Biology subjects

Didelot, X.

Publications and source records attributed to Didelot, X..

At least 19 recordsLinked to original sources

bioRxiv2026

An unconstrained Euclidean parametrisation of phylogenetic tree space

Phylogenetic trees combine a discrete topology with continuous branch lengths, which makes them difficult to work with. We introduce a recursive contour-grafting construction that encodes both components using unconstrained real-valued coordinates. After fixing an order of the leaves, each new leaf is attached at a point selected by a contour traversal of the tree constructed so far. Moving a graft coordinate continuously along the contour enables changes of topology when internal branches contract to zero. For trees with n labelled leaves, we apply the construction to rooted ultrametric trees, rooted trees with unrestricted branch lengths, and unrooted trees, obtaining parametrisations of dimensions n-1, 2n-2, and 2n-3, respectively. We show that the resulting construction functions are surjective and continuous onto the corresponding spaces of phylogenetic trees. We also determine the number of representations of a generic tree and calculate the Jacobian of each parametrisation. These results provide explicit change-of-variables formulae between Euclidean coordinates and the phylogenetic tree spaces, and we show how probability distributions on tree space can be sampled using generic algorithms that target unconstrained Euclidean coordinates. This approach paves the way for the development and application of new methodology in Bayesian phylogenetic inference.

evolutionary biology↗

bioRxiv2025

Quantifying the predictability of evolution by analysis of coalescent rate variation

We investigate the predictability of evolution in terms of the phylogenetic placement of new lineages. This leads us to develop a class of coalescent models that relax neutrality by allowing the rate of coalescence to vary as a continuous heritable trait. In this setting, each lineage has a relative propensity to coalesce, with coalescent odds ratios defined from the product of pairwise propensities. Estimated coalescent odds provide a statistic that captures variation in lineage growth and are informative about the strength of natural selection acting on individual lineages. A number of practical statistical methods are then developed: techniques to adjust for biased and non-uniform sampling; procedures to automatically calibrate hyperparameters governing the evolution of coalescent propensity; and, methods for clustering phylogenies into sets that delineate clades according to coalescent propensity. Simulations show sensitivity of these methods to detecting small selective effects acting on rare variants, and strong robustness to imbalanced sampling. We demonstrate these methods using two datasets reflecting microbial populations evolving under strong selection. First, we examine a large set of Neisseria gonorrhoeae genomes, and show that lineages with high coalescent odds feature a unique antibiotic resistance pattern which presaged its subsequent expansion. We then re-analyse SARS-CoV-2 data in combination with independent estimates of reproduction numbers corresponding to major variants of concern 2020-2023. This indicates that coalescent odds can function as an excellent tree-based proxy for relative fitness of major SARS-CoV-2 lineages.

evolutionary biology↗

bioRxiv2025

Diagnostics of dated phylogenies in microbial population genetics

Microbial population genetic studies often involve the use of a dated phylogeny to show how the genomes are related over a relevant timescale. Many tools have recently been developed to date the nodes of a standard phylogeny, but all make underlying assumptions that may not be realistic for a given dataset, making the results potentially unreliable. Model comparison is sometimes used to remedy this issue, whereby inference under several models is compared to establish which result can be trusted. Although such comparison is clearly useful to assess the relative merits of several inference attempts, here instead we focus on the problem of evaluating how good an inference is in absolute terms, without comparison. We consider several approaches for diagnosing potential issues in a reconstructed dated phylogeny, including outlier detection, posterior predictive checking and residual analysis. These methods are well-established diagnostics tools in other areas of statistics, but here we show how they can be applied to the specific inference of dated phylogenies. We illustrate their use on many simulated datasets, with inference being performed either from the correct model to quantify the specificity or from an incorrect model to quantify the sensitivity of the diagnostics methods. We also applied the methods to three real-life datasets to showcase the range of issues that they can detect. We advocate the use of these diagnostics tools for all microbial population genetic studies that involve the reconstruction of a dated phylogeny.

evolutionary biology↗

bioRxiv2025

Large-scale genomic characterisation of phage-plasmids in clinical Enterobacteriaceae isolates

The life cycle of certain bacteriophages involves their maintenance within the bacterial cell as extrachromosomal elements, complete with replication and partitioning systems. These "phage-plasmids" (P-Ps) are distributed widely among bacterial phyla but are not typically included during genomic surveillance studies, and previous reports do not consider the context of their host strain diversity. We recently identified a P1-like P-P carrying the blaCTX-M-15 resistance gene in Salmonella enterica serovar Typhi, prompting a subsequent investigation into the overall frequency of P-Ps among gastrointestinal bacteria under routine genomic surveillance within England. We expanded our study to include P-P groups known to be associated with Enterobacteriaceae (P1, D6, SSU5, N15) and scanned a collection of 66,856 genomes of diarrhoeagenic Escherichia spp., Shigella spp. and S. enterica. All four P-P groups were detected in our dataset, totalling 9% of E. coli and Shigella genomes and 2% of S. enterica genomes. A small subset harboured two distinct P-P groups. P1-like P-Ps, some of which carried a cytotoxic necrotising factor, were predominantly associated with Shiga toxin-producing E. coli strains of public health concern, including clonal complexes CC11 (O157:H7), CC29 (O26:H11) and CC165. In contrast, SSU5 group P-Ps were linked with multidrug-resistant lineages of both S. Typhi and Shigella sonnei. We found multiple antimicrobial resistance genes inserted into P-Ps via transposons, integrons and insertion sequences, and numerous defence/anti-defence systems. There was evidence of vertical transmission but also many links across species, time and geography, indicating that horizontal transfer is occurring regularly. Phage-plasmids are often described only as cryptic elements or not detected during genomic surveillance. We show that P-Ps are associated with clinically relevant lineages of human pathogens and can acquire accessory genes that may impact on disease severity and therefore should play a more prominent role in pathogen surveillance and epidemiology.

genomics↗

bioRxiv2025

Macrogenetic atlas of prokaryotes

Macrogenetics investigates patterns and predictors of intraspecific genetic variation across diverse taxa, offering a framework for understanding species diversity and addressing evolutionary hypotheses. Here, we present a macrogenetic atlas of prokaryotes (MAP), integrating genomic data from 15,235 prokaryotic species (30 parameters in 12 categories) and population genetic data from 786 species (22 parameters in 7 categories), together with phylogenetic, phenotypic, and ecological information. MAP enables quantitative species characterization, straightforward cross-species comparisons, and easy generation, testing, and refinement of evolutionary hypotheses. For example, our analyses show that long- and short-range genetic linkage capture distinct evolutionary dynamics and independently shape genetic diversity. Further, we propose that as within-species diversity increases, epistatic selection increasingly contributes to shaping genetic diversity, exemplified by convergent selection-driven structures (ecospecies) identified in Streptococcus mitis and S. oralis. Overall, MAP represents a widely applicable resource (www.genomap.cn) and offers novel insights into the drivers of macroevolution and the life cycle of prokaryotic species.

microbiology↗

bioRxiv2024

Bayesian Inference of Pathogen Phylogeography using the Structured Coalescent Model

Over the past decade, pathogen genome sequencing has become well established as a powerful approach to study infectious disease epidemiology. In particular, when multiple genomes are available from several geographical locations, comparing them is informative about the relative size of the local pathogen populations as well as past migration rates and events between locations. The structured coalescent model has a long history of being used as the underlying process for such phylogeographic analysis. However, the computational cost of using this model does not scale well to the large number of genomes frequently analysed in pathogen genomic epidemiology studies. Several approximations of the structured coalescent model have been proposed, but their effects are difficult to predict. Here we show how the exact structured coalescent model can be used to analyse a precomputed dated phylogeny, in order to perform Bayesian inference on the past migration history, the effective population sizes in each location, and the directed migration rates from any location to another. We describe an efficient reversible jump Markov Chain Monte Carlo scheme which is implemented in a new R package. We use simulations to demonstrate the scalability and correctness of our method and to compare it with existing comparable software. We also applied our new method to several state-of-the-art datasets on the population structure of real pathogens to showcase the relevance of our method to current data scales and research questions.

evolutionary biology↗

bioRxiv2024

Presence of phage-plasmids in multiple serovars of Salmonella enterica

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterised a circular P1-like phage-plasmid harbouring a blaCTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47,784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8-98.2kb in size, with an average length of 92.1kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterisation with long-read sequencing. Data SummaryAll of the FASTQ files examined in this study have been uploaded to the Sequence Read Archive under BioProject PRJNA248792. Accessions of individual isolates which were found to contain phage plasmids are listed in Supplementary Table S1. Impact StatementBacteriophage-like plasmids are increasingly being recognised as important mobile elements in many species of bacteria, particularly due to their involvement in the transmission of antimicrobial resistance (AMR); however, few studies of their overall prevalence in clinical datasets have been undertaken to date. In this study we have performed the first large-scale surveillance of human disease-associated Salmonella genomes for the presence of P1-like phage-plasmids, showing that they are more common than previously thought. Furthermore, we highlight how valuable information about the evolution and transmission of phage-plasmids in Salmonella and other Enterobacterales can be revealed by linking phage-plasmid prevalence and genetic diversity to epidemiologically relevant metadata such as S. enterica serovar, outbreak clusters, time, and geography. Our work shows the ability to use sequencing data and scalable bioinformatics workflows for the detection and characterisation of these extrachromosomal elements, highlights the importance of screening for novel mechanisms of AMR transmission, and provides a foundation for further surveillance studies of phage-plasmid prevalence.

microbiology↗

bioRxiv2023

Emergence of Acinetobacter baumannii International Clone 10 predominantly found in the Middle East

Acinetobacter baumannii is a globally distributed human pathogen. Infections caused by carbapenem-resistant isolates of A. baumannii (CRAB) are of great concern, as treatment options are very limited. Despite having among the highest rates reported worldwide, there exists limited genomic data from CRAB strains isolated in the Middle East. Here we report epidemiological, phenotypic, and genome sequencing data (short reads and long reads) on a set of 60 A. baumannii isolates belonging to Sequence Type ST158 (Pasteur MLST scheme). They represent a novel international clone (IC), designated IC10, with limited geographic spread beyond the Middle East. Specific antibiotic-resistance genes associated with this clone were identified and data on the plasmid content associated with this lineage are presented.

microbiology↗

bioRxiv2023

Inference of multiple mergers while dating a pathogen phylogeny

The vast majority of pathogen phylogenetic studies do not consider the possibility of multiple merger events being present, where a single node of the tree leads to more than two descendent branches. These events are however likely to occur when studying a relatively small population or if there is high variability in the reproductive chances. Here we consider the problem of detecting the presence of multiple mergers in the context of dating a phylogeny, that is determining the date of each of the nodes. We use the Lambda-coalescent theory as a modelling framework and show how Bayesian inference can be efficiently performed using a Billera-Holmes-Vogtmann space embedding and a customised Markov Chain Monte Carlo sampling scheme. We applied this new analysis methodology to a large number of simulated datasets to show that it is possible to infer if and when multiple merger events occurred, and that the phylogenetic dating is improved as a result of taking this information into account. We also analysed real datasets of Vibrio cholerae and Mycobacterium tuberculosis to demonstrate the relevance of our approach to real pathogen evolutionary epidemiology. We have implemented our new methodology in a R package which is freely available at https://github.com/dhelekal/MMCTime.

evolutionary biology↗

bioRxiv2023

The inter-continental population dynamics of Neisseria gonorrhoeae

Neisseria gonorrhoeae is a globally distributed sexually transmitted bacterial pathogen. Recent studies have revealed that its evolution has been shaped by antibiotic use, while molecular surveillance efforts have demonstrated large changes in lineage composition over relatively short time-spans. However, the global population dynamics of N. gonorrhoeae remain unsatisfyingly characterized. To reconstruct recent large-scale population dynamics, we generated a dated phylogeny from 9,732 N. gonorrhoeae genomes and found the effective population size of the species to have expanded gradually over the past two centuries. While the effective population size of clades with reduced susceptibility to extended-spectrum cephalosporins started declining around 2010, a major clade containing a mosaic mtr operon associated with cephalosporin susceptibility and decreased azithromycin did not display any reduction in population size. Using ancestral trait reconstruction analyses, we delineated transmission lineages, defined as groups of sequences in which all the samples can be traced back to the same import event to a given location. Import, export and local transmission dynamics across two densely sampled locations (Norway and Victoria, Australia) were investigated in detail. Norway exhibited substantially higher rates of strain import and export compared to Victoria, where incidence was to a larger extent fuelled by locally transmitted lineages. Taken together, our work highlights the power of large-scale phylogenomic analyses to uncover the complex dynamics of lineage transmission in N. gonorrhoeae.

microbiology↗

bioRxiv2023

Inference of infectious disease transmission using multiple genomes per host

In recent times, pathogen genome sequencing has become increasingly used to investigate infectious disease outbreaks. When genomic data is sampled densely enough amongst infected individuals, it can help resolve who infected whom. However, transmission analysis cannot rely solely on a phylogeny of the genomes but must account for the within-host evolution of the pathogen, which blurs the relationship between phylogenetic and transmission trees. When only a single genome is sampled for each host, the uncertainty about who infected whom can be quite high. Consequently, transmission analysis based on multiple genomes of the same pathogen per host has a clear potential for delivering more precise results, even though it is more laborious to achieve. Here we present a new methodology that can use any number of genomes sampled from a set of individuals to reconstruct their transmission network. We use simulated data to show that our method becomes more accurate as more genomes per host are provided, and that it can infer key infectious disease parameters such as the size of the transmission bottleneck, within-host growth rate, basic reproduction number and sampling fraction. We demonstrate the usefulness of our method in applications to real datasets from an outbreak of Pseudomonas aeruginosa amongst cystic fibrosis patients and a nosocomial outbreak of Klebsiella pneumoniae.

microbiology↗

bioRxiv2022

Estimating the fitness cost and benefit of antimicrobial resistance from pathogen genomic data

Increasing levels of antibiotic resistance in many bacterial pathogen populations is a major threat to public health. Resistance to an antibiotic provides a fitness benefit when the bacteria is exposed to this antibiotic, but resistance also often comes at a cost to the resistant pathogen relative to susceptible counterparts. We lack a good understanding of these benefits and costs of resistance for many bacterial pathogens and antibiotics, but estimating them could lead to better use of antibiotics in a way that reduces or prevents the spread of resistance. Here, we propose a new model for the joint epidemiology of susceptible and resistant variants, which includes explicit parameters for the cost and benefit of resistance. We show how Bayesian inference can be performed under this model using phylogenetic data from susceptible and resistant lineages and that by combining data from both we are able to disentangle and estimate the resistance cost and benefit parameters separately. We applied our inferential methodology to several simulated datasets to demonstrate good scalability and accuracy. We analysed a dataset of Neisseria gonorrhoeae genomes collected between 2000 and 2013 in the USA. We found that two unrelated lineages resistant to fluoroquinolones shared similar epidemic dynamics and resistance parameters. Fluoroquinolones were abandoned for the treatment of gonorrhoea due to increasing levels of resistance, but our results suggest that they could be used to treat a minority of around 10% of cases without causing resistance to grow again.

evolutionary biology↗

bioRxiv2022

KmerAperture: Retaining k-mer synteny for alignment-free estimation of within-lineage core and accessory differences

By decomposing genome sequences into k-mers, it is possible to estimate genome differences without alignment. Techniques such as k-mer minimisers (MinHash), have been developed and are often accurate approximations of distances based on full k-mer sets. These and other alignment-free methods avoid the large temporal and computational expense of alignment or mapping. However, these k-mer set comparisons are not entirely accurate within-species and can be completely inaccurate within-lineage. This is due, in part, to their inability to distinguish core polymorphism from accessory differences. Here we present a new approach, KmerAperture, which uses information on the k-mer relative genomic positions to determine the type of polymorphism causing differences in k-mer presence and absence between pairs of genomes. Single SNPs are expected to result in contiguous series of relative unique k-mers of length L= k. On the other hand, series of length L > k may be caused by accessory differences of length L-k+1; when the start and end of the sequence are contiguous with homologous sequence. Alternatively, they may be caused by multiple SNPs within k bp from each other and KmerAperture can determine whether that is the case. To demonstrate use cases KmerAperture was benchmarked using datasets including a very low diversity simulated population with accessory content independent from the number of SNPs, a simulated population were SNPs are spatially dense, a moderately diverse real cluster of genomes (Escherichia coli ST1193) with a large accessory genome and a low diversity real genome cluster (Salmonella Typhimurium ST34). We show that KmerAperture can accurately distinguish both core and accessory sequence diversity without alignment, outperforming other k-mer based tools.

bioinformatics↗

bioRxiv2022

Distinguishing imported cases from locally acquired cases within a geographically limited genomic sample of an infectious disease

The ability to distinguish imported cases from locally acquired cases has important consequences for the selection of public health control strategies. Genomic data can be useful for this, for example using a phylogeographic analysis in which genomic data from multiple locations is compared to determine likely migration events between locations. However, these methods typically require good samples of genomes from all locations, which is rarely available. Here we propose an alternative approach that only uses genomic data from a location of interest. By comparing each new case with previous cases from the same location we are able to detect imported cases, as they have a different genealogical distribution than that of locally acquired cases. We show that, when variations in the size of the local population are accounted for, our method has good sensitivity and excellent specificity for the detection of imports. We applied our method to data simulated under the structured coalescent model and demonstrate relatively good performance even when the local population has the same size as the external population. Finally, we applied our method to several recent genomic datasets from both bacterial and viral pathogens, and show that it can, in a matter of seconds or minutes, deliver important insights on the number of imports to a geographically limited sample of a pathogen population.

evolutionary biology↗

bioRxiv2022

KPop: An assembly-free and scalable method for the comparative analysis of microbial genomes

The recent explosion in the amount of available sequencing data challenges existing analysis techniques. Here we introduce KPop, a novel versatile method based on full k-mer spectra and dataset-specific transformations, through which thousands of assembled or unassembled microbial genomes can be quickly compared. Unlike minimizer-based methods that produce distances and have lower resolution, KPop is able to accurately map sequences onto a low-dimensional space. Extensive validation on simulated and real-life viral and bacterial datasets shows that KPop can correctly separate sequences at both species and sub-species levels even when the overall genomic diversity is low. KPop also rapidly identifies related sequences and systematically outperforms minimizer-based methods. KPops code is open-source and available on GitHub at https://github.com/PaoloRibeca/KPop.

bioinformatics↗

bioRxiv2022

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

evolutionary biology↗

bioRxiv2022

Penicillin Binding Protein Substitutions Co-occur with Fluoroquinolone Resistance in 'Epidemic Lineages of Multi Drug-Resistant Clostridioides difficile

Clostridioides difficile remains a key cause of healthcare-associated infection, with multi-drug-resistant (MDR) lineages causing high mortality ([≥]20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species resistance is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins, PBPs). Here, we investigated five C. difficile transpeptidases (PBP1-5) for recent substitutions. Previously published genome assemblies (n=7096) were obtained, representing sixteen geographically widespread lineages, including healthcare-associated MDR ST1(027), ST3(001) and ST17(018). Recent amino acid substitutions were found within PBP1 (n=50) and PBP3 (n=48), ranging from 1-10 substitutions per genome. {beta}-lactam MICs were measured for closely related pairs of wild-type and PBP substituted isolates separated by 20-273 SNPs. Recombination-corrected, dated phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1-4 doubling dilutions >wild-type up to [≤]1506g/ml. Substitution patterns varied by lineage and clade, showed geographic structure, and notably occurred post-1990, coincident with the acquisition of gyrA/B substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. The co-occurrence of resistance to cephalosporins and fluoroquinolones hinders attempts to understand their relative importance in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. IMPORTANCEFluoroquinolone and cephalosporin prescribing in healthcare settings have triggered outbreaks of high-mortality, multi-drug resistant C. difficile infection. Here, we identify a mechanism of acquired cephalosporin resistance in C. difficile, comprising amino acid substitutions in two cell-wall transpeptidase enzymes (penicillin binding proteins). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed that resistance to both cephalosporins and fluoroquinolones was co-acquired immediately before clinically important, outbreak strains emerged. PBP substitutions were geographically structured within genetic lineages, suggesting adaptation to local antimicrobial prescribing. Antimicrobial stewardship of cephalosporins and fluoroquinolones is an effective means of C. difficile outbreak control. Genetic changes conferring resistance likely impart a fitness-cost after antibiotic withdrawal. Our study identifies a mechanism that may explain the contribution of cephalosporin stewardship to resolving outbreak conditions. However, due to the co-occurrence of cephalosporin and fluoroquinolone resistance, further work is needed to determine the relative importance of each.

microbiology↗

bioRxiv2022

Phylogenetic analysis of the origin and spread of plague in Madagascar

BackgroundPlague is a zoonotic disease caused by the bacterium Yersinia pestis, highly prevalent in the Central Highlands, a mountainous region in the center of Madagascar. After a plague-free period of over 60 years in the northwestern coast city of Mahajanga, the disease reappeared in 1991 and caused several outbreaks until 1999. Previous research indicates that the disease was reintroduced to the city of Mahajanga from the Central Highlands instead of reemerging from a local reservoir. However, it is not clear how many reintroductions occurred and when they took place. Methodology/Principal findingsIn this study we applied a Bayesian phylogeographic model to detect and date migrations of Y. pestis between the two locations that could be linked to the re-emergence of plague in Mahajanga. Genome sequences of 300 Y. pestis strains sampled between 1964 and 2012 were analyzed. Four migrations from the Central Highlands to Mahajanga were detected. Two resulted in persistent transmission in humans, one was responsible for most of the human cases recorded between 1995 and 1999, while the other produced plague cases in 1991 and 1992. We dated the emergence of the Y. pestis sub-branch 1.ORI3, which is only present in Madagascar and Turkey, to the beginning of the 20th century, using a Bayesian molecular dating analysis. The split between 1.ORI3 and its ancestor lineage 1.ORI2 was dated to the second half of the 19th century. Conclusions/SignificanceOur results indicate that two independent migrations from the Central Highlands caused the plague outbreaks in Mahajanga during the 1990s, with both introductions occurring during the early 1980s. They happened over a decade before the detection of human cases, thus the pathogen likely survived in wild reservoirs until the spillover to humans was possible. This study demonstrates the value of Bayesian phylogenetics in elucidating the re-emergence of infectious diseases. Author summaryIn 1991 human cases of plague were reported in the city of Mahajanga, located in the west coast of Madagascar, after 60 years without human cases. Existing evidence suggests that Yersinia pestis, the causal agent of the disease, was reintroduced to the city from a mountainous region known as the Central Highlands. We performed a phylogeographic analysis on 300 Y. pestis genome sequences to determine how many migrations of the pathogen between the two locations were related to the reappearance of the disease in Mahajanga. The results revealed that two migrations from the Central Highlands were the cause of the outbreaks of plague in the west coast of the country during the 1990s. We also aimed to date the emergence of the Y.pestis variant that circulates in Madagascar and is also present in Turkey. To do this, we conducted a molecular dating analysis using an extended data set of 445 sequences, which contained sequences from Turkey and India (the country from which the pathogen was exported to Madagascar for the first time). The analysis indicated that this particular variant emerged in the first decade of the 20th century.

microbiology↗