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Background: Mixed infections of Mycobacterium tuberculosis and antibiotic heteroresistance continue to complicate tuberculosis (TB) diagnosis and treatment. Detection of mixed infections has been limited to molecular genotyping techniques, which lack the sensitivity and resolution to accurately estimate the multiplicity of TB infections. In contrast, whole genome sequencing offers sensitive views of the genetic differences between strains of M. tuberculosis within a sample. Although metagenomic tools exist to classify strains in a metagenomic sample, most tools have been developed for more divergent species, and therefore cannot provide the sensitivity required to disentangle strains within closely related bacterial species such as M. tuberculosis. Here we present QuantTB, a method to identify and quantify individual M. tuberculosis strains in whole genome sequencing data. QuantTB uses SNP markers to determine the combination of strains that best explain the allelic variation observed in a sample. QuantTB outputs a list of identified strains, their corresponding relative abundances, and a list of drugs for which resistance-conferring mutations (or heteroresistance) have been predicted within the sample., Results: We show that QuantTB has a high degree of resolution and is capable of differentiating communities differing by less than 25 SNPs and identifying strains down to 1× coverage. Using simulated data, we found QuantTB outperformed other metagenomic strain identification tools at detecting strains and quantifying strain multiplicity. In a real-world scenario, using a dataset of 50 paired clinical isolates from a study of patients with either reinfections or relapses, we found that QuantTB could detect mixed infections and reinfections at rates concordant with a manually curated approach., Conclusion: QuantTB can determine infection multiplicity, identify hetero-resistance patterns, enable differentiation between relapse and re-infection, and clarify transmission events across seemingly unrelated patients - even in low-coverage (1×) samples. QuantTB outperforms existing tools and promises to serve as a valuable resource for both clinicians and researchers working with clinical TB samples.

Background: Prophages play a significant role in prokaryotic evolution, often altering the function of the cell that they infect via transfer of new genes e.g., virulence or antibiotic resistance factors, inactivation of existing genes or by modifying gene expression. Recently, phage therapy has gathered renewed interest as a promising alternative to control bacterial infections. Cataloging the repertoire of prophages in large collections of species' genomes is an important initial step in understanding their evolution and potential therapeutic utility. However, current widely-used tools for identifying prophages within bacterial genome sequences are mainly web-based, can have long response times, and do not scale to keep pace with the many thousands of genomes currently being sequenced routinely., Methodology: In this work, we present ProphET, an easy to install prophage predictor to be used in Linux operation system, without the constraints associated with a web-based tool. ProphET predictions rely on similarity searches against a database of prophage genes, taking as input a bacterial genome sequence in FASTA format and its corresponding gene annotation in GFF. ProphET identifies prophages in three steps: similarity search, calculation of the density of prophage genes, and edge refinement. ProphET performance was evaluated and compared with other phage predictors based on a set of 54 bacterial genomes containing 267 manually annotated prophages., Findings and Conclusions: ProphET identifies prophages in bacterial genomes with high precision and offers a fast, highly scalable alternative to widely-used web-based applications for prophage detection., Competing Interests: GCC is an employee of Personal Genome Diagnostics. Personal Genome Diagnostics provided support in the form of salary to GCC as this manuscript was being finalized. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Tuberculosis (TB) is a global infectious threat that is intensified by an increasing incidence of highly drug-resistant disease. Whole-genome sequencing (WGS) studies of Mycobacterium tuberculosis, the causative agent of TB, have greatly increased our understanding of this pathogen. Since the first M. tuberculosis genome was published in 1998, WGS has provided a more complete account of the genomic features that cause resistance in populations of M. tuberculosis, has helped to fill gaps in our knowledge of how both classical and new antitubercular drugs work, and has identified specific mutations that allow M. tuberculosis to escape the effects of these drugs. WGS studies have also revealed how resistance evolves both within an individual patient and within patient populations, including the important roles of de novo acquisition of resistance and clonal spread. These findings have informed decisions about which drug-resistance mutations should be included on extended diagnostic panels. From its origins as a basic science technique, WGS of M. tuberculosis is becoming part of the modern clinical microbiology laboratory, promising rapid and improved detection of drug resistance, and detailed and real-time epidemiology of TB outbreaks. We review the successes and highlight the challenges that remain in applying WGS to improve the control of drug-resistant TB through monitoring its evolution and spread, and to inform more rapid and effective diagnostic and therapeutic strategies.

Background: Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection., Results: The genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems., Conclusion: Genomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances.

Fusobacterium nucleatum is a strictly anaerobic, Gram negative bacterial species that has been associated with dental infections, pre-term labor, appendicitis, inflammatory bowel disease, and, more recently, colorectal cancer. The species is unusual in its phenotypic and genotypic heterogeneity, with some strains demonstrating a more virulent phenotype than others; however, as yet the genetic basis for these differences is not understood. Bacteriophage are known to contribute to the virulence phenotype of several bacterial species. In this work, we set out to characterize the bacteriophage associated with F. nucleatum subsp. animalis strain 7-1, a highly invasive isolate from the human gastrointestinal tract. As well, we used computational approaches to predict and compare bacteriophage signatures across available sequenced F. nucleatum genomes., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Although the composition of the human microbiome is now well-studied, the microbiota's >8 million genes and their regulation remain largely uncharacterized. This knowledge gap is in part because of the difficulty of acquiring large numbers of samples amenable to functional studies of the microbiota. We conducted what is, to our knowledge, one of the first human microbiome studies in a well-phenotyped prospective cohort incorporating taxonomic, metagenomic, and metatranscriptomic profiling at multiple body sites using self-collected samples. Stool and saliva were provided by eight healthy subjects, with the former preserved by three different methods (freezing, ethanol, and RNAlater) to validate self-collection. Within-subject microbial species, gene, and transcript abundances were highly concordant across sampling methods, with only a small fraction of transcripts (<5%) displaying between-method variation. Next, we investigated relationships between the oral and gut microbial communities, identifying a subset of abundant oral microbes that routinely survive transit to the gut, but with minimal transcriptional activity there. Finally, systematic comparison of the gut metagenome and metatranscriptome revealed that a substantial fraction (41%) of microbial transcripts were not differentially regulated relative to their genomic abundances. Of the remainder, consistently underexpressed pathways included sporulation and amino acid biosynthesis, whereas up-regulated pathways included ribosome biogenesis and methanogenesis. Across subjects, metatranscriptional profiles were significantly more individualized than DNA-level functional profiles, but less variable than microbial composition, indicative of subject-specific whole-community regulation. The results thus detail relationships between community genomic potential and gene expression in the gut, and establish the feasibility of metatranscriptomic investigations in subject-collected and shipped samples.

Bacillus subtilis is a remarkably diverse bacterial species that is capable of growth within many environments. Recent microarray-based comparative genomic analyses have revealed that members of this species also exhibit considerable genomic diversity. The identification of strain-specific genes might explain how B. subtilis has become so broadly adapted. The goal of identifying ecologically adaptive genes could soon be realized with the imminent release of several new B. subtilis genome sequences. As we embark upon this exciting new era of B. subtilis comparative genomics we review what is currently known about the ecology and evolution of this species.

Vaccines that confer protection through induction of adaptive T-cell immunity rely on understanding T-cell epitope (TCE) evolution induced by immune escape. This is poorly understood in tuberculosis (TB), an ancient, chronic disease, where CD4 T-cell immunity is of recognized importance. We probed 905 functionally validated, curated human CD4 T cell epitopes in 79 Mycobacterium tuberculosis (Mtb) whole genomes from India. This screen resulted in identifying 64 mutated epitopes in these strains initially using a computational pipeline and subsequently verified by single nucleotide polymorphism (SNP) analysis. SNP based phylogeny revealed the 79 Mtb strains to cluster to East African Indian (EAI), Central Asian Strain (CAS), and Beijing (BEI) lineages. Eighty-nine percent of the mutated T-cell epitopes (mTCEs) identified in the 79 Mtb strains from India has not previously been reported. These mTCEs were encoded by genes with high nucleotide diversity scores including seven mTCEs encoded by six antigens in the top 10% of rapidly divergent Mtb genes encoded by these strains. Using a T cell functional assay readout, we demonstrate 62% of mTCEs tested to significantly alter CD4 T-cell IFNγ and/or IL2 secretion with associated changes in predicted HLA-DR binding affinity: the gain of function mutations displayed higher predicted HLA-DR binding affinity and conversely mutations resulting in loss of function displayed lower predicted HLA-DR binding affinity. Most mutated antigens belonged to the cell wall/cell processes, and, intermediary metabolism and respiration families though all known Mtb proteins encoded mutations. Analysis of the mTCEs in an SNP database of 5,310 global Mtb strains identified 82% mTCEs to be significantly more prevalent in Mtb strains isolated from India, including 36 mTCEs identified exclusively in strains from India. These epitopes had a significantly higher predicted binding affinity to HLA-DR alleles that were highly prevalent in India compared to HLA-DR alleles rare in India, highlighting HLA-DR maybe an important driver of these mutations. This first evidence of region-specific TCE mutations potentially employed by Mtb to escape host immunity has important implications for TB vaccine design.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedented^ broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found. [ABSTRACT FROM AUTHOR]

Several bacterial pathogens, including Salmonella enterica , can cause persistent infections in humans by mechanisms that are poorly understood. By comparing genomes of isolates longitudinally collected from 256 prolonged salmonellosis patients, we identified repeated mutations in global regulators, including the barA/sirA two-component regulatory system, across multiple patients and Salmonella serovars. Comparative RNA-seq analysis revealed that distinct mutations in barA/sirA led to diminished expression of Salmonella pathogenicity islands 1 and 4 genes, which are required for Salmonella invasion and enteritis. Moreover, barA/sirA mutants were attenuated in an acute salmonellosis mouse model and induced weaker transcription of host immune responses. In contrast, in a persistent infection mouse model, these mutants exhibited long-term colonization and prolonged shedding. Taken together, these findings suggest that selection of mutations in global virulence regulators facilitates persistent Salmonella infection in humans, by attenuating Salmonella virulence and inducing a weaker host inflammatory response. [Display omitted] • Salmonella global regulators are frequently mutated during persistent human infection • The BarA/SirA virulence regulatory pathway was most frequently mutated • barA/sirA mutants were less virulent and elicited a weakened host immune response • barA / sirA mutants colonized mice and were shed during persistent salmonellosis Grote et al. show that adaptations in global regulatory genes arise during persistent Salmonella infections in humans. These adaptations lead to decreased virulence gene expression, attenuated pathogenicity, and a weaker immune response in an acute salmonellosis model but to long-term colonization and shedding in a persistent mouse model. [ABSTRACT FROM AUTHOR]

Advances in modern sequencing technologies allow us to generate sufficient data to analyze hundreds of bacterial genomes from a single machine in a single day. This potential for sequencing massive numbers of genomes calls for fully automated methods to produce high-quality assemblies and variant calls. We introduce Pilon, a fully automated, all-in-one tool for correcting draft assemblies and calling sequence variants of multiple sizes, including very large insertions and deletions. Pilon works with many types of sequence data, but is particularly strong when supplied with paired end data from two Illumina libraries with small e.g., 180 bp and large e.g., 3–5 Kb inserts. Pilon significantly improves draft genome assemblies by correcting bases, fixing mis-assemblies and filling gaps. For both haploid and diploid genomes, Pilon produces more contiguous genomes with fewer errors, enabling identification of more biologically relevant genes. Furthermore, Pilon identifies small variants with high accuracy as compared to state-of-the-art tools and is unique in its ability to accurately identify large sequence variants including duplications and resolve large insertions. Pilon is being used to improve the assemblies of thousands of new genomes and to identify variants from thousands of clinically relevant bacterial strains. Pilon is freely available as open source software. [ABSTRACT FROM AUTHOR]

An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606T was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606T, A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606T and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species. [ABSTRACT FROM AUTHOR]