Need for speed: Key driver of host cell migration varies among mycobacteria.

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.

Evolution of Tuberculosis Pathogenesis.

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.

Adaptation of the Mycobacterium tuberculosis transcriptome to biofilm growth.

Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), is a leading global cause of death from infectious disease. Biofilms are increasingly recognized as a relevant growth form during M. tb infection and may impede treatment by enabling bacterial drug and immune tolerance. M. tb has a complicated regulatory network that has been well-characterized for many relevant disease states, including dormancy and hypoxia. However, despite its importance, our knowledge of the genes and pathways involved in biofilm formation is limited. Here we characterize the biofilm transcriptomes of fully virulent clinical isolates and find that the regulatory systems underlying biofilm growth vary widely between strains and are also distinct from regulatory programs associated with other environmental cues. We used experimental evolution to investigate changes to the transcriptome during adaptation to biofilm growth and found that the application of a uniform selection pressure resulted in loss of strain-to-strain variation in gene expression, resulting in a more uniform biofilm transcriptome. The adaptive trajectories of transcriptomes were shaped by the genetic background of the M. tb population leading to convergence on a sub-lineage specific transcriptome. We identified widespread upregulation of non-coding RNA (ncRNA) as a common feature of the biofilm transcriptome and hypothesize that ncRNA function in genome-wide modulation of gene expression, thereby facilitating rapid regulatory responses to new environments. These results reveal a new facet of the M. tb regulatory system and provide valuable insight into how M. tb adapts to new environments.

Genetic susceptibility to severe childhood asthma and rhinovirus-C maintained by balancing selection in humans for 150 000 years.

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.

Transcriptional and Translational Responsiveness of the Neisseria gonorrhoeae Type IV Secretion System to Conditions of Host Infections.

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. The 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.

Tuberculosis sanatorium treatment at the advent of the chemotherapy era.

BACKGROUND: The discovery of antibiotics in the mid-twentieth century marked a major transition in tuberculosis (TB) treatment and control. There are few studies describing the duration of TB disease and its treatment from the pre-chemotherapy era and little data on how these treatments changed in response to the development of effective antibiotics. The goal of this research is to understand how inpatient treatment for high incidence populations, the First Nations peoples of Saskatchewan, Canada, changed in response to increasing availability of antibiotics effective against TB. We expected that as treatment regimens transitioned from convalescence-only to triple antibiotic therapy, the length of inpatient treatment would shorten. METHODS: Analyses were performed on records of sanatoria admissions and discharges occurring between 1933 and 1959 in Saskatchewan, Canada. Year of antibiotic discovery was taken as a proxy for treatment regimen: no chemotherapy (pre-1944), mono-therapy (Streptomycin, 1944-1946), dual-therapy (Streptomycin and PAS, 1946-1952), and triple-therapy (Streptomycin, PAS, and INH 1952-). A pooled linear regression of log-transformed length of first admission as predicted by year of admission was modeled to assess the relationship between admission length and year of admission, corrected for clinical and demographic variables. RESULTS: First admission length increased 19% in the triple-therapy era as compared to the pre-chemotherapy era, from 316 days (10.4 months) to 377 days (12.4 months). After the discovery of INH (1952), we find statistically significant increases in the proportion of successfully completed therapies (0.55 versus 0.60, p = 0.035), but also in patients who left hospital against medical advice (0.19 versus 0.29, p < 0.0001), indicating that as hospitalizations lengthened, more patients chose to discharge without the sanction of their physician. The readmission rate increased from 10 to 50% of all admissions while the province-level TB-specific death rate fell from 63.1 per 10,000 in 1933 to 4.7 per 10,000 in 1958. CONCLUSION: Counterintuitively, we find that the length of first admissions increased with the discovery of TB-treating antibiotics. Increasing admission volume and readmission rate indicate an intensification of inpatient TB treatment during this era. These analyses provide a novel estimate of the effect of changing treatment policy on sanatorium admissions in this population.

Lineage specific histories of Mycobacterium tuberculosis dispersal in Africa and Eurasia.

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.

Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium 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.

Epidemiological and genomic determinants of tuberculosis outbreaks in First Nations communities in Canada.

BACKGROUND: In Canada, tuberculosis disproportionately affects foreign-born and First Nations populations. Within First Nations' peoples, a high proportion of cases occur in association with outbreaks. Tuberculosis transmission in the context of outbreaks is thought to result from the convergence of several factors including characteristics of the cases, contacts, the environment, and the pathogen. METHODS: We examined the epidemiological and genomic determinants of two well-characterized tuberculosis outbreaks attributed to two super-spreaders among First Nations in the province of Alberta. These outbreaks were associated with two distinct DNA fingerprints (restriction fragment-length polymorphisms or RFLPs 0.0142 and 0.0728). We compared outbreak isolates with endemic isolates not spatio-temporarily linked to outbreak cases. We extracted epidemiological variables pertaining to tuberculosis cases and contacts from individual public health records and the provincial tuberculosis registry. We conducted group analyses using parametric and non-parametric statistical tests. We carried out whole-genome sequencing and bioinformatic analysis using validated protocols. RESULTS: We observed differences between outbreak and endemic groups in the mean number of total and child-aged contacts and the number of contacts with new positive and converted tuberculin skin tests in all group comparisons (p < 0.05). Differences were also detected in the proportion of cases with cavitation on a chest radiograph and the mean number of close contacts in selected group comparisons (p < 0.02). A phylogenetic network analysis of whole-genome sequencing data indicated that most outbreak and endemic strains were closely related to the source case for the 0.0142 fingerprint. For the 0.0728 fingerprint, the source case haplotype was circulating among endemic cases prior to the outbreak. Genetic and temporal distances were not correlated for either RFLP 0.0142 (r2 = - 0.05) or RFLP 0.0728 (r2 = 0.09) when all isolates were analyzed. CONCLUSIONS: We found no evidence that endemic strains acquired mutations resulting in their emergence in outbreak form. We conclude that the propagation of these outbreaks was likely driven by the combination of characteristics of the source cases, contacts, and the environment. The role of whole-genome sequencing in understanding mycobacterial evolution and in assisting public health authorities in conducting contact investigations and managing outbreaks is important and expected to grow in the future.

Diversity of Mycobacterium tuberculosis across Evolutionary Scales.

Tuberculosis (TB) is a global public health emergency. Increasingly drug resistant strains of Mycobacterium tuberculosis (M.tb) continue to emerge and spread, highlighting adaptability of this pathogen. Most studies of M.tb evolution have relied on 'between-host' samples, in which each person with TB is represented by a single M.tb isolate. However, individuals with TB commonly harbor populations of M.tb numbering in the billions. Here, we use analyses of M.tb genomic data from within and between hosts to gain insight into influences shaping genetic diversity of this pathogen. We find that the amount of M.tb genetic diversity harbored by individuals with TB can vary dramatically, likely as a function of disease severity. Surprisingly, we did not find an appreciable impact of TB treatment on M.tb diversity. In examining genomic data from M.tb samples within and between hosts with TB, we find that genes involved in the regulation, synthesis, and transportation of immunomodulatory cell envelope lipids appear repeatedly in the extremes of various statistical measures of diversity. Many of these genes have been identified as possible targets of selection in other studies employing different methods and data sets. Taken together, these observations suggest that M.tb cell envelope lipids are targets of selection within hosts. Many of these lipids are specific to pathogenic mycobacteria and, in some cases, human-pathogenic mycobacteria. We speculate that rapid adaptation of cell envelope lipids is facilitated by functional redundancy, flexibility in their metabolism, and their roles mediating interactions with the host.

The role of selection in shaping diversity of natural M. tuberculosis populations.

Mycobacterium tuberculosis (M.tb), the cause of tuberculosis (TB), is estimated to infect a new host every second. While analyses of genetic data from natural populations of M.tb have emphasized the role of genetic drift in shaping patterns of diversity, the influence of natural selection on this successful pathogen is less well understood. We investigated the effects of natural selection on patterns of diversity in 63 globally extant genomes of M.tb and related pathogenic mycobacteria. We found evidence of strong purifying selection, with an estimated genome-wide selection coefficient equal to -9.5 × 10(-4) (95% CI -1.1 × 10(-3) to -6.8 × 10(-4)); this is several orders of magnitude higher than recent estimates for eukaryotic and prokaryotic organisms. We also identified different patterns of variation across categories of gene function. Genes involved in transport and metabolism of inorganic ions exhibited very low levels of non-synonymous polymorphism, equivalent to categories under strong purifying selection (essential and translation-associated genes). The highest levels of non-synonymous variation were seen in a group of transporter genes, likely due to either diversifying selection or local selective sweeps. In addition to selection, we identified other important influences on M.tb genetic diversity, such as a 25-fold expansion of global M.tb populations coincident with explosive growth in human populations (estimated timing 1684 C.E., 95% CI 1620-1713 C.E.). These results emphasize the parallel demographic histories of this obligate pathogen and its human host, and suggest that the dominant effect of selection on M.tb is removal of novel variants, with exceptions in an interesting group of genes involved in transportation and defense. We speculate that the hostile environment within a host imposes strict demands on M.tb physiology, and thus a substantial fitness cost for most new mutations. In this respect, obligate bacterial pathogens may differ from other host-associated microbes such as symbionts.

Dispersal of Mycobacterium tuberculosis via the Canadian fur trade.

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.

Phase variable colony variants are conserved across Gardnerella spp. and exhibit different virulence-associated phenotypes.

The Gardnerella genus, comprising at least 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, including Gardnerella spp., 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 tested Gardnerella strains, representing at least 10 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 phenotypic, proteomic, and genomic differences. Beyond increased colony size, large colony variants showed reduced vaginolysin secretion and faster growth rate relative to small colony variants. The ability to inhibit the growth of Neisseria gonorrhoeae and commensal Lactobacillus species varied by strain and, in some instances, differed between variants. Proteomics analyses indicated that 127-173 proteins were differentially expressed between variants. Proteins with increased expression in large variants of both strains were associated with amino acid and protein synthesis and protein folding, whereas those increased in small variants were related to nucleotide synthesis, phosphate transport, ABC transport, and glycogen breakdown. Furthermore, whole genome sequencing analyses revealed an abundance of genes associated with variable homopolymer tracts, implicating slipped strand mispairing in Gardnerella phase variation and illuminating the potential for previously unrecognized heterogeneity within clonal populations. Collectively, these results suggest that phase variants may be primed to serve different roles in BV pathogenesis.IMPORTANCEBacterial vaginosis is the most common gynecological disorder in women of childbearing age. Gardnerella species are crucial to the development of this dysbiosis, but the mechanisms involved in the infection are not understood. We discovered that Gardnerella species vary between two different forms, reflected in bacterial colony size. A slow-growing form makes large amounts of the toxin vaginolysin and is better able to survive in human cervix tissue. A fast-growing form is likely the one that proliferates to high numbers just prior to symptom onset and forms the biofilm that serves as a scaffold for multiple BV-associated anaerobic bacteria. Identification of the proteins that vary between different forms of the bacteria as well as those that vary randomly provides insight into the factors important for Gardnerella infection and immune avoidance.

Adaptation of the Mycobacterium tuberculosis transcriptome to biofilm growth.

Mycobacterium tuberculosis ( M. tb ), the causative agent of tuberculosis (TB), is a leading global cause of death from infectious disease. Biofilms are increasingly recognized as a relevant growth form during M. tb infection and may impede treatment by enabling bacterial drug and immune tolerance. M. tb has a complicated regulatory network that has been well-characterized for many relevant disease states, including dormancy and hypoxia. However, despite its importance, our knowledge of the genes and pathways involved in biofilm formation is limited. Here we characterize the biofilm transcriptomes of fully virulent clinical isolates and find that the regulatory systems underlying biofilm growth vary widely between strains and are also distinct from regulatory programs associated with other environmental cues. We used experimental evolution to investigate changes to the transcriptome during adaptation to biofilm growth and found that the application of a uniform selection pressure resulted in loss of strain-to-strain variation in gene expression, resulting in a more uniform biofilm transcriptome. The adaptive trajectories of transcriptomes were shaped by the genetic background of the M. tb population leading to convergence on a sub-lineage specific transcriptome. We identified widespread upregulation of non-coding RNA (ncRNA) as a common feature of the biofilm transcriptome and hypothesize that ncRNA function in genome-wide modulation of gene expression, thereby facilitating rapid regulatory responses to new environments. These results reveal a new facet of the M. tb regulatory system and provide valuable insight into how M. tb adapts to new environments. Importance: Understanding mechanisms of resistance and tolerance in Mycobacterium tuberculosis ( M. tb ) can help us develop new treatments that capitalize on M. tb 's vulnerabilities. Here we used transcriptomics to study both the regulation of biofilm formation in clinical isolates as well as how those regulatory systems adapt to new environments. We find that closely related clinical populations have diverse strategies for growth under biofilm conditions, and that genetic background plays a large role in determining the trajectory of evolution. These results have implications for future treatment strategies that may be informed by our knowledge of the evolutionary constraints on strain(s) from an individual infection. This work provides new information about the mechanisms of biofilm formation in M. tb and outlines a framework for population level approaches for studying bacterial adaptation.

Portrait of a generalist bacterium: pathoadaptation, metabolic specialization and extreme environments shape diversity of Staphylococcus saprophyticus.

Staphylococcus saprophyticus is a Gram-positive, coagulase-negative staphylococcus found in diverse environments including soil and freshwater, meat, and dairy foods. S. saprophyticus is also an important cause of urinary tract infections (UTIs) in humans, and mastitis in cattle. However, the genetic determinants of virulence have not yet been identified, and it remains unclear whether there are distinct sub-populations adapted to human and animal hosts. Using a diverse sample of S. saprophyticus isolates from food, animals, environmental sources, and human infections, we characterized the population structure and diversity of global populations of S. saprophyticus . We found that divergence of the two major clades of S. saprophyticus is likely facilitated by barriers to horizontal gene transfer (HGT) and differences in metabolism. Using genome-wide association study (GWAS) tools we identified the first Type VII secretion system (T7SS) described in S. saprophyticus and its association with bovine mastitis. Finally, we found that in general, strains of S. saprophyticus from different niches are genetically similar with the exception of built environments, which function as a 'sink' for S. saprophyticus populations. This work increases our understanding of the ecology of S. saprophyticus and of the genomics of bacterial generalists. Data summary: Raw sequencing data for newly sequenced S. saprophyticus isolates have been deposited to the NCBI SRA under the project accession PRJNA928770. A list of all genomes used in this work and their associated metadata are available in the supplementary material. Custom scripts used in the comparative genomics and GWAS analyses are available here: https://github.com/myoungblom/sapro_genomics . Impact statement: It is not known whether human and cattle diseases caused by S. saprophyticus represent spillover events from a generalist adapted to survive in a range of environments, or whether the capacity to cause disease represents a specific adaptation. Seasonal cycles of S. saprophyticus UTIs and molecular epidemiological evidence suggest that these infections may be environmentally-acquired rather than via transmission from person to person. Using comparative genomics and genome wide association study tools, we found that S. saprophyticus appears adapted to inhabit a wide range of environments (generalist), with isolates from animals, food, natural environments and human infections being closely related. Bacteria that routinely switch environments, particularly between humans and animals, are of particular concern when it comes to the spread of antibiotic resistance from farm environments into human populations. This work provides a framework for comparative genomic analyses of bacterial generalists and furthers our understanding of how bacterial populations move between humans, animals, and the environment.

The Gonococcal Genetic Island defines distinct sub-populations of Neisseria gonorrhoeae.

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.

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile.

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 using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

Comparative Genomics of Streptococcus oralis Identifies Large Scale Homologous Recombination and a Genetic Variant Associated with Infection.

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 that were isolated from blood cultures in patients with invasive infections and oral swabs of healthy volunteers and then determined the best-performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample of Streptococcus oralis isolates 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 was within a previously undescribed gene that was conserved across the majority of VGS species. Using the 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 highlighted the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS. IMPORTANCE The 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 of Streptococcus oralis genomes 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 shared homology with the anaerobic ribonucleoside triphosphate reductase, nrdD, and was highly conserved among VGS. This work increased our knowledge of VGS genomics and indicated that differences in virulence potential among S. oralis isolates were, at least in part, genetically determined.

Rapid adaptation of a complex trait during experimental evolution of Mycobacterium tuberculosis.

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.

In many environments, bacteria live together in structures called biofilms. Cells in biofilms coordinate with each other to protect the group and allow it to survive difficult conditions. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, forms biofilms when it infects the human body. Biofilms make the infection a lot more difficult to treat, which may be one of the reasons why tuberculosis is the deadliest bacterial infection in the world. Bacteria evolve rapidly over the course of a single infection, but bacteria forming biofilms evolve differently to bacteria living alone. This evolution happens through mutations to the bacterial DNA, which can be small (a single base in a DNA sequence changes to a different base) or larger changes (such as the deletion or insertion of several bases). Smith, Youngblom et al. studied the evolution of tuberculosis growing in biofilms in the lab. As the bacteria evolved, they tended to form thicker biofilms, an effect linked to 14 mutations involving single base DNA changes and four larger ones. Most of the changes were in regulatory regions of DNA, which control whether genes are 'read' by cells to produce proteins. These regions often change more though evolution than regions coding for proteins, because they have a coordinated effect on a group of related genes rather than randomly altering individual genes. Smith, Youngblom et al. also showed that biofilms made from different strains of tuberculosis evolved in different ways. Smith Youngblom et al.'s findings provide more information regarding how bacteria adapt to living in biofilms, which may reveal new ways to control them. This could have applications in water treatment, food production and healthcare. Learning how to treat bacteria growing in biofilms could also improve the outcomes for patients infected with tuberculosis.