Mapping the Kitchen Microbiota in Five European Countries Reveals a Set of Core Bacteria across Countries, Kitchen Surfaces, and Cleaning Utensils.

The residential kitchen is often heavily colonized by microbes originating from different sources, including food and human contact. Although a few studies have reported the bacterial composition in cleaning utensils and surface samples there is limited knowledge of the bacterial diversity across different sample types, households, and countries. As part of a large European study, we have identified the microbiota of 302 samples from cleaning utensils (sponges and cloths), kitchen surfaces (sinks, cutting boards, countertops, tap handles, and a pooled sample of other handles) in 74 households across 5 countries (France, Hungary, Norway, Portugal, and Romania). In total, 31 bacterial phyla were identified, with Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria being the most abundant. Despite large variations in households with respect to kitchen standards, kitchen practices, cleaning regimes, and diet and considerable differences in bacterial diversity between samples, eight bacterial genera/families commonly associated with environmental sources were identified in most samples and defined as a core microbiota: Acinetobacter, Pseudomonas, Enhydrobacter, Enterobacteriaceae, Psychrobacter, Chryseobacterium, Bacillus, and Staphylococcus. These genera/families were also among the bacteria with the highest relative abundance across all samples, in addition to Yersiniaceae, Kocuria, Pantoea, and Streptococcus. Taxa associated with potential pathogens and fecal indicators were low in abundance but broadly distributed throughout the households. The microbial composition of surface samples indicated that the microbial composition on kitchen surfaces is more characteristic for the particular country than the object type, while the microbiota of cleaning utensils was similar across countries but differed between types (sponge or cloth). IMPORTANCE There is limited knowledge of the characteristics, differences, and similarities of the bacterial composition in residential kitchens. Here, we report the microbiota of cleaning utensils (sponges and cloths) and five different surface samples in 74 households across five European countries. In addition to increasing the knowledge of the kitchen microbiota from many geographical areas, this study identified a core microbiota in European residential kitchens despite large variations in kitchen practices and kitchen design and standards across countries and households.

Hand hygiene practices during meal preparation-a ranking among ten European countries.

BACKGROUND: The aim of this paper was to map consumers' food hygiene practices from 10 European countries and evaluate which demographic groups are more likely to be exposed to foodborne pathogens and establish a ranking of adherence to food hygiene practices in 10 European countries. METHODS: The research design consisted of a cross-national quantitative consumer survey regarding food safety and hygiene practices during meal preparation (SafeConsume project) and was conducted in ten European countries (France, Denmark, Germany, Greece, Hungary, Norway, Portugal, Romania, Spain and UK). The survey questions were based on recommended hand hygiene practices and on observed practices from a field study performed in 90 European households from six of the countries covered by the survey (France, Hungary, Norway, Portugal, Romania, and UK). SPSS Statistics 26 (IBM Software Group, Chicago, IL) was used for the descriptive and regression analyses of the data. Regression analyses were used to check the relation between demographic characteristics, country of origin and self-reported hand hygiene practices. RESULTS: According to the regression models, families with elderly members aged over 65 showed a higher tendency to follow proper hand washing practices compared to families without elderly members. Meanwhile, families with children under the age of 6 reported being up to twice as likely to wash their hands at critical moments compared to families without children. Overall, taking into consideration the likelihood of washing hands after touching raw chicken and the percentages scores for proper hand cleaning methods and key moments for hand washing, the rank of the countries regarding proper hand hygiene practices was the following: Denmark, Greece, Norway, Romania, Hungary, Germany, UK, Portugal, France, and Spain. CONCLUSIONS: Information and education should point both at the key moments as suggested by the Royal Society for Public Health (RSPH) and the International Scientific Forum on Home Hygiene (IFH) and safe practices. Public health burden generated by improper hand washing may be significantly reduced if education is targeted on consumers' behaviour and practices.

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.

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.

Bacterial levels and diversity in kitchen sponges and dishwashing brushes used by consumers.

AIMS: The purpose of the work was to investigate bacterial levels and diversity as well as survival of Salmonella in used dish washing sponges and brushes and identify consumer practices that can potentially explain bacterial status of these items. METHODS AND RESULTS: Used washing up utensils were collected from consumers. The bacterial numbers (TVC) were very variable with an extremely high median level (10.3 log cfu/item) in Portuguese sponges and lower levels in Norwegian items (7.3 and 7.0 cfu/item for sponges and brushes). No self-reported practices or household composition could explain differences found in TVC levels among the collected sponges. Lower mean TVC levels were found in unworn brushes and brushes regularly cleaned with soap, but the differences were modest (1.5 log or less). A common set of bacteria was found in brushes and sponges, dominated by Acinetobacter, Chryseobacterium, Enhydrobacter, Enterobacteriaceae and Pseudomonas. There was no difference in TVC or bacterial diversity between conventional and antimicrobial sponges containing silver after 4 weeks of use. For used brushes inoculated with Salmonella and allowed to dry overnight, a significant reduction in Salmonella numbers was observed. No reduction was observed for brushes stored in humid conditions (in a plastic bag) or for sponges regardless of storing conditions. CONCLUSIONS: Overall, lower bacterial levels were observed in used brushes than in sponges, and Salmonella died more rapidly in brushes. A common set of non-pathogenic bacteria dominated in brushes and sponges. SIGNIFICANCE AND IMPACT OF STUDY: The study demonstrates that the use of brushes may be more hygienic than the use of sponges.

Kitchen layouts and consumers' food hygiene practices: Ergonomics versus safety.

Our paper emphasizes the importance of the kitchen layout in facilitating consumers' food hygiene practices. A significant correlation was found between the sink placement (inside or outside the kitchen) and hygienic practices during food handling based on a survey performed on consumers from ten European countries, indicating that those who had the sink in the kitchen were more likely to perform proper hygiene practices than those who have not. The self-reported practices were supported by observed practices in 64 households from five European countries. The observational study combined with the examination of kitchen layouts revealed that the kitchen work triangle with its apexes represented by the kitchen sink, cooking stove and refrigerator, which is recommended for ergonomic reasons by architects and designers, did not necessarily support food hygiene practices in kitchens. Cross-contamination events were associated with the sink - countertop distances longer than 1 m. Based on this, a new kitchen triangle with its apexes represented by the kitchen sink, working place (usually countertop) and cooking stove, with the distance between the sink and the working place less than 1 m is proposed to be used as norm in kitchen designs for combining ergonomics with safety. This triangle is proposedly named the food safety triangle and is aimed to mitigate the risks of foodborne illnesses by creating an arrangement that facilitates hygiene practices. This study is the first to highlight the importance of implementing the concept of food safety in the kitchen design based on significant correlations between kitchen equipment placement and consumers' food safety practices.

Surveillance of Listeria monocytogenes: Early Detection, Population Dynamics, and Quasimetagenomic Sequencing during Selective Enrichment.

In this study, we addressed different aspects regarding the implementation of quasimetagenomic sequencing as a hybrid surveillance method in combination with enrichment for early detection of Listeria monocytogenes in the food industry. Different experimental enrichment cultures were used, comprising seven L. monocytogenes strains of different sequence types (STs), with and without a background microbiota community. To assess whether the proportions of the different STs changed over time during enrichment, the growth and population dynamics were assessed using dapE colony sequencing and dapE and 16S rRNA amplicon sequencing. There was a tendency of some STs to have a higher relative abundance during the late stage of enrichment when L. monocytogenes was enriched without background microbiota. When coenriched with background microbiota, the population dynamics of the different STs was more consistent over time. To evaluate the earliest possible time point during enrichment that allows the detection of L. monocytogenes and at the same time the generation of genetic information that enables an estimation regarding the strain diversity in a sample, quasimetagenomic sequencing was performed early during enrichment in the presence of the background microbiota using Oxford Nanopore Technologies Flongle and Illumina MiSeq sequencing. The application of multiple displacement amplification (MDA) enabled detection of L. monocytogenes (and the background microbiota) after only 4 h of enrichment using both applied sequencing approaches. The MiSeq sequencing data additionally enabled the prediction of cooccurring L. monocytogenes strains in the samples. IMPORTANCE We showed that a combination of a short primary enrichment combined with MDA and Nanopore sequencing can accelerate the traditional process of cultivation and identification of L. monocytogenes. The use of Illumina MiSeq sequencing additionally allowed us to predict the presence of cooccurring L. monocytogenes strains. Our results suggest quasimetagenomic sequencing is a valuable and promising hybrid surveillance tool for the food industry that enables faster identification of L. monocytogenes during early enrichment. Routine application of this approach could lead to more efficient and proactive actions in the food industry that prevent contamination and subsequent product recalls and food destruction, economic and reputational losses, and human listeriosis cases.

Salmonella in eggs: From shopping to consumption-A review providing an evidence-based analysis of risk factors.

Nontyphoidal salmonellae are among the most prevalent foodborne pathogens causing gastrointestinal infections worldwide. A high number of cases and outbreaks of salmonellosis are associated with the consumption of eggs and egg products, and several of these occur at the household level. The aim of the current study is to critically evaluate the current status of knowledge on Salmonella in eggs from a consumer's perspective, analyzing the hazard occurrence and the good practices that should be applied to reduce salmonellosis risk. Following a HACCP (Hazard Analysis and Critical Control Point) based approach, some steps along the food journey were identified as Critical Consumer Handling (CCH)-steps in which consumers, through their behavior or choice, can significantly reduce the level of Salmonella in eggs and egg products. From shopping/collecting to consumption, each of these steps is discussed in this review to provide an evidence-based overview of risk factors of human salmonellosis related to egg consumption. The main message to consumers is to choose Salmonella-free eggs (those that some official entity or producer guarantees that does not contain Salmonella), when available, especially for dishes that are not fully heat treated. Second, as guaranteed Salmonella-free eggs are only available in a few countries, refrigerated storage from the point of collection and proper cooking will significantly reduce the risk of salmonellosis. This will require a revision of the actual recommendations/regulations, as not all ensure that eggs are maintained at temperatures that prevent growth of Salmonella from collection until the time of purchasing.

In-Depth Longitudinal Study of Listeria monocytogenes ST9 Isolates from the Meat Processing Industry: Resolving Diversity and Transmission Patterns Using Whole-Genome Sequencing.

Listeria monocytogenes is a pathogen mostly associated with the consumption of ready-to-eat foods and can cause severe disease and death. It can be introduced into food chains from raw materials, but often the contamination source is the food production environment, where certain clones can persist for years. In the meat chain, ST9 is one of the most commonly encountered L. monocytogenes sequence types, and for effective source tracking, the divergence and spread of ST9 must be understood. In this study, whole-genome sequencing (WGS) was used to characterize and track 252 L. monocytogenes ST9 isolates collected from four Norwegian meat processing plants between 2009 and 2017. The isolates formed distinct clusters relative to genomes found in public databases, and all but three isolates clustered into two major clonal populations. Different contamination patterns were revealed, e.g., evidence of contamination of two factories with a clone that diverged from its ancestor in the late 1990s through a common source of raw materials; breach of hygienic barriers within a factory, leading to repeated detection of two clones in the high-risk zone during a 4- to 6-year period; entry through the purchase and installation of second-hand equipment harboring a previously established clonal population; and spreading and diversification of two clones from two reservoirs within the same production room over a 9-year period. The present work provides data on the diversity of ST9, which is crucial for epidemiological investigations and highlights how WGS can be used for source tracking within food processing factories.IMPORTANCEListeria monocytogenes is a deadly foodborne pathogen that is widespread in the environment, and certain types can be established in food factories. The sequence type ST9 dominates in meat processing environments, and this work was undertaken to obtain data needed for the tracking of this subtype. By using whole-genome sequencing (WGS), we revealed the presence of cross-contamination routes between meat factories as well as within a single factory, including the spread from different reservoirs within the same room. It was also possible to estimate the time frame of persistence in the factory, as well as when and how new clones had entered. The present work contributes valuable information about the diversity of ST9 and exemplifies the potential power of WGS in food safety management, allowing the determination of relationships between strains both in an international context and locally between and within factories.

Situated food safety behavior.

Previous studies indicate that many consumers eat rare hamburgers and that information about microbiological hazards related to undercooked meat does not necessarily lead to changed behavior. With this study we aim to investigate whether consumers' willingness to eat hamburgers, both risky and safe, depends on the situation where they are confronted with the food. A representative sample of 1046 Norwegian consumers participated in a web experiment. Participants were randomly divided into four groups. Each group was told to imagine a specific eating situation (at their friend's place, at home, at a restaurant abroad, at a domestic restaurant). Four pictures of hamburgers (rare, medium rare, medium, well-done) were presented in randomized order, and participants rated their intentions to eat each hamburger. Situated risk perception was measured as the stated likelihood of food poisoning from consuming hamburgers in eight different situations. The results show that both risk perception and risk taking vary depending on the situation. In general, participants perceive their own home to be the safest place to consume a hamburger, but they are significantly more likely to consume an undercooked hamburger when at a friend's place. These findings indicate that situations play an important role for consumers' likelihood of eating unsafe food, and that risk taking does not always follow risk perception. That risk taking is elevated in situations that may have social consequences should be taken into consideration when developing food safety strategies.

Situated Food Safety Risk and the Influence of Social Norms.

Previous studies of risk behavior observed weak or inconsistent relationships between risk perception and risk-taking. One aspect that has often been neglected in such studies is the situational context in which risk behavior is embedded: Even though a person may perceive a behavior as risky, the social norms governing the situation may work as a counteracting force, overriding the influence of risk perception. Three food context studies are reported. In Study 1 (N = 200), we assess how norm strength varies across different social situations, relate the variation in norm strength to the social characteristics of the situation, and identify situations with consistently low and high levels of pressure to comply with the social norm. In Study 2 (N = 502), we investigate how willingness to accept 15 different foods that vary in terms of objective risk relates to perceived risk in situations with low and high pressure to comply with a social norm. In Study 3 (N = 1,200), we test how risk-taking is jointly influenced by the perceived risk associated with the products and the social norms governing the situations in which the products are served. The results indicate that the effects of risk perception and social norm are additive, influencing risk-taking simultaneously but as counteracting forces. Social norm had a slightly stronger absolute effect, leading to a net effect of increased risk-taking. The relationships were stable over different social situations and food safety risks and did not disappear when detailed risk information was presented.

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.

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.

Residential Bacteria on Surfaces in the Food Industry and Their Implications for Food Safety and Quality.

Surface hygiene is commonly measured as a part of the quality system of food processing plants, but as the bacteria present are commonly not identified, their roles for food quality and safety are not known. Here, we review the identity of residential bacteria and characteristics relevant for survival and growth in the food industry along with potential implications for food safety and quality. Sampling after cleaning and disinfection increases the likelihood of targeting residential bacteria. The increasing use of sequencing technologies to identify bacteria has improved knowledge about the bacteria present in food premises. Overall, nonpathogenic Gram-negative bacteria, especially Pseudomonas spp., followed by Enterobacteriaceae and Acinetobacter spp. dominate on food processing surfaces. Pseudomonas spp. persistence is likely due to growth at low temperatures, biofilm formation, tolerance to biocides, and low growth requirements. Gram-positive bacteria are most frequently found in dairies and in dry production environments. The residential bacteria may end up in the final products through cross-contamination and may affect food quality. Such effects can be negative and lead to spoilage, but the bacteria may also contribute positively, as through spontaneous fermentation. Pathogenic bacteria present in food processing environments may interact with residential bacteria, resulting in both inhibitory and stimulatory effects on pathogens in multispecies biofilms. The residential bacterial population, or bacteriota, does not seem to be an important source for the transfer of antibiotic resistance genes to humans, but more knowledge is needed to verify this. If residential bacteria occur in high numbers, they may influence processes such as membrane filtration and corrosion.

Microbiota formed on attached stainless steel coupons correlates with the natural biofilm of the sink surface in domestic kitchens.

Stainless steel coupons are frequently used in biofilm studies in the laboratory, as this material is commonly used in the food industry. The coupons are attached to different surfaces to create a "natural" biofilm to be studied further in laboratory trials. However, little has been done to investigate how well the microbiota on such coupons represents the surrounding environment. The microbiota on sink wall surfaces and on new stainless steel coupons attached to the sink wall for 3 months in 8 domestic kitchen sinks was investigated by next-generation sequencing (MiSeq) of the 16S rRNA gene derived from DNA and RNA (cDNA), and by plating and identification of colonies. The mean number of colony-forming units was about 10-fold higher for coupons than sink surfaces, and more variation in bacterial counts between kitchens was seen on sink surfaces than coupons. The microbiota in the majority of biofilms was dominated by Moraxellaceae (genus Moraxella/Enhydrobacter) and Micrococcaceae (genus Kocuria). The results demonstrated that the variation in the microbiota was mainly due to differences between kitchens (38.2%), followed by the different nucleic acid template (DNA vs RNA) (10.8%), and that only 5.1% of the variation was a result of differences between coupons and sink surfaces. The microbiota variation between sink surfaces and coupons was smaller for samples based on their RNA than on their DNA. Overall, our results suggest that new stainless steel coupons are suited to model the dominating part of the natural microbiota of the surrounding environment and, furthermore, are suitable for different downstream studies.

Coaggregation between Rhodococcus and Acinetobacter strains isolated from the food industry.

In this study, coaggregation interactions between Rhodococcus and Acinetobacter strains isolated from food-processing surfaces were characterized. Rhodococcus sp. strain MF3727 formed intrageneric coaggregates with Rhodococcus sp. strain MF3803 and intergeneric coaggregates with 2 strains of Acinetobacter calcoaceticus (MF3293, MF3627). Stronger coaggregation between A. calcoaceticus MF3727 and Rhodococcus sp. MF3293 was observed after growth in batch culture at 30 °C than at 20 °C, after growth in tryptic soy broth than in liquid R2A medium, and between cells in exponential and early stationary phases than cells in late stationary phase. The coaggregation ability of Rhodococcus sp. MF3727 was maintained even after heat and Proteinase K treatment, suggesting its ability to coaggregate was protein independent whereas the coaggregation determinants of the other strains involved proteinaceous cell-surface-associated polymers. Coaggregation was stable at pH 5-9. The mechanisms of coaggregation among Acinetobacter and Rhodococcus strains bare similarity to those displayed by coaggregating bacteria of oral and freshwater origin, with respect to binding between proteinaceous and nonproteinaceous determinants and the effect of environmental factors on coaggregation. Coaggregation may contribute to biofilm formation on industrial food surfaces, protecting bacteria against cleaning and disinfection.

Hamburger hazards and emotions.

Previous studies indicate that many consumers eat rare hamburgers and that information about microbiological hazards related to undercooked meat not necessarily leads to more responsible behavior. With this study we aim to investigate whether consumers' willingness to eat hamburgers depends on the emotions they experience when confronted with the food. A representative sample of 1046 Norwegian consumers participated in an online experiment. In the first part, participants were randomly divided into two groups. One group was confronted with a picture of a rare hamburger, whereas the other group was confronted with a picture of a well-done hamburger. The respondents were instructed to imagine that they were served the hamburger on the picture and then to indicate which emotions they experienced: fear, disgust, surprise, interest, pleasure, or none of these. In part two, all respondents were confronted with four pictures of hamburgers cooked to different degrees of doneness (rare, medium rare, medium well-done, well-done), and were asked to state their likelihood of eating. We analyzed the data by means of a multivariate probit model and two linear fixed-effect models. The results show that confrontation with rare hamburgers evokes more fear and disgust than confrontation with well-done hamburgers, that all hamburgers trigger pleasure and interest, and that a consumer's willingness to eat rare hamburgers depends on the particular type of emotion evoked. These findings indicate that emotions play an important role in a consumer's likelihood of eating risky food, and should be considered when developing food safety strategies.

Use of used vs. fresh cheese brines and the effect of pH and salt concentration on the survival of Listeria monocytogenes.

The aim of the study was to investigate how the use of fresh cheese brines compared with used brines and various combinations of pH and NaCl concentrations affected the survival of Listeria monocytogenes. Cheese brines from five Norwegian small scale cheese producers were analysed and showed great variations in pH (4·54-6·01) and NaCl concentrations (14·1-26·9 %). The survival of five strains of List. monocytogenes (two clinical isolates, two food isolates and one animal isolate) in four different cheese brines (three used and one fresh) was investigated. Results showed significant differences in survival both depending on the strains and the brines. Strains of human outbreak listeriosis cases showed greater ability to survive in the brines compared with food isolates and a List. monocytogenes reference strain (1-2 log10 difference after 200 d). All strains showed highest survival in the freshly prepared brine compared with the used brines. Molecular typing by multiple locus variable number tandem repeats analysis (MLVA) showed that there were no detectable alterations in the examined variable number tandem repeats of the genome in five strains after 200 d storage in any of the salt brines. Combined effects of pH (4·5, 5·25 and 6·0) and NaCl (15, 20 and 25 %) in fresh, filter sterilised brines on the survival of List. monocytogenes were examined and results showed that pathogen populations decreased over time in all brines. Death rates at any given NaCl concentration were highest at low pH (4·5) and death rates at any given pH were highest at low NaCl concentrations (15 %). In conclusion, the use of used brines reduced the survival of List. monocytogenes and a combination of low pH (4·5) and low salt concentrations (15 %) decreased the risk of List. monocytogenes survival compared with higher pH (5·25 or 6·0) and higher NaCl concentrations (20 or 25 %).

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.