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Abstract Background Mycobacterium ulcerans is an unusual bacterial pathogen with elusive origins. While closely related to the aquatic dwelling M. marinum, M. ulcerans has evolved the ability to produce the immunosuppressive polyketide toxin mycolactone and cause the neglected tropical disease Buruli ulcer. Other mycolactone-producing mycobacteria (MPM) have been identified in fish and frogs and given distinct species designations (M. pseudoshottsii, M. shinshuense, M. liflandii and M. marinum), however the evolution of M. ulcerans and its relationship to other MPM has not been defined. Here we report the comparative analysis of whole genome sequences from 30 MPM and five M. marinum. Results A high-resolution phylogeny based on genome-wide single nucleotide polymorphisms (SNPs) showed that M. ulcerans and all other MPM represent a single clonal group that evolved from a common M. marinum progenitor. The emergence of the MPM was driven by the acquisition of the pMUM plasmid encoding genes for the biosynthesis of mycolactones. This change was accompanied by the loss of at least 185 genes, with a significant overrepresentation of genes associated with cell wall functions. Cell wall associated genes also showed evidence of substantial adaptive selection, suggesting cell wall remodeling has been critical for the survival of MPM. Fine-grain analysis of the MPM complex revealed at least three distinct lineages, one of which comprised a highly clonal group, responsible for Buruli ulcer in Africa and Australia. This indicates relatively recent transfer of M. ulcerans between these continents, which represent the vast majority of the global Buruli ulcer burden. Our data provide SNPs and gene sequences that can differentiate M. ulcerans lineages, suitable for use in the diagnosis and surveillance of Buruli ulcer. Conclusions M. ulcerans and all mycolactone-producing mycobacteria are specialized variants of a common Mycobacterium marinum progenitor that have adapted to live in restricted environments. Examination of genes lost or retained and now under selective pressure suggests these environments might be aerobic, and extracellular, where slow growth, production of an immune suppressor, cell wall remodeling, loss or modification of cell wall antigens, and biofilm-forming ability provide a survival advantage. These insights will guide our efforts to find the elusive reservoir(s) of M. ulcerans and to understand transmission of Buruli ulcer.

Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Mycobacterium africanum Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range.

Background: The origin and spread of tuberculosis (TB) in Tasmania and the types of strains of Mycobacterium tuberculosis complex (MTBC) present in the population are largely unknown., Objective: The aim of this study was to perform the first genomic analysis of MTBC isolates from Tasmania to better understand the epidemiology of TB in the state., Methods: Whole-genome sequencing was performed on cultured isolates of MTBC collected from 2014-2016. Single-locus variant analysis was applied to determine the phylogeny of the isolates and the presence of drug-resistance mutations. The genomic data were then cross-referenced against public health surveillance records on each of the cases., Results: We determined that 83.3% of TB cases in Tasmania from 2014-2016 occurred in non-Australian born individuals. Two possible TB clusters were identified based on single locus variant analysis, one from November-December 2014 (n = 2), with the second from May-August 2015 (n = 4). We report here the first known isolate of multi-drug resistant (MDR) M. tuberculosis in Tasmania from 2016 for which we established its drug resistance mutations and potential overseas origin. In addition, we characterised a case of M. bovis TB in a Tasmanian-born person who presented in 2014, approximately 40 years after the last confirmed case in the state's bovids., Conclusions: TB in Tasmania is predominantly of overseas origin with genotypically-unique drug-susceptible isolates of M. tuberculosis. However, the state also exhibits features of TB that are observed in other jurisdictions, namely, the clustering of cases, and drug resistance. Early detection of TB and contact tracing, particularly of overseas-born cases, coordinated with rapid laboratory drug-susceptibility testing and molecular typing, will be essential for Tasmania to reach the World Health Organisation's TB eradication goals for low-incidence settings.

Objectives: This paper, the last in a series of three on 'feline leprosy', provides a detailed description of disease referable to the previously unnamed species, Candidatus 'Mycobacterium lepraefelis', a close relative of the human pathogens Mycobacterium leprae and Mycobacterium lepromatosis., Methods: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M lepraefelis' infection., Results: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory (VIDRL) records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Thirty-eight cats were definitively diagnosed with Candidatus 'M lepraefelis' infection. Typically, cats tended to be middle-aged or older when first infected, with a male predilection. Affected cats typically had widespread cutaneous lesions, in some cases after initially localised disease. Advanced cases were often systemically unwell. All cats had outdoor access. The histological picture was lepromatous in the majority of patients, although two cases had tuberculoid disease. In one case that underwent necropsy, lesions were evident in the liver, spleen and lungs. Treatment was varied, although most cats received a combination of oral clarithromycin and rifampicin. Prognosis for recovery was variable, but typically poor., Conclusions and Relevance: Candidatus 'M lepraefelis' typically causes high bacterial index (lepromatous) feline leprosy that in some cases progresses to systemic mycobacteriosis. The disease has a variable clinical course and prognosis. Many cases either died or were euthanased due to the infection. Multilocus sequence analysis reveals a heterogeneous picture and further analysis of draft genome sequencing may give clues to the taxonomy and epidemiology of this organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations. Comparative aspects: This paper finishes with a discussion of comparative aspects of infection caused by the three feline leproid disease agents that have been the subject of this series: Candidatus 'Mycobacterium tarwinense', Mycobacterium lepraemurium and Candidatus 'M lepraefelis'.

Objectives: This paper, the second in a series of three on 'feline leprosy', provides a detailed description of disease referable to Mycobacterium lepraemurium, the most common cause of feline leprosy worldwide., Methods: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with M lepraemurium infection., Results: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Sixty-five cats were definitively diagnosed with M lepraemurium infection. Typically, cats were 1-3 years of age when first infected, with a male gender predilection. Affected cats were generally systemically well. All had outdoor access. Lesions tended to consist of one or more cutaneous/subcutaneous nodules, typically located on the head and/or forelimbs, possibly reflecting the most likely locations for a rodent bite as the site of inoculation for organisms. Nodules had the propensity to ulcerate at some stage in the clinical course. The cytological and histological picture varied from tuberculoid, with relatively low bacterial numbers, to lepromatous with moderate to high bacterial numbers. Treatment was varied, although most cats underwent surgical resection of lesions with adjunctive medical therapy, most often using a combination of oral clarithromycin and rifampicin. Prognosis for recovery was generally good, and in two cases there was spontaneous remission without the requirement for medical intervention. Untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but had no apparent tendency to disseminate to internal organs., Conclusions and Relevance: M lepraemurium causes high bacterial index (lepromatous) or low bacterial index (tuberculoid) feline leprosy. The infection typically causes nodules of the skin and/or subcutis (which tend towards ulceration) on the head and/or forelimbs. The disease usually has an indolent clinical course and infected cats have a generally favourable response to therapeutic interventions, with rare cases undergoing spontaneous resolution. Genomic analysis may yield clues as to the environmental niche and culture requirements of this elusive organism. Prospective treatment trials and/or additional drug susceptibility testing in specialised systems would further inform treatment recommendations.

Objectives: This paper, the first in a series of three on 'feline leprosy', provides a detailed description of disease referable to Candidatus 'Mycobacterium tarwinense', the most common cause of feline leprosy in Victoria, Australia., Methods: Cases were sourced retrospectively and prospectively for this observational study, describing clinical, geographical and molecular microbiological data for cats definitively diagnosed with Candidatus 'M tarwinense' infection., Results: A total of 145 cases of feline leprosy were scrutinised; 114 'new' cases were sourced from the Victorian Infectious Diseases Reference Laboratory records, veterinary pathology laboratories or veterinarians, and 31 cases were derived from six published studies. Forty-two cats were definitively diagnosed with Candidatus 'M tarwinense' infection. Typically, cats were between 3 and 11 years of age, with no gender predilection, and were generally systemically well. All had outdoor access. Most cats underwent surgical resection of lesions with adjunctive medical therapy, often utilising a combination of oral clarithromycin and rifampicin for at least 3 months. Prognosis for recovery was generally good. Resolution of lesions was not observed in the absence of treatment, but a number of untreated cats continued to enjoy an acceptable quality of life despite persistence of the disease, which extended locally but did not appear to disseminate to internal organs. Preliminary results of draft genome sequencing confirmed that the species is a member of the Mycobacterium simiae complex., Conclusions and Relevance: Candidatus 'M tarwinense', a fastidious member of the M simiae complex, is capable of causing feline leprosy with a tendency to produce lesions on the head, particularly involving the eyes and periocular skin. The disease has an indolent clinical course and generally responds favourably to therapy despite lesions often containing large numbers of organisms. Detailed genomic analysis may yield clues as to the environmental niche and culture requirement of this elusive organism. Prospective treatment trials and/or drug susceptibility testing in specialised systems would further inform treatment recommendations.

This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

Background: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans, with endemicity predominantly in sub-Saharan Africa and south-eastern Australia. The mode of transmission and the environmental reservoir(s) of the bacterium and remain elusive. Real-time PCR investigations have detected M. ulcerans DNA in a variety of Australian environmental samples, including the faeces of native possums with and without clinical evidence of infection. This report seeks to expand on previously published findings by the authors' investigative group with regards to clinical and subclinical disease in selected wild possum species in BU-endemic areas of Victoria, Australia., Methodology/principal Findings: Twenty-seven clinical cases of M. ulcerans infection in free-ranging possums from southeastern Australia were identified retrospectively and prospectively between 1998-2011. Common ringtail possums (Pseudocheirus peregrinus), a common brushtail possum (Trichosurus vulpecula) and a mountain brushtail possum (Trichosurus cunninghami) were included in the clinically affected cohort. Most clinically apparent cases were adults with solitary or multiple ulcerative cutaneous lesions, generally confined to the face, limbs and/or tail. The disease was minor and self-limiting in the case of both Trichosurus spp. possums. In contrast, many of the common ringtail possums had cutaneous disease involving disparate anatomical sites, and in four cases there was evidence of systemic disease at post mortem examination. Where tested using real-time PCR targeted at IS2404, animals typically had significant levels of M. ulcerans DNA throughout the gut and/or faeces. A further 12 possums without cutaneous lesions were found to have PCR-positive gut contents and/or faeces (subclinical cases), and in one of these the organism was cultured from liver tissue. Comparisons were made between clinically and subclinically affected possums, and 61 PCR-negative, non-affected individuals, with regards to disease category and the categorical variables of species (common ringtail possums v others) and sex. Animals with clinical lesions were significantly more likely to be male common ringtail possums., Conclusions/significance: There is significant disease burden in common ringtail possums (especially males) in some areas of Victoria endemic for M. ulcerans disease. The natural history of the disease generally remains unknown, however it appears that some mildly affected common brushtail and mountain brushtail possums can spontaneously overcome the infection, whereas some severely affected animals, especially common ringtail possums, may become systemically, and potentially fatally affected. Subclinical gut carriage of M. ulcerans DNA in possums is quite common and in some common brushtail and mountain brushtail possums this is transient. Further work is required to determine whether M. ulcerans infection poses a potential threat to possum populations, and whether these animals are acting as environmental reservoirs in certain geographical areas.

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥10(6) organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.

Introduction: Buruli Ulcer (BU) is caused by the environmental microbe Mycobacterium ulcerans. Despite unclear transmission, contact with a BU endemic region is the key known risk factor. In Victoria, Australia, where endemic areas have been carefully mapped, we aimed to estimate the Incubation Period (IP) of BU by interviewing patients who reported defined periods of contact with an endemic area prior to BU diagnosis., Method: A retrospective review was undertaken of 408 notifications of BU in Victoria from 2002 to 2012. Telephone interviews using a structured questionnaire and review of notification records were performed. Patients with a single visit exposure to a defined endemic area were included and the period from exposure to disease onset determined (IP)., Results: We identified 111 of 408 notified patients (27%) who had a residential address outside a known endemic area, of whom 23 (6%) reported a single visit exposure within the previous 24 months. The median age of included patients was 30 years (range: 6 to 73) and 65% were male. 61% had visited the Bellarine Peninsula, currently the most active endemic area. The median time from symptom onset to diagnosis was 71 days (range: 34-204 days). The midpoint of the reported IP range was utilized to calculate a point estimate of the IP for each case. Subsequently, the mean IP for the cohort was calculated at 135 days (IQR: 109-160; CI 95%: 113.9-156), corresponding to 4.5 months or 19.2 weeks. The shortest IP recorded was 32 days and longest 264 days (Figure 1 & 2). IP did not vary for variables investigated., Conclusions: The estimated mean IP of BU in Victoria is 135 days (IQR: 109-160 days), 4.5 months. The shortest recorded was IP 34 days and longest 264 days. A greater understanding of BU IP will aid clinical risk assessment and future research.

Setting: Victoria, Australia., Objective: To describe the epidemiology and control of tuberculosis (TB) in Victoria, 2005-2010., Design: Retrospective review of laboratory-confirmed TB in Victoria, 2005-2010. State TB reference laboratory records were matched with Department of Health notification records to obtain laboratory, demographic, clinical and treatment data., Results: The incidence of TB fell in the Australian-born population but increased overall, reflecting an increase in the proportion of overseas-born cases from 88.9% to 95.8% between 2005 and 2010 (P = 0.03). Patients from India and Viet Nam accounted for over one third of all cases. For overseas-born cases, the median time between arrival and diagnosis was 4 years. Half of all diagnoses were pulmonary disease, of which 45.4% were Ziehl-Neelsen smear-positive. Treatment was most commonly self-administered (76.9%), and very few patients defaulted or failed treatment (1.1%). Only 4.1% of cases were linked to another laboratory-confirmed case. Multidrug-resistant TB remained uncommon (1.7% of cases)., Conclusions: TB in Victoria remains low by global standards and continues to overwhelmingly affect the overseas-born population. Current TB control strategies in Victoria are effective, but strengthened control in high-burden countries will also improve TB control locally.

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. Rapid detection of M. ulcerans in clinical specimens is essential to ensure early diagnosis and prevention of disability. This chapter describes a real-time PCR method for the direct detection of M. ulcerans from swabs, fresh tissue biopsies, and fixed tissue sections, which are the most common types of specimens used in the diagnosis of Buruli ulcer. The chapter also briefly describes methods for PCR detection of M. ulcerans in environmental samples, as reliable detection of M. ulcerans in the environment is becoming increasingly important for understanding the ecology and transmission of this important pathogen.

Background: Canine leproid granuloma (CLG) characteristically presents as single to multiple circumscribed dermal to subcutaneous nodules in haired skin. An unidentified mycobacterium is considered be the aetiological agent of this entity., Animals: Several cases of canine leproid granulomas occurred in dogs in New Zealand during 2010 and 2011. Cases appeared in clusters, affecting multiple closely related foxhounds domiciled in the same kennels. All affected hounds recovered after topical and/or systemic antimicrobial therapy. Two similar outbreaks that occurred in foxhounds near Melbourne, Australia are also reported., Methods: Cases were investigated using cytological, histological, microbiological and several molecular techniques. An environmental epidemiological study was also performed., Results: A diagnosis of CLG was established in 11 dogs. Molecular identification of the causative agent confirmed that it was a mycobacterial species with 100% sequence homology within the amplified regions of the 16S rRNA gene and internal transcribed spacer (ITS1) with that found in association with similar infections from the USA, Brazil and Australia., Conclusion and Clinical Importance: This report details the first occurrence of multiple cases of CLG occurring in in-contact dogs and the first proven case of CLG in dogs in New Zealand., (© 2012 The Authors. Veterinary Dermatology © 2012 ESVD and ACVD.)

Buruli ulcer (BU) is a necrotizing infection of skin and soft tissue caused by Mycobacterium ulcerans. In Australia, most cases of BU are linked to temperate, coastal Victoria and tropical, northern Queensland, and strains from these regions are distinguishable by variable-number tandem repeat (VNTR) typing. We present an epidemiological investigation of five patients found to have been infected during interstate travel and describe two nucleotide polymorphisms that differentiate M. ulcerans strains from northern Australia.

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

Background: Buruli ulcer (BU), caused by Mycobacterium ulcerans (M. ulcerans), is a necrotizing skin disease found in more than 30 countries worldwide. BU incidence is highest in West Africa; however, cases have substantially increased in coastal regions of southern Australia over the past 30 years. Although the mode of transmission remains uncertain, the spatial pattern of BU emergence in recent years seems to suggest that there is an environmental niche for M. ulcerans and BU prevalence., Methodology/principal Findings: Network analysis was applied to BU cases in Victoria, Australia, from 1981-2008. Results revealed a non-random spatio-temporal pattern at the regional scale as well as a stable and efficient BU disease network, indicating that deterministic factors influence the occurrence of this disease. Monthly BU incidence reported by locality was analyzed with landscape and climate data using a multilevel Poisson regression approach. The results suggest the highest BU risk areas occur at low elevations with forested land cover, similar to previous studies of BU risk in West Africa. Additionally, climate conditions as far as 1.5 years in advance appear to impact disease incidence. Warmer and wetter conditions 18-19 months prior to case emergence, followed by a dry period approximately 5 months prior to case emergence seem to favor the occurrence of BU., Conclusions/significance: The BU network structure in Victoria, Australia, suggests external environmental factors favor M. ulcerans transmission and, therefore, BU incidence. A unique combination of environmental conditions, including land cover type, temperature and a wet-dry sequence, may produce habitat characteristics that support M. ulcerans transmission and BU prevalence. These findings imply that future BU research efforts on transmission mechanisms should focus on potential vectors/reservoirs found in those environmental niches. Further, this study is the first to quantitatively estimate environmental lag times associated with BU outbreaks, providing insights for future transmission investigations.

Background: Buruli ulcer (BU) is a destructive skin condition caused by infection with the environmental bacterium, Mycobacterium ulcerans. The mode of transmission of M. ulcerans is not completely understood, but several studies have explored the role of biting insects. In this study, we tested for an association between the detection of M. ulcerans in mosquitoes and the risk of BU disease in humans in an endemic area of southeastern Australia., Methodology/principal Findings: Adult mosquitoes were trapped in seven towns on the Bellarine Peninsula in Victoria, Australia, from December 2004 to December 2009 and screened for M. ulcerans by real-time PCR. The number of laboratory-confirmed cases of BU in permanent residents of these towns diagnosed during the same period was tallied to determine the average cumulative incidence of BU in each location. Pearson's correlation coefficient (r) was calculated for the proportion of M. ulcerans-positive mosquitoes per town correlated with the incidence of BU per town. We found a strong dose-response relationship between the detection of M. ulcerans in mosquitoes and the risk of human disease (r, 0.99; 95% CI, 0.92-0.99; p < 0.001)., Conclusions/significance: 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 intervention strategies to control and prevent BU.

It has recently been shown that in a Buruli ulcer (BU) endemic region of southeastern Australia, significant numbers of possums (native tree-dwelling marsupials) have clinical BU disease. Furthermore, based on quantitative PCR (qPCR) analysis, animals with BU lesions (and some without) shed M. ulcerans DNA in their faeces, indicative of bacterial loads of up to 10(8) organisms/gram. These findings led us to propose that humans might also harbour M. ulcerans in their gastrointestinal tract and shed the bacterium in their faeces. We conducted a pilot study and collected faecal swabs from 26 patients with confirmed BU and 31 healthy household controls. Faecal samples were also collected from 10 healthy controls from non-endemic regions in Ghana. All 67 specimens were negative when tested by IS2404 PCR. The detection sensitivity of this method was ≥10(4) bacteria per gram (wet-weight) of human faecal material. We conclude that the human gastrointestinal tract is unlikely to be a significant reservoir of M. ulcerans.

Background: Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a destructive skin disease found predominantly in sub-Saharan Africa and south-eastern Australia. The precise mode(s) of transmission and environmental reservoir(s) remain unknown, but several studies have explored the role of aquatic invertebrate species. The purpose of this study was to investigate the environmental distribution of M. ulcerans in south-eastern Australia., Methodology/principal Findings: A range of environmental samples was collected from Point Lonsdale (a small coastal town southwest of Melbourne, Australia, endemic for BU) and from areas with fewer or no reported incident cases of BU. Mycobacterium ulcerans DNA was detected at low levels by real-time PCR in soil, sediment, water residue, aquatic plant biofilm and terrestrial vegetation collected in Point Lonsdale. Higher levels of M. ulcerans DNA were detected in the faeces of common ringtail (Pseudocheirus peregrinus) and common brushtail (Trichosurus vulpecula) possums. Systematic testing of possum faeces revealed that M. ulcerans DNA could be detected in 41% of faecal samples collected in Point Lonsdale compared with less than 1% of faecal samples collected from non-endemic areas (p<0.0001). Capture and clinical examination of live possums in Point Lonsdale validated the accuracy of the predictive value of the faecal surveys by revealing that 38% of ringtail possums and 24% of brushtail possums had laboratory-confirmed M. ulcerans skin lesions and/or M. ulcerans PCR positive faeces. Whole genome sequencing revealed an extremely close genetic relationship between human and possum M. ulcerans isolates., Conclusions/significance: The prevailing wisdom is that M. ulcerans is an aquatic pathogen and that BU is acquired by contact with certain aquatic environments (swamps, slow-flowing water). Now, after 70 years of research, we propose a transmission model for BU in which terrestrial mammals are implicated as reservoirs for M. ulcerans.

Two horses were diagnosed as having Mycobacterium ulcerans infections. The first was a 21-year-old Quarterhorse-cross mare living in Mallacoota (a coastal town near the border of New South Wales and Victoria, Australia) that presented with lichenification, hair-loss and oedema on a fetlock, which subsequently ulcerated, as well as a non-healing ulcer on the wither. The second horse was a 32 year-old Standardbred gelding from Nicholson, near Bairnsdale, Victoria, that had an ulcerated lesion on its caudal thigh. Histologically, there were characteristic changes seen with M. ulcerans infections in other species, including extensive necrosis without associated granulomatous inflammation. The organisms were seen in Ziehl-Neelsen-stained smears or sections of the lesions from both horses and were isolated in culture from the first horse. A definitive diagnosis was provided by real-time polymerase chain reaction targeting the M. ulcerans-specific insertion sequence, IS2404. Delayed identification of the infectious agent in the first case led to the use of suboptimal antimicrobial therapy, resulting in failure to control the infection and the horse was subsequently euthanased. The second horse was successfully treated following surgical debulking of the centre of the lesion and one session of aggressive cryosurgery. Mycobacterium ulcerans should be considered in the differential diagnosis of unexplained lichenification with oedematous and ulcerated skin lesions in horses living in regions where this organism is endemic.

Reliable molecular detection of Mycobacterium ulcerans in environmental samples is essential to study the ecology and transmission of this important human pathogen. Variable number tandem repeat (VNTR) typing is a valuable method for distinguishing M. ulcerans isolates from different geographic regions and for distinguishing M. ulcerans from other members of the Mycobacterium marinum/M. ulcerans complex, but its application to environmental samples has not yet been evaluated systematically. This study compares the sensitivity and specificity of PCR detection of 13 VNTR loci to determine the best loci for the analysis of environmental samples. This study demonstrates that VNTR typing using selected loci can be a useful addition to established molecular methods for detecting M. ulcerans in the environment and highlights some of the issues encountered when using molecular methods to detect microorganisms in environmental samples. When applied to environmental samples collected from an endemic region in Victoria, Australia, VNTR typing confirmed that the strain of M. ulcerans being detected was indistinguishable from the strain causing disease in humans in that region.

A 10-year-old castrated male domestic cat domiciled in eastern Victoria (Australia) was presented for a subcutaneous mass on its nasal bridge in November 2006. Cytological examination of an aspirate demonstrated pyogranulomatous inflammation. At surgery, the lesion consisted of an encapsulated mass containing viscid fluid. Histological examination of the resected lesion revealed pyogranulomatous inflammation surrounding a central zone of necrosis. Sections stained with the Ziehl-Neelsen method revealed numerous acid-fast bacilli, intracellularly within macrophages and extracellularly. Molecular studies established the infection was caused by Mycobacterium ulcerans. As histology demonstrated that the infection extended to the margin of the excised tissues, the cat was treated subsequently with clarithromycin (62.5mg orally once daily for 7 days, then twice daily for 3 months). The surgical wound healed unremarkably. The infection has not recurred at the time of writing, 1 year following discontinuation of treatment. Although M ulcerans infections have been recorded in variety of mammals, this is the first known case in a cat.

Between 1999 and 2006, 15 cats were diagnosed with disease attributable to a novel mycobacterial species. The infections consisted of granulomatous lesions in the skin, subcutis, and ocular or periocular tissues with an indolent but progressive clinical course. Lesions typically were found in facial regions or on the distal limbs. Cats of all ages and both sexes were affected. Infections often were challenging to treat, although they could be cured using surgery in concert with combination antimicrobial therapy. Microscopically, lesions were granulomatous to pyogranulomatous and contained numerous acid-fast bacilli. Scanty cultures of the causal microorganisms occasionally could be obtained in mycobacterial broth, but subculture to solid media failed. When cultures were not available, DNA was extracted from fresh tissue, lyophilized material, and formalin-fixed, paraffin-embedded tissues from lesions. PCR amplification of the 5' end of the 16S rRNA gene and regions within four additional loci (ITS1, hsp65, rpoB, and sodA) was performed with various efficiencies using mycobacterial primers. Nucleotide sequences were unique for each locus tested. Nucleotide sequences obtained from individual cases were identical for each locus for which the amplification was successful. Phylogenetic analysis performed using concatenated partial 16S rRNA and hsp65 gene sequences indicated that this novel mycobacterial species from Victoria is a member of the Mycobacterium simiae-related group, taxonomically related to the mycobacterium causing leproid granulomas in dogs throughout the world. Based on the clustering of cases, we refer to this novel species as Mycobacterium sp. strain Tarwin.

Buruli ulcer (BU) occurs in >30 countries. The causative organism, Mycobacterium ulcerans, is acquired from the environment, but the exact mode of transmission is unknown. We investigated an outbreak of BU in a small coastal town in southeastern Australia and screened by PCR mosquitoes caught there. All cases of BU were confirmed by culture or PCR. Mosquitoes were trapped in multiple locations during a 26-month period. BU developed in 48 residents of Point Lonsdale/Queenscliff and 31 visitors from January 2001 through April 2007. We tested 11,504 mosquitoes trapped at Point Lonsdale (predominantly Aedes camptorhynchus). Forty-eight pools (5 species) were positive for insertion sequence IS2404 (maximum likelihood estimate 4.3/1,000), and we confirmed the presence of M. ulcerans in a subset of pools by detection of 3 additional PCR targets.

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer. PCR has become a reliable and rapid method for the diagnosis of M. ulcerans infection in humans and has been used for the detection of M. ulcerans in the environment. This paper describes the development of a TaqMan assay targeting IS2404 multiplexed with an internal positive control to monitor inhibition with a detection limit of less than 1 genome equivalent of DNA. The assay improves the turnaround time for diagnosis and replaces conventional gel-based PCR as the routine method for laboratory confirmation of M. ulcerans infection in Victoria, Australia. Following analysis of 415 clinical specimens, the new test demonstrated 100% sensitivity and specificity compared with culture. Another multiplex TaqMan assay targeting IS2606 and the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes was designed to augment the specificity of the IS2404 PCR for the analysis of a variety of environmental samples. Assaying for these three targets enabled the detection of M. ulcerans DNA in soil, sediment, and mosquito extracts collected from an area of endemicity for Buruli ulcer in Victoria with a high degree of confidence. Final confirmation was obtained by the detection and sequencing of variable-number tandem repeat (VNTR) locus 9, which matched the VNTR locus 9 sequence obtained from the clinical isolates in this region. This suite of new methods is enabling rapid progress in the understanding of the ecology of this important human pathogen.

It had been assumed that production of the cytotoxic polyketide mycolactone was strictly associated with Mycobacterium ulcerans, the causative agent of Buruli ulcer. However, a recent study has uncovered a broader distribution of mycolactone-producing mycobacteria (MPM) that includes mycobacteria cultured from diseased fish and frogs in the United States and from diseased fish in the Red and Mediterranean Seas. All of these mycobacteria contain versions of the M. ulcerans pMUM plasmid, produce mycolactones, and show a high degree of genetic relatedness to both M. ulcerans and Mycobacterium marinum. Here, we show by multiple genetic methods, including multilocus sequence analysis and DNA-DNA hybridization, that all MPM have evolved from a common M. marinum progenitor to form a genetically cohesive group among a more diverse assemblage of M. marinum strains. Like M. ulcerans, the fish and frog MPM show multiple copies of the insertion sequence IS2404. Comparisons of pMUM and chromosomal gene sequences demonstrate that plasmid acquisition and the subsequent ability to produce mycolactone were probably the key drivers of speciation. Ongoing evolution among MPM has since produced at least two genetically distinct ecotypes that can be broadly divided into those typically causing disease in ectotherms (but also having a high zoonotic potential) and those causing disease in endotherms, such as humans.

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes Buruli ulcer (also known as Bairnsdale ulcer in Victoria and Daintree ulcer in northern Queensland). We describe two patients with laboratory-confirmed Buruli ulcer who were infected either in New South Wales or overseas. A molecular epidemiological investigation demonstrated that, while one case was probably acquired in Papua New Guinea, the other was most likely to have been acquired in southern NSW. To our knowledge, this is the first case of M. ulcerans infection acquired in NSW.

Mycobacterium ulcerans causes slowly progressive, destructive skin and soft tissue infections, known as Bairnsdale or Buruli ulcer (BU). Forty-six delegates with experience in the management of BU attended a 1-day conference in Melbourne on 10 February 2006, with the aim of developing a consensus approach to the diagnosis, treatment and control of BU. An initial draft document was extended and improved during a facilitated round table discussion. BU is an environmental infection that occurs in specific locations. The main risk factor for infection is contact with an endemic area. Prompt cleaning of abrasions sustained outdoors, wearing protective clothing, and avoiding mosquito bites may reduce an individual's risk of infection. BU can be rapidly and accurately diagnosed by polymerase chain reaction testing of ulcer swabs or biopsies. Best outcomes are obtained when the diagnosis is made early. To aid early diagnosis, health authorities should keep local populations informed of new outbreaks. BU is best treated with surgical excision, which, if possible, should include a small rim of healthy tissue. For small lesions this may be all that is required. However, there is a role for antibiotics for more extensive disease, and their use may allow more conservative surgery.

The general stress response in Neisseria gonorrhoeae was investigated. Transcriptional analyses of the genes encoding the molecular chaperones DnaK, DnaJ, and GrpE suggested that they are transcribed from sigma32 (RpoH)-dependent promoters upon exposure to stress. This was confirmed by mutational analysis of the sigma32 promoter of dnaK. The gene encoding the gonococcal RpoH sigma factor appears to be essential, as we could not isolate viable mutants. Deletion of an unusually long rpoH leader sequence resulted in elevated levels of transcription, suggesting that this region is involved in negative regulation of RpoH expression during normal growth. Transcriptional analyses and protein studies determined that regulation of the RpoH-mediated stress response is different from that observed in most other species, in which regulation occurs predominantly at the transcriptional and translational levels. We suggest that an increase in the activity of preformed RpoH is primarily responsible for induction of the stress response in N. gonorrhoeae.

An intact activator-binding site upstream of the sigma(54) promoter of the pilin-encoding pilE gene of Neisseria gonorrhoeae suggests gonococci produce a protein capable of binding this sequence. We cloned a chimeric gene, rsp, that has sequence similarity to both the pilS and pilR genes of Pseudomonas aeruginosa encoding a two-component regulatory system that controls piliation. This gene is transcribed in N. gonorrhoeae and indirect evidence suggests that Rsp binds to the activator-binding site of the pilE gene. Despite this, mutation of rsp has no effect on piliation in N. gonorrhoeae, suggesting that the remnants of this regulatory system have persisted in the genome, despite the loss of its original function.

A 960-bp ORF potentially encoding a site-specific recombinase has been cloned from Neisseria gonorrhoeae MS11-A. This ORF was designated pivNg on the basis of similarity of the deduced amino acid sequence to the Piv proteins of Moraxella spp. that are site-specific invertases. Southern hybridization and sequence analysis revealed that there were multiple copies of pivNg sequence within the genomes of N. gonorrhoeae strains tested, but not in several other neisserial species. Southern hybridization and sequence analysis further suggested that pivNg sequences may be associated with genomic rearrangements.

Our prior studies showed that certain cobalamins inhibit productive HIV-1 infection of primary cultures of blood lymphocytes and monocytes. We demonstrate here that this antiviral activity may be mediated by an inhibition of HIV-1 integrase, an enzyme required for productive infection. Purified recombinant HIV-1 integrase activity was inhibited in vitro by hydroxocobalamin (OH-Cbl), methylcobalamin (Me-Cbl), adenosylcobalamin (Ado-Cbl), and dicyanocobinamide (CN2-Cbi) with IC50 values of approximately 17, 17, 17, and 4 microM, respectively. The agents inhibited HIV-1 infection of cultured monocytes (IC50 values for OH-Cbl, Me-Cbl, Ado-Cbl, and CN2-Cbi of 6, 7, 4, and 1 microM, respectively) and of cultured lymphocytes (IC50 values of 60, 50, 60, and 11 microM, respectively). Experiments using cultured monocytes or lymphocytes demonstrated that OH-Cbl inhibited integration of HIV-1 DNA into cellular DNA. Thus, cobalamins and cobinamides represent novel inhibitors of HIV-1 integrase. These or related agents may be useful as anti-viral treatments that target HIV-1 integrase.

The pilE gene of Neisseria gonorrhoeae is transcribed from a sigma70 promoter (pilEp1) with an AT-rich tract extending 65 nucleotides upstream of the -35 box. Within this region is an integration host factor (IHF)-binding core consensus sequence. We have performed a detailed analysis to determine which upstream sequences are required for efficient transcription from pilEp1 in N. gonorrhoeae. Deletion of sequences upstream of the AT-rich tract had no effect on the level of transcription. However, the IHF-binding core consensus sequence and the AT-rich sequence further upstream were both required for enhanced levels of transcription from this promoter in both N. gonorrhoeae and an Escherichia coli strain producing IHF. In addition, an UP-like element positioned between the -35 box and the IHF-binding site was required for maximal transcription. The AT-rich region upstream of the IHF-binding core consensus sequence can also act as an UP-like element when appropriately repositioned upstream of the -35 box.

The sigma54 promoter (P3) upstream of the pilE gene in Neisseria gonorrhoeae was shown to be non-functional by transcriptional analysis of a PpilE::lacZ fusion containing only P3. A region on the chromosome of N. gonorrhoeae strain MS11-A was identified that potentially encodes a protein with a significant similarity to the Escherichia coli RpoN protein. However, this region (designated RLS for rpoN-like sequence) does not contain a single open reading frame (ORF) capable of encoding a functional RpoN protein. It appears that RLS may have arisen from an ancestral rpoN homologue that underwent a deletion removing the sequence encoding the essential helix-turn-helix (HTH) motif, and changing the subsequent reading frame. An RLS has been identified in several strains of N. gonorrhoeae and N. meningitidis. A 90-kDa gonococcal protein has previously been shown to react with a monoclonal antibody raised against the RpoN from Salmonella typhimurium. However, mutagenesis and Western blot analysis confirmed that the gene encoding this protein is not contained within RLS.

The DNA repair capacities of Neisseria gonorrhoeae have not been well characterised, however, it is known that the gonococcus possesses an excision repair system. The fact that genes in this system are part of the SOS regulon in Escherichia coli prompted this investigation into the transcriptional regulation of genes involved in DNA repair in N. gonorrhoeae. Northern (RNA-DNA) dot blot hybridisation was used to investigate potential DNA damage-mediated induction of the gonococcal recA, uvrA and uvrB genes. In contrast to the situation in E. coli, transcription of these genes in N. gonorrhoeae was not induced in response to treatment with methyl methanesulfonate (MMS) and UV light. These data indicated that the gonococcus does not possess an SOS-like system that is induced in response to DNA damage.

The pilE gene encodes the pilin subunit in Neisseria gonorrhoeae and Neisseria meningitidis. Transcriptional analysis of promoters upstream of pilE in N. gonorrhoeae has been described previously (Fyfe et al. (1995) J. Bacteriol. 177, 3781-3787). Transcription from the sigma54-dependent promoter P3 was detected in Pseudomonas aeruginosa. Here we show that this transcription is dependent on the P. aeruginosa transcriptional activator PilR, and a specific upstream sequence with a high degree of similarity to the PilR-binding site found upstream of the P. aeruginosa pilin gene. This implies there is an upstream activator site (UAS) present 5' of pilE. Sequencing upstream of the N. meningitidis MC58 c2 pilE gene shows this region to be very similar to that in N. gonorrhoeae. P3 and the UAS are conserved, although insertions were noted on either side of the UAS. Transcriptional analysis has shown that the N. meningitidis P3 promoter is used in P. aeruginosa, provided PilR and an upstream region that includes sequence similar to the UAS are present. Transcription from the N. meningitidis PpilE is stronger than from the N. gonorrhoeae equivalent. N. meningitidis uses the sigma70 promoter P1 to transcribe pilE.

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrA mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrA proteins of other bacterial species. A second open reading frame (ORF259) was identified upstream from, and in the opposite orientation to the gonococcal uvrA gene. Transcriptional fusions between portions of the gonococcal uvrA upstream region and a reporter gene were used to localise promoter activity in both E. coli and N. gonorrhoeae. The transcriptional starting points of uvrA and ORF259 were mapped in E. coli by primer extension analysis, and corresponding sigma70 promoters were identified. The arrangement of the uvrA-ORF259 intergenic region is similar to that of the gonococcal recA-aroD intergenic region. Both contain inverted copies of the 10 bp neisserial DNA uptake sequence situated between divergently transcribed genes. However, there is no evidence that either the uptake sequence or the proximity of the promoters influences expression of these genes.

The nucleotide sequence upstream of the groEL gene of Neisseria gonorrhoeae has been determined. Upstream of groEL is a homolog of groES and the divergently transcribed frpB gene. The promoter region of groES lacks the inverted repeat sequences (IR) that act as a regulatory element controlling the expression of similar operons in many other bacterial species. This region contains overlapping consensus sequences for sigma 32-dependent and sigma 70-dependent promoters, and an appropriately placed transcription start point was mapped downstream of these promoters. Northern hybridization demonstrated that synthesis of a full-length groES-groEL transcript was induced in heat-stressed cells. These experiments also revealed the presence of a shorter groES-specific transcript, apparently the result of the premature termination of transcription at an IR situated between the groES and groEL genes.

The activity, metabolism, and mode of action of (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (H2G) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) were studied. Compared to acyclovir (ACV), H2G has superior activity against VZV (50% inhibitory concentration of 2.3 microM) and Epstein-Barr virus (50% inhibitory concentration of 0.9 microM), comparable activity against HSV-1, and weaker activity against HSV-2. The antiviral effect on HSV-1 showed persistence after removal of compound. H2G was metabolized to its mono-, di- and triphosphate derivatives in virus-infected cells, with H2G-triphosphate being the predominant product. Only small amounts of H2G-triphosphate were detected in uninfected cells (1 to 10 pmol/10(6) cells), whereas the level in HSV-1-infected cells reached 1,900 pmol/10(6) cells. H2G was a substrate for all three viral thymidine kinases and could also be phosphorylated by mitochondrial deoxyguanosine kinase. The intracellular half-life of H2G-triphosphate varied in uninfected (2.5 h) and infected (HSV-1, 14 h; VZV, 3.7 h) cells but was always longer than the half-life of ACV-triphosphate (1 to 2 h). H2G-triphosphate inhibited HSV-1, HSV-2, and VZV DNA polymerases competitively with dGTP (Ki of 2.8, 2.2, and 0.3 microM, respectively) but could not replace dGTP as a substrate in a polymerase assay. H2G was not an obligate chain terminator but would only support limited DNA chain extension. Only very small amounts of radioactivity, which were too low to be identified by high-performance liquid chromatography analysis of the digested DNA, could be detected in purified DNA from uninfected cells incubated with [3H]H2G. Thus, H2G acts as an anti-herpesvirus agent, particularly potent against VZV, by formation of high concentrations of relatively stable H2G-triphosphate, which is a potent inhibitor of the viral DNA polymerases.

Type 4 pili are essential for virulence in Neisseria gonorrhoeae. The gonococcal pilin subunit is encoded by pilE, upstream of which three putative promoter sequences (P1, P2, and P3) have been identified. P1 and P2 are sigma 70-like promoters and are functional when a PpiE::cat transcriptional fusion is expressed in Escherichia coli DH5 alpha. P3 is sigma 54 dependent and overlaps the P1 sequence. Site-directed mutagenesis of the pilE promoters followed by transcriptional analysis in E. coli indicated that in the absence of an appropriate activator protein, binding of RNA polymerase-sigma 54 to P3 inhibits transcription from P1 on the order of 30-fold. Transcription from P3 was undetectable in E. coli. However, PilR-dependent, P3-associated expression was detected in Pseudomonas aeruginosa PAK containing a PpilE::cat fusion, with P3 the only intact promoter. A similar analysis was performed on gonococcal reporter strains containing wild-type and mutated PpilE::cat cassettes recombined into the chromosome. In such piliated gonococcal recombinants cultured in vitro, P1 was responsible for cat expression and almost certainly for transcription of pilE. Transcription from P2 and P3 was not detectable under these conditions. Inhibition of transcription from P1 by sigma 54 binding to P3 was not apparent in N. gonorrhoeae MS11-A, suggesting that sigma 54 was either absent or unable to bind to P3 in these cells.