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.

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.

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.

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.

High nutrient availability reduces the diversity and stability of the equine caecal microbiota.

BACKGROUND: It is well known that nutrient availability can alter the gut microbiota composition, while the effect on diversity and temporal stability remains largely unknown. METHODS: Here we address the equine caecal microbiota temporal stability, diversity, and functionality in response to diets with different levels of nutrient availability. Hay (low and slower nutrient availability) versus a mixture of hay and whole oats (high and more rapid nutrient availability) were used as experimental diets. RESULTS: We found major effects on the microbiota despite that the caecal pH was far from sub-clinical acidosis. We found that the low nutrient availability diet was associated with a higher level of both diversity and temporal stability of the caecal microbiota than the high nutrient availability diet. These observations concur with general ecological theories, suggesting a stabilising effect of biological diversity and that high nutrient availability has a destabilising effect through reduced diversity. CONCLUSION: Nutrient availability does not only change the composition but also the ecology of the caecal microbiota.

Novel developmental analyses identify longitudinal patterns of early gut microbiota that affect infant growth.

It is acknowledged that some obesity trajectories are set early in life, and that rapid weight gain in infancy is a risk factor for later development of obesity. Identifying modifiable factors associated with early rapid weight gain is a prerequisite for curtailing the growing worldwide obesity epidemic. Recently, much attention has been given to findings indicating that gut microbiota may play a role in obesity development. We aim at identifying how the development of early gut microbiota is associated with expected infant growth. We developed a novel procedure that allows for the identification of longitudinal gut microbiota patterns (corresponding to the gut ecosystem developing), which are associated with an outcome of interest, while appropriately controlling for the false discovery rate. Our method identified developmental pathways of Staphylococcus species and Escherichia coli that were associated with expected growth, and traditional methods indicated that the detection of Bacteroides species at day 30 was associated with growth. Our method should have wide future applicability for studying gut microbiota, and is particularly important for translational considerations, as it is critical to understand the timing of microbiome transitions prior to attempting to manipulate gut microbiota in early life.

Extending the Shelf Life of Atlantic Salmon (Salmo salar) with Sub-Chilled Storage and Modified Atmosphere Packaging in Recyclable Mono-Material Trays.

This study investigated the effect of sub-chilling whole gutted salmon and sub-chilled storage at -1 °C in modified-atmosphere packaging in two recyclable mono-material trays (CPET, HDPE). Quality parameters were measured, including water-holding properties, salt content, color, texture, lipid oxidation, and sensory and microbiological shelf life. The oxygen transmission rate was measured for the packages. Compared to traditional fish storage on ice, sub-chilling gave a 0.4% weight gain, better water-holding capacity, and higher salt content. The sub-chilled fish gave a significantly better sensory quality and microbiological shelf life of up to 49 days. Photobacterium was the dominating bacteria during storage. Salmon packaged in CPET trays had a higher drip loss than HDPE trays, but a lower rate of lipid oxidation (1-penten-3-ol). Our results showed the feasibility of significantly extending shelf life with sub-chilling, removing the need for ice. Moreover, using recyclable trays for packaging contributes to a circular economy without compromising food quality.

Subminimal inhibitory concentrations of the disinfectant benzalkonium chloride select for a tolerant subpopulation of Escherichia coli with inheritable characteristics.

Exposure of Escherichia coli to a subminimal inhibitory concentration (25% below MIC) of benzalkonium chloride (BC), an antimicrobial membrane-active agent commonly used in medical and food-processing environments, resulted in cell death and changes in cell morphology (filamentation). A small subpopulation (1-5% of the initial population) survived and regained similar morphology and growth rate as non-exposed cells. This subpopulation maintained tolerance to BC after serial transfers in medium without BC. To withstand BC during regrowth the cells up regulated a drug efflux associated gene (the acrB gene, member of the AcrAB-TolC efflux system) and changed expression of outer membrane porin genes (ompFW) and several genes involved in protecting the cell from the osmotic- and oxidative stress. Cells pre-exposed to osmotic- and oxidative stress (sodium chloride, salicylic acid and methyl viologen) showed higher tolerance to BC. A control and two selected isolates showing increased BC-tolerance after regrowth in BC was genome sequenced. No common point mutations were found in the BC- isolates but one point mutation in gene rpsA (Ribosomal protein S1) was observed in one of the isolates. The observed tolerance can therefore not solely be explained by the observed point mutation. The results indicate that there are several different mechanisms responsible for the regrowth of a tolerant subpopulation in BC, both BC-specific and general stress responses, and that sub-MIC of BC may select for phenotypic variants in a sensitive E. coli culture.

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.

Direct detection of single-nucleotide polymorphisms in bacterial DNA by SNPtrap.

A major challenge with single-nucleotide polymorphism (SNP) fingerprinting of bacteria and higher organisms is the combination of genome-wide screenings with the potential of multiplexing and accurate SNP detection. Single-nucleotide extension by the minisequencing principle represents a technology that both is highly accurate and enables multiplexing. A current bottleneck for direct genome analyses by minisequencing, however, is the sensitivity, since minisequencing relies on linear signal amplification. Here, we present SNPtrap, which is a novel approach that combines the specificity and possibility of multiplexing by minisequencing with the sensitivity obtained by logarithmic signal amplification by polymerase chain reaction (PCR). We show a SNPtrap proof of principle in a model system for two polymorphic SNP sites in the Salmonella tetrathionate reductase gene (ttrC).

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.

High Oxygen Packaging of Atlantic Cod Fillets Inhibits Known Spoilage Organisms, but Sensory Quality Is Not Improved Due to the Growth of Carnobacterium/Carnobacteriaceae.

Improved quality control and prolonged shelf life are important actions in preventing food waste. To get an overview of the bacterial diversity of fillets from live stored mature Atlantic cod, bacterial isolates were identified before and after storage (air and vacuum) and freezing/thawing. Based on the load of dominating bacteria, the effect of different packaging methods and a short freezing/thawing process on prolonged shelf-life was evaluated (total viable counts, bacteriota, sensory attributes, and volatile components). Hand filleted (strict hygiene) cod fillets had a low initial bacterial load dominated by the spoilage organism Photobacterium, whereas industrially produced fillets had higher bacterial loads and diversity (Pseudomonas, Arthrobacter, Psychrobacter, Shewanella). The identified bacteria after storage in vacuum or air were similar to the initially identified bacteria. Bacteriota analysis showed that a short time freezing/thawing process reduced Photobacterium while modified atmosphere packaging (MAP; 60% CO2/40% O2 or 60% CO2/40% N2) inhibited the growth of important spoilage bacteria (Photobacterium,Shewanella, Pseudomonas) and allowed the growth of Carnobacterium/Carnobacteriaceae and Acinetobacter. Despite being dominated by Photobacterium, fresh fillets stored in MAP 60% CO2/40% N2 demonstrated better sensory quality after 13 days of storage than fillets stored in MAP 60% CO2/40% O2 (dominated by Carnobacterium/Carnobacteriaceae). Carnobacterium spp. or other members of Carnobacteriaceae may therefore be potential spoilage organisms in cod when other spoilage bacteria are reduced or inhibited.

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.

Dishwashing sponges and brushes: Consumer practices and bacterial growth and survival.

Sponges are frequently used in kitchens and have been shown to harbor large numbers of bacteria, occasionally also pathogens. Less is known about kitchen brushes regarding usage and presence of bacteria. In the present study, the use of sponges and brushes was studied in a survey among 9966 European consumers in ten countries, and growth and survival of bacteria in sponges and brushes were examined in laboratory experiments. Sponges were the preferred hand-cleaning utensils for washing-up in the majority of countries, while brushes were most frequently used in Denmark and Norway. Consumers mostly change their sponges at regular times, but also sensory cues (looks dirty, smelly, slimy) and usage occurrences such as wiping up meat juices may trigger replacement. Besides cleaning the dishes, over a quarter of the dish brush users also use it to clean a chopping board after soilage from chicken meat juices. The water uptake and drying rate varied considerably, both between different sponges and between brushes and sponges, where brushes dried fastest. Campylobacter survived one day in all sponges and Salmonella more than seven days in two of three types of sponges. In the type of sponge that dried slowest, Salmonella grew on the first day and was always found in higher levels than in the other sponges. Non-pathogenic bacteria grew in the sponges and reached levels around 9 log CFU/sponge. In brushes all types of bacteria died over time. Campylobacter and Salmonella were reduced by more than 2.5 log to below the detection limit after one and three days, respectively. Bacteriota studies revealed a tendency for a dominance by Gram-negative bacteria and a shift to high relative prevalence of Pseudomonas over time in sponges. Both enumeration by agar plating and bacteriota analysis confirmed that the pathogens were in a minority compared to the other bacteria. Treatments of sponges and brushes with chlorine, boiling or in the dishwasher were effective to reduce Salmonella. We conclude that brushes are more hygienic than sponges and that their use should be encouraged. Contaminated sponges or brushes should be replaced or cleaned when they may have been in contact with pathogenic microorganisms, e.g. used on raw food spills. Cleaning of sponges and brushes with chlorine, boiling or dishwasher may be a safe alternative to replacing them with new ones.

Global responses of Escherichia coli to adverse conditions determined by microarrays and FT-IR spectroscopy.

The global gene expression and biomolecular composition in an Escherichia coli model strain exposed to 10 adverse conditions (sodium chloride, ethanol, glycerol, hydrochloric and acetic acid, sodium hydroxide, heat (46 degrees C), and cold (15 degrees C), as well as ethidium bromide and the disinfectant benzalkonium chloride) were determined using DNA microarrays and Fourier transform infrared (FT-IR) spectroscopy. In total, approximately 40% of all investigated genes (1682/4279 genes) significantly changed expression, compared with a nonstressed control. There were, however, only 3 genes (ygaW (unknown function), rmf (encoding a ribosomal modification factor), and ghrA (encoding a glyoxylate/hydroxypyruvate reductase)) that significantly changed expression under all conditions (not including benzalkonium chloride). The FT-IR analysis showed an increase in unsaturated fatty acids during ethanol and cold exposure, and a decrease during acid and heat exposure. Cold conditions induced changes in the carbohydrate composition of the cell, possibly related to the upregulation of outer membrane genes (glgAP and rcsA). Although some covariance was observed between the 2 data sets, principle component analysis and regression analyses revealed that the gene expression and the biomolecular responses are not well correlated in stressed populations of E. coli, underlining the importance of multiple strategies to begin to understand the effect on the whole cell.

Exploring the Brine Microbiota of a Traditional Norwegian Fermented Fish Product (Rakfisk) from Six Different Producers during Two Consecutive Seasonal Productions.

The purpose of this study was to explore the microbiota of Norwegian fermented fish (rakfisk), a traditional product popular in the Norwegian market. Brine samples, collected from six producers during two subsequent years, were used. The producers applied different salt concentrations (between 3.8% and 7.2% NaCl), ripening temperatures (between 3.5 and 7.5 °C), fish species (trout or char), and fish upbringing (wild trout, on-shore farmed trout or char, and off-shore farmed char). The microbiota in the brine during the ripening process was mainly characterized by DNA-based, culture-independent methods. In total, 1710 samples were processed and of these 1342 were used for the final analysis. The microbiota was dominated by Gammaproteobacteria and Bacilli with the largest variance between samples associated with the genera Psychrobacter and Lactobacillus. The variance in the material was mainly determined by the origin of the samples, i.e., the different producers. The microbiota from the individual producers was to a large extent reproducible from one year to the next and appeared to be determined by the relatively small differences in the salinity and the ripening temperature. This is the first study exploring the microbiota in rakfisk brine and it provides insights into environmental factors affecting the rakfisk ecosystems.

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.

Analysis of covariance patterns in gene expression data and FT-IR spectra.

The aim of this study was to detect and interpret correlation patterns in several large data matrices from the same biological system using Partial Least Squares Regression (PLSR) in order to get information on the system under investigation. To do this, DNA microarray data and Fourier Transform Infrared (FT-IR) spectra from a designed study where Campylobacter jejuni was exposed to environmental stress conditions, were used. The experimental design included variation in atmospheric conditions, temperature and time. PLSR was first used to analyse each of the two data types separately in order to explore the effect of the experimental parameters on the data. The results showed that both the gene expression and FT-IR spectra were affected by the variations in atmosphere, temperature and time, but that the effect was different for the two types of data. When the DNA microarray data and FT-IR spectra were linked together by PLSR, covariation due to temperature was seen. Both specific genes and ranges in the FT-IR spectra that were connected to the variation in temperature were detected. Some of these are possibly connected to properties of the cell wall of the bacteria. The results in this study show the potential of PLSR for investigation of covariance structures in biological data. By doing this, valuable information about the biological system can be detected and interpreted. It was also shown that the use of FT-IR spectroscopy provided important information about the stress responses in the bacteria, information that was not detected from the DNA microarray data.

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.