Abstract
The food processing environments of salmon and broiler are sources of contaminating bacteria which affect both food safety and quality. The hygiene in these environments is strongly dependent on effective cleaning and disinfection (C&D) routines that eliminate foodborne pathogens, biofilms, and keep bacterial loads to a minimum. Bacteria with high tolerance to disinfectants (DIs) may survive C&D routines. Studies have also revealed a correlation between disinfectant tolerance and antibiotic resistance, and there is a growing concern that food production systems can act as a reservoir of antimicrobial resistance (AMR). As the AMR crisis is estimated to cause more than a million deaths annually on a global basis, more knowledge is urgently needed to implement prevention strategies.
The aim of this work was to investigate the effect of commercial DIs on bacterial load, microbial community composition, and diversity in salmon and broiler processing plants. The in-vitro effect of these DIs was also determined towards both planktonic cells and biofilms of relevant foodborne pathogens and spoilage bacteria. This included exploratory analyses to investigate the development of DI-tolerance, DI-antibiotic cross-resistance, and pathogen protecting effects in biofilms. Moreover, the occurrence of AMR bacteria and genes was explored in processing environments, sidestream materials, and waste discharges from salmon and broiler processing. A comprehensive sampling campaign was carried out in processing plants located both in Norway and Romania. More than 1000 bacterial isolates were collected, in addition to environmental DNA samples (> 100). These were analyzed using advanced technologies including robotic high-throughput screening, biochemical assays with firefly luciferase (BacTiter-Glo), metataxonomic sequencing, and targeted hybrid capture-based sequencing.
The results demonstrated that C&D routines in salmon and broiler processing environments effectively reduced bacterial loads by more than 90% (> 1 log CFU/cm2), on average. In the salmon processing plant, the average count before C&D was lower (3 log CFU/cm2) than in the two broiler processing plants (3.7 and 7.0 log CFU/cm2). After C&D, the average bacterial load was similar in the Norwegian salmon and broiler processing plant (1.9 and 1.8 log CFU/cm2), with higher loads observed in the Romanian broiler processing plant (3.8 log CFU/cm2). The bacterial surface microbiota was dominated by Pseudomonas, Acinetobacter and Enterobacteriaceae across all processing plants. Differences were observed between broiler and salmon processing environments. High relative abundances of Citrobacter, Shewanella, and Staphylococcus were found in the former, while Mycoplasmataceae, Aliivibrio, and Janthinobacterium were among the most abundant taxa in the latter. Interestingly, the relative abundance of Pseudomonas increased sharply after C&D in all processing plants. The presence of viable Pseudomonas spp. after C&D was also confirmed by culture-based analyses. Phylogenetic classification of these isolates (n = 244) revealed a diverse consortium of species. Members of the P. fluorescens group dominated among isolates from both salmon and broiler processing environments in subgroups of P. fluorescens, P. fragi, P. gessardii and P. koreensis. Foodborne pathogens such as Listeria monocytogenes, Escherichia coli, Yersinia enterocolitica, Acinetobacter baumannii, and P. aeruginosa were also detected in processing environments in this study.
DI susceptibility testing with a selection of bacteria relevant to both food safety and spoilage revealed that industry-standard DIs effectively inhibit these bacteria in planktonic state at the recommended user concentration. A case study with L. monocytogenes highlighted the importance of using concentrations well above minimum inhibition concentrations (MICs) to sufficiently eliminate planktonic cells. Minimum bactericidal concentrations (MBCs) were up to eight times higher than MICs for some of the DIs tested against L. monocytogenes. Nevertheless, MBCs were still below the recommended user concentrations.
Furthermore, mono-species biofilms assays with Pseudomonas, Enterobacterales, and other taxa demonstrated that 20 – 30 min exposure to commercial DIs resulted in a mean luminescence reduction of more than 95% across the biofilms (n = 121). However, several biofilms survived the highest DI-concentrations tested, suggesting that biofilm presence may explain the detection of viable bacteria after C&D on-site in processing environments. Biofilms formed by non-pathogenic bacteria such as P. fluorescens may indirectly affect food safety by providing protection for pathogens. An exploratory analysis showed that mono-species biofilms of L. monocytogenes on steel-coupons did not survive the exposure to the DI Aqua Des Foam (1%). Whereas L. monocytogenes cultivated in a multi-species biofilm together with Pseudomonas spp. survived the DI-exposure in most of the experimental repetitions. Pathogens surviving disinfection may ultimately contaminate food products and contribute to foodborne illness outbreaks.
Antibiotics play a vital role for the treatment of serious human infections. In this work, the occurrence of antibiotic resistance was generally low among the bacterial isolates collected from the salmon and broiler industries. L. monocytogenes isolates (n = 44) were fully susceptible towards 12 out of 18 antibiotics, including first-line antibiotics used to treat listeriosis. Likewise, antibiotic resistance was low among Enterococcus isolates (n = 67), which were fully susceptible to seven out of twelve antibiotics. For the remaining six antibiotics, the percentage of resistant isolates was < 32%. Nevertheless, multidrug resistant E. coli harboring multiple resistance genes such as sul2, qnrS1, and blaCTX-M-1 was detected in sludge from the Romanian broiler processing plant. A diverse range of antibiotic resistance genes (ARGs) was also found in eDNA samples from processing environments, sidestream materials, and waste discharges of salmon and broiler. The number of unique ARGs ranged from 32 – 330, with the presence of many high-risk ARGs. Ultimately, these results show that salmon and broiler production systems contribute to the spread of AMR within the food value chain and to the environment.