 28. Histologically, the footpads showed epidermal erosion, moderate stromal edema, and moderate neutrophilic infiltration up to day 14, which gradually resolved. Nerve bundles showed intraneural hemorrhage, neutrophilic infiltration, and loss of Schwann cell nuclei on days 7 and 14. Ultrastructurally, vacuolar change of myelin started on day 14 and gradually subsided by day 42, but the density of myelinated fibers remained low. This study demonstrated that initial hyperesthesia is followed by sensory recovery and final hypoesthesia. Our present study suggests that mycolactone directly damages nerves and is responsible for the absence of pain characteristic of Buruli ulcer. Furthermore, mice injected with 200 microg of mycolactone showed pulmonary hemorrhage. This is the first study to demonstrate the systemic effects of mycolactone.
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http://dx.doi.org/10.1128/IAI.01588-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2346717PMC
May 2008

Mycolactone-mediated inhibition of tumor necrosis factor production by macrophages infected with Mycobacterium ulcerans has implications for the control of infection.

Infect Immun 2007 Aug 21;75(8):3979-88. Epub 2007 May 21.

Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, 4710-057 Braga, Portugal.

The pathogenicity of Mycobacterium ulcerans, the agent of Buruli ulcer, depends on the cytotoxic exotoxin mycolactone. Little is known about the immune response to this pathogen. Following the demonstration of an intracellular growth phase in the life cycle of M. ulcerans, we investigated the production of tumor necrosis factor (TNF) induced by intramacrophage bacilli of diverse toxigenesis/virulence, as well as the biological relevance of TNF during M. ulcerans experimental infections. Our data show that murine bone marrow-derived macrophages infected with mycolactone-negative strains of M. ulcerans (nonvirulent) produce high amounts of TNF, while macrophages infected with mycolactone-positive strains of intermediate or high virulence produce intermediate or low amounts of TNF, respectively. These results are in accordance with the finding that TNF receptor P55-deficient (TNF-P55 KO) mice are not more susceptible than wild-type mice to infection by the highly virulent strains but are more susceptible to nonvirulent and intermediately virulent strains, demonstrating that TNF is required to control the proliferation of these strains in animals experimentally infected by M. ulcerans. We also show that mycolactone produced by intramacrophage M. ulcerans bacilli inhibits, in a dose-dependent manner, but does not abrogate, the production of macrophage inflammatory protein 2, which is consistent with the persistent inflammatory responses observed in experimentally infected mice.
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http://dx.doi.org/10.1128/IAI.00290-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1951989PMC
August 2007

Newly identified Mycobacterium species in a Xenopus laevis colony.

Comp Med 2007 Feb;57(1):97-104

Department of Animal Medicine, University of Massachusetts Medical School, Worcester, USA.

The University of Massachusetts Medical School maintains 3 separate research colonies of Xenopus laevis, with each colony located in a separate building on campus. After a 5-wk in-house quarantine period, 34 wild-caught X. laevis were transferred into one of the existing colonies. As a result, this colony grew from 51 to 85 frogs. All animals were housed in a recirculating frog housing system. During the first 2 mo, 6 frogs died suddenly, and health reports were generated for another 10 frogs in this colony. The majority of health reports were written in response to acute coelomic distention. These patterns continued until, after 1 y, only 25 of the original 85 animals remained. Necropsies performed showed large accumulations of serosanguinous fluid in the subcutaneous space or body cavity. Granulomatous inflammatory lesions with acid-fast bacilli were generally present in the liver, lung, or spleen. Culture of affected tissues grew Mycobacterium sp. within 40 d. Polymerase chain reaction analysis confirmed the isolated organism to be the same species of Mycobacterium (provisionally named M. liflandii) recently reported by 2 other groups. However, previous clinical publications suggested that this bacterium originated only from X. tropicalis. The cases we present highlight the rapidly lethal effects of M. liflandii in a colony of wild-caught X. laevis and illustrate the need to dedicate further attention to this emerging Xenopus disease.
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February 2007

Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer.

Genome Res 2007 Feb 8;17(2):192-200. Epub 2007 Jan 8.

Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 75725 Paris Cedex 15, France.

Mycobacterium ulcerans is found in aquatic ecosystems and causes Buruli ulcer in humans, a neglected but devastating necrotic disease of subcutaneous tissue that is rampant throughout West and Central Africa. Here, we report the complete 5.8-Mb genome sequence of M. ulcerans and show that it comprises two circular replicons, a chromosome of 5632 kb and a virulence plasmid of 174 kb. The plasmid is required for production of the polyketide toxin mycolactone, which provokes necrosis. Comparisons with the recently completed 6.6-Mb genome of Mycobacterium marinum revealed >98% nucleotide sequence identity and genome-wide synteny. However, as well as the plasmid, M. ulcerans has accumulated 213 copies of the insertion sequence IS2404, 91 copies of IS2606, 771 pseudogenes, two bacteriophages, and multiple DNA deletions and rearrangements. These data indicate that M. ulcerans has recently evolved via lateral gene transfer and reductive evolution from the generalist, more rapid-growing environmental species M. marinum to become a niche-adapted specialist. Predictions based on genome inspection for the production of modified mycobacterial virulence factors, such as the highly abundant phthiodiolone lipids, were confirmed by structural analyses. Similarly, 11 protein-coding sequences identified as M. ulcerans-specific by comparative genomics were verified as such by PCR screening a diverse collection of 33 strains of M. ulcerans and M. marinum. This work offers significant insight into the biology and evolution of mycobacterial pathogens and is an important component of international efforts to counter Buruli ulcer.
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http://dx.doi.org/10.1101/gr.5942807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1781351PMC
February 2007

Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin.

Cell Microbiol 2005 Aug;7(8):1187-96

Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, Paris, France.

Mycobacterium ulcerans (Mu), the aetiological agent of Buruli ulcer, is an extracellular pathogen producing the macrolide toxin mycolactone. Using a mouse model of intradermal infection, we found that Mu was initially captured by phagocytes and transported to draining lymph nodes (DLN) within host cells. Similar to Buruli ulcers in humans, the infection site eventually became ulcerated with tissue necrosis and extracellular bacteria, at later stages. In contrast to Mycobacterium bovis BCG (BCG), Mu did not disseminate to the spleen. However, mice infected with Mu or BCG developed comparable primary cellular responses to mycobacterial antigens in DLN and spleen. The role of mycolactone in this sequence of events was examined with a mycolactone-deficient (mup045) mutant of Mu. Mup045 bacilli were better internalized than wild-type (wt) bacteria by mouse phagocytes in vitro. Moreover, infection with wt but not mup045 Mu led to inhibition of TNF-alpha expression, upregulation of MIP-2 chemokine, and host cell death within 1 day. Our results suggest that mycolactone expression during the intracellular life of Mu may contribute to immune evasion by inhibiting phagocytosis, provoking apoptosis of antigen presenting cells and altering the establishment of an appropriate inflammatory reaction.
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http://dx.doi.org/10.1111/j.1462-5822.2005.00546.xDOI Listing
August 2005

Structure determination of mycolactone C via total synthesis.

Org Lett 2004 Dec;6(26):4901-4

Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.

[structure: see text] By total synthesis, mycolactone C has been established as an approximately 1:1 mixture of Z-Delta4'5'- and E-Delta4'5'-geometric isomers of C12'-deoxymycolactones A and B.
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http://dx.doi.org/10.1021/ol0479996DOI Listing
December 2004

Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans.

Proc Natl Acad Sci U S A 2004 Feb 21;101(5):1345-9. Epub 2004 Jan 21.

Unité de Génétique Moléculaire Bactérienne, Génopole, Institut Pasteur, 28 Rue du Docteur Roux, 75725 Paris Cedex 15, France.

Mycobacterium ulcerans (MU), an emerging human pathogen harbored by aquatic insects, is the causative agent of Buruli ulcer, a devastating skin disease rife throughout Central and West Africa. Mycolactone, an unusual macrolide with cytotoxic and immunosuppressive properties, is responsible for the massive s.c. tissue destruction seen in Buruli ulcer. Here, we show that MU contains a 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant polyketide synthases (PKSs), and polyketide-modifying enzymes, and demonstrate that these are necessary and sufficient for mycolactone synthesis. This is a previously uncharacterized example of plasmid-mediated virulence in a Mycobacterium, and the emergence of MU as a pathogen most likely reflects the acquisition of pMUM001 by horizontal transfer. The 12-membered core of mycolactone is produced by two giant, modular PKSs, MLSA1 (1.8 MDa) and MLSA2 (0.26 MDa), whereas its side chain is synthesized by MLSB (1.2 MDa), a third modular PKS highly related to MLSA1. There is an extreme level of sequence identity within the different domains of the MLS cluster (>97% amino acid identity), so much so that the 16 ketosynthase domains seem functionally identical. This is a finding of significant consequence for our understanding of polyketide biochemistry. Such detailed knowledge of mycolactone will further the investigation of its mode of action and the development of urgently needed therapeutic strategies to combat Buruli ulcer.
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http://dx.doi.org/10.1073/pnas.0305877101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC337055PMC
February 2004
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