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.

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.

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.

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.

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.

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.

Persistence of microbiological hazards in food and feed production and processing environments.

Listeria monocytogenes (in the meat, fish and seafood, dairy and fruit and vegetable sectors), Salmonella enterica (in the feed, meat, egg and low moisture food sectors) and Cronobacter sakazakii (in the low moisture food sector) were identified as the bacterial food safety hazards most relevant to public health that are associated with persistence in the food and feed processing environment (FFPE). There is a wide range of subtypes of these hazards involved in persistence in the FFPE. While some specific subtypes are more commonly reported as persistent, it is currently not possible to identify universal markers (i.e. genetic determinants) for this trait. Common risk factors for persistence in the FFPE are inadequate zoning and hygiene barriers; lack of hygienic design of equipment and machines; and inadequate cleaning and disinfection. A well-designed environmental sampling and testing programme is the most effective strategy to identify contamination sources and detect potentially persistent hazards. The establishment of hygienic barriers and measures within the food safety management system, during implementation of hazard analysis and critical control points, is key to prevent and/or control bacterial persistence in the FFPE. Once persistence is suspected in a plant, a 'seek-and-destroy' approach is frequently recommended, including intensified monitoring, the introduction of control measures and the continuation of the intensified monitoring. Successful actions triggered by persistence of L. monocytogenes are described, as well as interventions with direct bactericidal activity. These interventions could be efficient if properly validated, correctly applied and verified under industrial conditions. Perspectives are provided for performing a risk assessment for relevant combinations of hazard and food sector to assess the relative public health risk that can be associated with persistence, based on bottom-up and top-down approaches. Knowledge gaps related to bacterial food safety hazards associated with persistence in the FFPE and priorities for future research are provided.

Assessment of ATP-Bioluminescence and Dipslide Sampling to Determine the Efficacy of Slaughterhouse Cleaning and Disinfection Compared with Total Aerobic and Enterobacterales Counts.

Inadequate cleaning and disinfection (C&D) in slaughterhouses can cause bacterial contamination of meat, resulting in foodborne disease and reduced meat quality. Different methods for monitoring the efficacy of C&D procedures are available, but few studies have assessed their reliability. This study examined C&D efficacy in slaughterhouses and evaluated the diagnostic performance of methods for measuring surface hygiene. One red meat and one poultry slaughterhouse in Sweden were each visited on six occasions before and six occasions after C&D. Sampling points were sampled with: swabbing and plating for total aerobic bacteria (TAB) and Enterobacterales (EB); dipslides for total viable count; and ATP-bioluminescence tests. To evaluate the diagnostic performance of the dipslide and ATP-bioluminescence methods, the results were compared with (TAB) as a reference. In total, 626 samples were collected. For the majority of samples, TAB was lower after than before C&D and EB were mainly detected before C&D, indicating C&D efficacy. Greater reductions in mean TAB were observed in processing areas (2.2 and 2.8 log CFU/100 cm2 in red meat and poultry slaughterhouse, respectively) than in slaughter areas (1.3 log CFU/100 cm2 in both slaughterhouses). Approximately half of all samples were assessed as non acceptably clean (52% for red meat and 46% for poultry slaughterhouse) according to previously published thresholds. Critical food contact surfaces that were insufficiently cleaned and disinfected were plucking fingers, shackles, and a post-dehairing table. Cleaning and disinfection of drains and floors were inadequate. The ATP-bioluminescence method showed low specificity compared with the reference (TAB) in both the red meat (0.30) and poultry slaughterhouses (0.64). The sensitivity of dipslides was low (0.26) in the red meat slaughterhouse compared with TAB. A combination of ATP-bioluminescence and dipslides could provide more accurate estimates of C&D efficacy.

Microarray-based transcriptome of Listeria monocytogenes adapted to sublethal concentrations of acetic acid, lactic acid, and hydrochloric acid.

Listeria monocytogenes , an important foodborne pathogen, commonly encounters organic acids in food-related environments. The transcriptome of L. monocytogenes L502 was analyzed after adaptation to pH 5 in the presence of acetic acid, lactic acid, or hydrochloric acid (HCl) at 25 °C, representing a condition encountered in mildly acidic ready-to-eat food kept at room temperature. The acid-treated cells were compared with a reference culture with a pH of 6.7 at the time of RNA harvesting. The number of genes and magnitude of transcriptional responses were higher for the organic acids than for HCl. Protein coding genes described for low pH stress, energy transport and metabolism, virulence determinates, and acid tolerance response were commonly regulated in the 3 acid-stressed cultures. Interestingly, the transcriptional levels of histidine and cell wall biosynthetic operons were upregulated, indicating possible universal response against low pH stress in L. monocytogenes. The opuCABCD operon, coding proteins for compatible solutes transport, and the transcriptional regulator sigL were significantly induced in the organic acids, strongly suggesting key roles during organic acid stress. The present study revealed the complex transcriptional responses of L. monocytogenes towards food-related acidulants and opens the roadmap for more specific and in-depth future studies.

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.