Biology subjects

Aarestrup, F. M.

Publications and source records attributed to Aarestrup, F. M..

26 records · Page 2Linked to original sources

bioRxiv2019

Specific Gut Microbiome Members are Associated with Distinct Immune Markers in Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundIncreasing evidence reveals the importance of the microbiome in health and disease and inseparable host-microbial dependencies. Host-microbe interactions are highly relevant in patients receiving allogeneic hematopoietic stem cell transplantation, (HSCT), i.e. a replacement of the cellular components of the patients immune system with that of a foreign donor. HSCT is employed as curative immunotherapy for a number of non-malignant and malignant hematologic conditions, including acute lymphoblastic leukemia. The procedure can be accompanied by severe side effects such as infections, acute graft-versus-host disease (aGvHD), and death. Here, we performed a longitudinal analysis of immunological markers, immune reconstitution and gut microbiota composition in relation to clinical outcomes in children undergoing HSCT. Such an analysis could reveal biomarkers, e.g. at the time point prior to HSCT, that in the future could be used to predict which patients are of high risk in relation to side effects and clinical outcomes and guide treatment strategies accordingly.\n\nResultsIn two multivariate analyses (sparse partial least squares regression and canonical correspondence analysis), we identified three consistent clusters: (1) High concentrations of the antimicrobial peptide human beta-defensin 2 (hBD2) prior to the transplantation in patients with high abundances of Lactobacillaceae, who later developed moderate or severe aGvHD and exhibited high mortality. (2) Rapid reconstitution of NK and B cells in patients with high abundances of obligate anaerobes such as Ruminococcaceae, who developed no or mild aGvHD and exhibited low mortality. (3) High inflammation, indicated by high levels of C-reactive protein, in patients with high abundances of facultative anaerobic bacteria such as Enterobacteriaceae. Furthermore, we observed that antibiotic treatment influenced the bacterial community state.\n\nConclusionsWe identify multivariate associations between specific microbial taxa, host immune markers, immune cell reconstitution and clinical outcomes in relation to HSCT. Our findings encourage further investigations into establishing longitudinal surveillance of the intestinal microbiome and relevant immune markers, such as hBD2, in HSCT patients. Profiling of the microbiome may prove useful as a prognostic tool that could help identify patients at risk of poor immune reconstitution and adverse outcomes, such as aGvHD and death, upon HSCT, providing actionable information in guiding precision medicine.

microbiology↗

bioRxiv2019

Library preparation and sequencing platform introduces bias in metagenomics characterisation of microbial communities

Metagenomics is increasingly used to describe microbial communities in biological specimens. Ideally, the steps involved in the processing of the biological specimens should not change the microbiome composition in a way that it could lead to false interpretations of inferred microbial community composition. Common steps in sample preparation include sample collection, storage, DNA isolation, library preparation, and DNA sequencing. Here we assess the effect of three library preparation kits and two DNA sequencing platforms. Of the library preparation kits, one involved a polymerase chain reaction (PCR) step (Nextera), and two were PCR-free (NEXTflex and KAPA). We sequenced the libraries on Illumina HiSeq and NextSeq platforms. As example microbiomes, we assessed two pig fecal samples and two sewage samples of which aliquots were stored at different storage conditions (immediate processing and storage at -80{degrees}C). All DNA isolations were performed in duplicate, totaling 80 samples excluding controls. We found that both library preparation and sequencing platform had systematic effects on the inferred microbial community composition. The different sequencing platforms introduced more variation than library preparation and freezing the samples. The results highlight that all sample processing steps need to be considered when comparing studies. Standardization of sample processing is key to generate comparable data within a study, and comparisons of differently generated data, such as in a meta-analysis, should be performed cautiously. ImportancePrevious research has reported effects of sample storage conditions and DNA isolation procedures on metagenomics-based microbiome composition; however, the effect of library preparation and DNA sequencing in metagenomics has not been thoroughly assessed. Here, we provide evidence that library preparation and sequencing platform introduce systematic biases in the metagenomic-based characterization of microbial communities. These findings suggest that library preparation and sequencing are important parameters to keep consistent when aiming to detect small changes in microbiome community structure. Overall, we recommend that all samples in a microbiome study are processed in the same way to limit unwanted variations that could lead to false conclusions. Furthermore, if we are to obtain a more holistic insight from microbiome data generated around the world, we will need to provide more detailed sample metadata, including information about the different sample processing procedures, together with the DNA sequencing data at the public repositories.

microbiology↗

bioRxiv2019

Large scale automated phylogenomical analysis of bacterial whole-genome isolates and the Evergreen platform

Public health authorities whole-genome sequence thousands of pathogenic isolates each month for microbial diagnostics and surveillance of pathogenic bacteria. The computational methods have not kept up with the deluge of data and need for real-time results. We have therefore created a bioinformatics pipeline for rapid subtyping and continuous phylogenomic analysis of bacterial samples, suited for large-scale surveillance. To decrease the computational burden, a two level clustering strategy is employed. The data is first divided into sets by matching each isolate to a closely related reference genome. The reads then are aligned to the reference to gain a consensus sequence and SNP based genetic distance is calculated between the sequences in each set. Isolates are clustered together with a threshold of 10 SNPs. Finally, phylogenetic trees are inferred from the non-redundant sequences and the clustered isolates are placed on a clade with the cluster representative sequence. The method was benchmarked and found to be accurate in grouping outbreak strains together, while discriminating from non-outbreak strains. The pipeline was applied in Evergreen Online, which processes publicly available sequencing data from foodborne bacterial pathogens on a daily basis, updating the phylogenetic trees as needed. It has so far placed more than 100,000 isolates into phylogenies, and has been able to keep up with the daily release of data. The trees are continuously published on https://cge.cbs.dtu.dk/services/Evergreen

bioinformatics↗

bioRxiv2018

Proof of concept: Malaria rapid diagnostic tests and massively parallel sequencing for surveillance of molecular markers of antimalarial resistance in Bissau, Guinea-Bissau during 2014-2017

Real-time and large-scale surveillance of molecular markers of antimalarial drug resistance is a potential method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging. This study investigates whether routinely used malaria rapid diagnostic tests (RDTs) can be used for massive parallel amplicon sequencing. RDTs used for malaria diagnosis were routinely collected together with patient age and sex between 2014 and 2017, from two health centres in Bissau, Guinea-Bissau. A subset of positive RDTs (n=2,184) were tested for Plasmodium DNA content. Those containing sufficient Plasmodium DNA (n=1,390) were used for library preparation, consisting of amplification of gene fragments from pfcrt, pfmdr1, pfdhfr, pfdhps and pfK13. A total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell. Pre-screening of samples for Plasmodium DNA content proved necessary and the nested PCR protocol applied for library preparation varied notably in PCR-positivity from 13-87%. We found a high frequency of the pfmdr1 codon 86N at 88%-97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33%-51% between 2014-2017. No polymorphisms indicating artemisinin tolerance were discovered. Lastly, the demographic data indicate a large proportion of young adults (66%, interquartile range 11-28 years) presenting with P. falciparum infections. With some caution, our findings suggest that routine collection of RDTs could facilitate large-scale molecular surveillance of antimalarial resistance.\n\nImportance (word count: 147)Continuous spread and repeated emergence of Plasmodium falciparum parasites resistant towards one or more antimalarials represents an enormous threat to current treatment efficacy levels, especially in sub-Saharan Africa, where 90% of malaria infections occur. In order to prevent substantial treatment failure, it is therefore recommended to monitor treatment efficacy every 2-3 years. Therapeutic efficacy studies, however, can present insurmountable logistical and financial challenges in some settings in sub-Saharan Africa. Molecular surveillance of antimalarial resistance is therefore an important proxy for treatment efficacy. However, the scale by which such studies can be performed depends on the development of high-throughput protocols and the accessibility of samples. If RDTs can be used in the high-throughput protocols available with Next Generation Sequencing (NGS)-technology, surveillance can be performed efficiently for any setting in which RDTs are already used for malaria diagnosis. The majority of settings in sub-Saharan Africa have access to RDTs.

molecular biology↗

bioRxiv2018

Proficiency of WHO Global Foodborne Infections Network External Quality Assurance System participants in the identification and susceptibility testing of thermo-tolerant Campylobacter spp. from 2003-2012.

Campylobacter spp. are food- and water borne pathogens. While rather accurate estimates for these pathogens are available in industrialized countries, a lack of diagnostic capacity in developing countries limits accurate assessments of prevalence in many regions. Proficiency in the identification and susceptibility testing of these organisms is critical for surveillance and control efforts. The aim of the study was to assess performance for identification and susceptibility testing of thermo-tolerant Campylobacter among laboratories participating in the World Health Organization (WHO) Global Foodborne Infections Network (GFN) External Quality Assurance System (EQAS) over a nine year period.\n\nParticipants (primarily national level laboratories) were encouraged to self-evaluate performance as part of continuous quality improvement.\n\nThe ability to correctly identify Campylobacter spp. varied by year and ranged from 61.9 % (2008) to 90.7 % (2012), and the ability to correctly perform antimicrobial susceptibility testing (AST) for Campylobacter spp. appeared to steadily increase from 91.4 % to 93.6 % in the test period (2009-2012).\n\nPoorest performance (60.0 % correct identification and 86.8 % correct AST results) was observed in African laboratories.\n\nOverall, approximately 10 % of laboratories reported either an incorrect identification or antibiogramme. As most participants were (supra)-national reference laboratories, these data raise significant concerns regarding capacity and proficiency at the local, clinical level. Addressing these diagnostic challenges is critical for both patient level management and broader surveillance and control efforts.

microbiology↗

bioRxiv2018

Megaphage infect Prevotella and variants are widespread in gut microbiomes

Bacteriophage (phage) dramatically shape microbial community composition, redistribute nutrients via host lysis, and drive evolution through horizontal gene transfer. Despite their importance, much remains to be learned about phage in the human microbiome. We investigated gut microbiomes of humans from Bangladesh and Tanzania, two African baboon social groups, and Danish pigs, and report that many contain phage belonging to a clade with genomes >540 kb in length, the largest yet reported in the human microbiome and close to the maximum size ever reported for phage. We refer to these as Lak phage. CRISPR spacer targeting indicates that the Lak phage infect bacteria of the genus Prevotella. We manually curated to completion 15 distinct Lak phage genomes recovered from metagenomes. The genomes display several interesting features, including use of an alternative genetic code, large intergenic regions that are highly expressed, and up to 35 putative tRNAs, some of which contain enigmatic introns. Different individuals have distinct phage genotypes, and shifts in variant frequencies over consecutive sampling days reflect changes in relative abundance of phage sub-populations. Recent homologous recombination has resulted in extensive genome admixture of nine baboon Lak phage populations. We infer that Lak phage are widespread in gut communities that contain Prevotella species, especially in individuals in the developing world, and conclude that megaphage, with fascinating and underexplored biology, may be common but largely overlooked components of human and animal gut microbiomes.

microbiology↗

bioRxiv2017

Abundance and diversity of the fecal resistome in slaughter pigs and broilers in nine European countries

EFFORT groupHaitske Graveland, Alieda van Essen, Bruno Gonzalez-Zorn, Gabriel Moyano, Pascal Sanders, Claire Chauvin, Julie David, Antonio Battisti, Andrea Caprioli, Jeroen Dewulf, Thomas Blaha, Katharina Wadepohl, Maximiliane Brandt, Dariusz Wasyl, Magdalena Skarzynska, Magdalena Zajac, Hristo Daskalov, Helmut W Saatkamp, Katharina D.C. Stark.\n\nAbstractAntimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. Use of antimicrobials in livestock selects for AMR that can subsequently be transferred to humans. This flow of AMR between reservoirs demands surveillance in livestock as well as in humans. As part of the EFFORT project (www.effort-against-amr.eu), we have quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, generating more than 5,000 gigabases of DNA sequence, using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a database constructed for this study, consisting of AMR genes identified through screening environmental DNA. The pig and poultry resistomes were very different in abundance and composition. There was a significant country effect on the resistomes, more so in pigs than poultry. We found higher AMR loads in pigs, while poultry resistomes were more diverse. We detected several recently described, critical AMR genes, including mcr-1 and optrA, the abundance of which differed both between host species and countries. We found that the total acquired AMR level, was associated with the overall country-specific antimicrobial usage in livestock and that countries with comparable usage patterns had similar resistomes. Novel, functionally-determined AMR genes were, however, not associated with total drug use.

microbiology↗

bioRxiv2017

Genomics-Based Identification of Microorganisms in Human Ocular Body Fluid

Advances in genomics have the potential to revolutionize clinical diagnostics. Here, we examine the microbiome of vitreous (intraocular body fluid) from patients who developed endophthalmitis following cataract surgery or intravitreal injection. Endophthalmitis is an inflammation of the intraocular cavity and can lead to a permanent loss of vision. As controls, we included vitreous from endophthalmitis-negative patients, balanced salt solution used during vitrectomy, and DNA extraction blanks. We compared two DNA isolation procedures and found that an ultraclean production of reagents appeared to reduce background DNA in these low microbial biomass samples. We created a curated microbial genome database (>5700 genomes) and designed a metagenomics workflow with filtering steps to reduce DNA sequences originating from: i) human hosts, ii) ambiguousness/contaminants in public microbial reference genomes, and iii) the environment. Our metagenomic read classification revealed in nearly all cases the same microorganism than was determined in cultivation- and mass spectrometry-based analyses. For some patients, we identified the sequence type of the microorganism and antibiotic resistance genes through analyses of whole genome sequence (WGS) assemblies of isolates and metagenomic assemblies. Together, we conclude that genomics-based analyses of human ocular body fluid specimens can provide actionable information relevant to infectious disease management.

microbiology↗