What can we learn from over 100,000 Escherichia coli genomes?

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Kaleb Z Abram, Zulema Udaondo, Carissa Bleker, Visanu Wanchai, Trudy M Wassenaar, Michael S Robeson, David W Ussery

bioRxiv 708131; doi: https://doi.org/10.1101/708131


The explosion of microbial genome sequences in public databases allows for large-scale population genomic studies of bacterial species, such as Escherichia coli. In this study, we examine and classify more than one hundred thousand E. coli and Shigella genomes. After removing outliers, a semi-automated Mash-based analysis of 10,667 assembled genomes reveals 14 distinct phylogroups. A representative genome or medoid identified for each phylogroup serves as a proxy to classify more than 95,000 unassembled genomes. This analysis shows that most sequenced E. coli genomes belong to 4 phylogroups (A, C, B1 and E2(O157)). Authenticity of the 14 phylogroups described is supported by pangenomic and phylogenetic analyses, which show differences in gene preservation between phylogroups. A phylogenetic tree constructed with 2,613 single copy core genes along with a matrix of phylogenetic profiles is used to confirm that the 14 phylogroups change at different rates of gene gain/loss/duplication. The methodology used in this work is able to identify previously uncharacterized phylogroups in E. coli species. Some of these new phylogroups harbor clonal strains that have undergone a process of genomic adaptation to the acquisition of new genomic elements related to virulence or antibiotic resistance. This is, to our knowledge, the largest E. coli genome dataset analyzed to date and provides valuable insights into the population structure of the species.

Read the publication here: https://www.biorxiv.org/content/10.1101/708131v2

2019_nCoV: Rapid classification of betacoronaviruses and identification of traditional Chinese medicine as potential origin of zoonotic coronaviruses

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Trudy M. Wassenaar and Ying Zou

Lett Appl Microbiol. 2020 Feb 14. doi: 10.1111/lam.13285. [Epub ahead of print]


The current outbreak of a novel SARS‐like coronavirus, 2019_nCoV, illustrated difficulties in identifying a novel coronavirus and its natural host, as the coding sequences of various Betacoronavirus species can be highly diverse. By means of whole‐genome sequence comparisons, we demonstrate that the non‐coding flanks of the viral genome can be used to correctly separate the recognized four betacoronavirus subspecies. The conservation would be sufficient to define target sequences that could, in theory, classify novel virus species into their subspecies. Only 253 upstream non‐coding sequences of Sarbecovirus are sufficient to identify genetic similarities between species of this subgenus. Further, it was investigated which bat species have commercial value in China, and would thus likely be handled for trading purposes. A number of coronavirus genomes have been published that were obtained from such bat species. These bats are used in Traditional Chinese Medicine, and their handling poses a potential risk to cause zoonotic coronavirus epidemics.

Keywords Sarbecovirus ; Coronavirus; Traditional Chinese Medicine; bats; epidemic; whole-genome comparison; zoonosis

Read the publication here: https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/lam.13285

Comment on “Enumeration of Escherichia coli in Probiotic Products. Microorganisms 2019, 7, 437”

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Wassenaar TM, von Bünau R, and Zimmermann

Microorganisms 2020, 8(2), 245; https://doi.org/10.3390/microorganisms8020245


Recently, Zimmer and Dorea published a communication on the enumeration of Escherichiaácoli in probiotic products containing this species [1]. They investigated the content of two commercial products, Mutaflor (produced by Pharma-Zentrale GmbH, Herdecke, Germany) and Symbioflor 2 (produced by SymbioPharm GmbH, Herborn, Germany). Mutaflor is available as viable, lyophilized E. ácoli bacteria inside an acid-resistant capsule, while Symbioflor 2 contains viable E. ácoliábacteria in a liquid suspension. The authors tested the viability of the bacteria for three batches of each product. They report that the products contained E. ácoli in numbers several orders of magnitude less than claimed on the product information [1].The authors assessed the number of viable bacteria by applying the methodology optimized for enumeration of coliform bacteria in surface waters. Serial dilutions were made in Ringers solution (the content of the Mutaflor capsules was resuspended for this) and these were enumerated in triplicate using a Colilert Quanti-train/2000 system, from which the most probable number (MPN) was obtained. This method is validated and recommended in multiple countries, including Germany [2], to estimate the number of coliform bacteria in samples expected to contain low numbers, for instance, surface water samples. For such samples the MPN methodology has been developed and optimized [3].

Read the publication here: https://www.mdpi.com/2076-2607/8/2/245

Comparative genomics of Hepatitis A virus, Hepatitis C virus and Hepatitis E virus provides insights into the evolutionary history of Hepatovirus species

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Trudy M. Wassenaar, Se-Ran Jun, Michael Robeson and David W. Ussery

MicrobiologyOpen. 2019 Nov 19:e973. doi: 10.1002/mbo3.973.


The intraspecies genomic diversity of the single-strand RNA (+) virus species hepatitis A virus (Hepatovirus), hepatitis C virus (Hepacivirus), and hepatitis E virus (Orthohepevirus) was compared. These viral species all can cause liver inflammation (hepatitis), but share no gene similarity. The codon usage of human hepatitis A virus (HAV) is suboptimal for replication in its host, a characteristic it shares with taxonomically related rodent, simian, and bat hepatitis A virus species. We found this codon usage to be strikingly similar to that of Triatoma virus that infects blood-sucking kissing bugs. The codon usage of that virus is well adapted to its insect host. The codon usage of HAV is also similar to other invertebrate viruses of various taxonomic families. An evolutionary ancestor of HAV and related virus species is hypothesized to be an insect virus that underwent a host jump to infect mammals. The similarity between HAV and invertebrate viruses goes beyond codon usage, as they also share amino acid composition characteristics, while not sharing direct sequence homology. In contrast, hepatitis C virus and hepatitis E virus are highly similar in codon usage preference, nucleotide composition, and amino acid composition, and share these characteristics with Human pegivirus A, West Nile virus, and Zika virus. We present evidence that these observations are only partly explained by differences in nucleotide composition of the complete viral codon regions. We consider the combination of nucleotide composition, amino acid composition, and codon usage preference suitable to provide information on possible evolutionary similarities between distant virus species that cannot be investigated by phylogeny.

Keywords Hepactovirus A; codon bias; comparative genomics; evolution; hepatitis A virus

Read the publication here: https://onlinelibrary.wiley.com/doi/full/10.1002/mbo3.973

Complete Genome Sequences of Four Isolates of Vancomycin-Resistant Enterococcus faecium with the vanA Gene and Two Daptomycin Resistance Mutations, Obtained from Two Inpatients with Prolonged Bacteremia.

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Piroon Jenjaroenpun, Thidathip Wongsurawat, Zulema Udaondo, Courtney Anderson, James Lopez, Meera Mohan, Ruslana Tytarenko, Brian Walker, Intawat Nookaew, David Ussery, Atul Kothari, Se-Ran Jun

Microbiol Resour Announc. 2020 Feb 6;9(6). pii: e01380-19. doi: 10.1128/MRA.01380-19.


Here, we present complete genome sequences of four Enterococcus faecium isolates, obtained from two patients with apparent vancomycin-resistant Enterococcus faecium bacteremia; these isolates also carried two mutations known to be associated with daptomycin resistance. Sequences were obtained using de novo and hybrid assembly of Oxford Nanopore and Illumina sequence data.

Read the publication here: https://mra.asm.org/content/9/6/e01380-19

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota

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Takeo Shibata, Mayumi Nakagawa, Hannah N Coleman, Sarah M Owens, William W Greenfield, Toshiyuki Sasagawa, Michael S Robeson

bioRxiv 2020.01.27.921619; doi: https://doi.org/10.1101/2020.01.27.921619


Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping, and can be used for long-term cytological biobanking. Herein, we investigate the feasibility of leveraging LBC specimens for use in CM surveys by amplicon sequencing. As methodological differences in DNA extraction protocols can potentially bias the composition of microbiota, we set out to determine the performance of four commonly used DNA extraction kits (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with cervical intraepithelial neoplasia (CIN) 2/3 or suspected CIN2/3 were each aliquoted for extraction by each of the four kits. We observed that, regardless of the extraction protocol used, all kits provided equivalent accessibility to the cervical microbiome, with some minor differences. For example, the ZymoBIOMICS kit appeared to differentially increase access of several more microbiota compared to the other kits. Potential kit contaminants were observed as well. Approximately 80% microbial genera were shared among all DNA extraction protocols. The variance of microbial composition per individual was larger than that of the DNA extraction protocol used. We also observed that HPV16 infection was significantly associated with community types that were not dominated by Lactobacillus iners.

Importance Collection of LBC specimens is routine for cervical cancer screening and HPV genotyping, and can be used for long-term cytological biobanking. We demonstrated that LBC samples, which had been under prolonged storage prior to DNA extraction, were able to provide a robust assessment of the CM and its relationship to HPV status, regardless of the extraction kit used. Being able to retroactively access the CM from biobanked LBC samples, will allow researchers to better interrogate historical interactions between the CM and its relationship to CIN and HPV. This alone has the potential to bring CM research one-step closer to the clinical practice.

Read the publication here: https://www.biorxiv.org/content/10.1101/2020.01.27.921619v1

The Interaction between 30b-5p miRNA and MBNL1 mRNA is Involved in Vascular Smooth Muscle Cell Differentiation in Patients with Coronary Atherosclerosis

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Chin Cheng Woo, Wenting Liu, Xiao Yun Lin, Rajkumar Dorajoo, Kee Wah Lee, A Mark Richards, Chuen Neng Lee, Thidathip Wongsurawat, Intawat Nookaew, Vitaly Sorokin

International Journal of Molecular Sciences 21 (1), 11


Vascular smooth muscle cells (VSMCs) in the arterial wall have diverse functions. In pathological states, the interplay between transcripts and microRNAs (miRNAs) leads to phenotypic changes. Understanding the regulatory role of miRNAs and their target genes may reveal how VSMCs modulate the pathogenesis of coronary artery disease. Laser capture microdissection was performed on aortic wall tissues obtained from coronary artery bypass graft patients with and without recent acute myocardial infarction (MI). The mSMRT-qPCR miRNA assay platform (MiRXES, Singapore) was used to profile miRNA. The miRNA data were co-analyzed with significant mRNA transcripts. TargetScan 7.1 was applied to evaluate miRNA-mRNA interactions. The miRNA profiles of 29 patients (16 MI and 13 non-MI) were evaluated. Thirteen VSMC-related miRNAs were differentially expressed between the MI and non-MI groups. Analysis revealed seven miRNA-targeted mRNAs related to muscular tissue differentiation and proliferation. TargetScan revealed that among the VSMC-related transcripts, MBNL1 had a recognition site that matched the hsa-miR-30b-5p target seed sequence. In addition to predicted analysis, our experiment in vitro with human VSMC culture confirmed that hsa-miR-30b-5p negatively correlated with MBNL1. Our data showed that overexpression of hsa-miR-30b-5p led to downregulation of MBNL1 in VSMCs. This process influences VSMC proliferation and might be involved in VSMC differentiation.

Keywords aortic wall; atherosclerosis; microRNA; muscle cell differentiation; vascular smooth muscle cells

Read the publication here: https://www.mdpi.com/1422-0067/21/1/11

Global metabolite profiles of rice brown planthopper-resistant traits reveal potential secondary metabolites for both constitutive and inducible defenses

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Uawisetwathana U, Chevallier OP, Xu Y, Kamolsukyeunyong W, Nookaew I, Somboon T, Toojinda T, Vanavichit A, Goodacre R, Elliott CT, Karoonuthaisiri N.

Metabolomics. 2019 Nov 19;15(12):151. doi: 10.1007/s11306-019-1616-0.


Introduction: Brown planthopper (BPH) is a phloem feeding insect that causes annual disease outbreaks, called hopper burn in many countries throughout Asia, resulting in severe damage to rice production. Currently, mechanistic understanding of BPH resistance in rice plant is limited, which has caused slow progression on developing effective rice varieties as well as effective farming practices against BPH infestation. Objective: To reveal rice metabolic responses during 8 days of BPH attack, this study examined polar metabolome extracts of BPH-susceptible (KD) and its BPH-resistant isogenic line (IL308) rice leaves. Methods: Ultra high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) was combined with multi-block PCA to analyze potential metabolites in response to BPH attack. Results: This multivariate statistical model revealed different metabolic response patterns between the BPH-susceptible and BPH-resistant varieties during BPH infestation. The metabolite responses of the resistant IL308 variety occurred on Day 1, which was significantly earlier than those of the susceptible KD variety which showed an induced response by Days 4 and 8. BPH infestation caused metabolic perturbations in purine, phenylpropanoid, flavonoid, and terpenoid pathways. While found in both susceptible and resistant rice varieties, schaftoside (1.8 fold), iso-schaftoside (1.7 fold), rhoifolin (3.4 fold) and apigenin 6-C-α-L-arabinoside-8-C-β-L-arabinoside levels (1.6 fold) were significantly increased in the resistant variety by Day 1 post-infestation. 20-hydroxyecdysone acetate (2.5 fold) and dicaffeoylquinic acid (4.7 fold) levels were considerably higher in the resistant rice variety than those in the susceptible variety, both before and after infestation, suggesting that these secondary metabolites play important roles in inducible and constitutive defenses against the BPH infestation. Conclusions: These potential secondary metabolites will be useful as metabolite markers and/or bioactive compounds for effective and durable approaches to address the BPH problem.

Keywords: Brown planthopper resistance; LC-HRMS; Metabolite profiling; Multi-block principal component analysis; Oryza sativa; Thai Jasmine rice

Read the publication here: https://link.springer.com/article/10.1007/s11306-019-1616-0

Metabolic profiling and compound-class identification reveal alterations in serum triglyceride levels in mice immunized with human vaccine adjuvant Alum.

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Khoomrung S, Nookaew I, Sen P, Olafsdottir TA, Persson J, Moritz T, Andersen P, Harandi AM, Nielsen J.

J Proteome Res. 2019 Oct 18. doi: 10.1021/acs.jproteome.9b00517. PMID: 31625748


Alum has been widely used as an adjuvant for human vaccines; however, the impact of Alum on host metabolism remains largely unknown. Herein, we applied mass spectrometry (LCMS)-based metabolic and lipid profiling to monitor the effects of Alum adjuvant on mouse serum at 6, 24, 72 and 168 h post-vaccination. We propose a new strategy termed Subclass Identification and Annotation for Metabolomics (SIAM) for class-wise identification of untargeted metabolomics data generated from high-resolution MS. Using this approach, we identified and validated the levels of several lipids in mouse serum that were significantly altered following Alum administration. These lipids showed a biphasic response even 168 h after vaccination. The majority of the lipids were triglycerides (TAGs), where TAGs with long chain unsaturated fatty acids were decreased at 24 h, and TAGs with short chain fatty acids were decreased at 168 h. To our knowledge, this is the first report on the impact of the human vaccine adjuvant Alum on host metabolome, and may provide new insights into the mechanism of action of Alum.

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