Liese Van Gompel, DVM Faculty of Veterinary Medicine Institute for Risk Assessment Sciences [email protected]

Liese Van Gompel, DVM Faculty of Veterinary Medicine Institute for Risk Assessment Sciences L.Vangompel@Uu.Nl

The faecal resistome and microbiome of farmers and slaughterhouse workers: A metagenome-wide cross-sectional study

1 Institute for Risk Assessment Sciences, Utrecht University, NL; 2 Intomics, DK; 3 National Food Institute, Danish Technical University, DK; 4 Vion Food Group, NL; 5 University Medical Centre Groningen, University of Groningen, NL; 6 Department of Infectious Diseases and Immunology, Utrecht University, NL; 7 Wageningen Bioveterinary Research, NL; 8 Centre for Infectious Disease Control, National Institute for Public Health and the Environment, NL

Introduction Methods By studying the entire human faecal resistome and associated bacteriome, the Faecal samples and questionnaires (e.g. regarding personal characteristics, diversity and abundance of faecal antimicrobial resistance genes (ARGs) can be antibiotic use & animal contact) were collected from persons working or living on comprehensively characterized. Prior culture-based studies have shown pig and broiler farms, working in a pig slaughterhouse (N=148) and from the associations between occupational exposure to livestock and carriage of specific general population (Lifelines cohort, N=46). Total faecal DNA was extracted and antimicrobial resistant bacteria. Using shotgun metagenomics, the present study sequenced using shotgun metagenomics (Illumina HiSeq4000). ARGs (resistome) investigated the abundance and diversity of 194 faecal resistomes and and bacterial genomes (bacteriome) were classified by mapping reads to the bacteriomes from humans occupationally exposed to ARGs in livestock and a ResFinder and NCBI bacterial reference genome databases. Alpha-diversity control population in the Netherlands. In addition, we sought to identify indices and Bray-Curtis (BC) dissimilarities were calculated and multivariate determinants for the resistome and bacteriome composition of these populations. analyses were performed (NMDS, PERMANOVA, SIMPER, DESeq2 analysis).

Results Figure 1: Pig farmers & slaughterhouse workers carry Pig slaughterhouse workers and pig farmers had a higher total ARG abundance significantly higher antimicrobial resistance gene (ARG) compared to broiler farmers and control subjects (Figure 1). Tetracycline,β- abundances in their faeces compared to broiler farmers lactam and macrolide resistance gene clusters dominatedA the5500 resistome of all & control subjects. studied groups. No significant resistome alpha diversity differences (species richness, Shannon diversity index) were found among the four5000 populations. p<0.0001 However, the resistome beta diversity showed a separation of the mean 5500 resistome composition of pig and pork exposed workers from broiler4500 farmers and p<0.0001 5000 controls, independent of their antimicrobial use (Figure 2). p<0.0001 4000 4500 p=0.08 We demonstrated differences in resistome and bacteriome composition3500 between 4000 Study population slaughter line positions, pig versus broiler exposed workers, as well as differences p=0.0001 3500 Pigpig slaughterhouse sh worker (WO) workers between farmers and employees versus family members (Table3000 1, PERMANOVA). p=0.6 3000 In addition, we found a significant correlation between the bacteriome and Welch test *: sample type, p =2 .3e-12 Pigpig farmers farmer (PF) 2500 resistome (Procrustes analysis) of the studied populations. Furthermore,2500 an in- Broilerbroiler farmersfarmer (BF) 2000 depth analysis of the pig and pork exposed workers compared2000 to the controls, Controlcontrol subjects population (CO) revealed significantly higher levels of 30 ARGs in pig and pork exposed workers 1500 compared to the control subjects (DESeq2 analysis). 1500 1000 ARG abundance (total FPKM) (total ARG abundance 1000 500 Figure 2: The distributions of the resistome (total FPKM) ARG abundance & bacteriome 0 N= 70 54 24 46 500 population means associated with pig-&-pork workers pig sh pig broiler control were-distinct from those of (broiler farmers) & controls. worker farmer farmer population 0 Study population Resistome: ARGs, stress=0.12 Bacteriome: genus, stress=0.13pig sh pig broiler control worker Tablefarmer 1: Significantfarmer resistomepopulation & bacteriome compositionalStudy population differences were identified-between & within populations independent-of-antimicrobial-use. B 2500 Resistome: Bacteriome: Categories used in the Study population Explained Explained PERMANOVA analysis 2250 variation variation

2000 Differences between populations

(1) Pig slaughterhouse workers, 1750 Full population 12.3% 14.9% (2) Pig & (3) Broiler farms, (4) (4 groups) (p=0.001)Type of farm (p=0.001) 1500 Controls farmer (1) Pig slaughterhouse workers, 1250 Full population excl. employee excl. workers from deboning, low risk categories 19.1% 26.1% (2) Pig & (3) Broiler farmers & other family 1000 (low animal contact) (p=0.001) (p=n.a.*) employees, excl. family spouse (4 groups) Study population 750 members, (4) Controls Pig slaughterhouse workers Pig farmers Broiler farmers Control subjects Differences within populations Ellipses represent the 95% CI of the centroid’s (population mean) SE per group. 500

R bnac (total FPKM) ARG abundance Workers from: (1) Lairage to Abattoir workers 7.3% 5.4% 250 dehairing, (2) Evisceration to (3 groups) (p=n.a.*) (p=0.09) Conclusion & Future plans Cooling, (3) Deboning We found an increased ARG carriage in persons working in the0 Dutch pork Farms 5.4% 4.7% production chain compared to broiler farmers and controls. Additionally, we (1) Pig farms, (2) Broiler farms showed significant differences in resistome and bacteriome composition of pig pig(2 farms groups) broiler farms (p=0.004) (p=0.008) and pork exposed workers compared to a control group, as well as within- Type of farm(1) Pig and broiler farmers & population (farms, slaughterhouse) compositional differences. Our results Farms 5.6% 14.3% employees, (2) Pig and broiler suggest direct or indirect livestock contact as a determinant for human faecal (2 groups) (p=0.001) (p=n.a.*) ARG carriage. Future work will focus on the integration of additional farm farmer’s family members (animal, dust) and pig slaughterhouse (carcass) resistomes and bacteriomes into *The assumption of homogeneity of variances was not met. However, a test at a higher the models. C 2750 resistome/bacteriome clustering level was found significant. 2500 2250 PKM)