Scientific publications

The following scientific publications are related to Global Surveillance and the associated projects.

One Day in Denmark: Nationwide point-prevalence survey of human bacterial isolates and comparison of classical and whole-genome sequence-based species identification methods
Rebelo et al. 2022

Implementing whole-genome sequencing (WGS) technologies in clinical microbiology laboratories can increase the amount and quality of information available for healthcare practitioners. In this study, we analysed the applicability of this method and determined the distribution of bacterial species processed in clinical settings in Denmark.

DOI:   11-02-2022

ResFinder – an open online resource for identification of antimicrobial resistance genes in next-generation sequencing data and prediction of phenotypes from genotypes
Florensa et al. 2022

Antimicrobial resistance (AMR) is one of the most important health threats globally. The ability to accurately identify resistant bacterial isolates and the individual antimicrobial resistance genes (ARGs) is essential for understanding the evolution and emergence of AMR and to provide appropriate treatment. The rapid developments in next-generation sequencing technologies have made this technology available to researchers and microbiologists at routine laboratories around the world. However, tools available for those with limited experience with bioinformatics are lacking, especially to enable researchers and microbiologists in low- and middle-income countries (LMICs) to perform their own studies. The CGE-tools (Center for Genomic Epidemiology) including ResFinder ( was developed to provide freely available easy to use online bioinformatic tools allowing inexperienced researchers and microbiologists to perform simple bioinformatic analyses. The main purpose was and is to provide these solutions for people involved in frontline diagnosis especially in LMICs. Since its original publication in 2012, ResFinder has undergone a number of improvements including improvement of the code and databases, inclusion of point mutations for selected bacterial species and predictions of phenotypes also for selected species.

DOI:    24-01-2022

Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life Years for 29 Cancer Groups From 2010 to 2019
A Systematic Analysis for the Global Burden of Disease Study 2019

Global Burden of Disease 2019 Cancer Collaboration

Question: What was the burden of cancer globally and across Sociodemographic Index (SDI) groupings in 2019, and how has incidence, morbidity, and mortality changed since 2010?
Findings: In this systematic analysis, there were 23.6 million new global cancer cases in 2019 (17.2 million when excluding those with nonmelanoma skin cancer), 10.0 million cancer deaths, and an estimated 250 million disability-adjusted life years estimated to be due to cancer; since 2010, these represent increases of 26.3%, 20.9%, and 16.0%, respectively. Absolute cancer burden increased in all SDI quintiles since 2010, but the largest percentage increases occurred in the low and low-middle SDI quintiles.
Meanings: The study results suggest that increased cancer prevention and control efforts are needed to equitably address the evolving and increasing burden of cancer across the SDI spectrum.

DOI:   30-12-2021

The global burden of adolescent and young adult cancer in 2019: a systematic analysis for the Global Burden of Disease Study 2019
GBD 2019 Adolescent Young Adult Cancer Collaborators

In estimating the global burden of cancer, adolescents and young adults with cancer are often overlooked, despite being a distinct subgroup with unique epidemiology, clinical care needs, and societal impact. Comprehensive estimates of the global cancer burden in adolescents and young adults (aged 15–39 years) are lacking. To address this gap, we analysed results from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019, with a focus on the outcome of disability-adjusted life-years (DALYs), to inform global cancer control measures in adolescents and young adults.

DOI:   03-12-2021

Danish Whole-Genome-Sequenced Candida albicans and Candida glabrata Samples Fit into Globally Prevalent Clades
Szarvas et al. 2021

Candida albicans and Candida glabrata are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the Candida population in the country. The majority of the 30 C. albicans isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight C. glabrata isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all C. albicans isolates to be susceptible to both azoles and echinocandins. Two C. glabrata isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species.

DOI:    12-11-2021

Pandemics– One Health preparedness for the next
Frank M. Aarestrup, Marc Bonten, Marion Koopmans

The majority of emerging infectious diseases originate in animals. Current routine surveillance is focused on known diseases and clinical syndromes, but the increasing likelihood of emerging disease outbreaks shows the critical importance of early detection of unusual illness or circulation of pathogens - prior to human disease manifestation. In this Viewpoint, we focus on one key pillar of preparedness—the need for early warning surveillance at the human, animal, environmental interface. The COVID-19 pandemic has revolutionized the scale of sequencing of pathogen genomes, and the current investments in global genomic surveillance offer great potential for a novel, truly integrated Disease X (with epidemic or pandemic potential) surveillance arm provided we do not make the mistake of developing them solely for the case at hand.

DOI:   07-10-2021

Standard Sample Storage Conditions Have an Impact on Inferred Microbiome Composition and Antimicrobial Resistance Patterns
Poulsen et al. 2021

Storage of biological specimens is crucial in the life and medical sciences. Storage conditions for samples can be different for a number of reasons, and it is unclear what effect this can have on the inferred microbiome composition in metagenomics analyses. Here, we assess the effect of common storage temperatures (deep freezer, −80°C; freezer, −20°C; refrigerator, 5°C; room temperature, 22°C) and storage times (immediate sample processing, 0 h; next day, 16 h; over weekend, 64 h; longer term, 4, 8, and 12 months) as well as repeated sample freezing and thawing (2 to 4 freeze-thaw cycles). We examined two different pig feces and sewage samples, unspiked and spiked with a mock community, in triplicate, respectively, amounting to a total of 438 samples (777 Gbp; 5.1 billion reads). Storage conditions had a significant and systematic effect on the taxonomic and functional composition of microbiomes.

DOI:   06-10-2021

Secrets of the Hospital Underbelly: Patterns of Abundance of Antimicrobial Resistance Genes in Hospital Wastewater Vary by Specific Antimicrobial and Bacterial Family
Perry et al. 2021

Background: Hospital wastewater is a major source of antimicrobial resistance (AMR) outflow into the environment. This study uses metagenomics to study how hospital clinical activity impacts antimicrobial resistance genes (ARGs) abundances in hospital wastewater.
Conclusion: We found that the relationship between hospital wastewater ARGs and antimicrobial usage or clinical isolate resistance varies by specific antimicrobial and bacterial family studied. One explanation, we consider is that relationships observed from multiple departments within a single hospital site will be detectable only for ARGs against parenteral antimicrobials uniquely used in the hospital setting. Our work highlights that using metagenomics to identify the full range of ARGs in hospital wastewater is a useful surveillance tool to monitor hospital ARG carriage and outflow and guide environmental policy on AMR.

DOI:  10-09-2021

Global, regional, and national burden of stroke and its risk factors, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019
GBD 2019 Stroke Collaborators

Regularly updated data on stroke and its pathological types, including data on their incidence, prevalence, mortality, disability, risk factors, and epidemiological trends, are important for evidence-based stroke care planning and resource allocation. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) aims to provide a standardised and comprehensive measurement of these metrics at global, regional, and national levels.

DOI:    03-09-2021

Genomic evolution of antimicrobial resistance in Escherichia coli
Leekitcharoenphon et al. 2021

The emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms.

DOI:   23-07-2021

Patterns of Gene Content and Co-occurrence Constrain the Evolutionary Path toward Animal Association in Candidate Phyla Radiation Bacteria
Jaffe et al. 2021

Candidate Phyla Radiation (CPR) bacteria are small, likely episymbiotic organisms found across Earth’s ecosystems. Despite their prevalence, the distribution of CPR lineages across habitats and the genomic signatures of transitions among these habitats remain unclear. Here, we expand the genome inventory for Absconditabacteria (SR1), Gracilibacteria, and Saccharibacteria (TM7), CPR bacteria known to occur in both animal-associated and environmental microbiomes, and investigate variation in gene content with habitat of origin. By overlaying phylogeny with habitat information, we show that bacteria from these three lineages have undergone multiple transitions from environmental habitats into animal microbiomes. Based on co-occurrence analyses of hundreds of metagenomes, we extend the prior suggestion that certain Saccharibacteria have broad bacterial host ranges and constrain possible host relationships for Absconditabacteria and Gracilibacteria.

DOI:   13-07-2021

Predicting Antimicrobial Resistance Using Partial Genome Alignments
Aytan-Aktug et al. 2021

Antimicrobial resistance (AMR) is an important global health threat that impacts millions of people worldwide each year. Developing methods that can detect and predict AMR phenotypes can help to mitigate the spread of AMR by informing clinical decision making and appropriate mitigation strategies. Many bioinformatic methods have been developed for predicting AMR phenotypes from whole-genome sequences and AMR genes, but recent studies have indicated that predictions can be made from incomplete genome sequence data. In order to more systematically understand this, we built random forest-based machine learning classifiers for predicting susceptible and resistant phenotypes for Klebsiella pneumoniae (1,640 strains), Mycobacterium tuberculosis (2,497 strains), and Salmonella enterica (1,981 strains).

DOI:   15-06-2021

A Peek into the Plasmidome of Global Sewage
Kirstahler et al. 2021

Plasmids can provide a selective advantage for microorganisms to survive and adapt to new environmental conditions. Plasmid-encoded traits, such as antimicrobial resistance (AMR) or virulence, impact the ecology and evolution of bacteria and can significantly influence the burden of infectious diseases. Insight about the identity and functions encoded on plasmids on the global scale are largely lacking. Here, we investigate the plasmidome of 24 samples (22 countries, 5 continents) from the global sewage surveillance project. We obtained 105-Gbp Oxford Nanopore and 167-Gbp Illumina NextSeq DNA sequences from plasmid DNA preparations and assembled 165,302 contigs (159,322 circular). Of these, 58,429 carried genes encoding for plasmid-related and 11,222 for virus/phage-related proteins. About 90% of the circular DNA elements did not have any similarity to known plasmids.

DOI:   26-05-2021

Risk factors for carbapenemase-producing organisms among inpatients in Scotland: A national matched case–control study
Zhao et al. 2020

To determine risk factors for carbapenemase-producing organisms (CPOs) and to determine the prognostic impact of CPOs.
A history of (prolonged) hospitalization, prolonged ICU or HDU stay; ENM diseases; and being immunocompromised increased risk for CPO. CPO infection was not associated with increased mortality but was associated with prolonged hospital stay.

DOI:    22-12-2020

Extended-spectrum beta-lactamase-producing Escherichia coli and antimicrobial resistance in municipal and hospital wastewaters in Czech Republic: Culture-based and metagenomic approaches
Kutilova et al. 2020

Wastewaters serve as important hot spots for antimicrobial resistance and monitoring can be used to analyse the abundance and diversity of antimicrobial resistance genes at the level of large bacterial and human populations. In this study, whole genome sequencing of beta-lactamase-producing Escherichia coli and metagenomic analysis of whole-community DNA were used to characterize the occurrence of antimicrobial resistance in hospital, municipal and river waters in the city of Brno (Czech Republic).

DOI:  21-11-2020

Setting a baseline for global urban virome surveillance in sewage
Nieuwenhuijse et al. 2020

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective.

DOI:    13-08-2020

ResFinder 4.0 for predictions of phenotypes from genotypes
Bortolaia et al. 2020

WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output.

The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n= 584),Salmonella spp. (n= 1081),Campylobacter jejuni (n= 239), Enterococcus faecium (n= 106), Enterococcus faecalis (n= 50) and Staphylococcus aureus (n= 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins.

DOI:  11-08-2020

The gut microbiome but not the resistome is associated with urogenital schistosomiasis in preschool-aged children
Osakunor et al. 2020

Helminth parasites have been shown to have systemic effects in the host. Using shotgun metagenomic sequencing, we characterise the gut microbiome and resistome of 113 Zimbabwean preschool-aged children (1–5 years). We test the hypothesis that infection with the human helminth parasite, Schistosoma haematobium, is associated with changes in gut microbial and antimicrobial resistance gene abundance/diversity. Here, we show that bacteria phyla Bacteroidetes, Firmicutes, Proteobacteria, and fungi phyla Ascomycota, Microsporidia, Zoopagomycota dominate the microbiome. The abundance of Proteobacteria, Ascomycota, and Basidiomycota differ between schistosome-infected versus uninfected children. Specifically, infection is associated with increases in Pseudomonas, Stenotrophomonas, Derxia, Thalassospira, Aspergillus, Tricholoma, and Periglandula, with a decrease in Azospirillum. We find 262 AMR genes, from 12 functional drug classes, but no association with individual-specific data. To our knowledge, we describe a novel metagenomic dataset of Zimbabwean preschool-aged children, indicating an association between urogenital schistosome infection and changes in the gut microbiome.

DOI:     02-04-2020

Metaphylogenetic analysis of global sewage reveals that bacterial strains associated with human disease show less degree of geographic clustering
Ahrenfeldt et al. 2020

Knowledge about the difference in the global distribution of pathogens and non-pathogens is limited. Here, we investigate it using a multi-sample metagenomics phylogeny approach based on short-read metagenomic sequencing of sewage from 79 sites around the world. For each metagenomic sample, bacterial template genomes were identified in a non-redundant database of whole genome sequences. Reads were mapped to the templates identified in each sample. Phylogenetic trees were constructed for each template identified in multiple samples.

DOI:     20-02-2020

Clades of huge phages from across Earth’s ecosystems
Basem Al-Shayeb et al. 2020

Bacteriophages typically have small genomes and depend on their bacterial hosts for replication. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is—to our knowledge—the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR–Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins.

DOI:   12-02-2020

Using sewage for surveillance of antimicrobial resistance
Frank M. Aarestrup and Mark E. J. Woolhouse

Antimicrobial resistance (AMR), a cross-cutting and increasing threat to global health, is a complex problem with multiple and interconnected drivers. Reliable surveillance data that accurately describe and characterize the global occurrence and distribution of AMR are essential for tracking changes in resistance over time, setting national and global priorities, assessing the impacts of interventions, identifying new kinds of resistance, and supporting investigation of (international) outbreaks of resistant pathogens. AMR surveillance data can also inform development of treatment guidelines. Yet it has proven difficult to achieve these objectives on a global scale, and especially in low- and middle-income countries (LMICs), largely because current surveillance systems deliver data that are extremely variable in quality and quantity and highly heterogeneous in terms of which population is sampled (usually a category of hospital patients) and what drug-bug combinations are included. Here, we outline a plan for a global AMR surveillance system based on applying next-generation sequencing (NGS) to human sewage that will be especially helpful for community AMR surveillance, which is difficult to achieve in other ways, and will provide an affordable surveillance option in resource-poor settings.

DOI:  10.1126/science.aba3432     07-02-2020

Prediction of Acquired Antimicrobial Resistance for Multiple Bacterial Species Using Neural Networks
Aytan-Aktug et al. 2020

Machine learning has proven to be a powerful method to predict antimicrobial resistance (AMR) without using prior knowledge for selected bacterial species-antimicrobial combinations. To date, only species-specific machine learning models have been developed, and to the best of our knowledge, the inclusion of information from multiple species has not been attempted. The aim of this study was to determine the feasibility of including information from multiple bacterial species to predict AMR for an individual species, since this may make it easier to train and update resistance predictions for multiple species and may lead to improved predictions.

DOI:  21-01-2020

Using Genomics to Track Global Antimicrobial Resistance
Hendriksen et al. 2019

The recent advancements in rapid and affordable DNA sequencing technologies have revolutionized diagnostic microbiology and microbial surveillance. The availability of bioinformatics tools and online accessible databases has been a prerequisite for this. We conducted a scientific literature review and here we present a description of examples of available tools and databases for antimicrobial resistance (AMR) detection and provide future perspectives and recommendations. At least 47 freely accessible bioinformatics resources for detection of AMR determinants in DNA or amino acid sequence data have been developed to date. These include, among others but not limited to, ARG-ANNOT, CARD, SRST2, MEGARes, Genefinder, ARIBA, KmerResistance, AMRFinder, and ResFinder.

DOI:   04-09-2019

Worldwide human mitochondrial haplogroup distribution from urban sewage
Pipek et al. 2019

Community level genetic information can be essential to direct health measures and study demographic tendencies but is subject to considerable ethical and legal challenges. These concerns become less pronounced when analyzing urban sewage samples, which are ab ovo anonymous by their pooled nature. We were able to detect traces of the human mitochondrial DNA (mtDNA) in urban sewage samples and to estimate the distribution of human mtDNA haplogroups. An expectation maximization approach was used to determine mtDNA haplogroup mixture proportions for samples collected at each different geographic location. Our results show reasonable agreement with both previous studies of ancient evolution or migration and current US census data; and are also readily reproducible and highly robust. Our approach presents a promising alternative for sample collection in studies focusing on the ethnic and genetic composition of populations or diseases associated with different mtDNA haplogroups and genotypes.

DOI:     12-08-2019

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage
Edwards et al. 2019

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals.

DOI:   08-07-2019

Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage
Hendriksen et al. 2019

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR.

DOI:   08-03-2019

Abundance and diversity of the faecal resistome in slaughter pigs and broilers in nine European countries
Munk et al. 2018

Antimicrobial resistance (AMR) in bacteria and associated human morbidity and mortality is increasing. The 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 and in humans. We quantified and characterized the acquired resistance gene pools (resistomes) of 181 pig and 178 poultry farms from nine European countries, sequencing more than 5,000 Gb of DNA using shotgun metagenomics. We quantified acquired AMR using the ResFinder database and a second 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 in poultry. We found higher AMR loads in pigs, whereas 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 between 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. However, functionally determined AMR genes were not associated with total drug use.

DOI:      23-07-2018

Meta-genomic analysis of toilet waste from long distance flights; a step towards global surveillance of infectious diseases and antimicrobial resistance
Petersen et al. 2015

Human populations worldwide are increasingly confronted with infectious diseases and antimicrobial resistance spreading faster and appearing more frequently. Knowledge regarding their occurrence and worldwide transmission is important to control outbreaks and prevent epidemics. Here, we performed shotgun sequencing of toilet waste from 18 international airplanes arriving in Copenhagen, Denmark, from nine cities in three world regions. An average of 18.6 Gb (14.8 to 25.7 Gb) of raw Illumina paired end sequence data was generated, cleaned, trimmed and mapped against reference sequence databases for bacteria and antimicrobial resistance genes. An average of 106,839 (0.06%) reads were assigned to resistance genes with genes encoding resistance to tetracycline, macrolide and beta-lactam resistance genes as the most abundant in all samples. We found significantly higher abundance and diversity of genes encoding antimicrobial resistance, including critical important resistance (e.g. blaCTX-M) carried on airplanes from South Asia compared to North America. Presence of Salmonella enterica and norovirus were also detected in higher amounts from South Asia, whereas Clostridium difficile was most abundant in samples from North America. Our study provides a first step towards a potential novel strategy for global surveillance enabling simultaneous detection of multiple human health threatening genetic elements, infectious agents and resistance genes.

DOI:       10-07-2015