A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Genomic analysis of Listeria monocytogenes CC7 associated with clinical infections and persistence in the food industry.

Listeria monocytogenes clonal complex 7 (CC7), belonging to lineage II, is the most common subtype among clinical listeriosis isolates in Norway, and is also commonly found in Norwegian food industry and outdoor environments. In the present study, the relative prevalence of CCs among clinical isolates of L. monocytogenes in European countries during 2010-2015 was determined. Then, phylogenomic and comparative genomic analyses was performed for 115 Norwegian and 255 international reference genomes from various sources, to examine potential explanations underlying the high prevalence of CC7 among Norwegian listeriosis cases. Selected isolates were also compared using in vitro virulence assays. The results showed a high relative prevalence of CC7 in clinical isolates from Norway and the neighboring Nordic countries Sweden and Finland. In contrast to in most other European countries, lineage II dominated among clinical isolates in these countries. Phylogenetic analysis of the 370 CC7 isolates separated the genomes into four clades, with the majority of Norwegian isolates (69 %) clustered in one of these clades, estimated to have diverged from the other clades around year 1830. The Norwegian isolates within this clade were widely distributed in different habitats; several (poultry) meat processing factories, a salmon processing plant, in nature, farms, and slugs, and among human clinical isolates. In particular, one pervasive CC7 clone was found across three poultry processing plants and one salmon processing plant, and also included three clinical isolates. All analysed CC7 isolates harbored the same set of 72 genes involved in both general and specific stress responses. Divergence was observed for plasmid-encoded genes including genes conferring resistance against arsenic (Tn554-arsCBADR), cadmium (cadA1C1 and cadA2C2), and the biocide benzalkonium chloride (bcrABC). No significant difference in prevalence of these genes was seen between isolates from different habitats or sources. Virulence attributes were highly conserved among the CC7 isolates. In vitro virulence studies of five representative CC7 isolates revealed a virulence potential that, in general, was not significantly lower than that of the control strain EGDe, with isolate-dependent differences that could not be correlated with genetic determinants. The study shows that CC7 is widespread in Norway, and that a pervasive CC7 clone was present in food processing plants. The study highlights the importance of CC7 and lineage II strains in causing listeriosis and shows that more research is needed to understand the reasons behind geographical differences in CC prevalence.

Reduction and Growth Inhibition of Listeria monocytogenes by Use of Anti-Listerial Nisin, P100 Phages and Buffered Dry Vinegar Fermentates in Standard and Sodium-Reduced Cold-Smoked Salmon.

Cold-smoked salmon are ready-to-eat products that may support the growth of pathogenic Listeria monocytogenes during their long shelf-life. Consumption of such contaminated products can cause fatal listeriosis infections. Another challenge and potential risk associated with CS salmon is their high levels of sodium salt. Excess dietary intake is associated with serious health complications. In the present study, anti-listerial bacteriocin (nisin), P100 bacteriophages (Phageguard L, PGL) and fermentates (Verdad N6, P-NDV) were evaluated as commercial bio-preservation strategies for increased control of L. monocytogenes in standard (with NaCl) and sodium-reduced (NaCl partially replaced with KCl) CS salmon. Treatments of CS salmon with nisin (1 ppm) and PGL (5 × 107 pfu/cm2) separately yielded significant initial reductions in L. monocytogenes (up to 0.7 log) compared to untreated samples. Enhanced additive reductions were achieved through the combined treatments of nisin and PGL. Fermentates in the CS salmon inhibited the growth of Listeria but did not lead to its eradication. The lowest levels of L. monocytogenes during storage were observed in nisin- and PGL-treated CS salmon containing preservative fermentates and stored at 4 °C, while enhanced growth was observed during storage at an abusive temperature of 8 °C. Evaluation of industry-processed standard and sodium-replaced CS salmon confirmed significant effects with up to 1.7 log reductions in L. monocytogenes levels after 34 days of storage of PGL- and nisin-treated CS salmon-containing fermentates. No differences in total aerobic plate counts were observed between treated (PGL and nisin) or non-treated standard and sodium-reduced CS salmon at the end of storage. The microbiota was dominated by Photobacterium, but with a shift showing dominance of Lactococcus spp. and Vagococcus spp. in fermentate-containing samples. Similar and robust reductions in L. monocytogenes can be achieved in both standard and sodium-replaced CS salmon using the bio-preservation strategies of nisin, PGL and fermentates under various and relevant processing and storage conditions.

Deciphering the virulence potential of Listeria monocytogenes in the Norwegian meat and salmon processing industry by combining whole genome sequencing and in vitro data.

Whole genome sequencing (WGS) of foodborne pathogens such as Listeria monocytogenes is globally on the rise in the food industry. It provides an improvement for proactive surveillance and source-tracking and allows in-depth genetic characterization of the pathogen. In the present study, the virulence gene profile including 99 virulence genes of 767 L. monocytogenes isolates from the Norwegian meat and salmon processing industry was characterized. The isolate collection comprised 28 clonal complexes (CCs) that occur globally. We additionally determined the in vitro virulence potential for 13 major CCs in human intestinal epithelial Caco2 cells using cocktails of three to six representative isolates. Our aim was to test whether the virulence potential could be predicted from the virulence gene profiles to estimate the application potential of WGS in risk assessment in the food industry. The virulence gene profiles were highly conserved within the individual CCs and similar among phylogenetically closely related CCs. We observed a CC-associated distribution of accessory virulence genes in addition to different length polymorphisms. Furthermore, we detected different premature stop codons (PMSC) in the inlA gene, which were mainly present in CC9, CC121 and CC5 isolates. Accordingly, CC9 and CC5 were unable to invade Caco2 cells, whereas CC121 showed moderate virulence potential due to the presence of an isolate harboring full-length inlA. The highest invasion was observed for CC403 and CC415, potentially due to the presence of accessory virulence genes. We demonstrated that CC14, which harbored full-length inlA, was unable to invade Caco2 cells due to a low inlA gene expression. Reconstruction of inlA in CC9 and CC121 isolates showed that without the presence of InlA on the cell wall (as detected in the CC9 isolates), invasion into host cells failed. Our study showed that predicting the virulence potential based on genetic virulence profiles provides valuable information for risk assessment in the food industry but also has its limitations. The mere presence of a full-length inlA gene is not sufficient for virulence, but gene expression and the presence of the protein on the cell wall is required for the successful invasion of L. monocytogenes into host cells. Moreover, hypovirulent CCs like CC121 were among the most abundant human clinical isolates in Norway despite harboring a PMSC mutation in the inlA gene. In conclusion, our study highlights that combining genotypic and phenotypic data is of great importance to improve the informative value of applying WGS in the food industry.

Pervasive Listeria monocytogenes Is Common in the Norwegian Food System and Is Associated with Increased Prevalence of Stress Survival and Resistance Determinants.

To investigate the diversity, distribution, persistence, and prevalence of stress survival and resistance genes of Listeria monocytogenes clones dominating in food processing environments in Norway, genome sequences from 769 L. monocytogenes isolates from food industry environments, foods, and raw materials (512 of which were sequenced in the present study) were subjected to whole-genome multilocus sequence typing (wgMLST), single-nucleotide polymorphism (SNP), and comparative genomic analyses. The data set comprised isolates from nine meat and six salmon processing facilities in Norway collected over a period of three decades. The most prevalent clonal complex (CC) was CC121, found in 10 factories, followed by CC7, CC8, and CC9, found in 7 factories each. Overall, 72% of the isolates were classified as persistent, showing 20 or fewer wgMLST allelic differences toward an isolate found in the same factory in a different calendar year. Moreover, over half of the isolates (56%) showed this level of genetic similarity toward an isolate collected from a different food processing facility. These were designated as pervasive strains, defined as clusters with the same level of genetic similarity as persistent strains but isolated from different factories. The prevalence of genetic determinants associated with increased survival in food processing environments, including heavy metal and biocide resistance determinants, stress response genes, and inlA truncation mutations, showed a highly significant increase among pervasive isolates but not among persistent isolates. Furthermore, these genes were significantly more prevalent among the isolates from food processing environments compared to in isolates from natural and rural environments (n = 218) and clinical isolates (n = 111) from Norway. IMPORTANCE Listeria monocytogenes can persist in food processing environments for months to decades and spread through the food system by, e.g., contaminated raw materials. Knowledge of the distribution and diversity of L. monocytogenes is important in outbreak investigations and is essential to effectively track and control this pathogen in the food system. The present study presents a comprehensive overview of the prevalence of persistent clones and of the diversity of L. monocytogenes in Norwegian food processing facilities. The results demonstrate extensive spread of highly similar strains throughout the Norwegian food system, in that 56% of the 769 collected isolates from food processing factories belonged to clusters of L. monocytogenes identified in more than one facility. These strains were associated with an overall increase in the prevalence of plasmids and determinants of heavy metal and biocide resistance, as well as other genetic elements associated with stress survival mechanisms and persistence.

Microbial Safety and Sensory Analyses of Cold-Smoked Salmon Produced with Sodium-Reduced Mineral Salts and Organic Acid Salts.

Cold-smoked (CS) salmon contains high levels of sodium salts, and excess dietary sodium intake is associated with an array of health complications. CS salmon may also represent a food safety risk due to possible presence and growth of the foodborne pathogen Listeria monocytogenes which may cause fatal human infections. Here we determine how reformulated CS salmon using commercial sodium-reduced salt replacers containing KCl (e.g., Nutek, Smart Salt, SOLO-LITE) and acetate-based preservative salts (Provian K, proviant NDV) affect sensory properties, quality, and microbial safety. Initial sensory screening of sodium-reduced CS salmon was followed by L. monocytogenes growth analyses in selected variants of reformulated CS salmon, and finally by analyses of CS salmon variants produced in an industrial smokehouse. Projective mapping indicated overall minor sensory changes in sodium-replaced samples compared with a conventional product with NaCl. Growth of L. monocytogenes was temperature-dependent (4 °C vs. 8 °C storage) with similar growth in sodium-reduced and conventional CS salmon. The addition of 0.9% of the preservative salts Provian K or Provian NDV gave up to 4 log lower L. monocytogenes counts in both sodium-reduced and conventional cold-smoked salmon after 29 days of chilled storage. No changes in pH (range 6.20−6.33), aw levels (range 0.960−0.973), or weight yield (96.8 ± 0.2%) were evident in CS salmon with salt replacers or Provian preservative salts. Analyses of CS salmon produced with selected mineral salt and preservative salt combinations in an industrial salmon smokery indicated marginal differences in sensory properties. Samples with the preservative salt Provian NDV provided L. monocytogenes growth inhibition and low-level total viable counts (<2.8 log/g) dominated by Photobacterium and Carnobacterium during storage. Production of sodium-reduced CS salmon with inhibiting salts provides a simple method to achieve a healthier food product with increased food safety.

Whole-Genome Sequencing Analysis of Listeria monocytogenes from Rural, Urban, and Farm Environments in Norway: Genetic Diversity, Persistence, and Relation to Clinical and Food Isolates.

Listeria monocytogenes is a ubiquitous environmental bacterium associated with a wide variety of natural and human-made environments, such as soil, vegetation, livestock, food processing environments, and urban areas. It is also among the deadliest foodborne pathogens, and knowledge about its presence and diversity in potential sources is crucial to effectively track and control it in the food chain. Isolation of L. monocytogenes from various rural and urban environments showed higher prevalence in agricultural and urban developments than in forest or mountain areas, and that detection was positively associated with rainfall. Whole-genome sequencing (WGS) was performed for the collected isolates and for L. monocytogenes from Norwegian dairy farms and slugs (218 isolates in total). The data were compared to available data sets from clinical and food-associated sources in Norway collected within the last decade. Multiple examples of clusters of isolates with 0 to 8 whole-genome multilocus sequence typing (wgMLST) allelic differences were collected over time in the same location, demonstrating persistence of L. monocytogenes in natural, urban, and farm environments. Furthermore, several clusters with 6 to 20 wgMLST allelic differences containing isolates collected across different locations, times, and habitats were identified, including nine clusters harboring clinical isolates. The most ubiquitous clones found in soil and other natural and animal ecosystems (CC91, CC11, and CC37) were distinct from clones predominating among both clinical (CC7, CC121, and CC1) and food (CC9, CC121, CC7, and CC8) isolates. The analyses indicated that ST91 was more prevalent in Norway than other countries and revealed a high proportion of the hypovirulent ST121 among Norwegian clinical cases. IMPORTANCE Listeria monocytogenes is a deadly foodborne pathogen that is widespread in the environment. For effective management, both public health authorities and food producers need reliable tools for source tracking, surveillance, and risk assessment. For this, whole-genome sequencing (WGS) is regarded as the present and future gold standard. In the current study, we use WGS to show that L. monocytogenes can persist for months and years in natural, urban, and dairy farm environments. Notably, clusters of almost identical isolates, with genetic distances within the thresholds often suggested for defining an outbreak cluster, can be collected from geographically and temporally unrelated sources. The work highlights the need for a greater knowledge of the genetic relationships between clinical isolates and isolates of L. monocytogenes from a wide range of environments, including natural, urban, agricultural, livestock, food production, and food processing environments, to correctly interpret and use results from WGS analyses.

Improved microbial and sensory quality of chicken meat by treatment with lactic acid, organic acid salts and modified atmosphere packaging.

Microbial contamination and growth play important roles in spoilage and quality loss of raw poultry products. We evaluated the suitability of three commercially available organic acid based antimicrobial compounds, Purac FCC80 (l-lactic acid), Verdad N6 (buffered vinegar fermentate) and Provian K (blend of potassium acetate and diacetate) to prevent growth of the innate microbiota, reduce spoilage and enhance the sensory quality of raw chicken under vacuum, high CO2 (60/40% CO2/N2), and high O2 (75/25% O2/CO2) modified atmosphere (MA) storage conditions. Solutions were applied warm (50 °C) or cold (4 °C) to reflect treatments prior to (Prechill) or after (Postchill) cooling of chicken carcasses, respectively. Single postchill treatments of raw chicken wings with 5% Verdad N6 or Provian K solutions and MA storage enabled complete growth inhibition during the first seven days of storage before growth resumed. Enhanced bacterial control was obtained by combining Prechill lactic acid and Postchill Verdad N6 or Provian K treatments which indicated initial reductions up to 1.1 log and where total bacterial increase after 20 days storage was limited to 1.8-2.1 log. Antibacterial effects were dependent on the concentration of the inhibiting salts used, pH and the storage conditions. Bacterial community analyses showed increased relative levels of Gram-positive bacteria and with reductions of potential spoilage organisms in samples treated with the organic acid salts Verdad N6 and Provian K. Sensory analyses of raw, treated wings showed prominent lower scores in several spoilage associated odour attributes when compared with untreated chicken wings after 13 days storage. For heat-treated chicken, only minor differences for 22 tested attributes were detected between seven antimicrobial treatments and untreated control chicken. Immersion in commercially available organic acid/salt solutions combined with MA storage can reduce bacterial levels, improve microbial and sensory quality, and potentially improve shelf life and reduce food waste of chicken products.

Antibiotic Resistance and Phylogeny of Pseudomonas spp. Isolated over Three Decades from Chicken Meat in the Norwegian Food Chain.

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.

Improved control of Listeria monocytogenes during storage of raw salmon by treatment with the fermentate Verdad N6 and nisin.

Fresh Atlantic salmon (Salmo salar) represents a healthy, nutritious food with global distribution and increasing consumption and economic value. Contaminating Listeria monocytogenes in fresh salmon represents a health hazard to consumers, is linked to extensive product recalls and is a major challenge for salmon processors. Verdad N6, a commercially available buffered vinegar, was evaluated as a treatment for raw salmon fillets either alone or in combination with the antimicrobial peptide nisin, with regard to anti-listerial effects under processing and storage, and influence on sensory quality and background microbiota. Salmon fillets were surface contaminated with L. monocytogenes and immersed in solutions of Verdad N6 or treated with nisin or a combination of these two treatments. Levels of L. monocytogenes were determined during vacuum-pack refrigerated storage. The use of Verdad N6 resulted in increased lag times and substantially reduced growth of L. monocytogenes. The inhibitory effects were dependent on Verdad N6 levels, immersion time, and storage time and temperature. A 5 s immersion in 10% Verdad N6 solution at 4 °C reduced growth of L. monocytogenes from log 2.8 to log 1 after 12 days of storage. Nisin (0.2-1 ppm) had listericidal effects up to 1 log but did not inhibit regrowth when used alone. Appropriate combinations of Verdad N6 and nisin led to L. monocytogenes levels no higher after 12 days of storage than the initial levels. The inhibitory effects were markedly lower at 7 °C than at 4 °C. Salmon with Verdad N6 showed reduced levels of total counts during storage indicating a longer shelf-life, and a shift in the dominating bacteria with reduced and increased relative levels of Enterobacteriaceae and lactic acid bacteria, respectively. Sensory analyses of raw and cooked Verdad N6 treated a non-treated salmon resulted in small differences. In summary, Verdad N6 is an option for production of high-quality raw salmon with increased shelf-life and enhanced food safety through its Listeria inhibiting effects. The application of Verdad N6 in combination with nisin treatment can further reduce the listeria-risks of these products.

Listeria Monocytogenes Biofilm Removal Using Different Commercial Cleaning Agents.

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.

Reduction and inhibition of Listeria monocytogenes in cold-smoked salmon by Verdad N6, a buffered vinegar fermentate, and UV-C treatments.

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.

Listeria monocytogenes strains show large variations in competitive growth in mixed culture biofilms and suspensions with bacteria from food processing environments.

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.

Complete Genome Sequences of Six Listeria monocytogenes Sequence Type 9 Isolates from Meat Processing Plants in Norway.

Listeria monocytogenes is a foodborne pathogen that causes the often-fatal disease listeriosis. We present here the complete genome sequences of six L. monocytogenes isolates of sequence type 9 (ST9) collected from two different meat processing facilities in Norway. The genomes were assembled using Illumina and Nanopore sequencing data.

Survival of Five Strains of Shiga Toxigenic Escherichia coli in a Sausage Fermentation Model and Subsequent Sensitivity to Stress from Gastric Acid and Intestinal Fluid.

The ability of foodborne pathogens to exhibit adaptive responses to stressful conditions in foods may enhance their survival when passing through the gastrointestinal system. We aimed to determine whether Escherichia coli surviving stresses encountered during a model dry-fermented sausage (DFS) production process exhibit enhanced tolerance and survival in an in vitro gastrointestinal model. Salami sausage batters spiked with five E. coli isolates, including enterohaemorrhagic E. coli strains isolated from different DFS outbreaks, were fermented in a model DFS process (20°C, 21 days). Control batters spiked with the same strains were stored at 4°C for the same period. Samples from matured model sausages and controls were thereafter exposed to an in vitro digestion challenge. Gastric exposure (pH 3) resulted in considerably reduced survival of the E. coli strains that had undergone the model DFS process. This reduction continued after entering intestinal challenge (pH 8), but growth resumed after 120 min. When subjected to gastric challenge for 120 min, E. coli that had undergone the DFS process showed about 2.3 log10⁡ lower survival compared with those kept in sausage batter at 4°C. Our results indicated that E. coli strains surviving a model DFS process exhibited reduced tolerance to subsequent gastric challenge at low pH.

Cleaning and Disinfection of Biofilms Composed of Listeria monocytogenes and Background Microbiota from Meat Processing Surfaces.

Surfaces of food processing premises are exposed to regular cleaning and disinfection (C&D) regimes, using biocides that are highly effective against bacteria growing as planktonic cells. However, bacteria growing in surface-associated communities (biofilms) are typically more tolerant toward C&D than their individual free-cell counterparts, and survival of pathogens such as Listeria monocytogenes may be affected by interspecies interactions within biofilms. In this study, Pseudomonas and Acinetobacter were the most frequently isolated genera surviving on conveyor belts subjected to C&D in meat processing plants. In the laboratory, Pseudomonas, Acinetobacter, and L. monocytogenes dominated the community, both in suspensions and in biofilms formed on conveyor belts, when cultures were inoculated with eleven-genus cocktails of representative bacterial strains from the identified background flora. When biofilms were exposed to daily C&D cycles mimicking treatments used in food industry, the levels of Acinetobacter and Pseudomonas mandelii diminished, and biofilms were instead dominated by Pseudomonas putida (65 to 76%), Pseudomonas fluorescens (11 to 15%) and L. monocytogenes (3 to 11%). The dominance of certain species after daily C&D correlated with high planktonic growth rates at 12°C and tolerance to C&D. In single-species biofilms, L. monocytogenes developed higher tolerance to C&D over time, for both the peracetic acid and quaternary ammonium disinfectants, indicating that a broad-spectrum mechanism was involved. Survival after C&D appeared to be a common property of L. monocytogenes strains, as persistent and sporadic subtypes showed equal survival rates in complex biofilms. Biofilms established preferentially in surface irregularities of conveyor belts, potentially constituting harborage sites for persistent contamination.IMPORTANCE In the food industry, efficient production hygiene is a key measure to avoid the accumulation of spoilage bacteria and eliminate pathogens. However, the persistence of bacteria is an enduring problem in food processing environments. This study demonstrated that environmental bacteria can survive foam cleaning and disinfection (C&D) at concentrations used in the industrial environment. The phenomenon was replicated in laboratory experiments. Important characteristics of persisting bacteria were a high growth rate at low temperature, a tolerance to the cleaning agent, and the ability to form biofilms. This study also supports other recent research suggesting that strain-to-strain variation cannot explain why certain subtypes of Listeria monocytogenes persist in food processing environments while others are found only sporadically. The present investigation highlights the failure of regular C&D and a need for research on improved agents that efficiently detach the biofilm matrix.

Transfer Potential of Plasmids Conferring Extended-Spectrum-Cephalosporin Resistance in Escherichia coli from Poultry.

Escherichia coli strains resistant to extended-spectrum cephalosporins (ESC) are widely distributed in Norwegian broiler production, and the majority harbor transferable IncK or IncI1 plasmids carrying blaCMY-2 Persistent occurrence in broiler farms may occur through the survival of ESC-resistant E. coli strains in the farm environment, or by transfer and maintenance of resistance plasmids within a population of environmental bacteria with high survival abilities. The aim of this study was to determine the transferability of two successful blaCMY-2-carrying plasmids belonging to the incompatibility groups IncK and IncI1 into E. coli and Serratia species recipients. Initially, conjugative plasmid transfer from two E. coli donors to potential recipients was tested in an agar assay. Conjugation was further investigated for selected mating pairs in surface and planktonic assays at temperatures from 12°C to 37°C. Transfer of plasmids was observed on agar, in broth, and in biofilm at temperatures down to 25°C. The IncK plasmid was able to transfer into Serratia marcescens, and transconjugants were able to act as secondary plasmid donors to different E. coli and Serratia species recipients. All transconjugants displayed an AmpC phenotype corresponding to the acquisition of blaCMY-2 In summary, the results indicate that the IncK plasmid may transfer between E. coli and Serratia spp. under conditions relevant for broiler production.IMPORTANCE Certain blaCMY-2-carrying plasmids are successful and disseminated in European broiler production. Traditionally, plasmid transferability has been studied under conditions that are optimal for bacterial growth. Plasmid transfer has previously been reported between E. coli bacteria in biofilms at 37°C and in broth at temperatures ranging from 8 to 37°C. However, intergenus transfer of blaCMY-2-carrying plasmids from E. coli to environmental bacteria in the food-processing chain has not been previously studied. We demonstrate that blaCMY-2-carrying plasmids are capable of conjugative transfer between different poultry-associated bacterial genera under conditions relevant for broiler production. Transfer to Serratia spp. and to hosts with good biofilm-forming abilities and with the potential to act as secondary plasmid donors to new hosts might contribute to the persistence of these resistance plasmids. These results contribute to increased knowledge of factors affecting the persistence of ESC resistance in broiler production and can provide a basis for improvement of routines and preventive measures.

Tolerance to quaternary ammonium compound disinfectants may enhance growth of Listeria monocytogenes in the food industry.

The antibacterial effect of disinfectants is crucial for the control of Listeria monocytogenes in food processing environments. Tolerance of L. monocytogenes to sublethal levels of disinfectants based on quaternary ammonium compounds (QAC) is conferred by the resistance determinants qacH and bcrABC. The presence and distribution of these genes have been anticipated to have a role in the survival and growth of L. monocytogenes in food processing environments where QAC based disinfectants are in common use. In this study, a panel of 680 L. monocytogenes from nine Norwegian meat- and salmon processing plants were grouped into 36 MLVA profiles. The presence of qacH and bcrABC was determined in 101 isolates from the 26 most common MLVA profiles. Five MLVA profiles contained qacH and two contained bcrABC. Isolates with qacH and bcrABC showed increased tolerance to the QAC Benzalkonium chloride (BC), with minimal inhibitory concentrations (MICs) of 5-12, 10-13 and <5ppm for strains with qacH (two allele variants observed), bcrABC, and neither gene, respectively. Isolates with qacH or bcrABC were not more tolerant to BC in bactericidal tests in suspension or in biofilms compared with isolates lacking the genes. Water residue samples collected from surfaces in meat processing plants after QAC disinfection had bactericidal effect against L. monocytogenes when the sample BC levels were high (>100ppm). A sample with lower BC concentrations (14ppm of chain length C-12 and 2.7ppm of chain length C-14) inhibited growth of L. monocytogenes not containing bcrABC or qacH, compared to strains with these genes. The study has shown that L. monocytogenes harbouring the QAC resistance genes qacH and bcrABC are prevalent in the food industry and that residuals of QAC may be present in concentrations after sanitation in the industry that result in a growth advantage for bacteria with such resistance genes.

Contamination of salmon fillets and processing plants with spoilage bacteria.

The processing environment of salmon processing plants represents a potential major source of bacteria causing spoilage of fresh salmon. In this study, we have identified major contamination routes of important spoilage associated species within the genera Pseudomonas, Shewanella and Photobacterium in pre-rigor processing of salmon. Bacterial counts and culture-independent 16S rRNA gene analysis on salmon fillet from seven processing plants showed higher levels of Pseudomonas spp. and Shewanella spp. in industrially processed fillets compared to salmon processed under strict hygienic conditions. Higher levels of Pseudomonas spp. and Shewanella spp. were found on fillets produced early on the production day compared to later processed fillets. The levels of Photobacterium spp. were not dependent on the processing method or time of processing. In follow-up studies of two plants, bacterial isolates (n=2101) from the in-plant processing environments (sanitized equipment/machines and seawater) and from salmon collected at different sites in the production were identified by partial 16S rRNA gene sequencing. Pseudomonas spp. dominated in equipment/machines after sanitation with 72 and 91% of samples from the two plants being Pseudomonas-positive. The phylogenetic analyses, based on partial 16S rRNA gene sequencing, showed 48 unique sequence profiles of Pseudomonas of which two were dominant. Only six profiles were found on both machines and in fillets in both plants. Shewanella spp. were found on machines after sanitation in the slaughter department while Photobacterium spp. were not detected after sanitation in any parts of the plants. Shewanella spp. and Photobacterium spp. were found on salmon in the slaughter departments. Shewanella was frequently present in seawater tanks used for bleeding/short term storage. In conclusion, this study provides new knowledge on the processing environment as a source of contamination of salmon fillets with Pseudomonas spp. and Shewanella spp., while Photobacterium spp. most likely originate from the live fish and seawater. The study show that strict hygiene during processing is a prerequisite for optimal shelf life of salmon fillets and that about 90% reductions in the initial levels of bacteria on salmon fillets can be obtained using optimal hygienic conditions.

Biofilm Matrix Composition Affects the Susceptibility of Food Associated Staphylococci to Cleaning and Disinfection Agents.

Staphylococci are frequently isolated from food processing environments, and it has been speculated whether survival after cleaning and disinfection with benzalkonium chloride (BC)-containing disinfectants is due to biofilm formation, matrix composition, or BC efflux mechanisms. Out of 35 food associated staphylococci, eight produced biofilm in a microtiter plate assay and were identified as Staphylococcus capitis (2), S. cohnii, S. epidermidis, S. lentus (2), and S. saprophyticus (2). The eight biofilm producing strains were characterized using whole genome sequencing. Three of these strains contained the ica operon responsible for production of a polysaccharide matrix, and formed a biofilm which was detached upon exposure to the polysaccharide degrading enzyme Dispersin B, but not Proteinase K or trypsin. These strains were more tolerant to the lethal effect of BC both in suspension and biofilm than the remaining five biofilm producing strains. The five BC susceptible strains were characterized by lack of the ica operon, and their biofilms were detached by Proteinase K or trypsin, but not Dispersin B, indicating that proteins were major structural components of their biofilm matrix. Several novel cell wall anchored repeat domain proteins with domain structures similar to that of MSCRAMM adhesins were identified in the genomes of these strains, potentially representing novel mechanisms of ica-independent biofilm accumulation. Biofilms from all strains showed similar levels of detachment after exposure to alkaline chlorine, which is used for cleaning in the food industry. Strains with qac genes encoding BC efflux pumps could grow at higher concentrations of BC than strains without these genes, but no differences were observed at biocidal concentrations. In conclusion, the biofilm matrix of food associated staphylococci varies with respect to protein or polysaccharide nature, and this may affect the sensitivity toward a commonly used disinfectant.