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The incidence of gonorrhoea is increasing at an alarming pace, and therapeutic options continue to narrow as a result of worsening drug resistance. Neisseria gonorrhoeae is naturally competent, allowing the organism to adapt rapidly to selection pressures including antibiotics. A sub-population of N. gonorrhoeae carries the Gonococcal Genetic Island (GGI), which encodes a type IV secretion system (T4SS) that secretes chromosomal DNA. Previous research has shown that the GGI increases transformation efficiency in vitro, but the extent to which it contributes to horizontal gene transfer (HGT) during infection is unknown. Here we analysed genomic data from clinical isolates of N. gonorrhoeae to better characterize GGI+ and GGI− sub-populations and to delineate patterns of variation at the locus itself. We found the element segregating at an intermediate frequency (61%), and it appears to act as a mobile genetic element with examples of gain, loss, exchange and intra-locus recombination within our sample. We further found evidence suggesting that GGI+ and GGI− sub-populations preferentially inhabit distinct niches with different opportunities for HGT. Previously, GGI+ isolates were reported to be associated with more severe clinical infections, and our results suggest this could be related to metal-ion trafficking and biofilm formation. The co-segregation of GGI+ and GGI− isolates despite mobility of the element suggests that both niches inhabited by N. gonorrhoeae remain important to its overall persistence as has been demonstrated previously for cervical- and urethral-adapted sub-populations. These data emphasize the complex population structure of N. gonorrhoeae and its capacity to adapt to diverse niches.

TheGardnerellagenus, now made up of more than 13 species, is associated with the polymicrobial disorder bacterial vaginosis (BV). However, the details of BV pathogenesis are poorly defined, and the contributions made by individual species are largely unknown. We report here that colony phenotypes characterized by size (large and small) and opacity (opaque and translucent) are phase variable and are conserved among all testedGardnerellastrains, representing at least ten different species. With the hypothesis that these different variants could be an important missing piece to the enigma of how BV develops in vivo, we characterized their differences. Beyond increased colony size, large colony variants (Lg) showed reduced vaginolysin secretion and faster growth rate relative to small colony variants (Sm). The ability to inhibit growth ofNeisseria gonorrhoeaeand commensal lactobacillus species varied by strain and in some instances differed between variants. Proteomics analyses indicate that 127-173 proteins are differentially expressed between variants. Further, whole genome sequencing analyses revealed an abundance of genes associated with variable poly-guanine tracts, implicating slipped strand mispairing inGardnerellaphase variation, and illuminating the potential for previously unrecognized variability within clonal populations. Collectively, these results suggest that colony variants may be primed to serve different roles in BV pathogenesis.

In this issue of Cell, Saelens et al. describe a new function for mycobacterial Type VII secretion systems: manipulation of host cell migration. They find that a substantial proportion of global TB cases arise from bacteria lacking this function, raising questions about its role in pathoadaptation of Mycobacterium tuberculosis.

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests utilizing molecularly intact heme. Here we describe the mechanism underlying this trait, which we discovered using molecular genetics, phylogenetics, and population analyses. Most metronidazole-resistant strains evolved a T to G mutation, we term PnimBG, in the -10 regulatory promoter of the 5-nitroimidazole reductase nimB, resulting in the gene being constitutively transcribed. Silencing or deleting nimB eliminated metronidazole resistance. We identified the protein as a heme-dependent nitroreductase that degraded nitro-drugs to an amine lacking antimicrobial activity. We further discovered that the metronidazole-resistant PnimBGmutation was strongly associated with the Thr82Ile substitution conferring fluoroquinolone resistance in epidemic strains. Re-analysis of published genomes from global isolates confirmed that all but one encoding PnimBG also carried the Thr82Ile mutation. Our findings suggest that fluoroquinolone and metronidazole resistance co-mediated the pandemic of healthcare-associated C. difficile that are associated with poorer treatment outcomes in CDI patients receiving metronidazole.

The viridans group streptococci (VGS) are a large consortium of commensal streptococci that colonize the human body. Many species within this group are opportunistic pathogens causing bacteremia and infective endocarditis (IE), yet little is known about why some strains cause invasive disease. Identification of virulence determinants is complicated by the difficulty of distinguishing between the closely related species of this group. Here, we analyzed genomic data from VGS isolated from patient blood cultures with invasive infections and from oral swabs of healthy volunteers and determined the best performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample ofStreptococcus oralisisolates and found evidence of frequent recombination. We used multiple genome-wide association study tools to identify candidate determinants of invasiveness. These tools gave consistent results, leading to the discovery of a single synonymous single nucleotide polymorphism (SNP) that was significantly associated with invasiveness. This SNP is within a previously undescribed gene that is conserved across the majority of VGS species. Using growth in the presence of human serum and a simulated infective endocarditis vegetation model, we were unable to identify a phenotype for the enriched allele in laboratory assays, suggesting a phenotype may be specific to natural infection. These data highlight the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS.ImportanceThe viridians group streptococci (VGS) are a large collection of closely related commensal streptococci, with many being opportunistic pathogens causing invasive diseases such as bacteremia and infective endocarditis. Little is known about virulence determinants in these species, and there is a distinct lack of genomic information available for the VGS. In this study, we collected VGS isolates from invasive infections and healthy volunteers and performed whole genome sequencing for a suite of downstream analyses. We focused on a diverse sample ofStreptococcus oralisgenomes and identified high rates of recombination in the population as well as a single genome variant highly enriched in invasive isolates. The variant lies within a previously uncharacterized gene,nrdM, which shares homology with the anaerobic ribonucleoside triphosphate reductase,nrdD, and is highly conserved among VGS. This work increases our knowledge of VGS genomics and indicates that differences in virulence potential amongS. oralisisolates is, at least in part, genetically determined.

Objective A recent study reported a tuberculosis (TB) outbreak in which, among newly infected individuals, exposure to additional active infections was associated with a higher probability of developing active disease. Referred to as complex contagion, multiple reexposures to TB within a short period after initial infection is hypothesized to confer a greater likelihood of developing active infection in 1 y. The purpose of this article is to develop and validate an agent-based simulation model (ABM) to study the effect of complex contagion on population-level TB transmission dynamics. Methods We built an ABM of a TB epidemic using data from a series of outbreaks recorded in the 20th century in Saskatchewan, Canada. We fit 3 dynamical schemes: base, with no complex contagion; additive, in which each reexposure confers an independent risk of activated infection; and threshold, in which a small number of reexposures confers a low risk and a high number of reexposures confers a high risk of activation. Results We find that the base model fits the mortality and incidence output targets best, followed by the threshold and then the additive models. The threshold model fits the incidence better than the base model does but overestimates mortality. All 3 models produce qualitatively realistic epidemic curves. Conclusion We find that complex contagion qualitatively changes the trajectory of a TB epidemic, although data from a high-incidence setting are reproduced better with the base model. Results from this model demonstrate the feasibility of using ABM to capture nuances in TB transmission.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), is a leading cause of death due to infectious disease. TB is not traditionally associated with biofilms, but M. tb biofilms are linked with drug and immune tolerance and there is increasing recognition of their contribution to the recalcitrance of TB infections. Here, we used M. tb experimental evolution to investigate this complex phenotype and identify candidate loci controlling biofilm formation. We identified novel candidate loci, adding to our understanding of the genetic architecture underlying M. tb biofilm development. Under selective pressure to grow as a biofilm, regulatory mutations rapidly swept to fixation and were associated with changes in multiple traits, including extracellular matrix production, cell size, and growth rate. Genetic and phenotypic paths to enhanced biofilm growth varied according to the genetic background of the parent strain, suggesting that epistatic interactions are important in M. tb adaptation to changing environments.

Mycobacterium tuberculosis is a globally distributed, lethal pathogen of humans. The virulence armamentarium of M. tuberculosis appears to have been developed on a scaffold of antiphagocytic defenses found among diverse, mostly free-living species of Mycobacterium. Pathoadaptation was further aided by the modularity, flexibility, and interactivity characterizing mycobacterial effectors and their regulators. During emergence of M. tuberculosis, novel genetic material was acquired, created, and integrated with existing tools. The major mutational mechanisms underlying these adaptations are discussed in this review, with examples. During its evolution, M. tuberculosis lost the ability and/or opportunity to engage in lateral gene transfer, but despite this it has retained the adaptability that characterizes mycobacteria. M. tuberculosis exemplifies the evolutionary genomic mechanisms underlying adoption of the pathogenic niche, and studies of its evolution have uncovered a rich array of discoveries about how new pathogens are made.

Mycobacterium abscessus is a rapid growing, free-living species of bacterium that also causes lung infections in humans. Human infections are usually acquired from the environment; however, dominant circulating clones (DCCs) have emerged recently in both M. abscessus subsp. massiliense and subsp. abscessus that appear to be transmitted among humans and are now globally distributed. These recently emerged clones are potentially informative about the ecological and evolutionary mechanisms of pathogen emergence and host adaptation. The geographical distribution of DCCs has been reported, but the genomic processes underlying their transition from environmental bacterium to human pathogen are not well characterized. To address this knowledge gap, we delineated the structure of M. abscessus subspecies abscessus and massiliense using genomic data from 200 clinical isolates of M. abscessus from seven geographical regions. We identified differences in overall patterns of lateral gene transfer (LGT) and barriers to LGT between subspecies and between environmental and host-adapted bacteria. We further characterized genome reorganization that accompanied bacterial host adaptation, inferring selection pressures acting at both genic and intergenic loci. We found that both subspecies encode an expansive pangenome with many genes at rare frequencies. Recombination appears more frequent in M. abscessus subsp. massiliense than in subsp. abscessus, consistent with prior reports. We found evidence suggesting that phage are exchanged between subspecies, despite genetic barriers evident elsewhere throughout the genome. Patterns of LGT differed according to niche, with less LGT observed among host-adapted DCCs versus environmental bacteria. We also found evidence suggesting that DCCs are under distinct selection pressures at both genic and intergenic sites. Our results indicate that host adaptation of M. abscessus was accompanied by major changes in genome evolution, including shifts in the apparent frequency of LGT and impacts of selection. Differences were evident among the DCCs as well, which varied in the degree of gene content remodelling, suggesting they were placed differently along the evolutionary trajectory toward host adaptation. These results provide insight into the evolutionary forces that reshape bacterial genomes as they emerge into the pathogenic niche.

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels, but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. These data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

11 páginas, 6 figuras, 2 tablas. Accesible en PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295914/, A small number of high-burden countries account for the majority of tuberculosis cases worldwide. Detailed data are lacking from these regions. To explore the evolutionary history of Mycobacterium tuberculosis in China-the country with the third highest tuberculosis burden-we analysed a countrywide collection of 4,578 isolates. Little genetic diversity was detected, with 99.4% of the bacterial population belonging to lineage 2 and three sublineages of lineage 4. The deeply rooted phylogenetic positions and geographic restriction of these four genotypes indicate that their populations expanded in situ following a small number of introductions to China. Coalescent analyses suggest that these bacterial subpopulations emerged in China around 1,000 years ago, and expanded in parallel from the twelfth century onwards, and that the whole population peaked in the late eighteenth century. More recently, sublineage L2.3, which is indigenous to China and exhibited relatively high transmissibility and extensive global dissemination, came to dominate the population dynamics of M. tuberculosis in China. Our results indicate that historical expansion of four M. tuberculosis strains shaped the current tuberculosis epidemic in China, and highlight the long-term genetic continuity of the indigenous M. tuberculosis population., This work was supported by the Natural Science Foundation of China (91631301 and 81661128043 to Q.G. and 81701975 to Q.L.). C.S.P. was supported by the National Institutes of Health (grant 1R01AI113287-01A1). S.G. was supported by the Swiss National Science Foundation (grants IZRJZ3_164171, 310030_166687, IZLSZ3_170834 and CRSII5_177163). This work was also supported by MINECO research grant SAF2016-77346-R (to I.C.), the European Research Council (638553-TB-ACCELERATE to I.C.), the National Science and Technology Major Project of China (2017ZX10201302 to Q.G., 2018ZX10103001 to Y.Z.), the Sanming Project of Medicine in Shenzhen (SZSM201611030 to Q.G.), JSGG20170413142559220 (to Q.G.), and National Basic Research programme of China (2014CB744403 to Y.Z.).

Mycobacterium abscessus is a rapid growing, free-living species of bacterium that also causes lung infections in humans. Human infections are usually acquired from the environment; however, dominant circulating clones (DCCs) have emerged recently in both M. abscessus subsp. massiliense and subsp. abscessus that appear to be transmitted among humans and are now globally distributed. These recently emerged clones are potentially informative about the ecological and evolutionary mechanisms of pathogen emergence and host adaptation. The geographical distribution of DCCs has been reported, but the genomic processes underlying their transition from environmental bacterium to human pathogen are not well characterized. To address this knowledge gap, we delineated the structure of M. abscessus subspecies abscessus and massiliense using genomic data from 200 clinical isolates of M. abscessus from seven geographical regions. We identified differences in overall patterns of lateral gene transfer (LGT) and barriers to LGT between subspecies and between environmental and host-adapted bacteria. We further characterized genome reorganization that accompanied bacterial host adaptation, inferring selection pressures acting at both genic and intergenic loci. We found that both subspecies encode an expansive pangenome with many genes at rare frequencies. Recombination appears more frequent in M. abscessus subsp. massiliense than in subsp. abscessus, consistent with prior reports. We found evidence suggesting that phage are exchanged between subspecies, despite genetic barriers evident elsewhere throughout the genome. Patterns of LGT differed according to niche, with less LGT observed among host-adapted DCCs versus environmental bacteria. We also found evidence suggesting that DCCs are under distinct selection pressures at both genic and intergenic sites. Our results indicate that host adaptation of M. abscessus was accompanied by major changes in genome evolution, including shifts in the apparent frequency of LGT and impacts of selection. Differences were evident among the DCCs as well, which varied in the degree of gene content remodelling, suggesting they were placed differently along the evolutionary trajectory toward host adaptation. These results provide insight into the evolutionary forces that reshape bacterial genomes as they emerge into the pathogenic niche.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties following the statewide “Safer at Home” order, which went into effect 25 March 2020. Our results suggest patterns of SARS-CoV-2 transmission may vary substantially even in nearby communities. Understanding these local patterns will enable better targeting of public health interventions., In this study, the authors present an analysis of 247 full-genome SARS-CoV-2 sequences obtained from two communities in Wisconsin, USA, and report distinct patterns of viral spread. Their results suggest that patterns of SARS-CoV-2 transmission and spread may vary substantially, even between neighbouring communities.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties, and limited viral transmission between counties, following the statewide “Safer at Home” public health order, which went into effect 25 March 2020. Our results suggest that early containment efforts suppressed the spread of SARS-CoV-2 within Wisconsin.

Selective pressures imposed by pathogens have varied among human populations throughout their evolution, leading to marked inter-population differences at some genes mediating susceptibility to infectious and immune-related diseases. Here, we investigated the evolutionary history of a common polymorphism resulting in a Y529 versus C529 change in the cadherin related family member 3 (CDHR3) receptor which underlies variable susceptibility to rhinovirus-C infection and is associated with severe childhood asthma. The protective variant is the derived allele and is found at high frequency worldwide (69–95%). We detected genome-wide significant signatures of natural selection consistent with a rapid increase of the haplotypes carrying the allele, suggesting that non-neutral processes have acted on this locus across all human populations. However, the allele has not fixed in any population despite multiple lines of evidence suggesting that the mutation predates human migrations out of Africa. Using an approximate Bayesian computation method, we estimate the age of the mutation while explicitly accounting for past demography and positive or frequency-dependent balancing selection. Our analyses indicate a single emergence of the mutation in anatomically modern humans ~150 000 years ago and indicate that balancing selection has maintained the beneficial allele at high equilibrium frequencies worldwide. Apart from the well-known cases of the MHC and ABO genes, this study provides the first evidence that negative frequency-dependent selection plausibly acted on a human disease susceptibility locus, a form of balancing selection compatible with typical transmission dynamics of communicable respiratory viruses that might exploit CDHR3.

Blastomycosis is a potentially life-threatening infection caused by the fungus Blastomyces dermatitidis. As with related fungal diseases, blastomycosis is noted to affect some populations more than others. These patterns of illness are often not related to predisposing conditions or exposure risks; thus, genetic differences are thought to underlie these health disparities. People of Hmong ancestry in Wisconsin are at elevated risk of blastomycosis compared to the general population. We studied the genetic codes of Hmong blastomycosis patients and identified candidate sites in their genomes that may explain their susceptibility to this infection. We further studied one particular region of the genome that is involved with the immune processes that fight B. dermatitidis. Our work revealed population differences in the response to fungi. A better understanding of the genetic underpinnings of susceptibility to infectious diseases has broader implications for community health, especially in the paradigm of personalized medicine., Genetic differences are hypothesized to underlie ethnic disparities in incidence rates of the endemic systemic mycoses, including blastomycosis. Individuals of Hmong ancestry display elevated risk for this serious fungal infection. Here, we interrogated the genomes of Wisconsin (WI) Hmong blastomycosis patients using homozygosity mapping to uncover regions of the genome that are likely shared among the greater Hmong population and filtered for variants with high potential to affect disease susceptibility. This approach uncovered 113 candidate susceptibility variants, and among the most promising are those in genes involved in the interleukin-17 (IL-17) response. In particular, we identified 25 linked variants near the gene encoding IL-6 (IL6). We validated differences in cytokine production between Hmong and European volunteers and formally demonstrated a critical role for IL-6 in the development of adaptive immunity to Blastomyces dermatitidis. Our findings suggest that the dysregulation of IL-17 responses underlies a recently reported and poorly understood ethnic health disparity.

The Mycobacterium tuberculosis complex lineage 4 (L4), also known as the “Euro-American” lineage, is the most widely dispersed of the seven human adapted lineages. L4 is comprised of ten sublineages including L4.4, which has a moderate global distribution and is the most common L4 sublineage in New Zealand. We have used a phylodynamics approach and a dataset of 236 global M. tuberculosis genomes to trace the origins and dispersal of L4.4 strains in New Zealand that are predominantly found in Māori and Pacific people. We identify an L4.4.1.1 sublineage clade of European origin, likely French, that is prevalent in indigenous populations in both New Zealand and Canada. Molecular dating suggests that expansion of European trade networks in the early 19th century led to dispersal of this clade to the South Pacific. We also identify historical and social factors within the region that have contributed to the local spread and expansion of these strains, including recent Pacific migrations to New Zealand and the rapid urbanization of Māori in the 20th century. Our results offer new insight into the dispersal of M. tuberculosis in the South Pacific region and provide a striking example of the role of historical European migrations in the dispersal of M. tuberculosis.Author SummaryTuberculosis kills more people worldwide than any other infectious disease and indigenous populations are disproportionately affected by the disease. Here, we have used a large global dataset of Mycobacterium tuberculosis bacterial genomes to trace the historical origins of tuberculosis strains in New Zealand that are most frequently found in Māori and Pacific people. These strains are locally known as the ‘Rangipo’ and ‘Otara’ strains (both Māori place names) and belong to the “Euro-American” lineage of M. tuberculosis. Via genome analysis, we find that these strains are closely related to M. tuberculosis strains found in indigenous populations in Canada that have a European origin. We used a molecular dating approach (a molecular clock) to infer the ages of these strains and date divergence events. The timing we infer corresponds to the introduction of these strains to Polynesia via expanding European trade networks in the South Pacific in the early 19th century and suggests that the Otara strain has migrated to New Zealand from the Pacific Islands multiple times. Our results provide insight into human social phenomena underlying the expansion and dispersal of M. tuberculosis and reassert the important role of European colonial migrations in the global dispersal of the M. tuberculosis Euro-American lineage. This work also highlights the pejorative and stigmatizing mislabelling of the New Zealand strains with indigenous Māori place names, suggesting that these strains should be renamed.

Mycobacterium tuberculosis (M.tb) is a globally distributed, obligate pathogen of humans that can be divided into seven clearly defined lineages. An emerging consensus places the origin and global dispersal of M.tb within the past 6,000 years: identifying how the ancestral clone of M.tb spread and differentiated within this timeframe is important for identifying the ecological drivers of the current pandemic. We used Bayesian phylogeographic inference to reconstruct the migratory history of M.tb in Africa and Eurasia and to investigate lineage specific patterns of spread from a geographically diverse sample of 552 M.tb genomes. Applying evolutionary rates inferred with ancient M.tb genome calibration, we estimated the timing of major events in the migratory history of the pathogen. Inferred timings contextualize M.tb dispersal within historical phenomena that altered patterns of connectivity throughout Africa and Eurasia: trans‐Indian Ocean trade in spices and other goods, the Silk Road and its predecessors, the expansion of the Roman Empire, and the European Age of Exploration. We found that Eastern Africa and Southeast Asia have been critical in the dispersal of M.tb. Our results further reveal that M.tb populations have grown through range expansion, as well as in situ, and delineate the independent evolutionary trajectories of bacterial subpopulations underlying the current pandemic.

Repeated emergence, not international dissemination, is behind the rise of multidrug-resistant lineage 4 tuberculosis., On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.

A recent study reported on a tuberculosis (TB) outbreak in a largely Inuit village. Among newly infected individuals, exposure to additional active cases was associated with an increasing probability of developing active disease within a year. Using binomial risk models, we evaluated two potential mechanisms by which multiple infections during the first year following initial infection could account for increasing disease risk with increasing exposures. In the reinfection model , each infectious contact confers an independent risk of an infection, and infections contribute independently to active disease. In the threshold model , disease risk follows a sigmoidal function with small numbers of infectious contacts conferring a low risk of active disease and large numbers of contacts conferring a high risk. To determine the dynamic impact of reinfection during the early phase of infection, we performed simulations from a modified Reed–Frost model of TB dynamics following spread from an initial number of cases. We parametrized this model with the maximum-likelihood estimates from the reinfection and threshold models in addition to the observed distribution of exposures among new infections. We find that both models can plausibly account for the observed increase in disease risk with increasing infectious contacts, but the threshold model confers a better fit than a nested model without a threshold ( p = 0.04). Our simulations indicate that multiple exposures to infectious individuals during this critical time period can lead to dramatic increases in outbreak size. In order to decrease TB burden in high-prevalence settings, it may be necessary to implement measures aimed at preventing repeated exposures, in addition to preventing primary infection.

Mycobacterium tuberculosis(M.tb) is a globally distributed, obligate pathogen of humans that can be divided into seven clearly defined lineages. Identifying how the ancestral clone ofM.tbspread and differentiated is important for identifying the ecological drivers of the current pandemic. We reconstructedM.tbmigration in Africa and Eurasia, and investigated lineage specific patterns of spread. Applying evolutionary rates inferred with ancientM.tbgenome calibration, we linkM.tbdispersal to historical phenomena that altered patterns of connectivity throughout Africa and Eurasia: trans-Indian Ocean trade in spices and other goods, the Silk Road and its predecessors, the expansion of the Roman Empire and, the European Age of Exploration. We find that Eastern Africa and Southeast Asia have been critical in the dispersal ofM.tb. Our results reveal complex relationships between spatial dispersal and expansion ofM.tbpopulations, and delineate the independent evolutionary trajectories of bacterial sub-populations underlying the current pandemic.

Selective pressures imposed by pathogens have varied among human populations throughout their evolution, leading to marked inter-population differences at some genes mediating susceptibility to infectious and immune-related diseases. A common polymorphism resulting in a C529 versus T529 change in the Cadherin-Related Family Member 3 (CDHR3) receptor is associated with rhinovirus-C (RV-C) susceptibility and severe childhood asthma. Given the morbidity and mortality associated with RV-C dependent respiratory infections and asthma, we hypothesized that the protective variant has been under selection in the human population. Supporting this idea, a recent cross-species outbreak of RV-C among chimpanzees in Uganda, which carry the ancestral ‘risk’ allele at this position, resulted in a mortality rate of 8.9%. Using publicly available genomic data, we sought to determine the evolutionary history and role of selection acting on this infectious disease susceptibility locus. The protective variant is the derived allele and is found at high frequency worldwide, with the lowest relative frequency in African populations and highest in East Asian populations. There is minimal population structure among haplotypes, and we detect genomic signatures consistent with a rapid increase in frequency of the protective allele across all human populations. However, given strong evidence that the protective allele arose in anatomically modern humans prior to their migrations out of Africa and that the allele has not fixed in any population, the patterns observed here are not consistent with a classical selective sweep. We hypothesize that patterns may indicate frequency-dependent selection worldwide. Irrespective of the mode of selection, our analyses show the derived allele has been subject to selection in recent human evolution.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure on M. tuberculosis populations. Identifying the mutations that cause drug resistance in M. tuberculosis is important for guiding TB treatment and halting the spread of drug resistance. Whole-genome sequencing (WGS) of M. tuberculosis isolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug-resistant M. tuberculosis to characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, in M. tuberculosis and other clonal pathogens., Tuberculosis (TB) is the leading cause of death by an infectious disease, and global TB control efforts are increasingly threatened by drug resistance in Mycobacterium tuberculosis. Unlike most bacteria, where lateral gene transfer is an important mechanism of resistance acquisition, resistant M. tuberculosis arises solely by de novo chromosomal mutation. Using whole-genome sequencing data from two natural populations of M. tuberculosis, we characterized the population genetics of known drug resistance loci using measures of diversity, population differentiation, and convergent evolution. We found resistant subpopulations to be less diverse than susceptible subpopulations, consistent with ongoing transmission of resistant M. tuberculosis. A subset of resistance genes (“sloppy targets”) were characterized by high diversity and multiple rare variants; we posit that a large genetic target for resistance and relaxation of purifying selection contribute to high diversity at these loci. For “tight targets” of selection, the path to resistance appeared narrower, evidenced by single favored mutations that arose numerous times in the phylogeny and segregated at markedly different frequencies in resistant and susceptible subpopulations. These results suggest that diverse genetic architectures underlie drug resistance in M. tuberculosis and that combined approaches are needed to identify causal mutations. Extrapolating from patterns observed for well-characterized genes, we identified novel candidate variants involved in resistance. The approach outlined here can be extended to identify resistance variants for new drugs, to investigate the genetic architecture of resistance, and when phenotypic data are available, to find candidate genetic loci underlying other positively selected traits in clonal bacteria. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure on M. tuberculosis populations. Identifying the mutations that cause drug resistance in M. tuberculosis is important for guiding TB treatment and halting the spread of drug resistance. Whole-genome sequencing (WGS) of M. tuberculosis isolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug-resistant M. tuberculosis to characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, in M. tuberculosis and other clonal pathogens.

Tuberculosis (TB) is the leading cause of death by an infectious disease, and global TB control efforts are increasingly threatened by drug resistance inMycobacterium tuberculosis (M. tb).Unlike most bacteria, where lateral gene transfer is an important mechanism of resistance acquisition, resistantM. tbarises solely byde novochromosomal mutation. Using whole genome sequencing data from two natural populations ofM. tb,we characterized the population genetics of known drug resistance loci using measures of diversity, population differentiation, and convergent evolution. We found resistant sub-populations to be less diverse than susceptible sub-populations, consistent with ongoing transmission of resistantM. tb.A subset of resistance genes (“sloppy targets”) were characterized by high diversity and multiple rare variants; we posit that a large genetic target for resistance and relaxation of purifying selection contribute to high diversity at these loci. For “tight targets” of selection, the path to resistance appeared narrower, evidenced by single favored mutations that arose numerous times on the phylogeny and segregated at markedly different frequencies in resistant and susceptible sub-populations. These results suggest that diverse genetic architectures underlie drug resistance inM. tb,and combined approaches are needed to identify causal mutations. Extrapolating from patterns observed in well-characterized genes, we identified novel candidate variants involved in resistance. The approach outlined here can be extended to identify resistance variants for new drugs, to investigate the genetic architecture of resistance, and, when phenotypic data are available, to find candidate genetic loci underlying other positively selected traits in clonal bacteria.ImportanceMycobacterium tuberculosis (M. tb),the causative agent of tuberculosis (TB), is a significant burden on global health. Antibiotic treatment imposes strong selective pressure onM. tbpopulations. Identifying the mutations that cause drug resistance inM. tbis important for guiding TB treatment and halting the spread of drug resistance. Whole genome sequencing (WGS) ofM. tbisolates can be used to identify novel mutations mediating drug resistance and to predict resistance patterns faster than traditional methods of drug susceptibility testing. We have used WGS from natural populations of drug resistantM. tbto characterize effects of selection for advantageous mutations on patterns of diversity at genes involved in drug resistance. The methods developed here can be used to identify novel advantageous mutations, including new resistance loci, inM. tband other clonal pathogens.

Pregnancy complications are poorly represented in the archeological record, despite their importance in contemporary and ancient societies. While excavating a Byzantine cemetery in Troy, we discovered calcified abscesses among a woman’s remains. Scanning electron microscopy of the tissue revealed ‘ghost cells’, resulting from dystrophic calcification, which preserved ancient maternal, fetal and bacterial DNA of a severe infection, likely chorioamnionitis. Gardnerella vaginalis and Staphylococcus saprophyticus dominated the abscesses. Phylogenomic analyses of ancient, historical, and contemporary data showed that G. vaginalis Troy fell within contemporary genetic diversity, whereas S. saprophyticus Troy belongs to a lineage that does not appear to be commonly associated with human disease today. We speculate that the ecology of S. saprophyticus infection may have differed in the ancient world as a result of close contacts between humans and domesticated animals. These results highlight the complex and dynamic interactions with our microbial milieu that underlie severe maternal infections. DOI: http://dx.doi.org/10.7554/eLife.20983.001, eLife digest Why and how have some bacteria evolved to cause illness in humans? One way to study bacterial evolution is to search for ancient samples of bacteria and use DNA sequencing technology to investigate how modern bacteria have changed from their ancestors. Understanding the evolution process may help researchers to understand how some bacteria become resistant to the antibiotics designed to kill them. Complications that occur during pregnancy, including bacterial infections, have long been a major cause of death for women. Now, Devault, Mortimer et al. have been able to sequence the DNA of bacteria found in tissue collected from a woman buried 800 years ago in a cemetery in Troy. Some of the woman’s tissues had been well preserved because they had calcified (probably as the result of infection), which preserved their structure in a mineralized layer. Two mineralized “nodules” in the body appear to be the remains of abscesses. Some of the human DNA in the nodules came from a male, suggesting that the woman was pregnant with a boy and that the abscesses formed in placental tissue. Sequencing the DNA of the bacteria in the abscess allowed Devault, Mortimer et al. to diagnose the woman’s infection, which was caused by two types of bacteria. One species, called Gardnerella vaginalis, is found in modern pregnancy-related infections. The DNA of the ancient samples was similar to that of modern bacteria. The other bacteria species was an ancient form of Staphylococcus saprophyticus, a type of bacteria that causes urinary tract infections. However, the DNA of the ancient S. saprophyticus bacteria is quite different to that of the bacteria found in modern humans. Instead, their DNA sequence appears more similar to forms of the bacteria that infect currently livestock. As humans lived closely with their livestock at the time the woman lived, her infection may be due to a type of bacteria that passed easily between humans and animals. Overall, the results suggest that the disease-causing properties of bacteria can arise from a wide range of sources. In addition, Devault, Mortimer et al. have demonstrated that certain types of tissue found in archeological remains are a potential gold mine of information about the evolution of bacteria and other microbes found in the human body. DOI: http://dx.doi.org/10.7554/eLife.20983.002

Distributive conjugal transfer (DCT) is a newly described mechanism of lateral gene transfer (LGT) that results in a mosaic transconjugant structure, similar to the products of meiosis. We have tested popular LGT detection methods on whole-genome sequence data from experimental DCT transconjugants and used the best performing methods to compare genomic signatures of DCT with those of LGT through natural transformation, conjugative plasmids, and mobile genetic elements (MGE). We found that DCT results in transfer of larger chromosomal segments, that these segments are distributed more broadly around the chromosome, and that a greater proportion of the chromosome is affected by DCT than by other mechanisms of LGT. We used the best performing methods to characterize LGT in Mycobacterium canettii, the mycobacterial species most closely related to Mycobacterium tuberculosis. Patterns of LGT among M. canettii were highly distinctive. Gene flow appeared unidirectional, from lineages with minimal evidence of LGT to isolates with a substantial proportion (6–13%) of sites identified as recombinant. Among M. canettii isolates with evidence of LGT, recombinant fragments were larger and more evenly distributed relative to bacteria that undergo LGT through natural transformation, conjugative plasmids, and MGE. Spatial bias in M. canettii was also unusual in that patterns of recombinant fragment sharing mirrored overall phylogenetic structure. Based on the proportion of recombinant sites, the size of recombinant fragments, their spatial distribution and lack of association with MGE, as well as unidirectionality of DNA transfer, we conclude that DCT is the predominant mechanism of LGT among M. canettii.

Genetic research with Hmong-ancestry populations has examined differentiation among other Southeast Asian groups and select health conditions; however, there have been few discussions of specific methodological approaches in the literature. Studies within ethnically diverse communities must conduct culturally competent research in order to avoid stigmatization and harm to the communities. We present recommendations for conducting culturally competent genetic research with Hmong-ancestry populations through insights from interviews and observations from a pilot study examining a potential genetic basis of susceptibility to a fungal infection within a Hmong community. Implications for future genetic-based health research and public health are discussed.

Lateral gene transfer is an important mechanism for evolution among bacteria. Here, genome-wide gene insertion and deletion rates are modeled in a maximum-likelihood framework with the additional flexibility of modeling potential missing data. The performance of the models is illustrated using simulations and a data set on gene family phyletic patterns from Gardnerella vaginalis that includes an ancient taxon. A novel application involving pseudogenization/genome reduction magnitudes is also illustrated, using gene family data from Mycobacterium spp. Finally, an R package called indelmiss is available from the Comprehensive R Archive Network at https://cran.r-project.org/package=indelmiss, with support documentation and examples.

Mycobacteria have a distinct secretion system, termed type VII (T7SS), which is encoded by paralogous chromosomal loci (ESX) and associated with pathogenesis, conjugation, and metal homeostasis. Evolution of paralogous gene families is of interest because duplication is an important mechanism by which novel genes evolve, but there are potential conflicts between adaptive forces that stabilize duplications and those that enable evolution of new functions. Our objective was to delineate the adaptive forces underlying diversification of T7SS. Plasmid-borne ESX were described recently, and we found evidence that the initial duplication and divergence of ESX systems occurred on plasmids and was driven by selection for advantageous mutations. Plasmid conjugation has been linked to T7SS and type IV secretion systems (T4SS) in mycobacteria, and we discovered that T7SS and T4SS genes evolved in concert on the plasmids. We hypothesize that differentiation of plasmid ESX helps to prevent conjugation among cells harboring incompatible plasmids. Plasmid ESX appear to have been repurposed following migration to the chromosome, and there is evidence of positive selection driving further differentiation of chromosomal ESX. We hypothesize that ESX loci were initially stabilized on the chromosome by mediating their own transfer. These results emphasize the diverse adaptive paths underlying evolution of novelty, which in this case involved plasmid duplications, selection for advantageous mutations in the mobile and core genomes, migration of the loci between plasmids and chromosomes, and lateral transfer among chromosomes. We discuss further implications for the choice of model organism to study ESX functions inMycobacterium tuberculosis.

The “Beijing” Mycobacterium tuberculosis (Mtb) lineage 2 (L2) is spreading globally and has been associated with accelerated disease progression and increased antibiotic resistance. Here we performed a phylodynamic reconstruction of one of the L2 sublineages, the central Asian clade (CAC), which has recently spread to western Europe. We find that recent historical events have contributed to the evolution and dispersal of the CAC. Our timing estimates indicate that the clade was likely introduced to Afghanistan during the 1979–1989 Soviet–Afghan war and spread further after population displacement in the wake of the American invasion in 2001. We also find that drug resistance mutations accumulated on a massive scale in Mtb isolates from former Soviet republics after the fall of the Soviet Union, a pattern that was not observed in CAC isolates from Afghanistan. Our results underscore the detrimental effects of political instability and population displacement on tuberculosis control and demonstrate the power of phylodynamic methods in exploring bacterial evolution in space and time.

Patterns of gene flow can have marked effects on the evolution of populations. To better understand the migration dynamics of Mycobacterium tuberculosis , we studied genetic data from European M. tuberculosis lineages currently circulating in Aboriginal and French Canadian communities. A single M. tuberculosis lineage, characterized by the DS6 Quebec genomic deletion, is at highest frequency among Aboriginal populations in Ontario, Saskatchewan, and Alberta; this bacterial lineage is also dominant among tuberculosis (TB) cases in French Canadians resident in Quebec. Substantial contact between these human populations is limited to a specific historical era (1710–1870), during which individuals from these populations met to barter furs. Statistical analyses of extant M. tuberculosis minisatellite data are consistent with Quebec as a source population for M. tuberculosis gene flow into Aboriginal populations during the fur trade era. Historical and genetic analyses suggest that tiny M. tuberculosis populations persisted for ∼100 y among indigenous populations and subsequently expanded in the late 19th century after environmental changes favoring the pathogen. Our study suggests that spread of TB can occur by two asynchronous processes: ( i ) dispersal of M. tuberculosis by minimal numbers of human migrants, during which small pathogen populations are sustained by ongoing migration and slow disease dynamics, and ( ii ) expansion of the M. tuberculosis population facilitated by shifts in host ecology. If generalizable, these migration dynamics can help explain the low DNA sequence diversity observed among isolates of M. tuberculosis and the difficulties in global elimination of tuberculosis, as small, widely dispersed pathogen populations are difficult both to detect and to eradicate.

Despite a widespread global distribution and highly variable disease phenotype, there is little DNA sequence diversity among isolates of Mycobacterium tuberculosis. In addition, many regional population genetic surveys have revealed a stereotypical structure in which a single clone, lineage, or clade makes up the majority of the population. It is often assumed that dominant clones are highly adapted, that is, the overall structure of M. tuberculosis populations is the result of positive selection. In order to test this assumption, we analyzed genetic data from extant populations of bacteria circulating in Aboriginal communities in Saskatchewan, Canada. Demographic parameters of the bacterial population were estimated from archival epidemiological data collected over approximately 130 years since the onset of epidemic tuberculosis in the host communities. Bacterial genetic data were tested against neutral theory expectations and the local evolutionary history of M. tuberculosis investigated by phylogenetic analysis. Our findings are not consistent with positive selection on the bacterial population. Instead, we uncovered founder effects persisting over decades and barriers to gene flow within the bacterial population. Simulation experiments suggested that a combination of these neutral influences could result in the stereotypical structure of M. tuberculosis populations. Some aspects of population structure were suggestive of background selection, and data were on the whole consistent with combined effects of population bottlenecks, subdivision, and background selection. Neutral phenomena, namely, bottlenecks and partitions within populations, are prominent influences on the evolution of M. tuberculosis and likely contribute to restricted genetic diversity observed within this species. Given these influences, a complex evolutionary model will be required to define the relative fitness of different M. tuberculosis lineages and, ultimately, to uncover the genetic basis for its success as a pathogen.