Campylobacter is the major bacterial cause of gastroenteritis in humans. Transmission to humans occurs via contaminated food, water, animals and environments. Most human Campylobacter infections are attributed to poultry, but cannot only be explained with foodborne transmission. Campylobacter is found in several animals and environments, where its genome is threatened by invading DNA from other microorganisms, bacteriophages or plasmids. Like many bacteria it has an adaptive immune system (CRISPR-Cas) that protects its DNA against this threat. As bacteriophages and plasmids are often specific to a particular niche, CRISPR offers the possibility to link the acquired spacers to specific environments. In PINNACLE, we will investigate how CRISPR dynamics are related to environmental niche adaptation and recalibrate source attribution models. Our outcomes will provide better estimates of the (non-food) sources of Campylobacter, facilitating novel interventions to reduce its disease burden.

Antimicrobials are drugs that help cure people and animals of infections with bacteria. The slogan: ‘the more you use, the faster you lose,’ applies to antimicrobials. When you use antimicrobials, more bacteria become resistant, meaning infection can no longer be treated. This is a worldwide challenge for treating diseases in animals and humans.

Like many other organizations, the World Health Organization (WHO) are advocating for reducing the use of antimicrobials when they are not needed. The COINCIDE project explores what will happen when the use of colistin in animals is banned in Indonesia. Colistin is a specific antimicrobial that is used as a last resort in humans if nothing else works, resistance against this antimicrobial means that resistant disease-causing bacteria will have free reign.

In animals, colistin was banned in 2020, and a group of human doctors, veterinarians, anthropologists, and DNA researchers will look if fewer bacteria become resistant. We also want to reduce the colistin use in humans, as it is suspected to be used in humans without good reason. We will work with farmers and their veterinarians, but also with people in the community, doctors, and pharmacies, to find out why they are still using colistin and if they can do without. The project's outcome will help governments, farmers, veterinarians, human doctors, and everybody who needs colistin to safeguard this antimicrobial for use only when we cannot do without.

Project partners
Anis Karuniawati, Universitas Indonesia, Indonesia 
Juliëtte Severin, Erasmus MC University Medical Center Rotterdam, Netherlands
Jaap Wagenaar, Utrecht University, Faculty of Veterinary Medicine, Netherlands
Sunandar Sunandar, Center for Indonesian Veterinary Analytical Studies, Indonesia
Herman Barkema, University of Calgary, Canada
Koen Peeters, Institute of Tropical Medicine, Belgium
Imron Suandy, Directorate of Veterinary Public Health, DG of Livestock and Animal Health Services, MoA-Indonesia, Indonesia

Ph.D. Candidate

Soe Yu Naing, Utrecht University

• prof. dr. J.A. (Jaap) Wagenaar
• Soe Yu Naing

Since 2015, under the auspices of WHO, a basic protocol for One Health Surveillance of AMR has been established. This “Tricycle” protocol integrates human, animal and environmental surveillance and focuses on a single indicator for AMR: ESBL-producing E. coli. To our knowledge, this is the first One Health AMR surveillance protocol that has consistently been piloted across six different countries across the world.

The TRIuMPH project builds on the Tricycle project and on the JPI network “NETESE” by adding new research elements and protocols, thereby extending the application of the Tricycle surveillance. This will be achieved in a collaborative approach with current Tricycle and NETESE partners (PK, MY and MG) and partners that contributed to the Tricycle protocol development (Utrecht University, INSERM and RIVM National Institute for Public Health and the Environment ). New One Health protocols will be developed and applied in a one-year surveillance campaign for the detection of carbapenemase-producing Enterobacteriaceae (CPE, WP2), and for whole-genome sequencing analysis of ESBL / CPE isolates (WP3).

Within one single country, the extension of surveillance to a broader scale is needed, as analyses are currently limited to single cities. This will be brought about by two activities: Inclusion of additional sites within participating countries through in-country training (WP4), and integration with existing monitoring campaigns, such as for water samples taken within the Polio Eradication campaign (WP5). These also offer the opportunity to validate the applicability of wastewater sampling as a proxy of community prevalence of ESBL and CPE.

RIVM coordinates TRIuMPH. Partner countries involved are France, Madagascar, Malaysia, Pakistan and the Netherlands. RIVM's Heike Schmitt is the project coordinator.

Funding
The project has been awarded funding within the JPIAMR 9th transnational call: “Call on Diagnostics and Surveillance 2019.

WP2 partner - Development and implementation of CPE One Health diagnostics

WP3 leader – Development and implementation of WGS standard operating procedures

• dr. L. (Linda) van der Graaf-van Bloois
• prof. dr. J.A. (Jaap) Wagenaar

• Coordinator: Heike Schmitt - National Institute for Public Health and the Environment (RIVM) The Netherlands
• Laurence Armand-Lefevre - University Paris-Diderot Medical School INSERM IAME UMR1137 France
• Luc Samison - University of Antananarivo Centre d’Infectiologie Charles Mérieux Faculty of Medicine Madagascar
• Rohaidah Hashim - Institute for Medical Research Infectious Disease Research Centre MOH Kuala Lumpur Malaysia
• Muhammad Salman - National Institute of Health Public Health Laboratories Division Islamabad Pakistan

Research project after the selection of ESBLs in dairy cows after intramammary application of cephapirin and cefalonium

• dr. D.C. (David) Speksnijder

In recent years, the human microbiome has been characterised in great detail in several large-scale studies as a key player in intestinal and non-intestinal diseases, e.g. inflammatory bowel disease, diabetes and liver cirrhosis, along with brain development and behaviour. As more associations between microbiome and phenotypes are elucidated, research focus is now shifting towards causality and clinical use for diagnostics, prognostics and therapeutics, where some promising applications have recently been showcased. Microbiome data are inherently convoluted, noisy and highly variable, and non-standard analytical methodologies are therefore required to unlock its clinical and scientific potential. While a range of statistical modelling and Machine Learning (ML) methods are now available, sub-optimal implementation often leads to errors, over-fitting and misleading results, due to a lack of good analytical practices and ML expertise in the microbiome community. Thus, this COST Action network will create productive symbiosis between discovery-oriented microbiome researchers and data-driven ML experts, through regular meetings, workshops and training courses. Together, it will first optimise and then standardise the use of said techniques, following the creation of publicly available benchmark datasets. Correct usage of these approaches will allow for better identification of predictive and discriminatory ‘omics’ features, increase study repeatability, and provide mechanistic insights into possible causal or contributing roles of the microbiome. This Action will also investigate automation opportunities and define priority areas for novel development of ML/Statistics methods targeting microbiome data. Thus, this COST Action will open novel and exciting avenues within the fields of both ML/Statistics and microbiome research.

https://www.cost.eu/actions/CA18131/#tabs|Name:overview

Vice Leader Working Group 4 

Working Group 4: Dissemination and training
Objectives:
To disseminate the results of the Action in peer-reviewed journals, on the web-portal, at international conferences and through end-user workshops. WG4 will also organise training courses and workshops in microbiome-related ML methodologies recommended by Action members.
Tasks:
T4.1: Disseminate evaluation, optimisation and standardisation progress.
T4.2: Organise training courses (e.g. Summer Schools) and workshops.
Activities:
A4.1: A dissemination strategy will be developed whereby regular the COST progress and outcomes will be shared within and outside the Action at the Web-portal, international conferences, COST reports, white-papers and peer-reviewed publications.
A4.2: The WG4 members will assess the need for ML/Stats training among the COST members and subsequently design, organise and implement regular training courses and workshops.
Milestones:
M4.1: Annual reports (Y2-4Q1), white-paper (Y4Q2), peer-reviewed publications (Y1Q4/Y4Q2).
M4.2: Initial workshop (Y1Q2), training courses (Y2Q2/Y3Q2).
Major Deliverables:
D4.1: COST reports, white-papers and peer-reviewed publications.
D4.2: Regular workshops & training courses of best practice in ML/Stats for microbiome studies.
D4.3: Educational material shared: tutorials on paper and video (including a YouTube channel).

• Dr Marcus CLAESSON; Prof David GOMEZ-CABRERO; Dr Debora SANTO; Dr Karina MARCUS; Ms Olga GORCZYCA and 2 MC members from every EU country

Pig farms act as reservoir of Livestock-Associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). Through occupational exposure to farm dust and contact with pigs, farm workers are at risk for acquiring LA-MRSA. Although health care institutions can cope with the current situation, it is a burden for patients, health care staff, and finances. In addition, the recent observed adaptation of LA-MRSA originating from pigs to humans in Denmark further highlights the need to reduce LA-MRSA colonization in pigs and subsequent transmission to humans. In a pilot study of the nasal microbiome we observed that piglets become LA-MRSA positive after a few days of birth. The presence of several other bacterial species, including coagulase-negative staphylococci was negatively associated with the presence of LA-MRSA. More evidence is needed regarding which bacterial species and/or strains compete with LAMRSA. The project aims to establish the effect of colonization resistance (bacterial competition) on the transmission of LA-MRSA from pigs to humans by i) identifying bacterial species that compete with LA-MRSA (S. aureus in general) in a systematic way using state of the art bioinformatics and metagenomics methods at strain level, ii) studying the efficacy of applying a nasal microflora for piglets which will be produced under GMP conditions by the industrial partner in the project and tested under field conditions at conventional farms, and iii) to estimate the risk reduction as a result from limiting MRSA transmission to humans by reducing shedding and consequently a more limited environmental contamination. Communication with farmers, veterinarians, public health workers and other stakeholders, with the help of our supporting organizations, will prepare the stakeholders for the outcome of the project, bringing it close to immediate use in practice. ExcludeMRSA will deliver a reduction of MRSA colonization or will lead to complete prevention of MRSA colonization by precolonization of piglets with microflora. The efficacy will be assessed in two countries by using proven environmental risk models for human exposure and evaluating changes in the exposure risk association. Because of the earlier performed successful pilot studies, the experience of the partners and the inclusion of an industrial partner experienced in production of live strains, this project is feasible in three years’ time

• prof. dr. ir. D.J.J. (Dick) Heederik
• A.A. (Abel) Vlasblom
• prof. dr. J.A. (Jaap) Wagenaar
• dr. B. (Birgitta) Duim

• Marcus Claesson (UCC)
• Peadar Lawlor (Teagasc)
• Fellipe Freitas Barbosa (EW-nutrition)

Livestock-Associated Methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged in pigs globally. Pig farms act as a reservoir of LA-MRSA. In the Netherlands, the prevalence of LA-MRSA in slaughter pigs was 99.5% in 2015. Through exposure to animals and dust, farms are at risk for acquiring LA-MRSA. In the Netherlands MRSA colonization in the general population and in hospitals is very low. Strict infection control measures are implemented in hospitals (‘search and destroy’) to prevent spread of MRSA of people who are at risk of being MRSA-positive. With the introduction of LA-MRSA, all people working with living pigs and veal calves are considered as risk for being LA-MRSA positive and are included in the search and destroy program with quarantine and additional diagnostics. Infections with multidrug-resistant microorganisms, including LA-MRSA are a burden for patients, health care staff, and finances of health care institutions.

 The recent adaptation of LA-MRSA to humans in Denmark, and the recently observed unexplained cases of LA-MRSA patients in Dutch hospitals, highlights the need to prevent LA-MRSA transmission. At this moment, there are no intervention measures in pig production to reduce LA-MRSA in pigs. In a pilot microbiome study, we identified several bacterial species negatively associated with colonization of LA-MRSA in piglets. To identify and isolate bacterial strains that will effectively outcompete LA-MRSA when used as a pre-colonization microflora, strain level metagenomics followed by high throughput strain identification will be used. Subsequently these competing strains should be produced and applied nasally as live bacteria in newborn piglets, in order to limit or eradicate LA-MRSA outgrowth in the piglet nasopharynx. Prevention of LA-MRSA colonization will reduce the transmission risk of LA-MRSA from piglets to farm workers through dust. We aim to study the effect of this bacterial inference on transmission of LA-MRSA from pigs to humans by i) identifying competing bacterial species that might be included in a microflora to be used in pigs, ii) studying the efficacy of pre-colonizing piglets, and iii) estimating the risk reduction for MRSA transmission to humans as a result of reduced environmental contamination.

In this project, an interdisciplinary group works together with an industrial partner to identify bacterial species that compete with LA-MRSA for production of a microflora that can be administered to pigs for modulation of the nasal microbiome. The outcome of this project will be a novel intervention procedure based on protecting the piglets for colonization with LA-MRSA by competitive-exclusion.

• prof. dr. ir. L.A. (Lidwien) Smit
• prof. dr. ir. D.J.J. (Dick) Heederik
• dr. A. (Arie) van Nes
• dr. A Fluit
• A.A. (Abel) Vlasblom
• prof. dr. J.A. (Jaap) Wagenaar
• dr. B. (Birgitta) Duim

• Marjolein Kluytmans-van den Bergh
• Gerard van Eijden

Sustainable control of antimicrobial resistance (AMR) requires a One Health approach, because humans and animals can exchange bacteria and genetic mobile elements encoding for AMR. Responsible use of antimicrobials in animals is essential, but AMR genes may also spread in the absence of selection pressure. Thus, AMR surveillance has been implemented to evaluate AMR trends in animal populations. Current surveillance programs are, however, not fit for early detection of newly emerging AMR. The latter is important in case of resistance to antimicrobials of critical importance to humans. When only a few farms are positive at the time of first detection, measures like quarantine or culling could be implemented to minimize exposure of humans. In case many farms are affected at first detection, implementation of such measures is not feasible. 

Methicilin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant enterococci (VRE) and Extended Spectrum Beta-Lactamase Enterobacteriaceae (ESBL) are all examples of AMR’s with a large impact on human health that emerged in animal populations in the Netherlands and many other countries and that were already wide spread at the time of detection. The goal of BEWARE is to develop a blueprint for detecting emerging AMR in livestock when the number of affected farms is still low. This early warning blueprint is unique, because it takes account of the most likely routes of introduction, the transmission of AMR between animals and between farms and the diagnostic quality of the assay used. Although the blueprint will be applicable to all AMR’s and animal sectors, BEWARE will focus on carbapenemase producing Enterobacteriaceae (CPE) in veal calves, pigs and broilers. These are the most relevant livestock sectors regarding AMR and CPE emerging in animals is of critical importance in human health care. Specific goals are: 1) identify AMR introduction routes, their association with human behavior, and rank them according to their importance; 2) calculate transmission parameters for AMR-carrying bacteria within and between animal populations; 3) develop a sensitive and specific assay for early detection of CP-genes; and 4) generate a dynamic mathematical model to integrate the results to predict the numbers of affected farms at first detection as function of the selected sampling strategy. 

Developing such an early warning blueprint requires an interdisciplinary project subdivided in 4 work packages (WP). In WP1 AMR introduction routes will be identified, including their association with human behavior, and combined into a risk model of AMR introduction into the livestock populations that allows to rank the sites of possible introduction according to their relevance. The model will include both national and international routes of introduction and information will be collected from literature, available databases and by expert elicitation. 

In WP2 transmission of AMR carrying bacteria within and between populations determines the speed of AMR emergence. Parameters for AMR transmission in veal calf, broiler and pig farms will be estimated from available data sets, assuming transmission of emerging CPE carrying bacteria is like transmission of ESBL carrying Enterobacteriaceae. We will test this assumption in vivo in broilers. The network of animal movements between farms will be created from data available to the research consortium. 

In WP3 an assay for sensitive and specific metagenomics detection of CPE will be made. Following enrichment, DNA will be isolated and used for RT-PCR and for targeted metagenomics. Furthermore, we will perform a second enrichment step using the ResCap platform and samples will be sequenced using Illumina and Nanopore sequencing. Finally, test quality parameters will be estimated. 

In WP4 an early detection surveillance framework will be developed using a dynamic mathematical model. The framework will consist of three parts that integrate the results of WP 1-3: introduction of AMR genes/plasmids in the livestock population, transmission between animals and between farms, and detection. Using the fully parameterized framework, the sensitivity and specificity of early warning is evaluated. By varying the sampling strategy (i.e. frequency and sample size) the effects on the performance of the early detection system are studied. The blueprint can be easily adapted to other types of emerging AMR, provided their transmission and test quality parameters are available. The flexibility of the model regarding selected sampling strategy will enable policy makers to develop an AMR early warning program according their needs.

• N. (Natcha) Dankittipong
• prof. dr. J.A. (Arjan) Stegeman
• prof. dr. J.A. (Jaap) Wagenaar

• Jantien Backer (RIVM)
• Alex Bossers (WUR)
• Clazien de Vos (WUR)
• Manon Swanenburg (WUR)

Iridescent nanostructures reflect light to create intense, angle-dependent hues and are of importance for the development of novel optical surfaces (via biomimetics) and using bacteria as a reporter in diagnostic assays. Nature has a rich variety in iridescence but little is known about the underlying genetics and the relevant genes have not been identified in any kingdom of life. However, for the first time in any organism we have recently found responsible gene clusters in the bacterium Flavobacterium IR1 using high-throughput transposon mutagenesis screening. Homologous genes will be identified in metagenomics datasets and other iridescent isolates to understand their ecological significance. Moreover we aim to identify novel bacteria with different iridescent properties and the evolutionary relationship from a dataset of > 100,000 genomes using information from IR1 as a starting point.

These work supports the use of iridescent bacteria as design studios to create new optical materials (for paints, clothing, security applications) and the use of iridescent cellular assays in diagnostics (suitable for resource limited setting). The project is also of academic significance given the widespread incidence of iridescence in the natural world but the near complete lack of information on genetics and evolution.

The project is a collaboration between NIOZ (primary applicant) providing culture experience and contributing strains and genome sequences of relevant Flavobacteria; Utrecht (Hotel) with the expertise to perform the genomics/metagenomics/evolutionary bioinformatics and Hoekmine BV (commercial partner) contributing a Flavobacterium (genome sequence, gene clusters, unsequenced mutants) that will allow the genetics of structural colour to be approached and applied commercially.

• dr. B.E. (Bas) Dutilh

• Colin Ingham (Hoekmine BV)
• Henk Bolhuis (NIOZ)

Campylobacteriosis is the primary zoonosis in Europe, causing over 1.6 M cases and €76 M costs annually in the Netherlands alone (~17 M people). Most cases are caused by Campylobacter jejuni and C. coli, which are widespread in livestock and wildlife, providing many ways for human exposure beyond just food. Despite all the research and control efforts in the food chain, there is no significant decrease in human campylobacteriosis. Up to 80% of human campylobacteriosis cases can be attributed to the poultry reservoir, but only ~40% of poultry-borne cases are attributable to poultry consumption; thus, many poultry-borne Campylobacter strains infect humans via other routes. Campylobacter is often found in environmental sources like surface water, indicating recent contamination with (animal) fecal material, but its environmental routes are still largely unexplored. While Campylobacter survives poorly outside the host, there are some environmentally adapted strains that play a key role in the transmission between animals and humans via the environment. Surface water is a ‘sink’ that collects Campylobacter strains from different hosts whose relative contributions are largely unknown, though wild birds are thought to play a major role. However, the devastating H7N7 bird flu epidemic hitting the Netherlands in 2003 showed that even without a huge drop in poultry consumption, the massive poultry culling and closure of poultry abattoirs to contain the epidemic was associated with a 44-50% drop in human campylobacteriosis where these measures were enforced, suggesting a major role of the environment in human exposure to poultry-borne strains. Moreover, while studies show that poultry and wild birds are the most important contributors to Campylobacter surface water pollution, their contributions vary with the size of the poultry production. The environment may also act as a source of Campylobacter (re)colonization in livestock. As the environment seems to be a key player in Campylobacter epidemiology and the non-foodborne side of campylobacteriosis receives little attention despite its potential to provide new targets for Campylobacter control and research, this project will discern the origins and spread of Campylobacter strains contaminating the environment and will determine their contribution to human campylobacteriosis morbidity, as well as the underlying (non-foodborne) transmission routes. As a collaboration of 4 leading institutes in public, animal and environmental health in the Netherlands (RIVM, CVI, UU and Alterra WUR), we will collect and type with a gene-by-gene approach (whole-genome multilocus sequence typing, wg-MLST) over 1200 C. jejuni/coli isolates representative of the Dutch eco-epidemiological situation from human cases, surface water (agricultural ditches, recreational waters, wastewater outlets), animals (broilers, layers, beef/dairy cattle, sheep/goats, pigs, pets), and wild birds (Anseriformers, Charadriformes, Columbiformes, Suliformes) using both well-established surveillance systems and ad hoc sampling schemes. We will: (i) characterize genotypically the strains circulating in surface water, animals and humans; (ii) quantify the contribution of different wild bird, farm and companion animals to Campylobacter pollution in different surface water types, seasons and areas with varying human, livestock and wild bird densities; (iii) quantify the contribution of different animal reservoirs and surface water to human campylobacteriosis in different seasons and areas with varying human, livestock and wild bird densities to estimate both the fraction of human cases attributable to the environment and the relation between human cases and reservoir density; (iv) determine the evolutionary history and relations of Campylobacter strains in the environment, humans and animals to understand their diversity and ecology, identify source-specific genetic markers, examine the role of the environment in the emergence of pathogenic strains, and perform source attribution at a high resolution; (v) conduct combined source attribution and case-control analyses to identify risk exposures for human campylobacteriosis of environmental origin and its underlying transmission routes. By depicting the epidemiology of non-foodborne campylobacteriosis, this project will allow for the delineation of more holistic control strategies, including those preventing Campylobacter dissemination into the environment. Besides standard scientific outputs, the results of this project will be translated into practical action points and advices for regulatory authorities on how to tackle environmental campylobacteriosis. The project members host national/international Campylobacter reference labs and participate since many years in advisory groups for the Dutch Ministries of Health and Economic Affairs, as well as the poultry industry, so through these forums knowledge gained in this project will be translated into interventions.

• prof. dr. J.A. (Jaap) Wagenaar
• dr. B. (Birgitta) Duim
• dr. L. (Lapo) Mughini Gras
• dr. A.C. (Annemieke) Mulder MSc

• Eelco Franz (RIVM)
• Wilfrid van Pelt (RIVM)
• Hetty Blaak (RIVM)
• Ciska Schets (RIVM)
• Ralf Buij (WUR Alterra)
• Miriam Koene (WBVR)

Bioinformatics consultant for Janssen Pharmaceuticals on bacterial vaccine development.