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The aim of this study was metagenomics analyses of acquired antibiotic-resistance genes (ARGs) in the intestinal microbiome of two important food-animal species in Hungary from a One Health perspective. Intestinal content samples were collected from 12 domestic pigs ( Sus scrofa ) and from a common carp ( Cyprinus carpio ). Shotgun metagenomic sequencing of DNA purified from the intestinal samples was performed on the Illumina platform. The ResFinder database was applied for detecting acquired ARGs in the assembled metagenomic contigs. Altogether, 59 acquired ARG types were identified, 51 genes from domestic pig and 12 genes from the carp intestinal microbiome. ARG types belonged to the antibiotic classes aminoglycosides (27.1%), tetracyclines (25.4%), β-lactams (16.9%), and others. Of the identified ARGs, tet (E), a bla OXA - 48-like β-lactamase gene, as well as cphA4 , ampS , aadA2 , qnrS2 , and sul1 , were identified only in carp but not in swine samples. Several of the detected acquired ARGs have not yet been described from food animals in Hungary. The tet (Q), tet (W), tet (O), and mef (A) genes detected in the intestinal microbiome of domestic pigs had also been identified from free-living wild boars in Hungary, suggesting a possible relationship between the occurrence of acquired ARGs in domestic and wild animal populations.

The purpose of this study was to investigate the L. monocytogenes occurrence and genetic diversity in three Belgian pork cutting plants. We specifically aim to identify harborage sites and niche locations where this pathogen might occur. A total of 868 samples were taken from a large diversity of food and non-food contact surfaces after cleaning and disinfection (C&D) and during processing. A total of 13% (110/868) of environmental samples tested positive for L. monocytogenes . When looking in more detail, zone 3 non-food contact surfaces were contaminated more often (26%; 72/278) at typical harborage sites, such as floors, drains, and cleaning materials. Food contact surfaces (zone 1) were less frequently contaminated (6%; 25/436), also after C&D. PFGE analysis exhibited low genetic heterogeneity, revealing 11 assigned clonal complexes (CC), four of which (CC8, CC9, CC31, and CC121) were predominant and widespread. Our data suggest (i) the occasional introduction and repeated contamination and/or (ii) the establishment of some persistent meat-adapted clones in all cutting plants. Further, we highlight the importance of well-designed extensive sampling programs combined with genetic characterization to help these facilities take corrective actions to prevent transfer of this pathogen from the environment to the meat.

Although refrigeration and modified-atmosphere packaging (MAP) allow for an extended shelf life of cooked charcuterie products, they are still susceptible to bacterial spoilage. To obtain better insights into factors that govern product deterioration, ample information is needed on the associated microbiota. In this study, sliced MAP cooked ham and cooked chicken samples were subjected to culture-dependent and culture-independent microbial analysis. In total, 683 bacterial isolates were obtained and identified from 60 samples collected throughout the storage period. For both charcuterie types, lactic acid bacteria (LAB) constituted the most abundant microbial group. In cooked ham, Brochothrix thermosphacta was highly abundant at the beginning of the shelf-life period, but was later overtaken by Leuconostoc carnosum and Lactococcus piscium . For cooked chicken products, Latilactobacillus sakei was most abundant throughout the entire period. Additionally, 13 cooked ham and 16 cooked chicken samples were analyzed using metabarcoding. Findings obtained with this method were generally in accordance with the results from the culture-dependent approach, yet they additionally demonstrated the presence of Photobacterium at the beginning of the shelf-life period in both product types. The results indicated that combining culture-dependent methods with metabarcoding can give complementary insights into the evolution of microorganisms in perishable foods.

Antibiotic resistant bacteria and antibiotic residues can enter the environment when using animal manure as fertilizer. Twenty-five mixed beef cattle farmyard manure samples and 9 mixed fattening calf slurry samples from different farms across Belgium were investigated for the presence of 69 antibiotic residues, antibiotic resistant Escherichia coli and Salmonella spp. Doxycycline, oxytetracycline, ciprofloxacin, enrofloxacin, flumequine and lincomycin were detected in all fattening calf slurry samples with mean concentrations of 2776, 4078, 48, 31, 536 and 36 µg/kg manure, respectively. Sulfadiazine was detected at a mean concentration of 10,895 µg/kg. Further, antibiotic residues were found in only 4 of the 25 beef cattle farmyard manure samples. Oxytetracycline was detected twice below 500 µg/kg. Paromomycin, ciprofloxacin and enrofloxacin were detected in a concentration below 100 µg/kg. Of E. coli isolates, 88% and 23% from fattening calf slurry and beef cattle farmyard manure, respectively, were resistant to at least one of the antibiotics tested. Multi-drug resistance was observed at a maximum of 10 and 7 antibiotics, respectively. The occurrence of antibiotic resistant E. coli and antibiotic residues is shown to be higher in fattening calf slurry than in beef cattle farmyard manure used for agricultural field fertilization.

Biosecurity seems to be the most promising tool for Campylobacter control on poultry farms. A longitudinal molecular epidemiological study was performed during two production cycles, in which the broilers, the poultry house, and the environment of 10 (mixed) broiler farms were monitored weekly. Cecal droppings from the second production cycle were also used for 16S metabarcoding to study the differences in the microbiota of colonized and uncolonized flocks. Results showed that 3 out of 10 farms were positive for Campylobacter in the first production cycle, and 4 out of 10 were positive in the second. Broilers became colonized at the earliest when they were four weeks old. The majority of the flocks (57%) became colonized after partial depopulation. Before colonization of the flocks, Campylobacter was rarely detected in the environment, but it was frequently isolated from cattle and swine. Although these animals appeared to be consistent carriers of Campylobacter , molecular typing revealed that they were not the source of flock colonization. In accordance with previous reports, this study suggests that partial depopulation appears to be an important risk factor for Campylobacter introduction into the broiler house. Metabarcoding indicated that two Campylobacter -free flocks carried high relative abundances of Megamonas in their ceca, suggesting potential competition with Campylobacter .

Pig manure may contain antibiotic residues, antibiotic-resistant bacteria or pathogens, which may reach the environment upon fertilization. During this study, 69 antibiotic residues belonging to 12 classes were quantified in 89 pig slurry samples. These samples were also studied for the presence of Salmonella and for E. coli resistant to meropenem, colistin, ciprofloxacin, or cefotaxim. The obtained isolates were further tested for antibacterial susceptibility. No antibiotic residues were detected in four samples, whereas in the other samples, up to 12 antibiotics were found. The most frequently detected antibiotic residues were doxycycline, sulfadiazine, and lincomycin. Doxycycline was found in the highest concentration with a mean of 1476 µg/kg manure (range: 18-13632 µg/kg). Tylosin and oxytetracycline were found with mean concentrations of 784 µg/kg (range: 17-5599 µg/kg) and 482 µg/kg (range: 11-3865 µg/kg), respectively. Lincomycin, had a mean concentration of 177 µg/kg manure (range: 9-3154 µg/kg). All other 18 antibiotic residues were found with mean concentrations of less than 100 µg/kg manure. Fifty-one slurry samples harbored Salmonella ; 35% of the Salmonella isolates were sensitive to a panel of 14 antibiotics, whereas the other 65% were resistant up to five antibiotics. For E. coli , 52 manure samples contained E. coli isolates which were resistant to ciprofloxacin and 22 resistant to cefotaxime. All ciprofloxacin and cefotaxime-resistant isolates were multi-resistant, with resistance up to nine and eight antibiotics, respectively. This research indicates that pig slurry used for fertilization often contains antibiotic residues and antibiotic-resistant bacteria, including pathogens., Competing Interests: The authors declare no conflict of interest.