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.

Abstract

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

Abstract

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

Abstract

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

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

Abstract

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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An Assessment of Oxford Nanopore Sequencing for Human Gut Metagenome Profiling: A Pilot Study of Head and Neck Cancer Patients

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Thidathip Wongsurawat, Mayumi Nakagawa, Omar Atiq, Hannah N. Coleman, Piroon Jenjaroenpun, James I. Allred, Angela Trammel, Pantakan Puengrang, David W. Ussery, Intawat Nookaew

J Microbiol Methods. 2019 Oct 15:105739. doi: 10.1016/j.mimet.2019.105739. PMID: 31626891

Abstract

Gut metagenome profiling using the Oxford Nanopore Technology (ONT) sequencer was assessed in a pilot-sized study of 10 subjects. The taxonomic abundance of gut microbiota derived from ONT was comparable with Illumina Technology (IT) for the high-abundance species. IT better detected low-abundance species through amplification, when material was limited.

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Gastrointestinal tract dysbiosis enhances distal tumor progression through suppression of leukocyte trafficking

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Samir V. Jenkins,Michael S. Robeson II, Robert J. Griffin, Charles M. Quick, Eric R. Siegel, Martin J. Cannon, Kieng B. Vang, and Ruud P.M. Dings

Cancer Research, published online 7 October, 2019; DOI: 10.1158/0008-5472.CAN-18-4108.

Abstract

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis Lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNF-α levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T-cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNF-α supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer development.

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Genome-Based Comparison of Clostridioides difficile : Average Amino Acid Identity Analysis of Core Genomes

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Adriana Cabal, Se-Ran Jun, Piroon Jenjaroenpun, Visanu Wanchai, Intawat Nookaew, Thidathip Wongsurawat, Mary J. Burgess, Atul Kothari, Trudy M. Wassenaar1, David W. Ussery

Received: 30 August 2017 /Accepted: 2 February 2018 # The Author(s) 2018. This article is an open access publication

Abstract

Infections due to Clostridioides difficile (previously known as Clostridium difficile) are a major problem in hospitals, where cases can be caused by community-acquired strains as well as by nosocomial spread. Whole genome sequences from clinical samples contain a lot of information but that needs to be analyzed and compared in such a way that the outcome is useful for clinicians or epidemiologists. Here, we compare 663 public available complete genome sequences of C. difficile using average amino acid identity (AAI) scores. This analysis revealed that most of these genomes (640, 96.5%) clearly belong to the same species, while the remaining 23 genomes produce four distinct clusters within the Clostridioides genus. The main C. difficile cluster can be further divided into sub-clusters, depending on the chosen cutoff. We demonstrate that MLST, either based on partial or full gene-length, results in biased estimates of genetic differences and does not capture the true degree of similarity or differences of complete genomes. Presence of genes coding for C. difficile toxins A and B (ToxA/B), as well as the binary C. difficile toxin (CDT), was deduced from their unique PfamA domain architectures. Out of the 663 C. difficile genomes, 535 (80.7%) contained at least one copy of ToxA or ToxB, while these genes were missing from 128 genomes. Although some clusters were enriched for toxin presence, these genes are variably present in a given genetic background. The CDT genes were found in 191 genomes, which were restricted to a few clusters only, and only one cluster lacked the toxin A/B genes consistently. A total of 310 genomes contained ToxA/B without CDT (47%). Further, published metagenomic data from stools were used to assess the presence of C. difficile sequences in blinded cases of C. difficile infection (CDI) and controls, to test if metagenomic analysis is sensitive enough to detect the pathogen, and to establish strain relationships between cases from the same hospital. We conclude that metagenomics can contribute to the identification of CDI and can assist in characterization of the most probable causative strain in CDI patients.

Keywords C. difficile, AAI .MLST, Community-acquired infections, Comparative genomics

Read the publication here: http://rdcu.be/GWYD

Interspecific plant interactions reflected in soil bacterial community structure and nitrogen cycling in primary succession.

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Front. Microbiology, in the press, January 2018  | doi: 10.3389/fmicb.2018.00128
https://www.frontiersin.org/articles/10.3389/fmicb.2018.00128/abstract

Joseph E. Knelman, Emily B. Graham, Janet S. Prevéy, Michael S. Robeson, Patrick Kelly, Eran Hood and Steve K. Schmidt

Past research demonstrating the importance plant-microbe interactions as drivers of ecosystem succession has focused on how plants condition soil microbial communities, impacting subsequent plant performance and plant community assembly. These studies, however, largely treat microbial communities as a black box. In this study we sought to examine how emblematic shifts from early-successional Alnus sinuata (alder) to late successional Picea sitchensis (spruce) in primary succession may be reflected in specific belowground changes in bacterial community structure and nitrogen cycling related to the interaction of these two plants. We examined early successional alder-conditioned soils in a glacial forefield to delineate how alders alter the soil microbial community with increasing dominance. Further, we assessed the impact of late-successional spruce plants on these early-successional alder-conditioned microbiomes and related nitrogen cycling through a leachate addition microcosm experiment. In total, we show how increasingly abundant alder select for particular bacterial taxa. Additionally, we found that spruce leachate significantly alters the composition of these microbial communities in large part by driving declines in taxa that are enriched by alder, including bacterial symbionts. We found these effects to be spruce-specific, beyond a general leachate effect. Our work also demonstrates a unique influence of spruce on ammonium availability. Such insights bolster theory relating the importance of plant-microbe interactions with late-successional plants and interspecific plant interactions more generally.

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Genome Characterization of Oleaginous Aspergillus oryzae BCC7051: A Potential Fungal-Based Platform for Lipid Production

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Curr Microbiol. 2018 Jan;75(1):57-70. doi: 10.1007/s00284-017-1350-7. Epub 2017 Sep 1.

Thammarongtham C, Nookaew I, Vorapreeda T, Srisuk T, Land ML, Jeennor S, Laoteng K

Abstract

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.

PMID: 28865010 DOI: 10.1007/s00284-017-1350-7