Deciphering Resistome and Virulome Diversity in a Porcine Slaughterhouse and Pork Products Through Its Production Chain

Abstract

We aimed to better understand resistome and virulome patterns on animal and process-area surfaces through a pig slaughterhouse to track possible contamination within the food production chain. Culture-dependent methods revealed high levels of microbial contamination, corresponding to mesophilic and pathogenic bacteria on both the animal and process-area surfaces mainly in the anesthesia (AA and AS) zone followed by “scorching and whip” (FA and FS) zone and also in the end products. To evaluate the potential risk of antibiotic resistance and virulence determinants, shotgun metagenomic DNA-sequencing of isolates from selected areas/products uncovered a high diversity and richness of antibiotic resistance genes (ARGs): 55–62 genes in the anesthesia area (AA and AS) and 35–40 in “animal-arrival zone” (MA and MS). The “scorching and whip” (FA and FS) area, however, exhibited lowered abundance of ARGs (1–6), indicating that the scalding and depilating process (an intermediate zone between “anesthesia” and “scorching and whip”) significantly decreased bacterial load by 1–3 log10 but also diminished the resistome. The high prevalence of antibiotic-inactivating enzyme genes in the “animal-arrival zone” (60–65%) and “anesthesia” area (56%) were mainly represented by those for aminoglycoside (46–51%) and lincosamide (14–19%) resistance, which did not reflect selective pressures by antibiotics most commonly used in pig therapy—tetracyclines and beta-lactams. Contrary to ARGs, greater number of virulence resistance genes were detected after evisceration in some products such as kidney, which reflected the poor hygienic practices. More than 19 general virulence features—mainly adherence, secretion system, chemotaxis and motility, invasion and motility were detected in some products. However, immune evasion determinants were detected in almost all samples analyzed from the beginning of the process, with highest amounts found from the anesthesia area. We conclude that there are two main sources of contamination in a pig slaughterhouse: the microorganisms carried on the animals’ hide, and those from the evisceration step. As such, focussing control measures, e.g., enhanced disinfection procedures, on these contamination-source areas may reduce risks to food safety and consumer health, since the antibiotic and virulence determinants may spread to end products and the environment; further, ARG and virulence traits can exacerbate pathogen treatments.

The sources and transmission routes of microbial populations throughout a meat processing facility

“…For example, Bridier et al investigated the impact of disinfection procedures on the microbial ecology and Salmonella prevalence in a pig slaughterhouse using a combination of traditional culturing techniques and 16S rRNA gene sequencing 17 . Metagenomic and 16S rRNA gene profiling has been used in a variety of other meat and foodprocessing facilities to describe the spoilage associated microbiota and resistome in different facilities [18][19][20] . Here, we advance our knowledge about microbial diversity and biogeography along the pig meat processing chain by utilizing the high-throughput, longread sequencing capability of the PacBio technology to obtain thousands of full-length 16S rRNA gene sequences.…”

Role played by the environment in the emergence and spread of antimicrobial resistance (AMR) through the food chain

“…In addition, workers, through their hands or equipment, may act as sources of AMR (Van Gompel et al, 2020). Resistome studies have revealed not only frequent abundance of ARGs along the slaughter line but also some variability in these ARGs, emphasising the importance of good hygiene in carcass processing as stated at the beginning of this point (Campos Calero et al, 2018;Van Gompel et al, 2020).…”

“…Occupational transmission of AMR may occur at slaughterhouses (Mulders et al, 2010;van Cleef et al, 2010;Gilbert et al, 2012;Van Gompel et al, 2020) and at the same time workers, through their hands or equipment, may act as sources of AMR (Van Gompel et al, 2020). Resistome studies reveal variations in ARGs along the slaughter line and further emphasise the importance of good hygiene in carcass processing (Campos Calero et al, 2018;Van Gompel et al, 2020).…”

“…In 2019, poultry meat production was largest in Poland, Spain, France, Germany and Italy (Eurostat). 65,66 ARB, including human-pathogenic strains from poultry, are regularly found in meat (EFSA BIOHAZ Panel, 2019b; EFSA and ECDC, 2021a), especially from poultry meat exporting regions where control of antimicrobials is less developed (Koga et al, 2015;Campos Calero et al, 2018;Ma z ka et al, 2018;Rozman et al, 2018;Tansawai et al, 2018;Rabello et al, 2020;Xu et al, 2020). Antimicrobials were commonly used in the EU, prior to Regulation (EC) No 1091/2005 67 , for suppression of Salmonella to avoid detection of infection in the breeding pyramid and in meat birds prior to slaughter.…”

Antibiotic Resistance: From Pig to Meat

“…Despite a lower prevalence of E. coli and of antibiotic-resistant E. coli in game meat compared to conventional meat, part of the resistant strains recovered may originate from the processing environment. Despite existing cleaning programs, microorganisms resistant to antibiotics have been recovered from equipment in slaughter and meat processing facilities [111,120]. As sampling goes down the processing line, detection of bacteria bearing resistance genes decline [120].…”