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April 21, 2020  |  

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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April 21, 2020  |  

The use of Online Tools for Antimicrobial Resistance Prediction by Whole Genome Sequencing in MRSA and VRE.

The antimicrobial resistance (AMR) crisis represents a serious threat to public health and has resulted in concentrated efforts to accelerate development of rapid molecular diagnostics for AMR. In combination with publicly-available web-based AMR databases, whole genome sequencing (WGS) offers the capacity for rapid detection of antibiotic resistance genes. Here we studied the concordance between WGS-based resistance prediction and phenotypic susceptibility testing results for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) clinical isolates using publicly-available tools and databases.Clinical isolates prospectively collected at the University of Pittsburgh Medical Center between December 2016 and December 2017 underwent WGS. Antibiotic resistance gene content was assessed from assembled genomes by BLASTn search of online databases. Concordance between WGS-predicted resistance profile and phenotypic susceptibility as well as sensitivity, specificity, positive and negative predictive values (NPV, PPV) were calculated for each antibiotic/organism combination, using the phenotypic results as the gold standard.Phenotypic susceptibility testing and WGS results were available for 1242 isolate/antibiotic combinations. Overall concordance was 99.3% with a sensitivity, specificity, PPV, NPV of 98.7% (95% CI, 97.2-99.5%), 99.6% (95 % CI, 98.8-99.9%), 99.3% (95% CI, 98.0-99.8%), 99.2% (95% CI, 98.3-99.7%), respectively. Additional identification of point mutations in housekeeping genes increased the concordance to 99.4% and the sensitivity to 99.3% (95% CI, 98.2-99.8%) and NPV to 99.4% (95% CI, 98.4-99.8%).WGS can be used as a reliable predicator of phenotypic resistance for both MRSA and VRE using readily-available online tools.Copyright © 2019. Published by Elsevier Ltd.

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April 21, 2020  |  

Increased prevalence of Escherichia coli strains from food carrying blaNDM and mcr-1-bearing plasmids that structurally resemble those of clinical strains, China, 2015 to 2017.

Introduction: Emergence of resistance determinants of blaNDM and mcr-1 has undermined the antimicrobial effectiveness of the last line drugs carbapenems and colistin. Aim: This work aimed to assess the prevalence of blaNDM and mcr-1 in E. coli strains collected from food in Shenzhen, China, during the period 2015 to 2017. Methods: Multidrug-resistant E. coli strains were isolated from food samples. Plasmids encoding mcr-1 or blaNDM genes were characterised and compared with plasmids found in clinical isolates.ResultsAmong 1,166 non-repeated cephalosporin-resistant E. coli strains isolated from 2,147 food samples, 390 and 42, respectively, were resistant to colistin and meropenem, with five strains being resistant to both agents. The rate of resistance to colistin increased significantly (p?

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April 21, 2020  |  

Heterologous Expression of Ilicicolin H Biosynthetic Gene Cluster and Production of a New Potent Antifungal Reagent, Ilicicolin J.

Ilicicolin H is a broad-spectrum antifungal agent targeting mitochondrial cytochrome bc1 reductase. Unfortunately, ilicicolin H shows reduced activities in vivo. Here, we report our effort on the identification of ilicicolin H biosynthetic gene cluster (BGC) by genomic sequencing a producing strain, Neonectria sp. DH2, and its heterologous production in Aspergillus nidulans. In addition, a shunt product with similar antifungal activities, ilicicolin J, was uncovered. This effort would provide a base for future combinatorial biosynthesis of ilicicolin H analogues. Bioinformatics analysis suggests that the backbone of ilicicolin H is assembled by a polyketide-nonribosomal peptide synthethase (IliA), and then offloaded with a tetramic acid moiety. Similar to tenellin biosynthesis, the tetramic acid is then converted to pyridone by a putative P450, IliC. The decalin portion is most possibly constructed by a S-adenosyl-l-methionine (SAM)-dependent Diels-Alderase (IliD).

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April 21, 2020  |  

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.

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April 21, 2020  |  

Complete Genome Sequences of Bacteriocin-Producing Streptococcus thermophilus Strains ST106 and ST109.

Streptococcus thermophilus strains ST106 and ST109 produce broad-spectrum bacteriocins encoded within a bacteriocin-like peptide (blp) gene cluster. This study reports the complete genome sequences for both strains, with the ST109 chromosome containing 1,788,866 nucleotides (nt) and 1,572 predicted genes, and ST106 having 1,856,083 nt and 1,601 predicted genes.

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April 21, 2020  |  

Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine. Copyright © 2019 Jung et al.

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April 21, 2020  |  

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities. © 2019 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment.

High-throughput studies of microbial communities suggest that Archaea are a widespread component of microbial diversity in various ecosystems. However, proper quantification of archaeal diversity and community ecology remains limited, as sequence coverage of Archaea is usually low owing to the inability of available prokaryotic primers to efficiently amplify archaeal compared to bacterial rRNA genes. To improve identification and quantification of Archaea, we designed and validated the utility of several primer pairs to efficiently amplify archaeal 16S rRNA genes based on up-to-date reference genes. We demonstrate that several of these primer pairs amplify phylogenetically diverse Archaea with high sequencing coverage, outperforming commonly used primers. Based on comparing the resulting long 16S rRNA gene fragments with public databases from all habitats, we found several novel family- to phylum-level archaeal taxa from topsoil and surface water. Our results suggest that archaeal diversity has been largely overlooked due to the limitations of available primers, and that improved primer pairs enable to estimate archaeal diversity more accurately. © 2018 The Authors. Environmental Microbiology Reports published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Complete Genome Sequence of Lactic Acid Bacterium Pediococcus acidilactici Strain ATCC 8042, an Autolytic Anti-bacterial Peptidoglycan Hydrolase Producer

Pediococcus acidilactici is a probiotic bacterium that is industrially utilized in the food industry and antibiotics development. Here, we determine the complete nucleotide sequence of the genome of Pediococcus acidilactici ATCC 8042. The genome was sequenced by the PacBio RSII to generate a single contig consisting of circular chromosome sequence. Illumina MiniSeq sequencing platform and Sanger sequencing method were additionally utilized to correct errors resulting from the long-read sequencing platform. The sequence consists of 2,009,598 bp with a G + C content of 42.1% and contains 1,865 protein-coding sequences. Based on the sequence information, we could confirm and predict the presence of four peptidoglycan hydrolases by HyPe software. This work, therefore, provides the complete genomic information of P. acidilactici ATCC 8042 with a profitable potential of genome-scale comprehension of anti-pathogenic activity, which can be applied in nutraceutical and pharmaceutical biotechnology field.

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April 21, 2020  |  

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.

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April 21, 2020  |  

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Genomic investigation of Staphylococcus aureus recovered from Gambian women and newborns following an oral dose of intra-partum azithromycin.

Oral azithromycin given during labour reduces carriage of bacteria responsible for neonatal sepsis, including Staphylococcus aureus. However, there is concern that this may promote drug resistance.Here, we combine genomic and epidemiological data on S. aureus isolated from mothers and babies in a randomized intra-partum azithromycin trial (PregnAnZI) to describe bacterial population dynamics and resistance mechanisms.Participants from both arms of the trial, who carried S. aureus in day 3 and day 28 samples post-intervention, were included. Sixty-six S. aureus isolates (from 7 mothers and 10 babies) underwent comparative genome analyses and the data were then combined with epidemiological data. Trial registration (main trial): ClinicalTrials.gov Identifier NCT01800942.Seven S. aureus STs were identified, with ST5 dominant (n?=?40, 61.0%), followed by ST15 (n?=?11, 17.0%). ST5 predominated in the placebo arm (73.0% versus 49.0%, P?=?0.039) and ST15 in the azithromycin arm (27.0% versus 6.0%, P?=?0.022). In azithromycin-resistant isolates, msr(A) was the main macrolide resistance gene (n?=?36, 80%). Ten study participants, from both trial arms, acquired azithromycin-resistant S. aureus after initially harbouring a susceptible isolate. In nine (90%) of these cases, the acquired clone was an msr(A)-containing ST5 S. aureus. Long-read sequencing demonstrated that in ST5, msr(A) was found on an MDR plasmid.Our data reveal in this Gambian population the presence of a dominant clone of S. aureus harbouring plasmid-encoded azithromycin resistance, which was acquired by participants in both arms of the study. Understanding these resistance dynamics is crucial to defining the public health drug resistance impacts of azithromycin prophylaxis given during labour in Africa. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

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April 21, 2020  |  

Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates.

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96 and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a =95?%?phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.

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