Molecular and Functional Characterization of Lymphoid Progenitor Subsets Reveals a Bipartite Architecture of Human Lymphopoiesis.

The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.

Population Structure of Mycobacterium bovis in Germany: a Long-Term Study Using Whole-Genome Sequencing Combined with Conventional Molecular Typing Methods.

Mycobacterium bovis is the primary cause of bovine tuberculosis (bTB) and infects a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 M. bovis strains from animals (n = 38) and humans (n = 93) in Germany from 1999 to 2017 by whole-genome sequencing (WGS), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 M. bovis strains were classified into 13 major clades, of which 6 contained strains from both human and animal cases and 7 only strains from human cases. Bayesian analyses suggest that the M. bovis population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950s. WGS-based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2 to 11 members and involving strains from distinct host types, e.g., only cattle and also mixed hosts. Animal strains of four clusters were obtained over a 9-year span, pointing toward autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics constitute the method of choice to implement prospective molecular epidemiological surveillance of M. bovis The population of M. bovis in Germany is diverse, with subtle, but existing, interactions between different host groups.

Longitudinal Outbreak of Multidrug-Resistant Tuberculosis in a Hospital Setting, Serbia.

A retrospective population-based molecular epidemiologic study of multidrug-resistant Mycobacterium tuberculosis complex strains in Serbia (2008-2014) revealed an outbreak of TUR genotype strains in a psychiatric hospital starting around 1990. Drug unavailability, poor infection control, and schizophrenia likely fueled acquisition of additional resistance and bacterial fitness-related mutations over 2 decades.

Emergence of enterovirus D68 clade D1, France, August to November 2018.

We report a seasonal increase of enterovirus D68 (EV-D68) cases in France, with 54 cases detected between 19 August and 14 November 2018. Molecular typing revealed that 20 of 32 of the isolates belonged to clade D1, only sporadically detected before in France. Median age of D1-cases was 42 years, 10 developed severe respiratory signs and one had neurological complications. The 2018-D1 viruses showed a genetic divergence of 3.34 % with D1 viruses identified previously.

Molecular diversity and biennial circulation of enterovirus D68: a systematic screening study in Lyon, France, 2010 to 2016.

BackgroundUnderstanding enterovirus D68 (EV-D68) circulation patterns as well as risk factors for severe respiratory and neurological illness is important for developing preventive strategies. Methods: Between 2010 and 2016, 11,132 respiratory specimens from hospitalised patients in Lyon, France, were screened for EV-D68 by PCR. Phylogenetic relationships of the viral-protein-1 sequences were reconstructed using maximum-likelihood and Bayesian-Markov-Chain-Monte-Carlo approaches. Results: Overall, 171 infections with a biennial pattern were detected, including seven, one, 55, none, 42, one and 65 cases annually during 2010-16. Children (< 16 years-old; n = 150) were mostly affected and 71% (n = 121) of the total patients were under 5 years-old. In 146 patients with medical reviews, 73% (n = 107) presented with acute respiratory distress. Among paediatric patients with medical reviews (n = 133), 55% (n=73) had an asthma/wheezing history, while among adults (n = 13), 11 had underlying diseases. In total, 45 patients had severe infections and 28 patients needed intensive care unit stays. No acute flaccid myelitis (AFM) was detected. We found genotypes A, B1, B2 B3 and D circulating, and no associations between these and clinical presentations. During the study, new genotypes continuously emerged, being replaced over time. We estimated that ancestors of currently circulating genotypes emerged in the late-1990s to 2010. Rises of the EV-D68 effective population size in Lyon coincided with infection upsurges. Phylogenetic analyses showed ongoing diversification of EV-D68 worldwide, coinciding with more infections in recent years and increases of reported AFM paediatric cases. Conclusions: Reinforcement of diagnostic capacities and clinical-based surveillance of EV-D68 infections is needed in Europe to assess the EV-D68 burden.

Genome-wide mosaicism within Mycobacterium abscessus: evolutionary and epidemiological implications.

BACKGROUND: In mycobacteria, conjugation differs from the canonical Hfr model, but is still poorly understood. Here, we quantified this evolutionary processe in a natural mycobacterial population, taking advantage of a large clinical strain collection of the emerging pathogen Mycobacterium abscessus (MAB). RESULTS: Multilocus sequence typing confirmed the existence of three M. abscessus subspecies, and unravelled extensive allelic exchange between them. Furthermore, an asymmetrical gene flow occurring between these main lineages was detected, resulting in highly admixed strains. Intriguingly, these mosaic strains were significantly associated with cystic fibrosis patients with lung infections or chronic colonization. Genome sequencing of those hybrid strains confirmed that half of their genomic content was remodelled in large genomic blocks, leading to original tri-modal 'patchwork' architecture. One of these hybrid strains acquired a locus conferring inducible macrolide resistance, and a large genomic insertion from a slowly growing pathogenic mycobacteria, suggesting an adaptive gene transfer. This atypical genomic architecture of the highly recombinogenic strains is consistent with the distributive conjugal transfer (DCT) observed in M. smegmatis. Intriguingly, no known DCT function was found in M. abscessus chromosome, however, a p-RAW-like genetic element was detected in one of the highly admixed strains. CONCLUSION: Taken together, our results strongly suggest that MAB evolution is sporadically punctuated by dramatic genome wide remodelling events. These findings might have far reaching epidemiological consequences for emerging mycobacterial pathogens survey in the context of increasing numbers of rapidly growing mycobacteria and M. tuberculosis co-infections.

Introgressive hybridization and latitudinal admixture clines in North Atlantic eels.

BACKGROUND: Hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. Eels, both from North America and Europe migrate through the Atlantic to mate in a vast, overlapping area in the Sargasso Sea. Due to the lack of direct observation, it is unknown how these species remain reproductively isolated. The detection of inter-species hybrids in Iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in North Atlantic eels. RESULTS: Here, we show that while mitochondrial lineages remain completely distinct on both sides of the Atlantic, limited hybridization is detectable with nuclear DNA markers. The nuclear hybridization signal peaks in the northern areas and decreases towards the southern range limits on both continents according to Bayesian assignment analyses. By simulating increasing proportions of both F1 hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the European eel. Finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism. CONCLUSIONS: Fluctuating degrees of introgressive hybridization between Atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the European eel.

Emergence of bedaquiline-resistant tuberculosis and of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains with rpoB Ile491Phe mutation not detected by Xpert MTB/RIF in Mozambique: a retrospective observational study.

BACKGROUND: In 2021, an estimated 4800 people developed rifampicin-resistant tuberculosis in Mozambique, 75% of which went undiagnosed. Detailed molecular data on rifampicin-resistant and multidrug-resistant (MDR) tuberculosis are not available. Here, we aimed at gaining precise data on the determinants of rifampicin-resistant and MDR tuberculosis in Mozambique. METHODS: In this retrospective observational study, we performed whole-genome sequencing of 704 rifampicin-resistant Mycobacterium tuberculosis complex (Mtbc) strains submitted to the National Tuberculosis Reference Laboratory (NTRL) in Maputo, Mozambique, between 2015 and 2021. Phylogenetic strain classification, genomic resistance prediction, and cluster analysis were performed. FINDINGS: Between Jan 1, 2015, and July 31, 2021, 2606 Mtbc isolates with an isoniazid or rifampicin resistance were identified in the NTRL biobank, of which, 1483 (56·9%) were from men, 1114 (42·7%) from women, and nine (0·4%) were unknown. Genome-based drug-resistant prediction classified 704 Mtbc strains as rifampicin resistant. 628 (89%) of the 704 Mtbc strains were classified MDR; of those, 146 (23%) were pre-extensively drug resistant (pre-XDR; additional fluoroquinolone resistance), and 24 (4%) extensively drug resistant (XDR; combined fluoroquinolone and bedaquiline resistance). Overall, 61 (9%) of 704 strains revealed resistance to bedaquiline: five (7%) of 76 rifampicin resistant plus bedaquiline resistant, 32 (7%) of 458 MDR plus bedaquiline resistant, and 24 (100%) of 24 XDR. Prevalence of bedaquiline resistance increased from 3% in 2016 to 14% in 2021. The cluster rate (12 single-nucleotide polymorphism threshold) was 42% for rifampicin-resistant strains, 78% for MDR strains, 94% for pre-XDR strains, and 96% for XDR Mtbc strains. 31 (4%) of 704 Mtbc strains, belonging to a diagnostic escape outbreak strain previously described in Eswatini (group_56), had an rpoB Ile491Phe mutation which is not detected by Xpert MTB/RIF (no other rpoB mutation). Of these, 23 (74%) showed additional resistance to bedaquiline, 13 (42%) had bedaquiline and fluoroquinolone resistance, and two (6%) were bedaquiline, fluoroquinolone, and delamanid resistant. INTERPRETATION: Pre-XDR resistance is highly prevalent among MDR Mtbc strains in Mozambique and so is bedaquiline resistance; and the frequency of bedaquiline resistance quadrupled over time and was found even in Mtbc strains without fluoroquinolone resistance. Importantly, strains with Ile491Phe mutation were frequent, accounting for 31% (n=10) of MDR plus bedaquiline-resistant strains and 54% (n=13) of XDR Mtbc strains. Given the current diagnostic algorithms and treatment regimens, both the emergence of rifampicin resistance due to Ile491Phe and bedaquiline resistance might jeopardise MDR tuberculosis prevention and care unless sequencing-based technology is rolled out. The potential cross border spread of diagnostic escape strains needs further investigation. FUNDING: The German Ministry of Health through the Seq_MDRTB-Net project, the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy Precision Medicine in Inflammation and the Research Training Group 2501 TransEvo, the Leibniz Science Campus Evolutionary Medicine of the Lung, and the German Ministry of Education and Research via the German Center for Infection Research.

Whole genome sequencing versus traditional genotyping for investigation of a Mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study.

BACKGROUND: Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. METHODS AND FINDINGS: During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed "Hamburg clone") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. CONCLUSIONS: Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.

"Epidemic clones" of Listeria monocytogenes are widespread and ancient clonal groups.

The food-borne pathogen Listeria monocytogenes is genetically heterogeneous. Although some clonal groups have been implicated in multiple outbreaks, there is currently no consensus on how "epidemic clones" should be defined. The objectives of this work were to compare the patterns of sequence diversity on two sets of genes that have been widely used to define L. monocytogenes clonal groups: multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Further, we evaluated the diversity within clonal groups by pulsed-field gel electrophoresis (PFGE). Based on 125 isolates of diverse temporal, geographical, and source origins, MLST and MvLST genes (i) had similar patterns of sequence polymorphisms, recombination, and selection, (ii) provided concordant phylogenetic clustering, and (iii) had similar discriminatory power, which was not improved when we combined both data sets. Inclusion of representative strains of previous outbreaks demonstrated the correspondence of epidemic clones with previously recognized MLST clonal complexes. PFGE analysis demonstrated heterogeneity within major clones, most of which were isolated decades before their involvement in outbreaks. We conclude that the "epidemic clone" denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space.

A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus.

Due to the lack of fossil evidence, the timescales of bacterial evolution are largely unknown. The speed with which genetic change accumulates in populations of pathogenic bacteria, however, is a key parameter that is crucial for understanding the emergence of traits such as increased virulence or antibiotic resistance, together with the forces driving pathogen spread. Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of hospital-acquired infections. We have investigated an MRSA strain (ST225) that is highly prevalent in hospitals in Central Europe. By using mutation discovery at 269 genetic loci (118,804 basepairs) within an international isolate collection, we ascertained extremely low diversity among European ST225 isolates, indicating that a recent population bottleneck had preceded the expansion of this clone. In contrast, US isolates were more divergent, suggesting they represent the ancestral population. While diversity was low, however, our results demonstrate that the short-term evolutionary rate in this natural population of MRSA resulted in the accumulation of measurable DNA sequence variation within two decades, which we could exploit to reconstruct its recent demographic history and the spatiotemporal dynamics of spread. By applying Bayesian coalescent methods on DNA sequences serially sampled through time, we estimated that ST225 had diverged since approximately 1990 (1987 to 1994), and that expansion of the European clade began in 1995 (1991 to 1999), several years before the new clone was recognized. Demographic analysis based on DNA sequence variation indicated a sharp increase of bacterial population size from 2001 to 2004, which is concordant with the reported prevalence of this strain in several European countries. A detailed ancestry-based reconstruction of the spatiotemporal dispersal dynamics suggested a pattern of frequent transmission of the ST225 clone among hospitals within Central Europe. In addition, comparative genomics indicated complex bacteriophage dynamics.

Pleistocene desiccation in East Africa bottlenecked but did not extirpate the adaptive radiation of Lake Victoria haplochromine cichlid fishes.

The Great Lakes region of East Africa, including Lake Victoria, is the center of diversity of the mega-diverse cichlid fishes (Perciformes: Teleostei). Paleolimnological evidence indicates dramatic desiccation of this lake ca. 18,000-15,000 years ago. Consequently, the hundreds of extant endemic haplochromine species in the lake must have either evolved since then or refugia must have existed, within that lake basin or elsewhere, from which Lake Victoria was recolonized. We studied the population history of the Lake Victoria region superflock (LVRS) of haplochromine cichlids based on nuclear genetic analysis (12 microsatellite loci from 400 haplochomines) of populations from Lake Kivu, Lake Victoria, and the connected and surrounding rivers and lakes. Population genetic analyses confirmed that Lake Kivu haplochromines colonized Lake Victoria. Coalescent analyses show a 30- to 50-fold decline in the haplochromine populations of Lake Victoria, Lake Kivu, and the region ca. 18,000-15,000 years ago. We suggest that this coincides with drastic climatic and geological changes in the late Pleistocene. The most recent common ancestor of the Lake Victoria region haplochromines was estimated to have existed about 4.5 million years ago, which corresponds to the first radiation of cichlids in Lake Tanganyika and the origin of the tribe Haplochrominii. This relatively old evolutionary origin may explain the high levels of polymorphism still found in modern haplochromines. This degree of polymorphism might have acted as a "genetic reservoir" that permitted the explosive radiation of hundreds of haplochromines and their array of contemporary adaptive morphologies.

Forecasting Staphylococcus aureus Infections Using Genome-Wide Association Studies, Machine Learning, and Transcriptomic Approaches.

Staphylococcus aureus is a major human and animal pathogen, colonizing diverse ecological niches within its hosts. Predicting whether an isolate will infect a specific host and its subsequent clinical fate remains unknown. In this study, we investigated the S. aureus pangenome using a curated set of 356 strains, spanning a wide range of hosts, origins, and clinical display and antibiotic resistance profiles. We used genome-wide association study (GWAS) and random forest (RF) algorithms to discriminate strains based on their origins and clinical sources. Here, we show that the presence of sak and scn can discriminate strains based on their host specificity, while other genes such as mecA are often associated with virulent outcomes. Both GWAS and RF indicated the importance of intergenic regions (IGRs) and coding DNA sequence (CDS) but not sRNAs in forecasting an outcome. Additional transcriptomic analyses performed on the most prevalent clonal complex 8 (CC8) clonal types, in media mimicking nasal colonization or bacteremia, indicated three RNAs as potential RNA markers to forecast infection, followed by 30 others that could serve as infection severity predictors. Our report shows that genetic association and transcriptomics are complementary approaches that will be combined in a single analytical framework to improve our understanding of bacterial pathogenesis and ultimately identify potential predictive molecular markers. IMPORTANCE Predicting the outcome of bacterial colonization and infections, based on extensive genomic and transcriptomic data from a given pathogen, would be of substantial help for clinicians in treating and curing patients. In this report, genome-wide association studies and random forest algorithms have defined gene combinations that differentiate human from animal strains, colonization from diseases, and nonsevere from severe diseases, while it revealed the importance of IGRs and CDS, but not small RNAs (sRNAs), in anticipating an outcome. In addition, transcriptomic analyses performed on the most prevalent clonal types, in media mimicking either nasal colonization or bacteremia, revealed significant differences and therefore potent RNA markers. Overall, the use of both genomic and transcriptomic data in a single analytical framework can enhance our understanding of bacterial pathogenesis.

High fluoroquinolone resistance proportions among multidrug-resistant tuberculosis driven by dominant L2 Mycobacterium tuberculosis clones in the Mumbai Metropolitan Region.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. METHODS: In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. RESULTS: In total, 1016 MTBC strains were MDR, out of which 703 (69.2%) were pre-XDR and 45 (4.4%) were XDR. Cluster rates were high among MDR (57.8%) and pre-XDR/XDR (79%) strains with three dominant L2 (Beijing) strain clusters (Cl 1-3) representing half of the pre-XDR and 40% of the XDR-TB cases. L2 strains were associated with pre-XDR/XDR-TB (P < 0.001) and, particularly Cl 1-3 strains, had high first-line and FQ resistance rates (81.6-90.6%). Epidemic success analysis using THD showed that L2 strains outperformed L1, L3, and L4 strains in short- and long-term time scales. More importantly, L2 MDR and MDR + strains had higher THD success indices than their not-MDR counterparts. Overall, compensatory mutation rates were highest in L2 strains and positive selection was detected in genes of L2 strains associated with drug tolerance (prpB and ppsA) and virulence (Rv2828c). Compensatory mutations in L2 strains were associated with a threefold increase of THD indices, suggesting improved transmissibility. CONCLUSIONS: Our data indicate a drastic increase of FQ resistance, as well as emerging bedaquiline resistance which endangers the success of newly endorsed MDR-TB treatment regimens. Rapid changes in treatment and control strategies are required to contain transmission of highly successful pre-XDR L2 strains in the Mumbai Metropolitan region but presumably also India-wide.

Transcontinental spread and evolution of Mycobacterium tuberculosis W148 European/Russian clade toward extensively drug resistant tuberculosis.

Transmission-driven multi-/extensively drug resistant (M/XDR) tuberculosis (TB) is the largest single contributor to human mortality due to antimicrobial resistance. A few major clades of the Mycobacterium tuberculosis complex belonging to lineage 2, responsible for high prevalence of MDR-TB in Eurasia, show outstanding transnational distributions. Here, we determined factors underlying the emergence and epidemic spread of the W148 clade by genome sequencing and Bayesian demogenetic analyses of 720 isolates from 23 countries. We dated a common ancestor around 1963 and identified two successive epidemic expansions in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet Era. These population expansions favored accumulation of resistance mutations to up to 11 anti-TB drugs, with MDR evolving toward additional resistances to fluoroquinolones and second-line injectable drugs within 20 years on average. Timescaled haplotypic density analysis revealed that widespread acquisition of compensatory mutations was associated with transmission success of XDR strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected. The outstanding genetic arsenal of this geographically widespread M/XDR strain clade represents a "perfect storm" that jeopardizes the successful introduction of new anti-M/XDR-TB antibiotic regimens.

Exploring Semi-Quantitative Metagenomic Studies Using Oxford Nanopore Sequencing: A Computational and Experimental Protocol.

The gut microbiome plays a major role in chronic diseases, of which several are characterized by an altered composition and diversity of bacterial communities. Large-scale sequencing projects allowed for characterizing the perturbations of these communities. However, translating these discoveries into clinical applications remains a challenge. To facilitate routine implementation of microbiome profiling in clinical settings, portable, real-time, and low-cost sequencing technologies are needed. Here, we propose a computational and experimental protocol for whole-genome semi-quantitative metagenomic studies of human gut microbiome with Oxford Nanopore sequencing technology (ONT) that could be applied to other microbial ecosystems. We developed a bioinformatics protocol to analyze ONT sequences taxonomically and functionally and optimized preanalytic protocols, including stool collection and DNA extraction methods to maximize read length. This is a critical parameter for the sequence alignment and classification. Our protocol was evaluated using simulations of metagenomic communities, which reflect naturally occurring compositional variations. Next, we validated both protocols using stool samples from a bariatric surgery cohort, sequenced with ONT, Illumina, and SOLiD technologies. Results revealed similar diversity and microbial composition profiles. This protocol can be implemented in a clinical or research setting, bringing rapid personalized whole-genome profiling of target microbiome species.

Emergence and global spread of Listeria monocytogenes main clinical clonal complex.

The bacterial foodborne pathogen Listeria monocytogenes clonal complex 1 (Lm-CC1) is the most prevalent clonal group associated with human listeriosis and is strongly associated with cattle and dairy products. Here, we analyze 2021 isolates collected from 40 countries, covering Lm-CC1 first isolation to present days, to define its evolutionary history and population dynamics. We show that Lm-CC1 spread worldwide from North America following the Industrial Revolution through two waves of expansion, coinciding with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century. In sharp contrast to its global spread over the past century, transmission chains are now mostly local, with limited inter- and intra-country spread. This study provides an unprecedented insight into L. monocytogenes phylogeography and population dynamics and highlights the importance of genome analyses for a better control of pathogen transmission.

A new perspective on Listeria monocytogenes evolution.

Listeria monocytogenes is a model organism for cellular microbiology and host-pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype (4b for clones 1, 2 and 4, 1/2b for clones 3 and 5, 1/2a for clone 7, and 1/2c for clone 9), with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited (r/m<1 for nucleotides), implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence.

Origin, spread and demography of the Mycobacterium tuberculosis complex.

The evolutionary timing and spread of the Mycobacterium tuberculosis complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterial tandem repeat sequences as genetic markers, we show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. Using Bayesian statistics and experimental data on the variability of the mycobacterial markers in infected patients, we estimated the age of the MTBC at 40,000 years, coinciding with the expansion of "modern" human populations out of Africa. Furthermore, coalescence analysis revealed a strong and recent demographic expansion in almost all M. tuberculosis lineages, which coincides with the human population explosion over the last two centuries. These findings thus unveil the dynamic dimension of the association between human host and pathogen populations.

Applied phyloepidemiology: Detecting drivers of pathogen transmission from genomic signatures using density measures.

Understanding the driving forces of an epidemic is key to inform intervention strategies against it. Correlating measures of the epidemic success of a pathogen with ancillary parameters such as its drug resistance profile provides a flexible tool to identify such driving forces. The recently described time-scaled haplotypic density (THD) method facilitates the inference of a pathogen's epidemic success from genetic data. Contrary to demogenetic approaches that define success in an aggregated fashion, the THD computes an independent index of success for each isolate in a collection. Modeling this index using multivariate regression, thus, allows us to control for various sources of bias and to identify independent predictors of success. We illustrate the use of THD to address key questions regarding three exemplary epidemics of multidrug-resistant (MDR) bacterial lineages, namely Mycobacterium tuberculosis Beijing, Salmonella Typhi H58, and Staphylococcus aureus ST8 (including ST8-USA300 MRSA), based on previously published, international genetic datasets. In each case, THD analysis allowed to identify the impact, or lack thereof, of various factors on the epidemic success, independent of confounding by population structure and geographic distribution. Our results suggest that rifampicin resistance drives the MDR Beijing epidemic and that fluoroquinolone resistance drives the S. aureus ST8/USA300 epidemic, in line with previous evidence of a lack of resistance-associated fitness cost in these pathogens. Conversely, fluoroquinolone resistance measurably hampered the success of S. Typhi H58 and non-H58. These findings illustrate how THD can help leverage the massive genomic datasets generated by molecular epidemiology studies to address new questions. THD implementation for the R platform is available at https://github.com/rasigadelab/thd.