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Aim: Volatile organic compounds (VOCs) are potential biomarkers for diagnosing colorectal cancer (CRC). We characterized urinary VOCs from CRC patients, their spouses/cohabitors (spouses) and first‐degree relatives (relatives) to determine any differences. Correlation with stool‐derived microbiomes was also undertaken. Methods: Urine from 56 CRC patients, 45 spouses and 37 relatives was assayed using liquid chromatography, field asymmetric ion mobility spectrometry (FAIMS), mass spectrometer technology. Analysis was performed using five‐fold cross‐validation and a random forest classifier. Faecal microbiome 16S rRNA was sequenced using Illumina MiSeq protocols and analysed using UPARSE and QIIME pipelines. VOC and microbiome profiles were also compared before and after cancer treatment. Results: Urinary VOC profiles of CRC patients were indistinguishable from either spouses or relatives. When spouses and relatives were grouped together to form a larger non‐cancer control group (n = 82), their VOC profiles became distinguishable from those of CRC patients (n = 56) with 69% sensitivity and specificity, area under the curve 0.72 (P < 0.001). Microbiome analysis identified > 1300 operational taxonomic units across all groups. The analysis of similarity R value was 0.067 (P < 0.001), with significantly different bacterial abundances in 82 operational taxonomic units (6.2%) by Kruskal–Wallis testing. CRC patients' VOC or stool microbiome profiles were unchanged after treatment. Conclusion: Although CRC patients' urinary VOC profiles cannot be differentiated from those of spouses or relatives they can be differentiated from a larger non‐cancer control group. Comparison of the groups' microbiomes confirmed differences in bacterial species abundance. The current FAIMS‐based assay can detect a unique, but modest, signal in CRC patients' urinary VOCs, which remains unaltered after treatment. [ABSTRACT FROM AUTHOR]

The oral manifestations of chronic graft-versus-host disease (cGVHD) in eight allogeneic bone marrow transplant (BMT) paediatric recipients were studied clinically, and lip biopsies were performed in seven of them. A prominent lichenoid reaction was observed in four patients, two with accompanying ulceration. Superficial mucoceles were present in three children. Clinically obvious xerostomia was seen in seven patients. Lip biopsies were positive and correlated with the clinical manifestations. Both clinical and histological findings confirmed the diagnosis of cGVHD. In three additional children, with systemic manifestations indicating cGVHD, the oral mucosa was clinically and histologically normal, and the systemic manifestations were, thus, attributed to drug reactions. The above findings indicate the high value of oral examination in diagnosing or confirming paediatric cGVHD. Superficial mucoceles, reported for the first time in paediatric recipients, seem to be important in the early diagnosis of cGVHD. [ABSTRACT FROM AUTHOR]

Abstract Background Microbial taxonomy remains a conservative discipline, relying on phenotypic information derived from growth in pure culture and techniques that are time-consuming and difficult to standardize, particularly when compared to the ease of modern high-throughput genome sequencing. Here, drawing on the genus Acinetobacter as a test case, we examine whether bacterial taxonomy could abandon phenotypic approaches and DNA-DNA hybridization and, instead, rely exclusively on analyses of genome sequence data. Results In pursuit of this goal, we generated a set of thirteen new draft genome sequences, representing ten species, combined them with other publically available genome sequences and analyzed these 38 strains belonging to the genus. We found that analyses based on 16S rRNA gene sequences were not capable of delineating accepted species. However, a core genome phylogenetic tree proved consistent with the currently accepted taxonomy of the genus, while also identifying three misclassifications of strains in collections or databases. Among rapid distance-based methods, we found average-nucleotide identity (ANI) analyses delivered results consistent with traditional and phylogenetic classifications, whereas gene content based approaches appear to be too strongly influenced by the effects of horizontal gene transfer to agree with previously accepted species. Conclusion We believe a combination of core genome phylogenetic analysis and ANI provides an appropriate method for bacterial species delineation, whereby bacterial species are defined as monophyletic groups of isolates with genomes that exhibit at least 95% pair-wise ANI. The proposed method is backwards compatible; it provides a scalable and uniform approach that works for both culturable and non-culturable species; is faster and cheaper than traditional taxonomic methods; is easily replicable and transferable among research institutions; and lastly, falls in line with Darwin’s vision of classification becoming, as far as is possible, genealogical.

Photorhabdus is a bacterial genus containing both insect and emerging human pathogens. Most insect-restricted species display temperature restriction, unable to grow above 34°C, while Photorhabdus asymbiotica can grow at 37°C to infect mammalian hosts and cause Photorhabdosis. Metabolic adaptations have been proposed to facilitate the survival of this pathogen at higher temperatures, yet the biological mechanisms underlying these are poorly understood. We have reconstructed an extensively manually curated genome-scale metabolic model of P. asymbiotica (iEC1073, BioModels ID MODEL2309110001), validated through in silico gene knockout and nutrient utilization experiments with an excellent agreement between experimental data and model predictions. Integration of iEC1073 with transcriptomics data obtained for P. asymbiotica at temperatures of 28°C and 37°C allowed the development of temperature-specific reconstructions representing metabolic adaptations the pathogen undergoes when shifting to a higher temperature in a mammalian compared to insect host. Analysis of these temperature-specific reconstructions reveals that nucleotide metabolism is enriched with predicted upregulated and downregulated reactions. iEC1073 could be used as a powerful tool to study the metabolism of P. asymbiotica, in different genetic or environmental conditions., Importance: Photorhabdus bacterial species contain both human and insect pathogens, and most of these species cannot grow in higher temperatures. However, Photorhabdus asymbiotica , which infects both humans and insects, can grow in higher temperatures and undergoes metabolic adaptations at a temperature of 37°C compared to that of insect body temperature. Therefore, it is important to examine how this bacterial species can metabolically adapt to survive in higher temperatures. In this work, using a mathematical model, we have examined the metabolic shift that takes place when the bacteria switch from growth conditions in 28°C to 37°C. We show that P. asymbiotica potentially experiences predicted temperature-induced metabolic adaptations at 37°C predominantly clustered within the nucleotide metabolism pathway., Competing Interests: The authors declare no conflict of interest.

Environmental perturbations are encountered by microorganisms regularly and will require metabolic adaptations to ensure an organism can survive in the newly presenting conditions. In order to study the mechanisms of metabolic adaptation in such conditions, various experimental and computational approaches have been used. Genome-scale metabolic models (GEMs) are one of the most powerful approaches to study metabolism, providing a platform to study the systems level adaptations of an organism to different environments which could otherwise be infeasible experimentally. In this review, we are describing the application of GEMs in understanding how microbes reprogram their metabolic system as a result of environmental variation. In particular, we provide the details of metabolic model reconstruction approaches, various algorithms and tools for model simulation, consequences of genetic perturbations, integration of '-omics' datasets for creating context-specific models and their application in studying metabolic adaptation due to the change in environmental conditions., (© The Author(s) 2023. Published by Oxford University Press.)

Inherited bone marrow failure associated with heterozygous mutations in GATA2 predisposes toward hematological malignancies, but the mechanisms remain poorly understood. Here, we investigate the mechanistic basis of marrow failure in a zebrafish model of GATA2 deficiency. Single-cell transcriptomics and chromatin accessibility assays reveal that loss of gata2a leads to skewing toward the erythroid lineage at the expense of myeloid cells, associated with loss of cebpa expression and decreased PU.1 and CEBPA transcription factor accessibility in hematopoietic stem and progenitor cells (HSPCs). Furthermore, gata2a mutants show impaired expression of npm1a, the zebrafish NPM1 ortholog. Progressive loss of npm1a in HSPCs is associated with elevated levels of DNA damage in gata2a mutants. Thus, Gata2a maintains myeloid lineage priming through cebpa and protects against genome instability and marrow failure by maintaining expression of npm1a. Our results establish a potential mechanism underlying bone marrow failure in GATA2 deficiency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The opportunistic human pathogen Candida glabrata has become an increasingly important threat to human health, with infections globally characterized by high mortality rates and multidrug resistance. To face this threat, more efficient diagnostic and therapeutic approaches are required, underpinning research to help define the intraspecies epidemiology, genetic variability, and therefore, diagnostic and therapeutic target stability. Previous comparative genetics studies conducted on limited numbers of strains only revealed partial resolution of chromosomal settings. In this study, by combining short- and long-read genome sequencing, phenotypic characterization, and comparative genomics over a large set of strains, we detected strict relationships between large chromosomal rearrangements and phylogenetic clades, genes subjected to different selective pressures, and new sets of genes associated with resistance to antifungals. Overall, these results not only provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology but may also lay the foundations for the future development of tailored therapeutic approaches. IMPORTANCE The human pathogen Candida glabrata has become a global threat to human health, with infections characterized by high mortality and multidrug resistance. We have obtained nine fully assembled genomes from clinical isolates through a combination of short- and long-read sequencing approaches. The quality and completeness of such genomes and their subsequent comparison to the broadest set of genomes so far allowed us to pinpoint chromosomal rearrangements in several genomes and detect phylogenetic clades that were not associated with geographic location or isolation source. We identified a new set of genes associated with resistance to antifungals coding for adhesin or adhesin-like proteins, suggesting C. glabrata resists antifungals by forming aggregates or adhering to the host tissue. These results, which provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology, may initiate the development of precision medicine interventions for patients with suspected or proven invasive fungal infections.

Hydrogels are cross-linked networks of hydrophilic polymer chains with a three-dimensional structure. Owing to their unique features, the application of hydrogels for bacterial/antibacterial studies and bacterial infection management has grown in importance in recent years. This trend is likely to continue due to the rise in bacterial infections and antimicrobial resistance. By exploiting their physicochemical characteristics and inherent nature, hydrogels have been developed to achieve bacterial capture and detection, bacterial growth or elimination, antibiotic delivery, or bacterial sensing. Traditionally, the development of hydrogels for bacterial/antibacterial studies has focused on achieving a single function such as antibiotic delivery, antibacterial activity, bacterial growth, or bacterial detection. However, recent studies demonstrate the fabrication of multifunctional hydrogels, where a single hydrogel is capable of performing more than one bacterial/antibacterial function, or composite hydrogels consisting of a number of single functionalized hydrogels, which exhibit bacterial/antibacterial function synergistically. In this review, we first highlight the hydrogel features critical for bacterial studies and infection management. Then, we specifically address unique hydrogel properties, their surface/network functionalization, and their mode of action for bacterial capture, adhesion/growth, antibacterial activity, and bacterial sensing, respectively. Finally, we provide insights into different strategies for developing multifunctional hydrogels and how such systems can help tackle, manage, and understand bacterial infections and antimicrobial resistance. We also note that the strategies highlighted in this review can be adapted to other cell types and are therefore likely to find applications beyond the field of microbiology.

The survival strategies that Campylobacter jejuni (C. jejuni) employ throughout its transmission and infection life cycles remain largely elusive. Specifically, there is a lack of understanding about the posttranscriptional regulation of stress adaptations resulting from small noncoding RNAs (sRNAs). Published C. jejuni sRNAs have been discovered in specific conditions but with limited insights into their biological activities. Many more sRNAs are yet to be discovered as they may be condition-dependent. Here, we have generated transcriptomic data from 21 host- and transmission-relevant conditions. The data uncovered transcription start sites, expression patterns and posttranscriptional regulation during various stress conditions. This data set helped predict a list of putative sRNAs. We further explored the sRNAs' biological functions by integrating differential gene expression analysis, coexpression analysis, and genome-wide sRNA target prediction. The results showed that the C. jejuni gene expression was influenced primarily by nutrient deprivation and food storage conditions. Further exploration revealed a putative sRNA (CjSA21) that targeted tlp1 to 4 under food processing conditions. tlp1 to 4 are transcripts that encode methyl-accepting chemotaxis proteins (MCPs), which are responsible for chemosensing. These results suggested CjSA21 inhibits chemotaxis and promotes survival under food processing conditions. This study presents the broader research community with a comprehensive data set and highlights a novel sRNA as a potential chemotaxis inhibitor. IMPORTANCE The foodborne pathogen C. jejuni is a significant challenge for the global health care system. It is crucial to investigate C. jejuni posttranscriptional regulation by small RNAs (sRNAs) in order to understand how it adapts to different stress conditions. However, limited data are available for investigating sRNA activity under stress. In this study, we generate gene expression data of C. jejuni under 21 stress conditions. Our data analysis indicates that one of the novel sRNAs mediates the adaptation to food processing conditions. Results from our work shed light on the posttranscriptional regulation of C. jejuni and identify an sRNA associated with food safety.

Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA-mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

There is currently a renaissance in research on bacteriophages as alternatives to antibiotics. Phage specificity to their bacterial host, in addition to a plethora of other advantages, makes them ideal candidates for a broad range of applications, including bacterial detection, drug delivery, and phage therapy in particular. One issue obstructing phage efficiency in phage therapy settings is their poor localization to the site of infection in the human body. Here, we engineered phage T7 with lung tissue targeting homing peptides. We then used in vitro studies to demonstrate that the engineered T7 phages had a more significant association with the lung epithelium cells than wild-type T7. In addition, we showed that, in general, there was a trend of increased association of engineered phages with the lung epithelium cells but not mouse fibroblast cells, allowing for targeted tissue specificity. These results indicate that appending phages with homing peptides would potentially allow for greater phage concentrations and greater efficacy at the infection site.

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trx A have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7., Competing Interests: The authors declare no conflict of interest.

Bacteria export proteins across membranes using a range of transport machineries. Type VII secretion systems (T7SSs), originally described in mycobacteria, are now known to be widespread across diverse bacterial phyla. Recent studies have characterized secretion components and mechanisms of type VII secretion in pathogenic and environmental bacteria. A variety of functions have been attributed to T7SS substrates, including interactions with eukaryotes and with other bacteria. Here, we evaluate the growing body of knowledge on T7SSs, with focus on the nonmycobacterial systems, reviewing their phylogenetic distribution, structure and function in diverse settings., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pandoraea species, in particular Pandoraea apista, are opportunistic, multidrug-resistant pathogens in persons with cystic fibrosis (CF). To aid in understanding the role of P. apista in CF lung disease, we used Illumina MiSeq and nanopore MinION technology to sequence the whole genome of the P. apista LMG 16407(T)., (Copyright © 2015 Millard et al.)

Objectives: Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition., Study Design: An observational prospective cohort study., Setting: Burns care ward and critical care ward in the UK., Participants: Patients with >7% total burns by surface area were recruited into the study., Methods: All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates., Results: WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment., Conclusions: This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

We report the draft genome assembly of Elizabethkingia meningoseptica strain 502. The sample was isolated from the wound of a repatriated military serviceperson who suffered major trauma from an improvised explosive device (IED), resulting in wounds with extensive environmental contamination. E. meningoseptica was isolated from wounds in both legs. The draft genome assembly has 21 contigs with a total size of 3,960,744 bases. The genome contains genes encoding 26 putative β-lactamases.

Chickens are major source of food and protein worldwide. Feed conversion and the health of chickens relies on the largely unexplored complex microbial community that inhabits the chicken gut, including the ceca. We have carried out deep microbial community profiling of the microbiota in twenty cecal samples via 16S rRNA gene sequences and an in-depth metagenomics analysis of a single cecal microbiota. We recovered 699 phylotypes, over half of which appear to represent previously unknown species. We obtained 648,251 environmental gene tags (EGTs), the majority of which represent new species. These were binned into over two-dozen draft genomes, which included Campylobacter jejuni and Helicobacter pullorum. We found numerous polysaccharide- and oligosaccharide-degrading enzymes encoding within the metagenome, some of which appeared to be part of polysaccharide utilization systems with genetic evidence for the co-ordination of polysaccharide degradation with sugar transport and utilization. The cecal metagenome encodes several fermentation pathways leading to the production of short-chain fatty acids, including some with novel features. We found a dozen uptake hydrogenases encoded in the metagenome and speculate that these provide major hydrogen sinks within this microbial community and might explain the high abundance of several genera within this microbiome, including Campylobacter, Helicobacter and Megamonas.

The type III protein secretion system is an important pathogenicity factor of enteropathogenic and enterohaemorrhagic Escherichia coli pathotypes. The genes encoding this apparatus are located on a pathogenicity island (the locus of enterocyte effacement) and are transcriptionally activated by the master regulator Ler. In each pathotype Ler is also known to regulate genes located elsewhere on the chromosome, but the full extent of the Ler regulon is unclear, especially for enteropathogenic E. coli. The Ler regulon was defined for two strains of E. coli: E2348/69 (enteropathogenic) and EDL933 (enterohaemorrhagic) in mid and late log phases of growth by DNA microarray analysis of the transcriptomes of wild-type and ler mutant versions of each strain. In both strains the Ler regulon is focused on the locus of enterocyte effacement - all major transcriptional units of which are activated by Ler, with the sole exception of the LEE1 operon during mid-log phase growth in E2348/69. However, the Ler regulon does extend more widely and also includes unlinked pathogenicity genes: in E2348/69 more than 50 genes outside of this locus were regulated, including a number of known or potential pathogenicity determinants; in EDL933 only 4 extra-LEE genes, again including known pathogenicity factors, were activated. In E2348/69, where the Ler regulon is clearly growth phase dependent, a number of genes including the plasmid-encoded regulator operon perABC, were found to be negatively regulated by Ler. Negative regulation by Ler of PerC, itself a positive regulator of the ler promoter, suggests a negative feedback loop involving these proteins.

Streptococcus pneumoniae causes invasive infections, primarily at the extremes of life. A seven-valent conjugate vaccine (PCV7) is used to protect against invasive pneumococcal disease in children. Within three years of PCV7 introduction, we observed a fourfold increase in serotype 6C carriage, predominantly due to a single clone. We determined the whole-genome sequences of nineteen S. pneumoniae serotype 6C isolates, from both carriage (n = 15) and disease (n = 4) states, to investigate the emergence of serotype 6C in our population, focusing on a single multi-locus sequence type (MLST) clonal complex 395 (CC395). A phylogenetic network was constructed to identify different lineages, followed by analysis of variability in gene sets and sequences. Serotype 6C isolates from this single geographical site fell into four broad phylogenetically distinct lineages. Variation was seen in the 6C capsular locus and in sequences of genes encoding surface proteins. The largest clonal complex was characterised by the presence of lantibiotic synthesis locus. In our population, the 6C capsular locus has been introduced into multiple lineages by independent capsular switching events. However, rapid clonal expansion has occurred within a single MLST clonal complex. Worryingly, plasticity exists within current and potential vaccine-associated loci, a consideration for future vaccine use, target selection and design.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.

Importance: Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain., Objective: To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture., Design, Setting, and Patients: In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain., Main Outcomes and Measures: The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples., Results: During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered., Conclusions and Relevance: These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs.

Here, we take a snapshot of the high-throughput sequencing platforms, together with the relevant analytical tools, that are available to microbiologists in 2012, and evaluate the strengths and weaknesses of these platforms in obtaining bacterial genome sequences. We also scan the horizon of future possibilities, speculating on how the availability of sequencing that is 'too cheap to metre' might change the face of microbiology forever.

Three benchtop high-throughput sequencing instruments are now available. The 454 GS Junior (Roche), MiSeq (Illumina) and Ion Torrent PGM (Life Technologies) are laser-printer sized and offer modest set-up and running costs. Each instrument can generate data required for a draft bacterial genome sequence in days, making them attractive for identifying and characterizing pathogens in the clinical setting. We compared the performance of these instruments by sequencing an isolate of Escherichia coli O104:H4, which caused an outbreak of food poisoning in Germany in 2011. The MiSeq had the highest throughput per run (1.6 Gb/run, 60 Mb/h) and lowest error rates. The 454 GS Junior generated the longest reads (up to 600 bases) and most contiguous assemblies but had the lowest throughput (70 Mb/run, 9 Mb/h). Run in 100-bp mode, the Ion Torrent PGM had the highest throughput (80–100 Mb/h). Unlike the MiSeq, the Ion Torrent PGM and 454 GS Junior both produced homopolymer-associated indel errors (1.5 and 0.38 errors per 100 bases, respectively).

Objectives: To determine variation at the genome level in Escherichia coli ST131 clinical isolates previously shown to be phenotypically diverse., Methods: The genomes of 10 ST131 isolates extensively characterized in previous studies were sequenced using combinations of Illumina and 454 sequencing technology. Whole-genome comparisons and phylogenetic comparisons were then performed across the strain set and with other closely related extraintestinal pathogenic E. coli (ExPEC) strain types., Results: E. coli ST131 is overrepresented in a collection of clinical isolates, and there is large phenotypic variation amongst isolates. In contrast, genome sequencing of a selection of non-related clinical isolates shows almost no genetic variation between ST131 strains, and E. coli ST131 shows evidence of a genetically monomorphic pathogen showing a similar evolutionary trend to hypervirulent Clostridium difficile., Conclusions: A dominant circulating clone of E. coli ST131 has been identified in unrelated clinical urine samples in the UK. The clone splits into two distinct subgroups on the basis of antimicrobial resistance levels and carriage of extended-spectrum β-lactamase plasmids. This provides the most comprehensive snapshot to date of the true molecular epidemiology of ST131 clinical isolates.

Since the 2006 discovery of the Acinetobacter baumannii strain AYE AbaR1 resistance island, similar elements have been reported in numerous members of this species. As AbaR1 is distantly related to Tn7, we have renamed it TnAbaR1. TnAbaR transposons are known to carry multiple antibiotic resistance- and efflux-associated genes, although none have been experimentally studied en bloc. We deleted the TnAbaR transposon in A. baumannii A424, which we have designated TnAbaR23, and characterized independent deletion mutants DCO163 and DCO174. The NotI pulsed-field gel electrophoresis (PFGE) profile of strain DCO174 was consistent with targeted deletion of TnAbaR23 alone, but strain DCO163 apparently harbored a second large genomic deletion. Nevertheless, "subtractive amplification" targeting 52 TnAbaR and/or resistance-associated loci yielded identical results for both mutants and highlighted genes lost relative to strain A424. PCR mapping and genome sequencing revealed the entire 48.3-kb sequence of TnAbaR23. Consistent with TnAbaR23 carrying two copies of sul1, both mutants exhibited markedly increased susceptibility to sulfamethoxazole. In contrast, loss of tetAR(A) resulted in only a minor and variable increase in tetracycline susceptibility. Despite not exhibiting a growth handicap, strain DCO163 was more susceptible than strain DCO174 to 9 of 10 antibiotics associated with mutant-to-mutant variation in susceptibility, suggesting impairment of an undefined resistance-associated function. Remarkably, despite all three strains sharing identical gyrA and parC sequences, the ciprofloxacin MIC of DCO174 was >8-fold that of DCO163 and A424, suggesting a possible paradoxical role for TnAbaR23 in promoting sensitivity to ciprofloxacin. This study highlights the importance of experimental scrutiny and challenges the assumption that resistance phenotypes can reliably be predicted from genotypes alone.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1-4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism (937)ACC>(937)GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

Adherent-invasive Escherichia coli strains are increasingly being associated with intestinal pathologies. Here we present the genome sequence of E. coli HM605, a strain isolated from colonic biopsy specimens of a patient with Crohn's disease.

Infections of avian pathogenic Escherichia coli (APEC) result in annual multimillion-dollar losses to the poultry industry. Despite this, little is known about the mechanisms by which APEC survives and grows in the bloodstream. Thus, the aim of this study was to identify molecular mechanisms enabling APEC to survive and grow in this critical host environment. To do so, we compared the transcriptome of APEC O1 during growth in Luria-Bertani broth and chicken serum. Several categories of genes, predicted to contribute to adaptation and growth in the avian host, were identified. These included several known virulence genes and genes involved in adaptive metabolism, protein transport, biosynthesis pathways, stress resistance, and virulence regulation. Several genes with unknown function, which were localized to pathogenicity islands or APEC O1's large virulence plasmid, pAPEC-O1-ColBM, were also identified, suggesting that they too contribute to survival in serum. The significantly upregulated genes dnaK, dnaJ, phoP, and ybtA were subsequently subjected to mutational analysis to confirm their role in conferring a competitive advantage during infection. This genome-wide analysis provides novel insight into processes that are important to the pathogenesis of APEC O1.