Unraveling the intricacies of host-pathogen interaction through single-cell genomics.

Single-cell genomics provide researchers with tools to assess host-pathogen interactions at a resolution previously inaccessible. Transcriptome analysis, epigenome analysis, and immune profiling techniques allow for a better comprehension of the heterogeneity underlying both the host response and infectious agents. Here, we highlight technological advancements and data analysis workflows that increase our understanding of host-pathogen interactions at the single-cell level. We review various studies that have used these tools to better understand host-pathogen dynamics in a variety of infectious disease contexts, including viral, bacterial, and parasitic diseases. We conclude by discussing how single-cell genomics can advance our understanding of host-pathogen interactions.

High performance Legionella pneumophila source attribution using genomics-based machine learning classification.

Fundamental to effective Legionnaires' disease outbreak control is the ability to rapidly identify the environmental source(s) of the causative agent, Legionella pneumophila. Genomics has revolutionized pathogen surveillance, but L. pneumophila has a complex ecology and population structure that can limit source inference based on standard core genome phylogenetics. Here, we present a powerful machine learning approach that assigns the geographical source of Legionnaires' disease outbreaks more accurately than current core genome comparisons. Models were developed upon 534 L. pneumophila genome sequences, including 149 genomes linked to 20 previously reported Legionnaires' disease outbreaks through detailed case investigations. Our classification models were developed in a cross-validation framework using only environmental L. pneumophila genomes. Assignments of clinical isolate geographic origins demonstrated high predictive sensitivity and specificity of the models, with no false positives or false negatives for 13 out of 20 outbreak groups, despite the presence of within-outbreak polyclonal population structure. Analysis of the same 534-genome panel with a conventional phylogenomic tree and a core genome multi-locus sequence type allelic distance-based classification approach revealed that our machine learning method had the highest overall classification performance-agreement with epidemiological information. Our multivariate statistical learning approach maximizes the use of genomic variation data and is thus well-suited for supporting Legionnaires' disease outbreak investigations.IMPORTANCEIdentifying the sources of Legionnaires' disease outbreaks is crucial for effective control. Current genomic methods, while useful, often fall short due to the complex ecology and population structure of Legionella pneumophila, the causative agent. Our study introduces a high-performing machine learning approach for more accurate geographical source attribution of Legionnaires' disease outbreaks. Developed using cross-validation on environmental L. pneumophila genomes, our models demonstrate excellent predictive sensitivity and specificity. Importantly, this new approach outperforms traditional methods like phylogenomic trees and core genome multi-locus sequence typing, proving more efficient at leveraging genomic variation data to infer outbreak sources. Our machine learning algorithms, harnessing both core and accessory genomic variation, offer significant promise in public health settings. By enabling rapid and precise source identification in Legionnaires' disease outbreaks, such approaches have the potential to expedite intervention efforts and curtail disease transmission.

Statistical modeling based on structured surveys of Australian native possum excreta harboring Mycobacterium ulcerans predicts Buruli ulcer occurrence in humans.

Background: 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, with most disease transmission occurring in the summer months. 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. Methods: 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 using statistical modeling if M. ulcerans surveillance of possum excreta provided useful information for predicting future human BU case locations. Results: Over two sampling campaigns in summer and winter, we collected 2,282 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 year's 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. Conclusions: 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 the efficient targeting of public health responses to stop BU. Funding: This research was supported by the National Health and Medical Research Council of Australia and the Victorian Government Department of Health (GNT1152807 and GNT1196396).

Phylogenomic investigation of an outbreak of fluoroquinolone-resistant Salmonella enterica subsp. enterica serovar Paratyphi A in Phnom Penh, Cambodia.

In early 2020, the Medical Biology Laboratory of the Pasteur Institute of Cambodia isolated an unusually high number of fluoroquinolone-resistant Salmonella enterica subspecies enterica serovar Paratyphi A strains during its routine bacteriological surveillance activities in Phnom Penh, Cambodia. A public-health investigation was supported by genome sequencing of these Paratyphi A strains to gain insights into the genetic diversity and population structure of a potential outbreak of fluoroquinolone-resistant paratyphoid fever. Comparative genomic and phylodynamic analyses revealed the 2020 strains were descended from a previously described 2013-2015 outbreak of Paratyphi A infections. Our analysis showed sub-lineage 2.3.1 had remained largely susceptible to fluoroquinolone drugs until 2015, but acquired chromosomal resistance to these drugs during six separate events between late 2012 and 2015. The emergence of fluoroquinolone resistance was rapidly followed by the replacement of the original susceptible Paratyphi A population, which led to a dramatic increase of fluoroquinolone-resistant blood-culture-confirmed cases in subsequent years (2016-2020). The rapid acquisition of resistance-conferring mutations in the Paratyphi A population over a 3 year period is suggestive of a strong selective pressure on that population, likely linked with fluoroquinolone use. In turn, emergence of fluoroquinolone resistance has led to increased use of extended-spectrum cephalosporins like ceftriaxone that are becoming the drug of choice for empirical treatment of paratyphoid fever in Cambodia.

Non-toxigenic Corynebacterium diphtheriae in hallux ulceration.

INTRODUCTION: Toxigenic Corynebacterium diphtheriae causes classical diphtheria. Skin infections by toxigenic or non-toxigenic Corynebacterium diphtheriae are prevalent in the tropics but are rarely reported. CASE PRESENTATION: We report the identification of a non-toxigenic Corynebacterium diphtheriae (biovar Gravis) isolate in a 52-year-old Cambodian male. The patient presented purulent and non-healing ulcerations on the right hallux. The wound has healed after 7 days of antibiotic therapy with a favourable outcome. CONCLUSIONS: This case represents, to our knowledge, the first report of Corynebacterium diphtheriae in Cambodia in the last 10 years, and highlights the lack of diagnosis and notifications of diphtheria. It is important to raise awareness among clinicians and to set up diphtheria surveillance in Cambodia.

Bacterial endosymbionts protect beneficial soil fungus from nematode attack.

Fungi of the genus Mortierella occur ubiquitously in soils where they play pivotal roles in carbon cycling, xenobiont degradation, and promoting plant growth. These important fungi are, however, threatened by micropredators such as fungivorous nematodes, and yet little is known about their protective tactics. We report that Mortierella verticillata NRRL 6337 harbors a bacterial endosymbiont that efficiently shields its host from nematode attacks with anthelmintic metabolites. Microscopic investigation and 16S ribosomal DNA analysis revealed that a previously overlooked bacterial symbiont belonging to the genus Mycoavidus dwells in M. verticillata hyphae. Metabolic profiling of the wild-type fungus and a symbiont-free strain obtained by antibiotic treatment as well as genome analyses revealed that highly cytotoxic macrolactones (CJ-12,950 and CJ-13,357, syn necroxime C and D), initially thought to be metabolites of the soil-inhabiting fungus, are actually biosynthesized by the endosymbiont. According to comparative genomics, the symbiont belongs to a new species (Candidatus Mycoavidus necroximicus) with 12% of its 2.2 Mb genome dedicated to natural product biosynthesis, including the modular polyketide-nonribosomal peptide synthetase for necroxime assembly. Using Caenorhabditis elegans and the fungivorous nematode Aphelenchus avenae as test strains, we show that necroximes exert highly potent anthelmintic activities. Effective host protection was demonstrated in cocultures of nematodes with symbiotic and chemically complemented aposymbiotic fungal strains. Image analysis and mathematical quantification of nematode movement enabled evaluation of the potency. Our work describes a relevant role for endofungal bacteria in protecting fungi against mycophagous nematodes.

Tuberculosis in Australia's tropical north: a population-based genomic epidemiological study.

BACKGROUND: The Northern Territory (NT) has the highest tuberculosis (TB) rate of all Australian jurisdictions. We combined TB public health surveillance data with genomic sequencing of Mycobacterium tuberculosis isolates in the tropical 'Top End' of the NT to investigate trends in TB incidence and transmission. METHODS: This retrospective observational study included all 741 culture-confirmed cases of TB in the Top End over three decades from 1989-2020. All 497 available M. tuberculosis isolates were sequenced. We used contact tracing data to define a threshold pairwise SNP distance for hierarchical single linkage clustering, and examined putative transmission clusters in the context of epidemiologic information. FINDINGS: There were 359 (48%) cases born overseas, 329 (44%) cases among Australian First Nations peoples, and 52 (7%) cases were Australian-born and non-Indigenous. The annual incidence in First Nations peoples from 1989-2019 fell from average 50.4 to 11.0 per 100,000 (P<0·001). First Nations cases were more likely to die from TB (41/329, 12·5%) than overseas-born cases (11/359, 3·1%; P<0·001). Using a threshold of ≤12 SNPs, 28 clusters of between 2-64 individuals were identified, totalling 250 cases; 214 (86%) were First Nations cases and 189 (76%) were from a remote region. The time between cases and past epidemiologically- and genomically-linked contacts ranged from 4·5 months to 24 years. INTERPRETATION: Our findings support prioritisation of timely case detection, contact tracing augmented by genomic sequencing, and latent TB treatment to break transmission chains in Top End remote hotspot regions.

Low-Cost, Open-Source Device for High-Performance Fluorescence Detection of Isothermal Nucleic Acid Amplification Reactions.

The ability to detect SARS-CoV-2 is critical to implementing evidence-based strategies to address the COVID-19 global pandemic. Expanding SARS-CoV-2 diagnostic ability beyond well-equipped laboratories widens the opportunity for surveillance and control efforts. However, such advances are predicated on the availability of rapid, scalable, accessible, yet high-performance diagnostic platforms. Methods to detect viral RNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP) show promise as rapid and field-deployable tests; however, the per-unit costs of the required diagnostic hardware can be a barrier for scaled deployment. Here, we describe a diagnostic hardware configuration for LAMP technology, named the FABL-8, that can be built for approximately US$380 per machine and provide results in under 30 min. Benchmarking showed that FABL-8 has a similar performance to a high-end commercial instrument for detecting fluorescence-based LAMP reactions. Performance testing of the instrument with RNA extracted from a SARS-CoV-2 virus dilution series revealed an analytical detection sensitivity of 50 virus copies per microliter-a detection threshold suitable to detect patient viral load in the first few days following symptom onset. In addition to the detection of SARS-CoV-2, we show that the system can be used to detect the presence of two bacterial pathogens, demonstrating the versatility of the platform for the detection of other pathogens. This cost-effective and scalable hardware alternative allows democratization of the instrumentation required for high-performance molecular diagnostics, such that it could be available to laboratories anywhere-supporting infectious diseases surveillance and research activities in resource-limited settings.

Accessible Platform for High-Throughput COVID-19 Molecular Diagnostics and Genome Sequencing Using a Repurposed 3D Printer for RNA Extraction.

The COVID-19 pandemic has exposed the dependence of diagnostic laboratories on a handful of large corporations with market monopolies on the worldwide supply of reagents, consumables, and hardware for molecular diagnostics. Global shortages of key consumables for RT-qPCR detection of SARS-CoV-2 RNA have impaired the ability to run essential, routine diagnostic services. Here, we describe a workflow for rapid detection of SARS-CoV-2 RNA in upper respiratory samples including nasal swabs and saliva, utilizing low-cost equipment and readily accessible reagents. Using repurposed Creality3D Ender-3 three-dimensional (3D) printers, we built a semiautomated paramagnetic bead RNA extraction platform. The hardware for the system was built for $300 USD, and the material cost per reaction was $1 USD. Named the Ender VX500, instrument performance when paired with RT-qPCR for SARS-CoV-2 detection in nasal and saliva specimens was two virus copies per microliter. There was a high-performance agreement (assessed using 458 COVID-19 nasal swab specimens) with the Aptima SARS-CoV-2 assay run on the Hologic Panther, a commercial automated RNA extraction and detection platform. Inter- and intrainstrument precision was excellent (coefficients of variation (CoV) of 1.10 and 0.66-1.32%, respectively) across four instruments. The platform is scalable with throughput ranging from 23 specimens on a single instrument run by one user in 50 min to 364 specimens on four instruments run by four users in 190 min. Step-by-step instructions and protocols for building and running the Ender VX500 have been made available without restriction.

Introduction of Mycobacterium ulcerans disease in the Bankim Health District of Cameroon follows damming of the Mapé River.

Buruli ulcer (BU) is an emerging ulcerative skin disease caused by infection with Mycobacterium ulcerans. Efforts to control its spread have been hampered by our limited understanding of M. ulcerans reservoirs and transmission, and the factors leading to the emergence of BU disease in a particular region. In this report we investigate an anecdotal link between damming the Mapé River in Cameroon and the emergence of BU in the Health Districts bordering Lake Bankim, the impoundment created by the Mapé dam. We used bacterial population genomics and molecular dating to find compelling support for a 2000 M. ulcerans introduction event that followed about 10 years after the filling of the newly created impoundment in 1988. We compared the genomic reconstructions with high-resolution satellite imagery to investigate what major environmental alterations might have driven the emergence of the new focus.

Unstable chromosome rearrangements in Staphylococcus aureus cause phenotype switching associated with persistent infections.

Staphylococcus aureus small-colony variants (SCVs) are associated with unusually chronic and persistent infections despite active antibiotic treatment. The molecular basis for this clinically important phenomenon is poorly understood, hampered by the instability of the SCV phenotype. Here we investigated the genetic basis for an unstable S. aureus SCV that arose spontaneously while studying rifampicin resistance. This SCV showed no nucleotide differences across its genome compared with a normal-colony variant (NCV) revertant, yet the SCV presented the hallmarks of S. aureus linked to persistent infection: down-regulation of virulence genes and reduced hemolysis and neutrophil chemotaxis, while exhibiting increased survival in blood and ability to invade host cells. Further genome analysis revealed chromosome structural variation uniquely associated with the SCV. These variations included an asymmetric inversion across half of the S. aureus chromosome via recombination between type I restriction modification system (T1RMS) genes, and the activation of a conserved prophage harboring the immune evasion cluster (IEC). Phenotypic reversion to the wild-type-like NCV state correlated with reversal of the chromosomal inversion (CI) and with prophage stabilization. Further analysis of 29 complete S. aureus genomes showed strong signatures of recombination between hsdMS genes, suggesting that analogous CI has repeatedly occurred during S. aureus evolution. Using qPCR and long-read amplicon deep sequencing, we detected subpopulations with T1RMS rearrangements causing CIs and prophage activation across major S. aureus lineages. Here, we have discovered a previously unrecognized and widespread mechanism of reversible genomic instability in S. aureus associated with SCV generation and persistent infections.

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

Mycobacterium ulcerans Population Genomics To Inform on the Spread of Buruli Ulcer across Central Africa.

Buruli ulcer is a neglected tropical disease of skin and subcutaneous tissue caused by infection with the pathogen Mycobacterium ulcerans Many critical issues for disease control, such as understanding the mode of transmission and identifying source reservoirs of M. ulcerans, are still largely unknown. Here, we used genomics to reconstruct in detail the evolutionary trajectory and dynamics of M. ulcerans populations at a central African scale and at smaller geographical village scales. Whole-genome sequencing (WGS) data were analyzed from 179 M. ulcerans strains isolated from all Buruli ulcer foci in the Democratic Republic of the Congo, The Republic of Congo, and Angola that have ever yielded positive M. ulcerans cultures. We used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our phylogeographic analysis revealed one almost exclusively predominant sublineage of M. ulcerans that arose in Central Africa and proliferated in its different regions of endemicity during the Age of Discovery. We observed how the best sampled endemic hot spot, the Songololo territory, became an area of endemicity while the region was being colonized by Belgium (1880s). We furthermore identified temporal parallels between the observed past population fluxes of M. ulcerans from the Songololo territory and the timing of health policy changes toward control of the Buruli ulcer epidemic in that region. These findings suggest that an intervention based on detecting and treating human cases in an area of endemicity might be sufficient to break disease transmission chains, irrespective of other reservoirs of the bacterium.IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans The disease is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Currently, the major hurdles facing disease control are incomplete understandings of both the mode of transmission and environmental reservoirs of M. ulcerans As decades of spasmodic environmental sampling surveys have not brought us much closer to overcoming these hurdles, the Buruli ulcer research community has recently switched to using comparative genomics. The significance of our research is in how we used both temporal associations and the study of the mycobacterial demographic history to estimate the contribution of humans as a reservoir in Buruli ulcer transmission. Our approach shows that it might be possible to use bacterial population genomics to assess the impact of health interventions, providing valuable feedback for managers of disease control programs in areas where health surveillance infrastructure is poor.

Comparative Genomics Shows That Mycobacterium ulcerans Migration and Expansion Preceded the Rise of Buruli Ulcer in Southeastern Australia.

Since 2000, cases of the neglected tropical disease Buruli ulcer, caused by infection with Mycobacterium ulcerans, have increased 100-fold around Melbourne (population 4.4 million), the capital of Victoria, in temperate southeastern Australia. The reasons for this increase are unclear. Here, we used whole-genome sequence comparisons of 178 M. ulcerans isolates obtained primarily from human clinical specimens, spanning 70 years, to model the population dynamics of this pathogen from this region. Using phylogeographic and advanced Bayesian phylogenetic approaches, we found that there has been a migration of the pathogen from the east end of the state, beginning in the 1980s, 300 km west to the major human population center around Melbourne. This move was then followed by a significant increase in M. ulcerans population size. These analyses inform our thinking around Buruli ulcer transmission and control, indicating that M. ulcerans is introduced to a new environment and then expands, rather than it being from the awakening of a quiescent pathogen reservoir.IMPORTANCE Buruli ulcer is a destructive skin and soft tissue infection caused by Mycobacterium ulcerans and is characterized by progressive skin ulceration, which can lead to permanent disfigurement and long-term disability. Despite the majority of disease burden occurring in regions of West and central Africa, Buruli ulcer is also becoming increasingly common in southeastern Australia. Major impediments to controlling disease spread are incomplete understandings of the environmental reservoirs and modes of transmission of M. ulcerans The significance of our research is that we used genomics to assess the population structure of this pathogen at the Australian continental scale. We have then reconstructed a historical bacterial spread and modeled demographic dynamics to reveal bacterial population expansion across southeastern Australia. These findings provide explanations for the observed epidemiological trends with Buruli ulcer and suggest possible management to control disease spread.

Isoniazid resistance levels of Mycobacterium tuberculosis can largely be predicted by high-confidence resistance-conferring mutations.

The majority of Mycobacterium tuberculosis isolates resistant to isoniazid harbour a mutation in katG. Since these mutations cause a wide range of minimum inhibitory concentrations (MICs), largely below the serum level reached with higher dosing (15 mg/L upon 15-20 mg/kg), the drug might still remain partly active in presence of a katG mutation. We therefore investigated which genetic mutations predict the level of phenotypic isoniazid resistance in clinical M. tuberculosis isolates. To this end, the association between known and unknown isoniazid resistance-conferring mutations in whole genome sequences, and the isoniazid MICs of 176 isolates was examined. We found mostly moderate-level resistance characterized by a mode of 6.4 mg/L for the very common katG Ser315Thr mutation, and always very high MICs (≥19.2 mg/L) for the combination of katG Ser315Thr and inhA c-15t. Contrary to common belief, isolates harbouring inhA c-15t alone, partly also showed moderate-level resistance, particularly when combined with inhA Ser94Ala. No overt association between low-confidence or unknown mutations, except in katG, and isoniazid resistance (level) was found. Except for the rare katG deletion, line probe assay is thus not sufficiently accurate to predict the level of isoniazid resistance for a single mutation in katG or inhA.

Bacterial diversity in Buruli ulcer skin lesions: Challenges in the clinical microbiome analysis of a skin disease.

BACKGROUND: Buruli ulcer (BU) is an infectious disease caused by Mycobacterium ulcerans and considered the third most prevalent mycobacterial disease in humans. Secondary bacterial infections in open BU lesions are the main cause of pain, delayed healing and systemic illness, resulting in prolonged hospital stay. Thus, understanding the diversity of bacteria, termed the microbiome, in these open lesions is important for proper treatment. However, adequately studying the human microbiome in a clinical setting can prove difficult when investigating a neglected tropical skin disease due to its rarity and the setting. METHODOLOGY/PRINCIPAL FINDINGS: Using 16S rRNA sequencing, we determined the microbial composition of 5 BU lesions, 3 non-BU lesions and 3 healthy skin samples. Although no significant differences in diversity were found between BU and non-BU lesions, the former were characterized by an increase of Bacteroidetes compared to the non-BU wounds and the BU lesions also contained significantly more obligate anaerobes. With this molecular-based study, we were also able to detect bacteria that were missed by culture-based methods in previous BU studies. CONCLUSIONS/SIGNIFICANCE: Our study suggests that BU may lead to changes in the skin bacterial community within the lesions. However, in order to determine if such changes hold true across all BU cases and are either a cause or consequence of a specific wound environment, further microbiome studies are necessary. Such skin microbiome analysis requires large sample sizes and lesions from the same body site in many patients, both of which can be difficult for a rare disease. Our study proposes a pipeline for such studies and highlights several drawbacks that must be considered if microbiome analysis is to be utilized for neglected tropical diseases.

Multiple Introductions and Recent Spread of the Emerging Human Pathogen Mycobacterium ulcerans across Africa.

Buruli ulcer (BU) is an insidious neglected tropical disease. Cases are reported around the world but the rural regions of West and Central Africa are most affected. How BU is transmitted and spreads has remained a mystery, even though the causative agent, Mycobacterium ulcerans, has been known for more than 70 years. Here, using the tools of population genomics, we reconstruct the evolutionary history of M. ulcerans by comparing 165 isolates spanning 48 years and representing 11 endemic countries across Africa. The genetic diversity of African M. ulcerans was found to be restricted due to the bacterium's slow substitution rate coupled with its relatively recent origin. We identified two specific M. ulcerans lineages within the African continent, and inferred that M. ulcerans lineage Mu_A1 existed in Africa for several hundreds of years, unlike lineage Mu_A2, which was introduced much more recently, approximately during the 19th century. Additionally, we observed that specific M. ulcerans epidemic Mu_A1 clones were introduced during the same time period in the three hydrological basins that were well covered in our panel. The estimated time span of the introduction events coincides with the Neo-imperialism period, during which time the European colonial powers divided the African continent among themselves. Using this temporal association, and in the absence of a known BU reservoir or-vector on the continent, we postulate that the so-called "Scramble for Africa" played a significant role in the spread of the disease across the continent.

A Genomic Approach to Resolving Relapse versus Reinfection among Four Cases of Buruli Ulcer.

BACKGROUND: Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. METHODOLOGY: We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. PRINCIPAL FINDINGS: The findings suggest that after surgical treatment-without antibiotics-the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. CONCLUSIONS: To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development.

Whole genome comparisons suggest random distribution of Mycobacterium ulcerans genotypes in a Buruli ulcer endemic region of Ghana.

Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.