Biology subjects

Porter, J. L.

Publications and source records attributed to Porter, J. L..

11 recordsLinked to original sources

bioRxiv2025

Defining new Buruli ulcer endemic areas in urban southeastern Australia using bacterial genomics-informed possum excreta surveys

Buruli ulcer (BU) in southeastern Australia is a zoonosis caused by infection with Mycobacterium ulcerans. Australian native possums are a major wildlife reservoir and infected possums shed M. ulcerans in their excreta, with mosquitoes being the major transmitting vector in this region. BU is geographically restricted and this feature, combined with an average 4.8-month incubation period, makes tracking M. ulcerans environmental spread and timely identification of new BU endemic areas challenging. While human movement complicates transmission tracing, we used the highly territorial behaviour of native possums and high-resolution pathogen genomics to confidently identify new BU endemic areas in Melbournes inner northwest and southern suburbs of Geelong. Using pathogen genomic phylodynamic modelling, we estimated that M. ulcerans was introduced to these areas 2-6 years before the emergence of human BU cases. This study shows how possum excreta surveys combined with pathogen genome data can pinpoint new BU endemic areas, thus providing critical local knowledge for targeted public health interventions to reduce exposure risk and ensure early diagnosis.

microbiology↗

bioRxiv2025

A refined low-dose murine model of Mycobacterium ulcerans infection to assess integrated immune networks in Buruli ulcer pathogenesis

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is a slow-growing zoonotic pathogen with distinctive pathogenesis linked primarily to its toxin mycolactone. Recent research has shown that the M. ulcerans infectious dose is very low (<10 colony forming units [CFU]). Buruli ulcer animal infection models traditionally use bacterial challenge doses in the range 104 - 106 CFU; a range orders of magnitude higher than natural infection. These large doses represent an unrealistic challenge for vaccine trials and studies of immunity. Here, we address this issue and describe a murine tail infection model in two genetically distinct mouse strains (BALB/c and C57BL/6) using quality-controlled, M. ulcerans challenge doses (10 - 20 CFU and 100 CFU). Over 24-weeks, we assessed host responses to infection by measuring >70 clinical, immunological and microbiological parameters. Principal findings included a 100% infection rate even at the lowest bacterial challenge, but with a dose-dependent delay in lesion onset and disease progression for both mouse strains. Bacterial growth kinetics were similar between mouse strains. There was a difference in immune profiles between mouse strains and between low (10 CFU) versus high (100 CFU) bacterial challenge doses. C57BL/6 mice exhibited more robust systemic cellular responses and more rapid lesion onset compared to BALB/c mice. There were dose-dependent cytokine and chemokine differences in C57BL/6 mice, while BALB/c mice displayed similar responses across both doses. Antibody responses were only detected late in the infection and were associated with the high-dose inoculum in both strains. Machine learning and other statistical analyses highlighted the importance of activated CD8+ T cells and dendritic cells in the immune response to low-dose infection in C57BL/6 mice. Murine low-dose M. ulcerans infection models provide confidence for future human Buruli ulcer challenge trials and will inform the development of effective vaccines and therapeutics.

microbiology↗

bioRxiv2024

Buruli ulcer surveillance in south-eastern Australian possums: infection status, lesion mapping and internal distribution of Mycobacterium ulcerans

Buruli ulcer (BU) is a neglected tropical disease of skin and subcutaneous tissues caused by Mycobacterium ulcerans. BU-endemic areas are highly focal, and M. ulcerans transmission dynamics vary in different settings. In the south-eastern Australian state of Victoria, BU is an endemic vector-borne zoonosis, with mosquitoes and native possums implicated in transmission, and humans as incidental hosts. Despite the importance of possums as wildlife reservoirs of M. ulcerans, knowledge of BU in these animals is limited. Opportunistic necropsy-based and active trap-and-release surveillance studies were conducted in and around Melbourne and Geelong, Victoria, to investigate BU in possums. Demographic data and biological samples were collected, and when present, cutaneous lesions suggestive of BU were mapped. Samples were tested for the presence of M. ulcerans DNA by IS2404 qPCR. The final dataset included 26 possums: 20 necropsied; 6 trapped and released. Most possums (77%) were common ringtails from inner Melbourne. Nine possums (eight ringtails, one brushtail) had skin lesions, ranging from single and mild, to multiple and severe, exposing bones and tendons in three cases. M. ulcerans was confirmed in 78% (7/9) of clinically affected possums and 65% of possums without lesions (11/17). Possums with moderate and severe disease had widespread systemic internal bacterial dissemination and were shedding M. ulcerans in their faeces. The anatomical distribution of cutaneous lesions and PCR positivity of biological samples suggests possums may be contracting BU from bites of M. ulcerans-harbouring mosquitoes, traumatic skin wounds, ingestion of an unknown environmental source, and/or during early development in the pouch. Ringtail possums appear highly susceptible to infection with M. ulcerans and are important reservoirs of the bacteria in Victoria. A One Health approach is needed to design and implement integrated interventions that reduce M. ulcerans transmission in Victoria, thereby protecting wildlife and humans from this emerging zoonotic disease. Author summaryBuruli ulcer (BU), a neglected tropical skin disease, is emerging as a public health concern in the temperate Australian state of Victoria. Here, BU is spread by mosquitoes, and native possums are wildlife reservoirs of the causative bacterium, Mycobacterium ulcerans. Possums can be infected by BU, but knowledge of infection and disease in these animals is limited. We conducted surveillance studies in the two largest cities of Victoria, examining live and deceased possums. We found skin lesions in a third of examined possums and confirmed presence of M. ulcerans in almost three-quarters of the animals. Mouth swabs were positive for the bacteria in nearly two thirds of possums, as were pouch swabs of almost half the females. We also conducted mapping of the bodily distribution of skin lesions and found that paws and the undersides of abdomens and tails were the most affected areas. Our findings add support to the concept that possums, particularly ringtails, are M. ulcerans reservoirs in Victoria, and suggest several possible routes of infection for free-living possums that warrant further research. Improved understanding of BU in possums may allow development of targeted interventions that reduce disease transmission and protect both animal and human health.

microbiology↗

bioRxiv2024

Mycobacterium ulcerans challenge strain selection for a Buruli ulcer controlled human infection model

Critical scientific questions remain regarding infection with Mycobacterium ulcerans, the organism responsible for the neglected tropical disease, Buruli ulcer (BU). A controlled human infection model has the potential to accelerate our knowledge of the immunological correlates of disease, to test prophylactic interventions and novel therapeutics. Here we present microbiological evidence supporting M. ulcerans JKD8049 as a suitable human challenge strain. This non-genetically modified Australian isolate is susceptible to clinically relevant antibiotics, can be cultured in animal-free and surfactant-free media, can be enumerated for precise dosing, and has stable viability following cryopreservation. Infectious challenge of humans with JKD8049 is anticipated to imitate natural infection, as M. ulcerans JKD8049 is genetically stable following in vitro passage and produces the key virulence factor, mycolactone. Also reported are considerations for the manufacture, storage, and administration of M. ulcerans JKD8049 for controlled human infection.

microbiology↗

bioRxiv2024

A marine sponge-associated mycobacterium closely related to Mycobacterium tuberculosis

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

microbiology↗

bioRxiv2023

A transmission chain linking Mycobacterium ulcerans with Aedes notoscriptus mosquitoes, possums and human Buruli ulcer cases in southeastern Australia

In temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused by Mycobacterium ulcerans. Native possums are the major local environmental reservoir of M. ulcerans as they not only develop Buruli lesions but they also shed M. ulcerans in their excreta. However the way humans acquire M. ulcerans from possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes in M. ulcerans transmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and four ad hoc field surveys across an area of 350km2 on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns of M. ulcerans-positive mosquito occurrence with M. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstruct M. ulcerans genomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period. Culex molestus and Aedes notoscriptus were the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association between M. ulcerans and Ae. notoscriptus (p<0.0001) with a maximum likelihood estimate (MLE) of 5.88 M. ulcerans positive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fed Ae. notoscriptus showed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed shared M. ulcerans genome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate that Ae. notoscriptus likely transmit M. ulcerans in southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

microbiology↗

bioRxiv2022

Structured surveys of Australian native possum excreta predict Buruli ulcer occurrence in humans

Buruli ulcer (BU) is a neglected tropical disease caused by infection of subcutaneous tissue with Mycobacterium ulcerans. BU is commonly reported across rural regions of Central and West Africa but has been increasing dramatically in temperate southeast Australia around the major metropolitan city of Melbourne. Previous research has shown that Australian native possums are reservoirs of M. ulcerans and that they shed the bacteria in their fecal material (excreta). Field surveys show that locales where possums harbor M. ulcerans overlap with human cases of BU, raising the possibility of using possum excreta surveys to predict the risk of disease occurrence in humans. We thus established a highly structured 12-month possum excreta surveillance program across an area of 350 km2 in the Mornington Peninsula area 70 km south of Melbourne, Australia. The primary objective of our study was to assess if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Over two sampling campaigns in summer and winter, we collected 2282 possum excreta specimens of which 11% were PCR positive for M. ulcerans-specific DNA. Using the spatial scanning statistical tool SatScan, we observed non-random, co-correlated clustering of both M. ulcerans positive possum excreta and human BU cases. We next trained a statistical model with the Mornington Peninsula excreta survey data to predict the future likelihood of human BU cases occurring in the region. By observing where human BU cases subsequently occurred, we show that the excreta model performance was superior to a null model trained using the previous years human BU case incidence data (AUC 0.66 vs 0.55). We then used data unseen by the excreta-informed model from a new survey of 661 possum excreta specimens in Geelong, a geographically separate BU endemic area to the southwest of Melbourne, to prospectively predict the location of human BU cases in that region. As for the Mornington Peninsula, the excreta-based BU prediction model outperformed the null model (AUC 0.75 vs 0.50) and pinpointed specific locations in Geelong where interventions could be deployed to interrupt disease spread. This study highlights the One Health nature of BU by confirming a quantitative relationship between possum excreta shedding of M. ulcerans and humans developing BU. The excreta survey-informed modeling we have described will be a powerful tool for efficient targeting of public health responses to stop BU.

microbiology↗

bioRxiv2020

High antibody titres induced by protein subunit vaccines against Buruli ulcer using Mycobacterium ulcerans antigens Hsp18 and MUL_3720

BackgroundMycobacterium ulcerans is the causative agent of a debilitating skin and soft tissue infection known as Buruli ulcer (BU). There is no vaccine against BU. The purpose of this study was to investigate the vaccine potential of two previously described immunogenic M. ulcerans proteins, MUL_3720 and Hsp18, using a mouse tail infection model of BU. MethodsRecombinant versions of the two proteins were each electrostatically coupled with a previously described lipopeptide adjuvant. Seven C57BL/6 and seven BALB/c mice were vaccinated and boosted with each of the formulations. Vaccinated mice were then challenged with M. ulcerans via subcutaneous tail inoculation. Vaccine performance was assessed by time-to-ulceration compared to unvaccinated mice. ResultsThe MUL_3720 and Hsp18 vaccines induced high titres of antigen-specific antibodies that were predominately subtype IgG1. However, all mice developed ulcers by day-40 post-M. ulcerans challenge. No significant difference was observed in the time-to-onset of ulceration between the experimental vaccine groups and unvaccinated animals. ConclusionsThese data align with previous vaccine experiments using Hsp18 and MUL_3720 that indicated these proteins may not be appropriate vaccine antigens. This work highlights the need to explore alternative vaccine targets and different approaches to understand the role antibodies might play in controlling BU.

microbiology↗

bioRxiv2019

Vaccine-specific immune responses against Mycobacterium ulcerans infection in a low-dose murine challenge model

The neglected tropical disease Buruli ulcer (BU) is an infection of subcutaneous tissue with Mycobacterium ulcerans. There is no effective BU vaccine. Here, we assessed an experimental prime-boost vaccine in a low-dose murine tail infection model. We used the enoyl-reductase (ER) domain of the M. ulcerans mycolactone polyketide synthases electrostatically coupled with a previously described TLR-2 agonist-based lipopeptide adjuvant, R4Pam2Cys. Mice were vaccinated and then challenged via tail inoculation with 14-20 colony forming units (CFU) of an engineered bioluminescent strain of M. ulcerans. Mice receiving either the experimental ER vaccine or Mycobacterium bovis Bacille Calmette-Guerin (BCG) were equally well protected, with both groups faring significantly better than non-vaccinated animals (p<0.05). A suite of 29 immune parameters were assessed in the mice at the end of the experimental period. Multivariate statistical approaches were then used to interrogate the immune response data to develop disease-prognostic models. High levels of IL-2 and low IFN-{gamma} produced in the spleen best predicted control of infection across all vaccine groups. Univariate logistic regression then revealed vaccine-specific profiles of protection. High titres of ER-specific IgG serum antibodies together with IL-2 and IL-4 in the draining lymph node (DLN) were associated with protection induced by the experimental ER vaccine. In contrast, high titres of IL-6, TNF-, IFN-{gamma} and IL-10 in the DLN and low IFN{gamma} titres in the spleen were associated with protection following BCG vaccination. This study suggests an effective BU vaccine must induce localized, tissue-specific immune profiles with controlled inflammatory responses at the site of infection.

immunology↗

bioRxiv2018

Bioluminscent Mycobacterium ulcerans, a tool to study host-pathogen interactions in a murine tail model of Buruli ulcer

Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans. In this study we used a previously reported strain of M. ulcerans, genetically engineered to constitutively produce bioluminescence, to follow the progression of Buruli ulcer in mice using an in-vivo imaging (IVIS(R)) system. We aimed to characterize a mouse tail infection model for pathogenesis, as well as for pre-clinical vaccine and drug development research for Buruli ulcer. Immune parameters, such as antibody titers and cytokine levels, were determined throughout the course of the infection and histology specimens were examined for comparison with human pathology. Nine out of ten (90%) BALB/c mice infected subcutaneously with 105 M. ulcerans JKD8049 (containing pMV306 hsp16+luxG13) exhibited light emission from the site of infection over the course of the experiment indicating M. ulcerans growth in-vivo. Five out of ten (50%) animals developed clinical signs of disease. Antibody titers were overall low and their onset was late, as measured by responses to both heterogenous (bacterial whole cell lysate) and single antigen (Hsp18) targets. IFN-{gamma}, and IL-10 are reported to play a vital role in host control of Buruli ulcer and these cytokines were elevated in animals with pathology. For mice with advanced pathology, histology revealed clusters of acid-fast bacilli within subcutaneous tissue 300-400 m beneath the epidermis of the tail, with macrophage infiltration and granuloma-formation resembling human Buruli ulcer. This study has shown the utility of using bioluminescent M. ulcerans and IVIS(R) in a mouse tail infection model to study Buruli ulcer infection.\n\nAuthor summaryBuruli ulcer is one of the so called neglected tropical diseases. It is an infectious disease, mainly occurring in West Africa but also in Australia. It manifests as skin lesion and ulcer. Up to date, the way of transmission is inadequately understood. Also, there is no vaccine to protect against the disease. Buruli ulcer is treatable with a course of antibiotics that need to be given for the duration of two months. More laboratory research is needed to elucidate the mechanism of transmission, develop a vaccine and improve and shorten antibiotic therapy. For this, animal (mouse) models of disease are used. The aim of this study was to refine and improve the mouse tail infection model of Buruli ulcer. For this, we used a genetically modified Mycobacterium ulcerans strain that emits light. After infection of animals, light emitted from the bacteria was read out with an in-vivo imaging (IVIS) camera. This allowed us to monitor the location of bacteria in the living animal over time without the need to kill the animal. We also measured parameters of the immune system such as antibodies and cytokines as a baseline for future studies into immunology, vaccine development and pathology of Buruli ulcer. We successfully improved and characterized the mouse tail infection model in Buruli ulcer with the use of modern technology using light emitting bacteria and the IVIS camera.

microbiology↗

bioRxiv2017

A target-specific assay for rapid and quantitative detection of Mycobacterium chimaera DNA in environmental and clinical specimens

Mycobacterium chimaera is an opportunistic environmental mycobacterium, belonging to the Mycobacterium intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest world-wide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays, specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan qPCR assay for M. chimaera with a detection limit of 10 CFU in whole blood. In vitro screening against DNA extracted from 40 other mycobacteria and 22 bacterial species from 21 diverse genera confirmed in silico predicted specificity for M. chimaera. Screening 33 water samples from heater cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera.

microbiology↗