Multidrug-Resistant Lineage of Enterotoxigenic Escherichia coli ST182 With Serotype O169:H41 in Airline Waste.

Enterotoxigenic Escherichia coli (ETEC) is the primary aetiologic agent of traveller's diarrhoea and a significant cause of diarrhoeal disease and death in developing countries. ETEC O169:H41 strains are known to cause both traveller's diarrhoea and foodborne outbreaks in developed countries and are cause for concern. Here, whole-genome sequencing (WGS) was used to assemble 46 O169:H41 (ST182) E. coli draft genomes derived from two airplane waste samples sourced from a German international airport. The ST182 genomes were compared with all 84 publicly available, geographically diverse ST182 genomes to construct a core genome-based phylogenetic tree. ST182 isolates were all phylogroup E, the majority serotype O169:H41 (n = 121, 93%) and formed five major clades. The airplane waste isolates differed by an average of 15 core SNPs (range 0-45) but their accessory genome content was diverse. While uncommon in other ST182 genomes, all airplane-derived ST182 isolates carried: (i) extended-spectrum β-lactamase gene bla CTX-M- 15 notably lacking the typical adjacent ISEcp1; (ii) qnrS1 and the S83L mutation in gyrA, both conferring resistance to fluoroquinolones; and (iii) a class 1 integron structure (IS26-intI1 Δ 648-dfrA17-aadA5-qacEΔ1-sul1-ORF-srpC-padR-IS6100-mphR-mrx-mphA-IS26) identified previously in major extraintestinal pathogenic E. coli STs but not in ETEC. ST182 isolates carried ETEC-specific virulence factors STp + CS6. Adhesin/invasin tia was identified in 89% of aircraft ST182 isolates (vs 23%) and was located on a putative genomic island within a hotspot region for various insertions including PAI I536 and plasmid-associated transposons. The most common plasmid replicons in this collection were IncFII (100%; F2:A-:B-) and IncB/O/K/Z (89%). Our data suggest that potentially through travel, E. coli ST182 are evolving a multidrug-resistant profile through the acquisition of class 1 integrons and different plasmids

Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction.

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US$5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial

Hackflex: low cost Illumina sequencing library construction for high sample counts

ABSTRACT We developed Hackflex, a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 11 times more libraries for high-throughput Illumina sequencing to be generated at a fixed cost. We call this new method Hackflex. Quality of library preparation was tested by constructing libraries from E. coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. We demonstrated that Hackflex can produce high quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. Using Hackflex, we were able to achieve a per sample reagent cost of library prep of A$8.66, which is 8.23 times lower than the Standard Nextera Flex protocol at advertised retail price. An additional simple modification to the protocol enables a further price reduction of up to 11 fold or about A$6.50/sample. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programs where sequencing large numbers of libraries is beneficial

A large-scale metagenomic survey dataset of the post-weaning piglet gut lumen

BackgroundEarly weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to modulate the gut microbial composition. As an alternative to antibiotic treatment, studies have previously investigated the potential of probiotics for the prevention of postweaning diarrhea. In order to describe the post-weaning gut microbiota, and to study the effects of two probiotics formulations and of intramuscular antibiotic treatment on the gut microbiota, we sampled and processed over 800 faecal time-series samples from 126 piglets and 42 sows.ResultsHere we report on the largest shotgun metagenomic dataset of the pig gut lumen microbiome to date, consisting of >8 Tbp of shotgun metagenomic sequencing data. The animal trial, the workflow from sample collection to sample processing, and the preparation of libraries for sequencing, are described in detail. We provide a preliminary analysis of the dataset, centered on a taxonomic profiling of the samples, and a 16S-based beta diversity analysis of the mothers and the piglets in the first 5 weeks after weaning.ConclusionsThis study was conducted to generate a publicly available databank of the faecal metagenome of weaner piglets aged between 3 and 9 weeks old, treated with different probiotic formulations and intramuscular antibiotic treatment. Besides investigating the effects of the probiotic and intramuscular antibiotic treatment, the dataset can be explored to assess a wide range of ecological questions with regards to antimicrobial resistance, host-associated microbial and phage communities, and their dynamics during the aging of the host

Community composition and development of the post-weaning piglet gut microbiome

ABSTRACT We report on the largest metagenomic analysis of the pig gut microbiome to date. By processing over 800 faecal time-series samples from 126 piglets and 42 sows, we generated over 8Tbp of metagenomic shotgun sequence data. Here we describe the animal trial procedures, the generation of our metagenomic dataset and the analysis of the microbial community composition using a phylogenetic framework. We assess the effects of intramuscular antibiotic treatment and probiotic oral treatment on the diversity of gut microbial communities. We found differences between individual hosts such as breed, litter, and age, to be important contributors to variation in the community composition. Treatment effects of the antibiotic and probiotic treatments were found but were subtle, while host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appears to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life

High-throughput Quant-iT PicoGreen assay using an automated liquid handling system

© 2019 Future Science. All rights reserved. Workflows in NGS facilities require high-standard practices and high-throughput pipelines to process the large number of samples received in a timely manner. Downstream protocols such as NGS library preparation require accurate estimation of nucleic acid concentrations, which can be achieved using fluorescent dye-based nucleic acid measurement. Here, we report a protocol for preparing a 384-well Quant-iT PicoGreen assay. The protocol allows the concentrations of 184 DNA samples to be measured simultaneously in duplicate in only 1 h using an Eppendorf epMotion 5075 liquid handling system. The advantages of this high-throughput approach include a reduction in both reagents (10x less reagents compared to a standard protocol) and time (3 h for 384 samples compared with 3 days)

Cap removal by honey bees leads to higher pollen rewards from grapevine flowers

Pollen of wind-pollinated plants such as grapevine rapidly dries out and is blown away after the anthers dehisce. Therefore, from the point of view of a pollen-collecting bee, pollen from wind-pollinated flowers is best collected soon after it becomes exposed. In grapevine, pollen becomes available immediately after the calyptra is shed, a process also referred to as capfall. We show, for the first time, that honey bees foraging on grapevine actively remove the calyptra from flowers. Using manual cap removal, we estimate that cap removal increases the pollen yield by 70 % compared to collecting pollen from flowers after capfall. The bees selectively foraged on inflorescences with high numbers of loose caps, thus further enhancing their pollen revenue. We discuss the possible benefits of cap removal by honey bees for the development of individual berries and grape bunches of certain varieties

Mediterranean fruit fly genes exhibit different expression patterns between heat and cold treatments.

Invasive Tephritid fruit flies are a global threat to both agriculture and horticulture industries. Biosecurity has played a critical role in reducing their damage but becomes more and more challenging after several key chemical pesticides were banned or withdrawn for health or environmental reasons. This has led to non-chemical approaches including heat and cold treatments being broadly utilized to get rid of fruit fly infestation. However, the molecular mechanisms to kill the flies underlying these stressors are not clear yet. This knowledge will certainly help refine current post-harvest treatment strategies and develop more efficient, cost-effective and environmentally friendly approaches for fruit fly management. Previously, the molecular response of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) to heat was examined thoroughly, in which 31 key genes were identified with significant changes in expression levels and their high-resolution expression timeline was constructed across 11 timepoints. However, whether these candidate genes respond to cold in the same way was unknown. Here, a temperature bioassay was conducted and the expression profiles of these genes were investigated across the same 11 timepoints using cold treatment. The results showed that most of candidate genes exhibited divergent expression profiles compared to heat treatment, suggesting that the fly molecular response to cold may be different from those to heat. This study provides new knowledge of Tephritid fruit fly response to cold at a molecular level, which could aid in improving current fruit fly management and facilitate the development of new strategies to control this serious horticultural insect pest

Mediterranean fruit fly genes exhibit different expression patterns between heat and cold treatments

Invasive Tephritid fruit flies are a global threat to both agriculture and horticulture industries. Biosecurity has played a critical role in reducing their damage but becomes more and more challenging after several key chemical pesticides were banned or withdrawn for health or environmental reasons. This has led to non-chemical approaches including heat and cold treatments being broadly utilized to get rid of fruit fly infestation. However, the molecular mechanisms to kill the flies underlying these stressors are not clear yet. This knowledge will certainly help refine current post-harvest treatment strategies and develop more efficient, cost-effective and environmentally friendly approaches for fruit fly management. Previously, the molecular response of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) to heat was examined thoroughly, in which 31 key genes were identified with significant changes in expression levels and their high-resolution expression timeline was constructed across 11 timepoints. However, whether these candidate genes respond to cold in the same way was unknown. Here, a temperature bioassay was conducted and the expression profiles of these genes were investigated across the same 11 timepoints using cold treatment. The results showed that most of candidate genes exhibited divergent expression profiles compared to heat treatment, suggesting that the fly molecular response to cold may be different from those to heat. This study provides new knowledge of Tephritid fruit fly response to cold at a molecular level, which could aid in improving current fruit fly management and facilitate the development of new strategies to control this serious horticultural insect pest