Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 57,441 bioRxiv papers from 264,501 authors.
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in category microbiology
4,470 results found. For more information, click each entry to expand.
14,205 downloads microbiology
Microplates are essential tools for biofilm research since it allows high throughput screening of biofilm forming strains or in the assay of anti-biofilm drugs. However, 96 well microtitre plate based assays share the issue of 'edge effect'. The primary cause of the 'edge effect' phenomenon is evaporation. As edge effect causes a significant increase in plate rejection rate by introducing experimental error, we improvised the classical crystal violet assay to reduce water loss from the peripheral wells. The improvised method showed a significant reduction in edge effect and minimised error in crystal violet assay
14,119 downloads microbiology
Zika virus (ZIKV) infections were more common in the zoonotic cycle until the end of the 20th century with few human cases in Africa and Southeastern Asia. Recently, the Asian lineage of ZIKV is spreading along human-to-human chains of transmission in the Pacific Islands and in South America. To better understand its recent urban expansion, we compared genetic differences among the lineages. Herein we show that the recent Asian lineage spread is associated with significant NS1 codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titers. These findings were supported by a significant correlation with growth in Malthusian fitness. Furthermore, we predicted several epitopes in the NS1 protein that are shared between ZIKV and Dengue. Our results imply in a significant dependence of the recent human ZIKV spread on NS1 translational selection.
7,915 downloads microbiology
John P Pribis, Libertad García-Villada, Yin Zhai, Ohad Lewin-Epstein, Anthony Wang, Jingjing Liu, Jun Xia, Qian Mei, Devon M Fitzgerald, Julia Bos, Robert Austin, Christophe Herman, David Bates, Lilach Hadany, P.J. Hastings, Susan M Rosenberg
Antibiotics can induce mutations that cause antibiotic resistance. Yet, despite their importance, mechanisms of antibiotic-promoted mutagenesis remain elusive. We report that the fluoroquinolone antibiotic ciprofloxacin (cipro) induces mutations that cause drug resistance by triggering differentiation of a mutant-generating cell subpopulation, using reactive oxygen species (ROS) to signal the sigma-S (σS) general-stress response. Cipro-generated DNA breaks activate the SOS DNA-damage response and error-prone DNA polymerases in all cells. However, mutagenesis is restricted to a cell subpopulation in which electron transfer and SOS induce ROS, which activate the σS response, allowing mutagenesis during DNA-break repair. When sorted, this small σS-response-'on' subpopulation produces most antibiotic cross-resistant mutants. An FDA-approved drug prevents σS induction specifically inhibiting antibiotic-promoted mutagenesis. Furthermore, SOS-inhibited cell division, causing multi-chromosome cells, is required for mutagenesis. The data support a model in which within-cell chromosome cooperation together with development of a 'gambler' cell subpopulation promote resistance evolution without risking most cells.
6,909 downloads microbiology
In the field of observation, chance favours only the prepared mind (Pasteur). Impressive developments in genomics have led microbiology to its third Golden Age. However, conventional metagenomics strategies necessitate retrograde transfer of samples from extreme or remote environments for later analysis, rendering the powerful insights gained retrospective in nature, striking a contrast with Pasteur's dictum. Here we implement highly portable USB-based nanopore DNA sequencing platforms coupled with field-adapted environmental DNA extraction, rapid sequence library generation and off-line analyses of shotgun metagenome and 16S ribosomal RNA gene amplicon profiles to characterize microbiota dwelling within cryoconite holes upon Svalbard glaciers, the Greenland Ice Sheet and the Austrian Alps. We show in-field nanopore sequencing of metagenomes captures taxonomic composition of supraglacial microbiota, while 16S rRNA Furthermore, comparison of nanopore data with prior 16S rRNA gene V1-V3 pyrosequencing from the same samples, demonstrates strong correlations between profiles obtained from nanopore sequencing and laboratory based sequencing approaches. 16S gene amplicon sequencing resolves bacterial community responses to habitat changes. Finally, we demonstrate the fidelity and sensitivity of in-field sequencing by analysis of mock communities using field protocols. Ultimately, in-field sequencing potentiated by nanopore devices raises the prospect of enhanced agility in exploring Earth's most remote microbiomes.
6,186 downloads microbiology
Gonorrhea is a sexually transmitted infection caused by the Gram-negative bacterium Neisseria gonorrhoeae. Resistance to first-line empirical monotherapy has emerged, so robust methods are needed to appropriately evaluate the activity of existing and novel antimicrobials against the bacterium. Pharmacodynamic functions, which describe the relationship between the concentration of antimicrobials and the bacterial net growth rate, provide more detailed information than the MIC only. In this study, a novel standardized in vitro time-kill curve assay was developed. The assay was validated using five World Health Organization N. gonorrhoeae reference strains and various concentrations of ciprofloxacin, and then the activity of nine antimicrobials with different target mechanisms were examined against a highly susceptible clinical wild type isolate (cultured in 1964). From the time-kill curves, the bacterial net growth rates at each antimicrobial concentration were estimated. Finally, a pharmacodynamic function was fitted to the data, resulting in four parameters that describe the pharmacodynamic properties of each antimicrobial. Ciprofloxacin resistance determinants shifted the pharmacodynamic MIC (zMIC) and attenuated the bactericidal effect at antimicrobial concentrations above the zMIC. Ciprofloxacin, spectinomycin and gentamicin had the strongest bactericidal effect during the first six hours of the assay. Only tetracycline and chloramphenicol showed a purely bacteriostatic effect. The pharmacodynamic functions differed between antimicrobials, showing that the effect of the drugs at concentrations below and above the MIC vary widely. In conclusion, N. gonorrhoeae time-kill curve experiments analyzed with pharmacodynamic functions have potential for in vitro evaluation of new and existing antimicrobials and dosing strategies to treat gonorrhea.
5,914 downloads microbiology
Taxonomy is a fundamental organizing principle of biology, which ideally should be based on evolutionary relationships. Microbial taxonomy has been greatly restricted by the inability to obtain most microorganisms in pure culture and, to a lesser degree, the historical use of phenotypic properties as the basis for classification. However, we are now at the point of obtaining genome sequences broadly representative of microbial diversity by using culture-independent techniques, which provide the opportunity to develop a comprehensive genome-based taxonomy. Here we propose a standardized bacterial taxonomy based on a concatenated protein phylogeny that conservatively removes polyphyletic groups and normalizes ranks based on relative evolutionary divergence. From 94,759 bacterial genomes, 99 phyla are described including six major normalized monophyletic units from the subdivision of the Proteobacteria, and amalgamation of the Candidate Phyla Radiation into the single phylum Patescibacteria. In total, 73% of taxa had one or more changes to their existing taxonomy.
5,610 downloads microbiology
In this manuscript we evaluate the potential for microbiome characterization by sequencing of near-full length 16S rRNA gene region fragments using the Oxford Nanopore MinION (hereafter Nanopore) sequencing platform. We analyzed pure-culture E. coli and P. fluorescens, as well as a low-diversity mixed community sample from hydraulic fracturing produced water. Both closed and open reference operational taxonomic unit (OTU) picking failed, necessitating the direct use of sequences without OTU picking. The Ribosomal Database Project classifier against the Green Genes database was found to be the optimal annotation approach, with average pure-culture annotation accuracies of 93.8% and 82.0% at the phyla and genus levels, respectively. Comparative analysis of an environmental sample using Nanopore and Illumina MiSeq sequencing identified high taxonomic similarity when using a weighted metric (Bray-Curtis), and significantly reduced similarity when using an unweighted metric (Jaccard). These results highlight the great potential of Nanopore sequencing to analyze broad microbial community trends, and the challenge of applying Nanopore sequencing to discern rare taxa in mixed microbial communities. Finally, we observed that between-run carryover following washes on the same flowcell accounted for >10% of sequence reads, necessitating future development to either prevent carryover or filter sequences of interest (e.g. barcoding).
5,363 downloads microbiology
Samples from sera and oral swabs from fifteen marmosets (Callithrix jacchus) and nine capuchin-monkeys (Sapajus libidinosus) captured in Ceara State in Brazil were tested for Zika virus. Samples were positive by Real time PCR and sequencing of the amplified product from a capuchin monkey showed 100% similarity to other ZIKV from South America. This is the first report on ZIKV detection among Neotropical primates.
4,588 downloads microbiology
Background: Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity, but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences platforms overcome this limitation, their application has been limited due to higher error rates or smaller data output. Results: In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the aforementioned protocol that reduces sample-processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain full-length 16S rRNA gene sequences. Conclusions: NanoAmpli-Seq accurately estimates the diversity of tested mock communities with average sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware basecalling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.
4,408 downloads microbiology
Themoula Charalampous, Hollian Richardson, Gemma Louise Kay, Rossella Baldan, Christopher Jeanes, Duncan Rae, Sara Grundy, Daniel J Turner, John Wain, Richard Mark Leggett, David M Livermore, Justin O'Grady
Lower respiratory infections (LRIs) accounted for three million deaths worldwide in 2016, the leading infectious cause of mortality. The 'gold standard' for investigation of bacterial LRIs is culture, which has poor sensitivity and is too slow to guide early antibiotic therapy. Metagenomic sequencing potentially could replace culture, providing rapid, sensitive and comprehensive results. We developed a metagenomics pipeline for the investigation of bacterial LRIs using saponin-based host DNA depletion combined with rapid nanopore sequencing. The first iteration of the pipeline was tested on respiratory samples from 40 patients. It was then refined to reduce turnaround and increase sensitivity, before testing a further 41 samples. The refined method was 96.6% concordant with culture for detection of pathogens and could accurately detect resistance genes with a turnaround time of six hours. This study demonstrates that nanopore metagenomics can rapidly and accurately characterise bacterial LRIs when combined with efficient human DNA depletion.
4,365 downloads microbiology
High-throughput sequencing of the 16S rRNA gene is widely used in microbial ecology, with Illumina platforms being widely used in recent studies. The MiniSeq, Illumina's latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger sequencing platforms (e.g. MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed an additional sequencing primer that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples as a quality control benchmark. After careful filtering procedures, we were able to recapture a realistic richness of the mock community, and identify meaningful differences in alpha and beta diversity in the environmental samples. These results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.
4,109 downloads microbiology
Water recreation, though increasing globally, is strongly associated with infectious diseases. Unexpectedly, artificial water recreation systems e.g. swimming pools account for 90% of these outbreaks. It is therefore essential that pool waters be regularly monitored for deviations from microbial water quality guidelines. To assess the sanitary quality of a club swimming pool in Ile-Ife, Nigeria, I used the multiple-tube fermentation technique to determine the most probable number (MPN) of coliform bacteria in 100 ml of pool water. MPN estimates ranged from 9 to 93 with geometric mean of 38. Escherichia coli was isolated from positive presumptive tubes, indicating recent faecal contamination. The isolate elicited similar biochemical reactions as reference E. coli (ATCC-25922), except that it utilized sucrose and liquefied gelatin, which probably indicates potential pathogenicity. Also, the E. coli isolate was resistant to 13 antibiotics from 9 different classes. Finally, coliform counts and detection of E. coli clearly violates international guidelines. I recommend that pool operators increase water disinfection efficiency and educate the public on the need for improved swimmer hygiene to reduce the risk of recreational water illness transmission.
3,821 downloads microbiology
Understanding the drivers of microbial diversity is a fundamental question in microbial ecology. Extensive literature discusses different methods for describing microbial diversity and documenting its effects on ecosystem function. However, it is widely believed that diversity depends on the number of reads that are sequenced. I discuss a statistical perspective on diversity, framing the diversity of an environment as an unknown parameter, and discussing the bias and variance of plug-in and rarefied estimates. I argue that by failing to account for both bias and variance, we invalidate analysis of alpha diversity. I describe the state of the statistical literature for addressing these problems, and suggest that measurement error modeling can address issues with variance, but bias corrections need to be utilized as well. I encourage microbial ecologists to avoid motivating their investigations with alpha diversity analyses that do not use valid statistical methodology.
3,348 downloads microbiology
Loes M Olde Loohuis, Serghei Mangul, Anil Ori, Guillaume Jospin, David Koslicki, Harry Taegyun Yang, Timothy Wu, Marco P. Boks, Catherine Lomen-Hoerth, Martina Wiedau-Pazos, Rita Cantor, Willem M de Vos, Rene S Kahn, Eleazar Eskin, Roel A. Ophoff
The role of the human microbiome in health and disease is increasingly appreciated. We studied the composition of microbial communities present in blood across 192 individuals, including healthy controls and patients with three disorders affecting the brain: schizophrenia, amyotrophic lateral sclerosis, and bipolar disorder. By using high-quality unmapped RNA sequencing reads as candidate microbial reads, we performed profiling of microbial transcripts detected in whole blood. We were able to detect a wide range of bacterial and archaeal phyla in blood. Interestingly, we observed an increased microbial diversity in schizophrenia patients compared to the three other groups. We replicated this finding in an independent schizophrenia case-control cohort. This increased diversity is inversely correlated with estimated cell abundance of a subpopulation of CD8+ memory T cells in healthy controls, supporting a link between microbial products found in blood, immunity, and schizophrenia.
3,276 downloads microbiology
Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New sequencing technologies produce long reads that can span the entire 16S rRNA gene, but have substantially higher error rates that 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 mixtures of known bacterial strains 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. E. coli strains in the mock communities were correctly classified to the O157:H7 and K12 sub-species clades from the 16S gene sequences recovered by our method. In human fecal samples, our method recovered the full complement of 16S rRNA gene variants in detected E. coli strains and showed strong technical replication. We discuss the promises and challenges of of classification based on the full complement of multi-copy marker genes such as the 16S rRNA gene. 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.
3,140 downloads microbiology
Zika virus (ZIKV) infection in utero might lead to microcephaly and other congenital defects. In adults, cases of Guillain-Barré syndrome and meningoencephalitis associated with ZIKV infection have been reported, but to date, no specific therapy is available. There is an urgency for the discovery of antiviral agents capable of inhibiting viral replication and its deleterious effects. Chloroquine is widely used as an antimalarial drug and anti-inflammatory agent that also shows antiviral activity against several viruses. Here we show that chloroquine exhibits antiviral activity against ZIKV in VERO, human brain microvascular endothelial, and neural stem cells. We demonstrated in vitro that chloroquine reduces the number of ZIKV-infected cells, virus production and cell death promoted by ZIKV infection without cytotoxic effects. Our results suggest that chloroquine is a promising candidate for ZIKV clinical trials, since it is already approved for clinical use and can be safely administered to pregnant woman.
3,019 downloads microbiology
Sacha J. Pidot, Wei Gao, Andrew H Buultjens, Ian R. Monk, Romain Guerillot, Glen P Carter, Jean Y.H. Lee, Margaret M. C. Lam, Susan A. Ballard, M. Lindsay Grayson, Andrew A. Mahony, Elizabeth A. Grabsch, Despina Kotsanas, Tony Korman, Geoffrey W. Coombs, J. Owen Robinson, Anders Goncalves da Silva, Torsten Seemann, Benjamin P. Howden, Paul D. R. Johnson, Timothy P. Stinear
Alcohol-based hand rubs are international pillars of hospital infection control, restricting transmission of pathogens such as Staphylococcus aureus. Despite this success, health care infections caused by Enterococcus faecium (Efm) - another multidrug resistant pathogen - are increasing. We tested alcohol tolerance of 139 hospital Efm isolates, obtained between 1997 and 2015 and found Efm post-2010 were 10-fold more tolerant to alcohol killing than older isolates. Using a mouse infection control model, we then showed that alcohol tolerant Efm resisted standard 70% isopropanol surface disinfection and led to gastrointestinal colonization significantly more often than alcohol sensitive Efm. We next looked for bacterial genomic signatures of adaptation. Tolerant Efm have independently accumulated mutations modifying genes involved in carbohydrate uptake and metabolism. Mutagenesis confirmed their roles in isopropanol tolerance. These findings suggest bacterial adaptation and complicate infection control recommendations. Additional policies and procedures to prevent Efm spread are required.
3,010 downloads microbiology
Basem Al-Shayeb, Rohan Sachdeva, Lin-Xing Chen, Fred Ward, Patrick Munk, Audra Devoto, Cindy J Castelle, Matthew R Olm, Keith Bouma-Gregson, Yuki Amano, Christine He, Raphael Meheust, Brandon Brooks, Alex Thomas, Adi Lavy, Paula Matheus-Carnevali, Christine Sun, Daniela Goltsman, Mikayla A Borton, Tara C Nelson, Rose Kantor, Alexander L. Jaffe, Ray Keren, Ibrahim F Farag, Shufei Lei, Kari Finstad, Ronald Amundson, Karthik Anantharaman, Jinglie Zhou, Alexander J Probst, Mary E Power, Susannah G Tringe, Wen-Jun Li, Kelly Wrighton, Sue Harrison, Michael Morowitz, David A. Relman, Jennifer A Doudna, Anne-Catherine Lehours, Lesley Warren, Jamie H.D. Cate, Joanne M Santini, Jill F Banfield
Phage typically have small genomes and depend on their bacterial hosts for replication. DNA sequenced from many diverse ecosystems revealed hundreds of huge phage genomes, between 200 kbp and 716 kbp in length. Thirty-four genomes were manually curated to completion, including the largest phage genomes yet reported. Expanded genetic repertoires include diverse and new CRISPR-Cas systems, tRNAs, tRNA synthetases, tRNA modification enzymes, translation initiation and elongation factors, and ribosomal proteins. Phage CRISPR-Cas systems have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phage may repurpose bacterial CRISPR-Cas systems to eliminate competing phage. We phylogenetically define major clades of huge phage from human and other animal microbiomes, oceans, lakes, sediments, soils and the built environment. We conclude that their large gene inventories reflect a conserved biological strategy, observed over a broad bacterial host range and across Earth's ecosystems.
2,931 downloads microbiology
Microbial communities are critical to ecosystem function. A key objective of metagenomic studies is to analyse organism-specific metabolic pathways and reconstruct community interaction networks. This requires accurate assignment of assembled genome fragments to genomes. Existing binning methods often fail to reconstruct a reasonable number of genomes and report many bins of low quality and completeness. Furthermore, the performance of existing algorithms varies between samples and biotopes. Here, we present a dereplication, aggregation and scoring strategy, DAS Tool, that combines the strengths of a flexible set of established binning algorithms. DAS Tool applied to a constructed community generated more accurate bins than any automated method. Further, when applied to environmental and host-associated samples of different complexity, DAS Tool recovered substantially more near-complete genomes, including novel lineages, than any single binning method alone. The ability to reconstruct many near-complete genomes from metagenomics data will greatly advance genome-centric analyses of ecosystems.
2,874 downloads microbiology
Daniel McDonald, Embriette Hyde, Justine W Debelius, James T Morton, Antonio Gonzalez, Gail Ackermann, Alexander A. Aksenov, Bahar Behsaz, Caitriona Brennan, Yingfeng Chen, Lindsay DeRight Goldasich, Pieter C Dorrestein, Robert R Dunn, Ashkaan K Fahimipour, James Gaffney, Jack A Gilbert, Grant Gogul, Jessica L Green, Philip Hugenholtz, Greg Humphrey, Curtis Huttenhower, Matthew A Jackson, Stefan Janssen, Dilip V Jeste, Lingjing Jiang, Scott T. Kelley, Dan Knights, Tomasz Kosciolek, Joshua Ladau, Jeff Leach, Clarisse Marotz, Dmitry Meleshko, Alexey V Melnik, Jessica L Metcalf, Hosein Mohimani, Emmanuel Montassier, Jose Navas-Molina, Tanya T Nguyen, Shyamal Peddada, Pavel Pevzner, Katherine S Pollard, Gholamali Rahnavard, Adam Robbins-Pianka, Naseer Sangwan, Joshua Shorenstein, Larry Smarr, Se Jin Song, Timothy Spector, Austin D Swafford, Varykina G Thackray, Luke R Thompson, Anupriya Tripathi, Yoshiki Vazquez-Baeza, Alison Vrbanac, Paul Wischmeyer, Elaine Wolfe, Qiyun Zhu, The American Gut Consortium, Rob Knight
Although much work has linked the human microbiome to specific phenotypes and lifestyle variables, data from different projects have been challenging to integrate and the extent of microbial and molecular diversity in human stool remains unknown. Using standardized protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-scientists, together with an open research network, we compare human microbiome specimens primarily from the USA, UK, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes as compared to environmental samples, demonstrate the utility of procedures for removing the effects of overgrowth during room-temperature shipping for revealing phenotype correlations, uncover new molecules and kinds of molecular communities in the human stool metabolome, and examine emergent associations among the microbiome, metabolome, and the diversity of plants that are consumed (rather than relying on reductive categorical variables such as veganism, which have little or no explanatory power). We also demonstrate the utility of the living data resource and cross-cohort comparison to confirm existing associations between the microbiome and psychiatric illness, and to reveal the extent of microbiome change within one individual during surgery, providing a paradigm for open microbiome research and education.
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