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. A.L. (Aldert) Zomer

• 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. A.L. (Aldert) Zomer

• Marjolein Kluytmans-van den Bergh
• Gerard van Eijden

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. A.L. (Aldert) Zomer
• 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)

• ing. M.J. (Marian) Broekhuizen-Stins
• dr. E.M. (Els) Broens
• prof. dr. J.A. (Jaap) Wagenaar
• H. (Heleen) Zweerus
• A.J. (Arjen) Timmerman