Background: Feed contaminated with Salmonella spp. constitutes a risk of Salmonella infections in animals, and subsequently in the consumers of animal products. Salmonella are occasionally isolated from the feed factory environment and some clones of Salmonella persist in the factory environment for several years. One hypothesis is that biofilm formation facilitates persistence by protecting bacteria against environmental stress, e.g. disinfection. The aim of this study was to investigate the biofilm forming potential of Salmonella strains from feed- and fishmeal factories. The study included 111 Salmonella strains isolated from Norwegian feed and fish meal factories in the period 1991–2006 of serovar Agona, serovar Montevideo, serovar Senftenberg and serovar Typhimurium. Results: Significant differences were found between serovars regarding the abilities to form biofilm on polystyrene (microtiter plate assay) and in the air-liquid interface of nutrient broth (pellicle assay). Strains of serovar Agona and serovar Montevideo were good biofilm producers. In Norwegian factories, clones of these serovars have been observed to persist for several years. Most serovar Senftenberg clones appear to persist for a shorter period, and strains of this serovar were medium biofilm producers in our test systems. Strains of the serovar Typhimurium were relatively poor biofilm producers. Salmonella ser. Typhimurium clones have not been observed to persist even though this serovar is resident in Norwegian wild life. When classifying strains according to persistence or presumed non-persistence, persistent strains produced more biofilm than presumed non-persisting strains. Conclusion: The results indicate a correlation between persistence and biofilm formation which suggests that biofilm forming ability may be an important factor for persistence of Salmonella in the factory environment.

Interactions and competition between resident bacteria in food processing environments could affect their ability to survive, grow and persist in microhabitats and niches in the food industry. In this study, the competitive ability of L. monocytogenes strains grown together in separate culture mixes with other L. monocytogenes (L. mono mix), L. innocua (Listeria mix), Gram-negative bacteria (Gram- mix) and with a multigenera mix (Listeria + Gram- mix) was investigated in biofilms on stainless steel and in suspensions at 12 °C. The mixed cultures included resident bacteria from processing surfaces in meat and salmon industry represented by L. monocytogenes (n = 6), L. innocua (n = 5) and Gram-negative bacteria (n = 6; Acinetobacter sp., Pseudomonas fragi, Pseudomonas fluorescens, Serratia liquefaciens, Stenotrophomonas maltophilia). Despite hampered in growth in mixed cultures, L. monocytogenes established in biofilms with counts at day nine between 7.3 and 9.0 log per coupon with the lowest counts in the Listeria + G- mix that was dominated by Pseudomonas. Specific L. innocua inhibited growth of L. monocytogenes strains differently; inhibition that was further enhanced by the background Gram-negative microbiota. In these multispecies and multibacteria cultures, the growth competitive effects lead to the dominance of a strong competitor L. monocytogenes strain that was only slightly inhibited by L. innocua and showed strong competitive abilities in mixed cultures with resident Gram-negative bacteria. The results indicates complex patterns of bacterial interactions and L. monocytogenes inhibition in the multibacteria cultures that only partially depend on cell contact and likely involve various antagonistic and bacterial tolerance mechanisms. The study indicates large variations among L. monocytogenes in their competitiveness under multibacterial culture conditions that should be considered in further studies towards understanding of L. monocytogenes persistence in food processing facilities.

Effective cleaning and disinfection (C&D) is pivotal for the control of Listeria monocytogenes in food processing environments. Bacteria in biofilms are protected from biocidal action, and effective strategies for the prevention and removal of biofilms are needed. In this study, different C&D biofilm control strategies on pre-formed L. monocytogenes biofilms on a conveyor belt material were evaluated and compared to the effect of a conventional chlorinated, alkaline cleaner (agent A). Bacterial reductions up to 1.8 log were obtained in biofilms exposed to daily C&D cycles with normal user concentrations of alkaline, acidic, or enzymatic cleaning agents, followed by disinfection using peracetic acid. No significant differences in bactericidal effects between the treatments were observed. Seven-day-old biofilms were more tolerant to C&D than four-day-old biofilms. Attempts to optimize biofilm eradication protocols for four alkaline, two acidic, and one enzymatic cleaning agent, in accordance with the manufacturers’ recommendations, were evaluated. Increased concentrations, the number of subsequent treatments, the exposure times, and the temperatures of the C&D agents provided between 4.0 and >5.5 log reductions in colony forming units (CFU) for seven-day-old L. monocytogenes biofilms. Enhanced protocols of conventional and enzymatic C&D protocols have the potential for improved biofilm control, although further optimizations and evaluations are needed.

The presence of Salmonella enterica serovars in feed ingredients, products and processing facilities is a well recognized problem worldwide. In Norwegian feed factories, strict control measures are implemented to avoid establishment and spreading of Salmonella throughout the processing chain. There is limited knowledge on the presence and survival of the resident microflora in feed production plants. Information on interactions between Salmonella and other bacteria in feed production plants and how they affect survival and biofilm formation of Salmonella is also limited. The aim of this study was to identify resident microbiota found in feed production environments, and to compare the survival of resident flora strains and Salmonella to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of Salmonella was determined. Surface microflora characterization from two feed productions plants, by means of 16 S rDNA sequencing, revealed a wide diversity of bacteria. Survival, disinfection and biofilm formation experiments were conducted on selected dominant resident flora strains and Salmonella. Results showed higher survival properties by resident flora isolates for desiccation, and disinfection compared to Salmonella isolates. Dual-species biofilms favored Salmonella growth compared to Salmonella in mono-species biofilms, with biovolume increases of 2.8-fold and 3.2-fold in the presence of Staphylococcus and Pseudomonas, respectively. These results offer an overview of the microflora composition found in feed industry processing environments, their survival under relevant stresses and their potential effect on biofilm formation in the presence of Salmonella. Eliminating the establishment of resident flora isolates in feed industry surfaces is therefore of interest for impeding conditions for Salmonella colonization and growth on feed industry surfaces. In-depth investigations are still needed to determine whether resident flora has a definite role in the persistence of Salmonella in feed processing environments.

Pseudomonas is ubiquitous in nature and a predominant genus in many foods and food processing environments, where it primarily represents major food spoilage organisms. The food chain has also been reported to be a potential reservoir of antibiotic-resistant Pseudomonas. The purpose of the current study was to determine the occurrence of antibiotic resistance in psychrotrophic Pseudomonas spp. collected over a time span of 26 years from retail chicken in Norway and characterize their genetic diversity, phylogenetic distribution and resistance genes through whole-genome sequence analyses. Among the 325 confirmed Pseudomonas spp. isolates by 16S rRNA gene sequencing, antibiotic susceptibility profiles of 175 isolates to 12 antibiotics were determined. A subset of 31 isolates being resistant to ≥3 antibiotics were whole-genome sequenced. The isolates were dominated by species of the P. fluorescens lineage. Isolates susceptible to all antibiotics or resistant to ≥3 antibiotics comprised 20.6% and 24.1%, respectively. The most common resistance was to aztreonam (72.6%), colistin (30.2%), imipenem (25.6%) and meropenem (12.6%). Resistance properties appeared relatively stable over the 26-year study period but with taxa-specific differences. Whole-genome sequencing showed high genome variability, where isolates resistant to ≥3 antibiotics belonged to seven species. A single metallo-betalactmase gene (cphA) was detected, though intrinsic resistance determinants dominated, including resistance–nodulation (RND), ATP-binding cassette (ABC) and small multidrug resistance (Smr) efflux pumps. This study provides further knowledge on the distribution of psychrotrophic Pseudomonas spp. in chicken meat and their antibiotic resistance properties. Further monitoring should be encouraged to determine food as a source of antibiotic resistance and maintain the overall favorable situation with regard to antibiotic resistance in the Norwegian food chain.

Contamination, survival and growth of Listeria monocytogenes in cold-smoked salmon represent serious health hazards to consumers and major challenges for salmon processors. Verdad N6, a commercially available buffered vinegar, was evaluated as an ingredient in cold-smoked salmon with regard to anti-listerial effects under processing and storage, sensory quality and consumer preference, effects on background microbiota and yield during production. Cold-smoked salmon with Verdad N6 added in the dry-salting process was produced. Salmon fillets were surface contaminated with a mix of L. monocytogenes. Levels of L. monocytogenes were determined during vacuum pack refrigerated storage for 29 days. The use of Verdad N6 resulted in increased lag times and reduced growth rates of L. monocytogenes. The inhibitory effects were dependent on Verdad N6 levels (0-2%), storage time and temperature (4 or 8 °C), type of contamination (between slices or on non-sliced salmon) and degree of smoking. The presence of dextrose (1%) in the recipe had no significant effects on L. monocytogenes levels after storage. On sliced salmon, complete growth inhibition at 4 °C storage could be obtained using 1% Verdad N6 compared to a 3 log increase in L. monocytogenes counts in control salmon. At abuse temperatures (8 °C), corresponding L. monocytogenes levels increased <2 log and 5-6 log during 29 days storage. On non-sliced salmon, 1% Verdad N6 provided complete growth reductions at 4 and 8 °C storage while L. monocytogenes in control salmon increased 2.3 and 4.6 log, respectively, in the same period. The use of Verdad N6 in combination with bactericidal UV-C treatments (fluence 50 mJ/cm2) provided an initial 0.8 log reduction and complete L. monocytogenes growth inhibition on subsequent storage at 4 and 8 °C. Salmon with Verdad N6 showed reduced levels of total counts during storage and a shift in the dominating bacteria with reduced and increased relative levels of Photobacterium and lactic acid bacteria, respectively. A consumer test showed no consistent differences in liking of salmon with and without Verdad N6. In summary, Verdad N6 is an option for the production of high quality cold-smoked salmon with enhanced food safety through its robust listeriostatic effects. The application of Verdad N6 in combination with listericidal UV-C light treatment can further reduce the listeria-risks of this ready-to-eat food product category.