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April 21, 2020

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020

Chromulinavorax destructans, a pathogen of microzooplankton that provides a window into the enigmatic candidate phylum Dependentiae.

Members of the major candidate phylum Dependentiae (a.k.a. TM6) are widespread across diverse environments from showerheads to peat bogs; yet, with the exception of two isolates infecting amoebae, they are only known from metagenomic data. The limited knowledge of their biology indicates that they have a long evolutionary history of parasitism. Here, we present Chromulinavorax destructans (Strain SeV1) the first isolate of this phylum to infect a representative from a widespread and ecologically significant group of heterotrophic flagellates, the microzooplankter Spumella elongata (Strain CCAP 955/1). Chromulinavorax destructans has a reduced 1.2 Mb genome that is so specialized for infection that it shows no evidence of complete metabolic pathways, but encodes an extensive transporter system for importing nutrients and energy in the form of ATP from the host. Its replication causes extensive reorganization and expansion of the mitochondrion, effectively surrounding the pathogen, consistent with its dependency on the host for energy. Nearly half (44%) of the inferred proteins contain signal sequences for secretion, including many without recognizable similarity to proteins of known function, as well as 98 copies of proteins with an ankyrin-repeat domain; ankyrin-repeats are known effectors of host modulation, suggesting the presence of an extensive host-manipulation apparatus. These observations help to cement members of this phylum as widespread and diverse parasites infecting a broad range of eukaryotic microbes.

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April 21, 2020

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

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April 21, 2020

Genomic characterization of Kerstersia gyiorum SWMUKG01, an isolate from a patient with respiratory infection in China.

The Gram-negative bacterium Kerstersia gyiorum, a potential etiological agent of clinical infections, was isolated from several human patients presenting clinical symptoms. Its significance as a possible pathogen has been previously overlooked as no disease has thus far been definitively associated with this bacterium. To better understand how the organism contributes to the infectious disease, we determined the complete genomic sequence of K. gyiorum SWMUKG01, the first clinical isolate from southwest China.The genomic data obtained displayed a single circular chromosome of 3, 945, 801 base pairs in length, which contains 3, 441 protein-coding genes, 55 tRNA genes and 9 rRNA genes. Analysis on the full spectrum of protein coding genes for cellular structures, two-component regulatory systems and iron uptake pathways that may be important for the success of the bacterial survival, colonization and establishment in the host conferred new insights into the virulence characteristics of K. gyiorum. Phylogenomic comparisons with Alcaligenaceae species indicated that K. gyiorum SWMUKG01 had a close evolutionary relationships with Alcaligenes aquatilis and Alcaligenes faecalis.The comprehensive analysis presented in this work determinates for the first time a complete genome sequence of K. gyiorum, which is expected to provide useful information for subsequent studies on pathogenesis of this species.

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April 21, 2020

Information about variations in multiple copies of bacterial 16S rRNA genes may aid in species identification.

Variable region analysis of 16S rRNA gene sequences is the most common tool in bacterial taxonomic studies. Although used for distinguishing bacterial species, its use remains limited due to the presence of variable copy numbers with sequence variation in the genomes. In this study, 16S rRNA gene sequences, obtained from completely assembled whole genome and Sanger electrophoresis sequencing of cloned PCR products from Serratia fonticola GS2, were compared. Sanger sequencing produced a combination of sequences from multiple copies of 16S rRNA genes. To determine whether the variant copies of 16S rRNA genes affected Sanger sequencing, two ratios (5:5 and 8:2) with different concentrations of cloned 16S rRNA genes were used; it was observed that the greater the number of copies with similar sequences the higher its chance of amplification. Effect of multiple copies for taxonomic classification of 16S rRNA gene sequences was investigated using the strain GS2 as a model. 16S rRNA copies with the maximum variation had 99.42% minimum pairwise similarity and this did not have an effect on species identification. Thus, PCR products from genomes containing variable 16S rRNA gene copies can provide sufficient information for species identification except from species which have high similarity of sequences in their 16S rRNA gene copies like the case of Bacillus thuringiensis and Bacillus cereus. In silico analysis of 1,616 bacterial genomes from long-read sequencing was also done. The average minimum pairwise similarity for each phylum was reported with their average genome size and average “unique copies” of 16S rRNA genes and we found that the phyla Proteobacteria and Firmicutes showed the highest amount of variation in their copies of their 16S rRNA genes. Overall, our results shed light on how the variations in the multiple copies of the 16S rRNA genes of bacteria can aid in appropriate species identification.

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April 21, 2020

Genome Sequence of the Black Yeast Exophiala lecanii-corni.

The genome sequence of Exophiala lecanii-corni, a melanized dimorphic fungus with the capability of degrading several volatile organic compounds, was sequenced using PacBio single-molecule real-time (SMRT) sequencing to assist with understanding the molecular basis of its uncommon morphological and metabolic characteristics. The assembled draft genome is presented here.

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April 21, 2020

Closed Genome Sequence of Escherichia coli K-12 Group Strain C600.

Escherichia coli strain C600 is a prototypical K-12 derived laboratory strain which has been broadly used for molecular microbiology and bacterial physiology studies since its isolation in 1954. Here, we present the closed genome sequence of E. coli strain C600, retrieved from the American Type Culture Collection (ATCC 23724).

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April 21, 2020

Decreased biofilm formation ability of Acinetobacter baumannii after spaceflight on China’s Shenzhou 11 spacecraft.

China has prepared for construction of a space station by the early 2020s. The mission will require astronauts to stay on the space station for at least 180 days. Microbes isolated from the International Space Station (ISS) have shown profound resistance to clinical antibiotics and environmental stresses. Previous studies have demonstrated that the space environment could affect microbial survival, growth, virulence, biofilms, metabolism, as well as their antibiotic-resistant phenotypes. Furthermore, several studies have reported that astronauts experience a decline in their immunity during long-duration spaceflights. Monitoring microbiomes in the ISS or the spacecraft will be beneficial for the prevention of infection among the astronauts during spaceflight. The development of a manned space program worldwide not only provides an opportunity to investigate the impact of this extreme environment on opportunistic pathogenic microbes, but also offers a unique platform to detect mutations in pathogenic bacteria. Various microorganisms have been carried on a spacecraft for academic purposes. Acinetobacter baumannii is a common multidrug-resistant bacterium often prevalent in hospitals. Variations in the ability to cope with environmental hazards increase the chances of microbial survival. Our study aimed to compare phenotypic variations and analyze genomic and transcriptomic variations in A. baumannii among three different groups: SS1 (33 days on the Shenzhou 11 spacecraft), GS1 (ground control), and Aba (reference strain). Consequently, the biofilm formation ability of the SS1 strain decreased after 33 days of spaceflight. Furthermore, high-throughput sequencing revealed that some differentially expressed genes were downregulated in the SS1 strain compared with those in the GS1 strain. In conclusion, this present study provides insights into the environmental adaptation of A. baumannii and might be useful for understanding changes in the opportunistic pathogenic microbes on our spacecraft and on China’s future ISS. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Genomic characterization of Nocardia seriolae strains isolated from diseased fish.

Members of the genus Nocardia are widespread in diverse environments; a wide range of Nocardia species are known to cause nocardiosis in several animals, including cat, dog, fish, and humans. Of the pathogenic Nocardia species, N. seriolae is known to cause disease in cultured fish, resulting in major economic loss. We isolated two N. seriolae strains, CK-14008 and EM15050, from diseased fish and sequenced their genomes using the PacBio sequencing platform. To identify their genomic features, we compared their genomes with those of other Nocardia species. Phylogenetic analysis showed that N. seriolae shares a common ancestor with a putative human pathogenic Nocardia species. Moreover, N. seriolae strains were phylogenetically divided into four clusters according to host fish families. Through genome comparison, we observed that the putative pathogenic Nocardia strains had additional genes for iron acquisition. Dozens of antibiotic resistance genes were detected in the genomes of N. seriolae strains; most of the antibiotics were involved in the inhibition of the biosynthesis of proteins or cell walls. Our results demonstrated the virulence features and antibiotic resistance of fish pathogenic N. seriolae strains at the genomic level. These results may be useful to develop strategies for the prevention of fish nocardiosis. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

A Controlled Human Infection Model of Group A Streptococcus Pharyngitis: Which Strain and Why?

Group A Streptococcus (GAS) is a major cause of global infection-related morbidity and mortality. A modern controlled human infection model (CHIM) of GAS pharyngitis can accelerate vaccine development and pathogenesis research. A robust rationale for strain selection is central to meeting ethical, scientific, and regulatory requirements. Multifaceted characterization studies were done to compare a preferred candidate emm75 (M75) GAS strain to three other strains: an alternative candidate emm12 (M12) strain, an M1 strain used in 1970s pharyngitis CHIM studies (SS-496), and a representative (5448) of the globally disseminated M1T1 clone. A range of approaches were used to explore strain growth, adherence, invasion, delivery characteristics, short- and long-term viability, phylogeny, virulence factors, vaccine antigens, resistance to killing by human neutrophils, and lethality in a murine invasive model. The strains grew reliably in a medium without animal-derived components, were consistently transferred using a swab method simulating the CHIM protocol, remained viable at -80°C, and carried genes for most candidate vaccine antigens. Considering GAS molecular epidemiology, virulence factors, in vitro assays, and results from the murine model, the contemporary strains show a spectrum of virulence, with M75 appearing the least virulent and 5448 the most. The virulence profile of SS-496, used safely in 1970s CHIM studies, was similar to that of 5448 in the animal model and virulence gene carriage. The results of this multifaceted characterization confirm the M75 strain as an appropriate choice for initial deployment in the CHIM, with the aim of safely and successfully causing pharyngitis in healthy adult volunteers. IMPORTANCE GAS (Streptococcus pyogenes) is a leading global cause of infection-related morbidity and mortality. A modern CHIM of GAS pharyngitis could help to accelerate vaccine development and drive pathogenesis research. Challenge strain selection is critical to the safety and success of any CHIM and especially so for an organism such as GAS, with its wide strain diversity and potential to cause severe life-threatening acute infections (e.g., toxic shock syndrome and necrotizing fasciitis) and postinfectious complications (e.g., acute rheumatic fever, rheumatic heart disease, and acute poststreptococcal glomerulonephritis). In this paper, we outline the rationale for selecting an emm75 strain for initial use in a GAS pharyngitis CHIM in healthy adult volunteers, drawing on the findings of a broad characterization effort spanning molecular epidemiology, in vitro assays, whole-genome sequencing, and animal model studies. Copyright © 2019 Osowicki et al.

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April 21, 2020

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.

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April 21, 2020

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections.

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads-the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process.

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