The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level.

Type 2 diabetes mellitus (T2D) is a growing health problem, but little is known about its early disease stages, its effects on biological processes or the transition to clinical T2D. To understand the earliest stages of T2D better, we obtained samples from 106 healthy individuals and individuals with prediabetes over approximately four years and performed deep profiling of transcriptomes, metabolomes, cytokines, and proteomes, as well as changes in the microbiome. This rich longitudinal data set revealed many insights: first, healthy profiles are distinct among individuals while displaying diverse patterns of intra- and/or inter-personal variability. Second, extensive host and microbial changes occur during respiratory viral infections and immunization, and immunization triggers potentially protective responses that are distinct from responses to respiratory viral infections. Moreover, during respiratory viral infections, insulin-resistant participants respond differently than insulin-sensitive participants. Third, global co-association analyses among the thousands of profiled molecules reveal specific host–microbe interactions that differ between insulin-resistant and insulin-sensitive individuals. Last, we identified early personal molecular signatures in one individual that preceded the onset of T2D, including the inflammation markers interleukin-1 receptor agonist (IL-1RA) and high-sensitivity C-reactive protein (CRP) paired with xenobiotic-induced immune signalling. Our study reveals insights into pathways and responses that differ between glucose-dysregulated and healthy individuals during health and disease and provides an open-access data resource to enable further research into healthy, prediabetic and T2D states.

BackgroundChanges in diet and exercise can alter the gut microbiome of humans and mice; however, few studies to date have assessed the microbiomes of highly fit athletes. In this pilot study, we used metagenomic whole genome shotgun (mWGS) and metatranscriptomic (RNA-Seq) sequencing to show what organisms are both present and active in the gut microbiomes of both professional and amateur level competitive cyclists and to determine if any significant differences exist between these two groups.ResultsUsing mWGS sequencing data, we showed that the gut microbiomes of 33 cyclists split into three taxonomic clusters, characterized by either high Prevotella, high Bacteroides or a mix of many genera including Bacteroides, Prevotella, Eubacterium, Ruminococcus, and Akkermansia. While no significant correlations could be found between taxonomic cluster and being either a professional or amateur level cyclist, high abundance of the genus Prevotella (≥2.5%) was significantly correlated with time reported exercising during an average week. Increased abundance of Prevotella was correlated with a number of amino acid and carbohydrate metabolism pathways, including branched chain amino acid metabolism. Further analysis of the metatranscriptome revealed significant taxonomic differences when compared to the metagenome. There was increased abundance of Methanobrevibacter smithii transcripts in a number of professional cyclists in comparison to amateur cyclists and this archaeon had upregulation of genes involved in the production of methane. Furthermore, when methane metabolism was upregulated, there was similar upregulation of energy and carbohydrate metabolism pathways.ConclusionsThese results provide a framework for common constituents of the gut community in individuals who follow an exercise-rich lifestyle. These data also suggest how certain organisms such as M. smithii may beneficially influence the metabolic efficiency of the gut community in professional cyclists due to synergistic metabolic cross-feeding events.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0320-4) contains supplementary material, which is available to authorized users.

Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

Found widespread around the globe, Serratia are Gram-negative bacteria capable of thriving in a diverse number of environments that include water, soil, and the digestive tracts of various animals. Known for their ability to produce a myriad of extracellular enzymes, these bacteria also produce various secondary metabolites that directly contribute to their survival. While the effects Serratia species have on other organisms range from parasitic to symbiotic, what these bacteria have in common is their ability to resist attack, respond appropriately to environmental conditions, and outcompete other microorganisms when colonizing their respective niche. This review highlights the mechanisms utilized by Serratia species that drive their ubiquitous nature, with emphasis on the latest findings. Also discussed is how secreted compounds drive these bacteria towards pathogenic, mutualistic, and antagonistic associations.

The physiology of a newly recognized Serratia species, termed South African Caenorhabditis briggsae Isolate (SCBI), which is both a nematode mutualist and an insect pathogen, was investigated and compared to that of Serratia marcescens Db11, a broadhost-range pathogen. The two Serratia strains had comparable levels of virulence for Manduca sexta and similar cytotoxic activity patterns, but motility and lipase and hemolytic activities differed significantly between them. M embers of the genus Serratia are found widespread around the globe and are well known for their roles as insect pathogens (14). A newly recognized Serratia species, termed South African Caenorhabditis briggsae Isolate (SCBI), was identified following its isolation from the nematode C. briggsae KT0001 (2). These C. briggsae KT0001 nematodes were recovered from soil samples through Galleria mellonella bait traps in three provinces in South Africa (2). Serratia sp. strain SCBI is lethal to G. mellonella. When directly injected into the hemocoel in numbers of less than 1,000 CFU, larvae die within 72 h. A distinct characteristic of Serratia sp. SCBI is that it forms an apparent association with C. briggsae KT0001 as well as other Caenorhabditis nematodes, including Caenorhabditis elegans, which results in entomopathogenicity of the nematode (1, 2). This microbe-nematode association between Serratia sp. SCBI and C. briggsae may represent a potential emerging entomopathogenic association. Only two other Serratia species are known to use a nematode partner to establish an infection in an invertebrate host (40,43).Based on 16S phylogeny, Serratia sp. SCBI is closely related to S. marcescens Db11 (3), a reported pathogen of C. elegans (24,34,36). We have sequenced the entire Serratia sp. SCBI genome and have performed an analysis comparing it to the S. marcescens Db11 genome (F. Abebe-Akele, L. S. Tisa, V. Cooper, P. J. Hatcher, E. Abebe, and W. K. Thomas, unpublished data). S. marcescens Db11 is a well-known pathogen of vertebrates and invertebrates, including Caenorhabditis nematodes. Although their 16S RNA genes are 99% identical, genome-wide assessment (23) supports the idea that Serratia sp. SCBI and S. marcescens Db11 represent two distinct species. However, the high (about 80%) similarity at the 16S rRNA gene and within open reading frames indicates that Serratia sp. SCBI and S. marcescens Db11 share a close evolutionary relationship despite their distinct associations with Caenorhabditis nematodes. Since mutualistic associations have the potential to evolve from parasitic relationships (10), it is possible that Serratia sp. SCBI diverged from S. marcescens, making the leap from Caenorhabditis pathogen to a mutualistic relationship.Although the virulence factors of S. marcescens have been well studied (6,8,24,26,33), there have been no studies on the physiological properties of Serratia sp. SCBI and their contribution to pathogenesis. Due to their close evolutionary history, a comparative study of Serratia sp. SCBI and S. marcescens Db11 was cond...

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.