Prevalence and types of Campylobacter on poultry farms and in their direct environment.

To study whether broiler and layer farms contribute to the environmental Campylobacter load, environmental matrices at or close to farms, and caecal material from chickens, were examined. Similarity between Campylobacter from poultry and environment was tested based on species identification and Multilocus Sequence Typing. Campylobacter prevalence in caecal samples was 97% at layer farms (n = 5), and 93% at broiler farms with Campylobacter-positive flocks (n = 2/3). Campylobacter prevalence in environmental samples was 24% at layer farms, and 29% at broiler farms with Campylobacter-positive flocks. Campylobacter was detected in soil and surface water, not in dust and flies. Campylobacter prevalence in adjacent and remote surface waters was not significantly (P > 0.1) different. Detected species were C. coli (52%), C. jejuni (40%) and C. lari (7%) in layers, and C. jejuni (100%) in broilers. Identical sequence types (STs) were detected in caecal material and soil. A deviating species distribution in surface water adjacent to farms indicated a high background level of environmental Campylobacter. STs from layer farms were completely deviant from surface water STs. The occasional detection of identical STs in broilers, wastewater at broiler farms and surface water in the farm environment suggested a possible contribution of broiler farms to the aquatic environmental Campylobacter load.

Environmental surveillance during an outbreak of tularaemia in hares, the Netherlands, 2015.

Tularaemia, a disease caused by the bacterium Francisella tularensis, is a re-emerging zoonosis in the Netherlands. After sporadic human and hare cases occurred in the period 2011 to 2014, a cluster of F. tularensis-infected hares was recognised in a region in the north of the Netherlands from February to May 2015. No human cases were identified, including after active case finding. Presence of F. tularensis was investigated in potential reservoirs and transmission routes, including common voles, arthropod vectors and surface waters. F. tularensis was not detected in common voles, mosquito larvae or adults, tabanids or ticks. However, the bacterium was detected in water and sediment samples collected in a limited geographical area where infected hares had also been found. These results demonstrate that water monitoring could provide valuable information regarding F. tularensis spread and persistence, and should be used in addition to disease surveillance in wildlife.

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P(1B)-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.

Molecular typing of Coxiella burnetii from animal and environmental matrices during Q fever epidemics in the Netherlands.

BACKGROUND: The bacterium Coxiella burnetii has caused unprecedented outbreaks of Q fever in the Netherlands between 2007 and 2010. Since 2007, over 4000 human cases have been reported, with 2354 cases in 2009 alone. Dairy goat farms were identified as most probable sources for emerging clusters of human Q fever cases in their vicinity. However, identifying individual farms as primary source for specific clusters of human cases remains a challenge, partly due to limited knowledge of the different C. burnetii strains circulating in livestock, the environment and humans. RESULTS: We used a multiplex multi-locus variable number of tandem repeats analysis (MLVA) assay to investigate the genotypic diversity of C. burnetii in different types of samples that were collected nationwide during the Dutch Q fever outbreaks between 2007 and 2010. Typing was performed on C. burnetii positive samples obtained from several independent studies investigating C. burnetii presence in animals and the environment. Six different genotypes were identified on 45 farm locations, based on sequence-confirmed estimates of repeat numbers of six MLVA markers. MLVA genotype A was observed on 38 of the 45 selected farm locations in animals and in environmental samples. CONCLUSIONS: Sequence confirmation of the numbers of tandem repeats within each locus and consensus about repeat identification is essential for accurate MLVA typing of C. burnetii. MLVA genotype A is the most common genotype in animal samples obtained from goat, sheep, and rats, as well as in environmental samples such as (aerosolized) dust, which is considered to be the major transmission route from animals via the environment to humans. The finding of a single dominant MLVA genotype in patients, the environment, and livestock complicates accurate source-finding. Pinpointing individual sources in the Netherlands requires discrimination of genotypes at a higher resolution than attained by using MLVA, as it is likely that the dominant C. burnetii MLVA type will be detected on several farms and in different patients in a particular area of interest.

Impact of long-term dietary habits on the human gut resistome in the Dutch population.

The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilus when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature.

Riverine microplastic and microbial community compositions: A field study in the Netherlands.

Plastic pollution in aquatic environments, particularly microplastics (<5 mm), is an emerging health threat. The buoyancy, hydrophobic hard surfaces, novel polymer carbon sources and long-distance transport make microplastics a unique substrate for biofilms, potentially harbouring pathogens and enabling antimicrobial resistance (AMR) gene exchange. Microplastic concentrations, their polymer types and the associated microbial communities were determined in paired, contemporaneous samples from the Dutch portion of the river Rhine. Microplastics were collected through a cascade of 500/100/10 µm sieves; filtrates and surface water were also analysed. Microplastics were characterized with infrared spectroscopy. Microbial communities and selected virulence and AMR genes were determined with 16S rRNA-sequencing and qPCR. Average microplastic concentration was 213,147 particles/m3; polyamide and polyvinylchloride were the most abundant polymers. Microbial composition on 100-500 µm samples differed significantly from surface water and 10-100 µm or smaller samples, with lower microbial diversity compared to surface water. An increasingly 'water-like' microbial community was observed as particles became smaller. Associations amongst specific microbial taxa, polymer types and particle sizes, as well as seasonal and methodological effects, were also observed. Known biofilm-forming and plastic-degrading taxa (e.g. Pseudomonas) and taxa harbouring potential pathogens (Pseudomonas, Acinetobacter, Arcobacter) were enriched in certain sample types, and other risk-conferring signatures like the sul1 and erm(B) AMR genes were almost ubiquitous. Results were generally compatible with the existence of taxon-selecting mechanisms and reduced microbial diversity in the biofilms of plastic substrates, varying over seasons, polymer types and particle sizes. This study provided updated field data and insights on microplastic pollution in a major riverine environment.

Determinants of presence and removal of antibiotic resistance genes during WWTP treatment: A cross-sectional study.

Wastewater treatment plants (WWTPs), linking human fecal residues and the environment, are considered as hotspots for the spread of antimicrobial resistance (AMR). In order to evaluate the role of WWTPs and underlying operational parameters for the removal of AMR, the presence and removal efficiency of a selected set of 6 antimicrobial resistance genes (ARGs) and 2 mobile genetic elements (MGEs) was evaluated by means of qPCR in influent and effluent samples from 62 Dutch WWTPs. The role of possible factors impacting the concentrations of ARGs and MGEs in the influent and their removal was identified through statistical analysis. ARGs and the class I integron-integrase gene (intI1) were, on average, removed to a similar extent (1.76 log reduction) or better (+0.30-1.90 logs) than the total bacteria (measured as 16S rRNA gene). In contrast, broad-host-range plasmids (IncP-1) had a significantly increased (p < 0.001) relative abundance after treatment. The presence of healthcare institutions in the area served did only slightly increase the concentrations of ARGs or MGEs in influent. From the extended panel of operational parameters, rainfall, increasing the hydraulic load of the plant, most significantly (p < 0.05) affected the treatment efficiency by decreasing it on average -0.38 logs per time the flow exceeded the average daily flow. Our results suggest that overall, WWTP treatments do not favor the proliferation of the assessed resistance genes but might increase the relative abundance of broad-host-range plasmids of the IncP-1 type.

Environmental Surveillance of Zoonotic Francisella tularensis in the Netherlands.

Tularemia is an emerging zoonosis caused by the Gram-negative bacterium Francisella tularensis, which is able to infect a range of animal species and humans. Human infections occur through contact with animals, ingestion of food, insect bites or exposure to aerosols or water, and may lead to serious disease. F. tularensis may persist in aquatic reservoirs. In the Netherland, no human tularemia cases were notified for over 60 years until in 2011 an endemic patient was diagnosed, followed by 17 cases in the 6 years since. The re-emergence of tularemia could be caused by changes in reservoirs or transmission routes. We performed environmental surveillance of F. tularensis in surface waters in the Netherlands by using two approaches. Firstly, 339 samples were obtained from routine monitoring -not related to tularemia- at 127 locations that were visited between 1 and 8 times in 2015 and 2016. Secondly, sampling efforts were performed after reported tularemia cases (n = 8) among hares or humans in the period 2013-2017. F. tularensis DNA was detected at 17% of randomly selected surface water locations from different parts of the country. At most of these positive locations, DNA was not detected at each time point and levels were very low, but at two locations contamination was clearly higher. From 7 out of the 8 investigated tularemia cases, F. tularensis DNA was detected in at least one surface water sample collected after the case. By using a protocol tailored for amplification of low amounts of environmental DNA, 10 gene targets were sequenced. Presence of F. tularensis subspecies holarctica was confirmed in 4 samples, and in 2 of these, clades B.12 and B.6 were identified. This study shows that for tularemia, information regarding the spatial and temporal distribution of its causative agent could be derived from environmental surveillance of surface waters. Tracking a particular strain in the environment as source of infection is feasible and could be substantiated by genotyping, which was achieved in water samples with only low levels of F. tularemia present. These techniques allow the establishment of a link between tularemia cases and environmental samples without the need for cultivation.

Distribution, Numbers, and Diversity of ESBL-Producing E. coli in the Poultry Farm Environment.

This study aimed to discern the contribution of poultry farms to the contamination of the environment with ESBL-producing Escherichia coli and therewith, potentially to the spread of these bacteria to humans and other animals. ESBL-producing E. coli were detected at all investigated laying hen farms (n = 5) and broiler farms (n = 3) in 65% (46/71) and 81% (57/70) of poultry faeces samples, respectively. They were detected in rinse water and run-off water (21/26; 81%), other farm animals (11/14; 79%), dust (21/35; 60%), surface water adjacent to farms (20/35; 57%), soil (48/87; 55%), on flies (11/73; 15%), and in barn air (2/33; 6%). The highest prevalence and concentrations in the outdoor environment were observed in soil of free-range areas at laying hen farms (100% of samples positive, geometric mean concentration 2.4×10(4) cfu/kg), and surface waters adjacent to broiler farms during, or shortly after, cleaning between production rounds (91% of samples positive, geometric mean concentration 1.9×10(2) cfu/l). The diversity of ESBL-producing E. coli variants with respect to sequence type, phylogenetic group, ESBL-genotype and antibiotic resistance profile was high, especially on broiler farms where on average 16 different variants were detected, and the average Simpson's Indices of diversity (SID; 1-D) were 0.93 and 0.94 among flock and environmental isolates respectively. At laying hen farms on average nine variants were detected, with SIDs of 0.63 (flock isolates) and 0.77 (environmental isolates). Sixty percent of environmental isolates were identical to flock isolates at the same farm. The highest proportions of 'flock variants' were observed in dust (94%), run-off gullies (82%), and barn air (67%), followed by surface water (57%), soil (56%), flies (50%) and other farm animals (35%).The introduction of ESBL-producing E. coli from poultry farms to the environment may pose a health risk if these bacteria reach places where people may become exposed.

Coxiella burnetii (Q fever) in Rattus norvegicus and Rattus rattus at livestock farms and urban locations in the Netherlands; could Rattus spp. represent reservoirs for (re)introduction?

The Q fever outbreak in the Netherlands in 2007-2010 prompted government interventions to reduce the human incidence by reduction of Q fever shedding at dairy goat farms. Mandatory hygiene measures were taken, including the control of animal reservoirs. It has been postulated that brown rats, through their commensal nature, form an important factor in the persistent dissemination of endemic circulating Coxiella burnetii in nature to domestic animals, livestock and humans. Here, the occurrence of C. burnetii in rats captured at different types of location during the Q fever outbreak in the Netherlands, viz. urban areas, nature areas and various types of farm has been determined. This is a first step towards the elucidation of the reservoir status of rats in veterinary and human Q fever epidemiology. C. burnetii DNA was detected in the spleen of 4.9% of the brown rats (Rattus norvegicus) and 3.0% of the black rats (Rattus rattus). Evidence for C. burnetii infection was also found in liver, kidney, lung and intestinal tissue but not in heart, brain and pancreas. C. burnetii IgGs were detected in 15.8% of the brown rats. Positive rats were collected at goat, pig, cattle and poultry farms, and urban locations; including locations outside the designated 5km "increased-risk" zones around bulk milk positive goat farms. The percentage of rat-positive locations was the highest for goat farms (50%) and cattle farms (14.3%). The presence of actively infected rats outside the lambing season and at multiple environmental settings including urban locations might suggest that rats are not merely a spill-over host due to infection by a contaminated environment but might represent true reservoirs, capable of independent maintenance of C. burnetii infection cycles and thereby contributing to spread and transmission of the pathogen. If frequent (re)introduction of C. burnetii to small ruminant farms can be caused by rats as maintenance reservoirs, mandatory wildlife control and lifelong vaccination of herds will be necessary.