Insight to the diversity of Photobacterium spp. isolated from European plaice (Pleuronectes platessa) based on phylogenetic analysis, phenotypic characterisation and spoilage potential.

This study aimed to explore the diversity of fifty-four Photobacterium strains isolated from muscle tissue of European plaice (Pleuronectes platessa) caught at different fishing seasons and stored 14-days under various conditions. Single phylogenetic markers (16S rRNA, gapA, gyrB and recA) and multilocus sequence analysis (MLSA) were employed to classify isolates at species level. Furthermore, intra- and interspecies variability in the phenotypic traits, maximum specific growth rate (µmax) and spoilage potential of the Photobacterium isolates were investigated. The isolates were classified into the P. iliopiscarium (53.7 %), P. phosphoreum (40.7 %) and P. piscicola (5.6 %) clades using MLSA. Two housekeeping genes, gyrB and recA, exhibited a consistent phylogenetic relationship with MLSA, suggesting that they might be used as individual phylogenetic markers for the Photobacterium genus. Intra- and interspecies variability in the expression of phenotypic characteristics and the production of trimethylamine (TMA), inosine (HxR), and hypoxanthine (Hx) were observed. A growth optimum temperature for P. iliopiscarium was approximately 20 °C, while those for P. phosphoreum and P. piscicola were closer to 15 °C. All isolates exhibited the highest growth density at 1.5 % NaCl, followed by 0.5 %, 3 %, and 6 % NaCl. However, P. phosphoreum demonstrated a higher NaCl tolerance than the other two species. Although, the high CO2 atmosphere significantly inhibited the growth of all strains at 4 °C, P. phosphoreum and P. piscicola showed higher growth density at 15 °C than P. iliopiscarium. Notably, all strains demonstrated H2S production. The µmax varied considerably within each species, highlighting the significance of strain-level variability. This study demonstrates that P. iliopiscarium and P. piscicola, alongside P. phosphoreum, are efficient TMA-, HxR-, Hx-, and H2S-producers, suggesting their potential contribution to synergistic off-odour generation and spoilage. Moreover, the Photobacterium isolates seem to exhibit diverse adaptations to their environments, resulting in fluctuated growth and spoilage potential. Understanding intra- and interspecies variability will facilitate modelling seafood spoilage in microbial risk assessments and developing targeted hurdles to prolong products' shelf-life.

Selection of lactic acid bacteria for biopreservation of salmon products applying processing-dependent growth kinetic parameters and antimicrobial mechanisms.

Biopreservation using lactic acid bacteria (LAB) is a promising technology to prevent the growth of pathogenic microorganisms in fresh and mildly processed food. The main aim of this study was to select LAB, originally isolated from ready-to-eat (RTE) seafood, for biopreservation of fresh salmon and processed salmon products. Ten LAB strains (five Carnobacterium and five Leuconostoc) were selected based on previously demonstrated bioprotective properties to investigate their antimicrobial mechanisms and temperature-dependent growth kinetics in a sterile salmon juice model system. Furthermore, five strains (three Carnobacterium and two Leuconostoc) were selected to test process-dependent growth kinetic parameters relevant to the secondary processing of salmon. Two strains (Carnobacterium maltaromaticum 35 and C. divergens 468) showed bacteriocin-like activity against Listeria innocua, while inhibitory effect of cell-free supernatants (CFS) was not observed against Escherichia coli. All selected strains were able to grow in sterile salmon juice at tested temperatures (4, 8, 12 and 16 °C), with specific growth rates (µ) ranging from 0.01 to 0.04/h at 4 °C and reaching a maximum population density of 8.4-9 log CFU/ml. All five strains tested for process-dependent growth kinetic parameters were able to grow in the range of 0.5-5% NaCl and 0.13-0.26% purified condensed smoke (VTABB and JJT01), with inter- and intraspecies variation in growth kinetics. According to the temperature-dependent growth kinetics and antimicrobial assay results, two strains, Leuconostoc mesenteroides 68 (Le.m.68) and C. divergens 468 (C d.468), were selected for in situ test to validate their ability to grow in vacuum-packed fresh salmon at 4 °C. Both strains were able to grow at maximum growth rates of 0.29 ± 0.04/d for Le. m.68 and 0.39 ± 0.06/d for C.d.468, and their final concentrations were 7.91 ± 0.31 and 8.02 ± 0.25 log CFU/g, respectively. This study shows that LAB, originally isolated from RTE seafood, have promising potential as bioprotective strains in fresh and processed salmon products.

Whole genome sequence analysis of Aeromonas spp. isolated from ready-to-eat seafood: antimicrobial resistance and virulence factors.

Aeromonas are widespread in aquatic environments and are considered emerging pathogens in humans and animals. Multidrug resistant (MDR) Aeromonas circulating in the aquatic environment and food production chain can potentially disseminate antimicrobial resistance (AMR) to humans via the foodborne route. In this study, we aimed to investigate AMR and virulence factors of 22 Aeromonas strains isolated from ready-to-eat (RTE) seafood. A multilocus phylogenetic analysis (MLPA) using the concatenated sequences of six housekeeping genes (gyrB, rpoD, gyrA, recA, dnaJ, and dnaX) in the 22 Aeromonas genomes and average nucleotide identity (ANI) analysis revealed eight different species; A. caviae, A. dhakensis, A. hydrophila, A. media, A. rivipollensis, A. salmonicida, A. bestiarum, and A. piscicola. The presence of virulence genes, AMR genes and mobile genetic elements (MGEs) in the Aeromonas genomes was predicted using different databases. Our data showed that the genes responsible for adherence and motility (Msh type IV pili, tap type IV pili, polar flagella), type II secretion system (T2SS) and hemolysins were present in all strains, while the genes encoding enterotoxins and type VI secretion system (T6SS) including major effectors were highly prevalent. Multiple AMR genes encoding ß-lactamases such as cphA and blaOXA were detected, and the distribution of those genes was species-specific. In addition, the quinolone resistance gene, qnrS2 was found in a IncQ type plasmid of the A. rivopollensis strain A539. Furthermore, we observed the co-localization of a class I integron (intl1) with two AMR genes (sul1 and aadA1), and a Tn521 transposon carrying a mercury operon in A. caviae strain SU4-2. Various MGEs including other transposons and insertion sequence (IS) elements were identified without strongly associating with detected AMR genes or virulence genes. In conclusion, Aeromonas strains in RTE seafood were potentially pathogenic, carrying several virulence-related genes. Aeromonas carrying multiple AMR genes and MGEs could potentially be involved in the dissemination and spread of AMR genes to other bacterial species residing in the same environment and possibly to humans. Considering a One-Health approach, we highlight the significance of monitoring AMR caused by Aeromonas circulating in the food chain.

Photobacterium predominate the microbial communities of muscle of European plaice (Pleuronectes platessa) caught in the Norwegian sea independent of skin and gills microbiota, fishing season, and storage conditions.

The aim of this study was to investigate seasonal variations (September, December and April) in the initial microbial communities of skin and gills' external mucosal tissues (EMT) and muscle of European plaice (Pleuronectes platessa). Moreover, a potential relationship between EMT and fresh muscle microbiota was examined. The microbial community succession in plaice muscle as a function of fishing season and storage conditions was also investigated. The selected seasons for the storage experiment were September and April. Investigated storage conditions were; fillets packaged in either vacuum or modified atmosphere (70 % CO2, 20 % N2, 10 % O2) and chilled/refrigerated conditions (4 °C). Whole fish stored on ice (0 °C) was selected as a commercial standard. Seasonal variations were detected in the initial microbial communities of EMT and plaice muscle. The highest microbial diversity was found in EMT and muscle of April-caught plaice, followed by December and September catch indicating the important role of environmental factors in shaping the initial EMT and muscle microbial communities. The EMT microbial communities were more diverse than fresh muscle samples. The low number of shared taxa between EMT and initial muscle microbial communities indicates that only a minor part of the muscle microbiota came from the EMT. Psychrobacter and Photobacterium were the predominant genera in the EMT microbial communities in all seasons. Photobacterium dominated the initial muscle microbial communities with a gradual seasonal reduction of its abundance from September to April. Storage time and storage conditions shaped a less diverse and distinct community compared to the fresh muscle. However, no clear separation was seen between the communities at the middle and end of storage time. Regardless of EMT microbiota, fishing season and storage conditions, Photobacterium dominated the microbial communities of stored muscle samples. The Photobacterium prevalence as the primary specific spoilage organism (SSO) could be attributed to its high relative abundance in the initial microbiota of muscle and its CO2-tolerance. The findings of this study indicate the important contribution of Photobacterium to the microbial spoilage of plaice. Thus, the development of innovative preservation techniques addressing the rapid growth of Photobacterium could contribute to the production of high-quality and shelf-stable convenient retail plaice products.

The effect of food processing factors on the growth kinetics of Aeromonas strains isolated from ready-to-eat seafood.

Aeromonas are ubiquitous aquatic bacteria and frequently isolated from seafood. There is growing awareness of Aeromonas as foodborne pathogens, particularly in connection with consumption of ready-to-eat (RTE) seafood. The aim of this study was to investigate the effect of food processing factors on the growth kinetics of eight Aeromonas strains (representing seven species) isolated from RTE seafood. The effect of low temperature (4 and 8 °C) in combination with different NaCl concentrations (0.5-6.5 %) or with two purified condensate smokes (PCSs; Red Arrow SmokEz VTABB and JJT01) at different concentrations (0-0.26 %) was studied in Trypton Soy Broth (TSB). In food processing, application of PCS is considered healthier than traditional smoking. Growth kinetics parameters of each strain were estimated by using a primary predictive model. Our result showed that the addition of 3.5 % NaCl at refrigeration temperature (4 °C) was not sufficient to inhibit the growth of A. media, A. bestiarum, A. piscicola, and A. salmonicida, while higher NaCl concentration (≥5.0 %) at 8 °C suppressed their growth. On the other hand, our result demonstrated the antimicrobial potential of using PCS at maximal allowed concentration (0.26 %) against Aeromonas. PCS concentration and phenol content were important factors influencing the growth kinetics parameters of Aeromonas. Moreover, the growth kinetics of three Aeromonas strains were further studied in commercially produced vacuum-packed fresh and cold-smoked salmon stored at 4 °C for 14 and 21 days, respectively. Our results demonstrate that vacuum packing combined with cold storage at 4 °C was insufficient to inhibit the growth of Aeromonas in fresh salmon, while the growth was inhibited in a minimally salted cold-smoked salmon (salt content of 1.8 %). Our study implies that mild food processing factors applied in the production of RTE seafood might not guarantee the total inhibition of Aeromonas. Even though further studies on evaluating the antimicrobial potential of PCSs in actual seafood matrixes are necessary, the present study suggests that PCS technology might be a promising approach to prevent the potential growth of Aeromonas.

Growth and Spoilage Potential of an Aeromonas salmonicida Strain in Refrigerated Atlantic Cod (Gadus morhua) Stored under Various Modified Atmospheres.

Aeromonas spp. are ubiquitous aquatic bacteria, frequently isolated from seafood. The growth and spoilage potential of an inoculated strain of Aeromonas salmonicida (SU2) in Atlantic cod (Gadus morhua) fillets were assessed as a function of various modified atmospheres (MA) (CO2 (67%) with O2 (33%) or N2 (33%), and vacuum packaging (VP)) under refrigerated storage (4 °C) for 13 days. Additionally, the general microbiological quality was assessed by quantifying total aerobic psychrotrophic count (PC), total aerobic plate count (APC), and H2S-producing bacteria. A selection of spoilage metabolites was quantified by nuclear magnetic resonance (NMR) spectroscopy. The MA containing CO2/O2 and CO2/N2 significantly inhibited the growth of the inoculated strain throughout storage (p < 0.001), whereas VP allowed for a 3-log increase of Aeromonas in 13 days. No significant effect of the inoculation with A. salmonicida on spoilage metabolite production was detected. Applying O2 instead of N2 in the CO2-enriched atmosphere resulted in a significantly lower PC for 10 days, and H2S-producing bacteria were inhibited in both MAs compared to vacuum. This study provides data that can be used as a basis to further elucidate the role of bacteria belonging to the genus Aeromonas as potential spoilage organisms in marine fish species.

Diversity and Antimicrobial Activity towards Listeria spp. and Escherichia coli among Lactic Acid Bacteria Isolated from Ready-to-Eat Seafood.

Biopreservation is a food preservation technology using microorganisms and/or their inherent antimicrobial metabolites to inhibit undesirable microorganisms. The aim of the present study was to explore the diversity and antimicrobial activity of lactic acid bacteria (LAB) strains (n = 99) isolated from ready-to-eat (RTE) seafood (cold-smoked salmon (CSS), gravlax, and sushi) towards two strains of Listeria monocytogenes (CCUG 15527, F11), Listeria innocua (CCUG 15531) and Escherichia coli (CCUG 38079). The LAB strains were assigned to five different genera (Carnobacterium spp., Lactobacillus spp., Leuconostoc spp., Weissella spp., and Enterococcus sp.) by sequencing a 1150 bp stretch of the 16S rRNA gene. A significant association between the seafood source and the distribution of LAB genera was found (p < 0.001), of which Leuconostoc spp. were most prevalent in sushi and Carnobacterium sp. and Lactobacillus sp. were most frequently isolated from CSS and gravlax. Antimicrobial activity among the LAB was significantly affected by LAB genera (F= 117.91, p < 0.001, one-way ANOVA), product of origin (F = 3.47, p < 0.05), and target (F = 4.64, p = 0.003). LAB isolated from sushi demonstrated a significantly higher antimicrobial effect than LAB from CSS and gravlax (p < 0.05). In general, a significantly higher antimicrobial activity was found towards Listeria spp. than E. coli (p < 0.05). However, Leuconostoc spp. demonstrated similar antimicrobial effects towards E. coli and Listeria spp., except for L. monocytogenes F11 being more sensitive (p < 0.05). This study suggested that seafood-derived LAB strains could be selected for technological application in RTE seafood systems.

The Significance of Mesophilic Aeromonas spp. in Minimally Processed Ready-to-Eat Seafood.

Minimally processed and ready-to-eat (RTE) seafood products are gaining popularity because of their availability in retail stores and the consumers' perception of convenience. Products that are subjected to mild processing and products that do not require additional heating prior to consumption are eaten by an increasing proportion of the population, including people that are more susceptible to foodborne disease. Worldwide, seafood is an important source of foodborne outbreaks, but the exact burden is not known. The increased interest in seafood products for raw consumption introduces new food safety issues that must be addressed by all actors in the food chain. Bacteria belonging to genus Aeromonas are ubiquitous in marine environments, and Aeromonas spp. has held the title "emerging foodborne pathogen" for more than a decade. Given its high prevalence in seafood and in vegetables included in many RTE seafood meals, the significance of Aeromonas as a potential foodborne pathogen and a food spoilage organism increases. Some Aeromonas spp. can grow relatively uninhibited in food during refrigeration under a broad range of pH and NaCl concentrations, and in various packaging atmospheres. Strains of several Aeromonas species have shown spoilage potential by the production of spoilage associated metabolites in various seafood products, but the knowledge on spoilage in cold water fish species is scarce. The question about the significance of Aeromonas spp. in RTE seafood products is challenged by the limited knowledge on how to identify the truly virulent strains. The limited information on clinically relevant strains is partly due to few registered outbreaks, and to the disputed role as a true foodborne pathogen. However, it is likely that illness caused by Aeromonas might go on undetected due to unreported cases and a lack of adequate identification schemes. A rather confusing taxonomy and inadequate biochemical tests for species identification has led to a biased focus towards some Aeromonas species. Over the last ten years, several housekeeping genes has replaced the 16S rRNA gene as suitable genetic markers for phylogenetic analysis. The result is a more clear and robust taxonomy and updated knowledge on the currently circulating environmental strains. Nevertheless, more knowledge on which factors that contribute to virulence and how to control the potential pathogenic strains of Aeromonas in perishable RTE seafood products are needed.

Comparative Evaluation on the Quality and Shelf life of Atlantic Salmon (Salmo salar L.) Filets Using Microwave and Conventional Pasteurization in Combination with Novel Packaging Methods.

A comparative evaluation on the effect of carbon dioxide (CO2 ) on quality and shelf life of Atlantic salmon loins pasteurized with microwave and conventional technology was conducted. The experimental design allowed CO2 to enter the salmon muscle before (soluble gas stabilization [SGS] + vacuum) or after pasteurization (CO2 emitter + vacuum), whereas the control samples (vacuum only) were not presented for CO2 . This setup resulted in six different groups; three heated with microwaves and three with conventional pasteurization. The core temperature of microwave samples was 58.8 ± 2.2 °C, whereas the surface temperature was equal to the oven temperature (62 °C) during conventional pasteurization and close to the core temperature during microwave pasteurization (57.6 ± 1.4 °C). Microwave-heated samples showed higher microbial growth; decreased shelf life; and darker (lower L* -value), more reddish (higher a* -value), and yellowish (higher b* -value) colors compared to conventional-heated salmon. Lowest liquid loss (LL) was observed in salmon packaged with the CO2 emitter, whereas a SGS step prior to pasteurization did not affect the LL negatively as compared to samples packaged in vacuum only. Treatment with CO2 , independent of the prestep using SGS or an emitter, resulted in increased shelf life. Protein denaturation, microbial growth, product color, product shelf life, and sensory properties of the salmon loin were significantly affected by the applied pasteurization method (microwave- or conventional pasteurization). However, the heat load was probably too high to detect differences resulting from the pretreatment using SGS or packaging with CO2 emitter. PRACTICAL APPLICATION: Recent developments with increased time pressure from both work and past time activities have led to a tremendous increase in the demand for convenient, tasty ready-to-use food options. Furthermore, contemporary trends for consumption of fresh or lightly processed seafood stress the need to develop processing methods that allow a fulfillment of these demands, while still offering a reasonable shelf life. Carbon dioxide in combination with either microwave or conventional pasteurization is innovative processing technology that can meet consumer's demand of such products.

Growth of mesophilic Aeromonas salmonicida in an experimental model of nigiri sushi during cold storage.

The genus Aeromonas includes human pathogenic bacteria frequently isolated from seafood, and the increased consumption of ready-to-eat seafood poses new food safety issues regarding the presence of potentially pathogenic Aeromonas spp. in stored products for raw consumption, such as retail sushi with a shelf life of up to three days. This study assessed 1) the growth kinetics of a mesophilic A. salmonicida strain during storage at 4 °C and 8 °C in a nigiri sushi model, and 2) the strain variability in growth at pH ranging from 3.5 to 10 for a subset of mesophilic Aeromonas strains previously isolated from sushi. Inoculated slices of raw salmon were compared with and without rice. A predictive model for A. hydrophila (ComBase Predictor) did not sufficiently predict growth of the tested strain under the intrinsic conditions of nigiri sushi or salmon at both temperatures. Refrigeration alone (4 °C) did not inhibit growth of A. salmonicida on salmon. Within the first 72 h, representing the typical shelf life of retail sushi products, we observed a 10-fold increase in the concentration of the inoculated strain (including a lag-phase of approximately 34 h). Contact with acidified rice, resulting in a pH drop in the salmon, was the reason for the decreased bacterial viability in the nigiri sushi samples. However, the effect of acidification decreased at 8 °C, resulting in a 2-fold increase in the growth rate and a reduced lag-phase compared to refrigeration. Variability in the ability to grow in different pH levels was observed between strains. The highest color formation rates, representing cellular respiration analyzed in a phenotypic microarray system, were observed between pH 5 and 8. A few strains, including the A. salmonicida strain applied in the nigiri sushi model, were able to grow at pH 4.5 (at optimal temperature). The results demonstrated that mesophilic Aeromonas spp. can represent a microbiological hazard in retail sushi products during cold storage. Rice acidification in combination with low storage temperature (≤4 °C) are prerequisites to prevent growth of potentially pathogenic Aeromonas species during the relatively short shelf life.

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). ß-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7-3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.

Assessment of microbiological quality of retail fresh sushi from selected sources in Norway.

Retail fresh sushi is gaining popularity in Europe. This study was conducted to investigate the microbiological quality of selected samples of fresh sushi with a shelf life of 2 to 3 days offered as complete meals in Norwegian supermarkets. Analysis of aerobic plate counts in 58 sushi samples from three producers revealed large variations in microbiological quality, and 48% of the analyzed sushi boxes were rated as unsatisfactory (> 6.0 log CFU/g). Mesophilic Aeromonas spp. was detected in 71% of the samples. In a follow-up study, we collected products and raw materials directly from the production facility of one producer and observed a significant decrease (P < 0.01) in aerobic plate counts compared with the initial sampling. The observed difference between products purchased in stores compared with those collected directly from the factory suggests that poor temperature control during distribution and display in stores leads to reduced microbiological quality. Microbiological analysis of the sushi ingredients revealed that potentially pathogenic bacteria such as mesophilic Aeromonas spp. or bacteria belonging to the Enterobacteriaceae can be introduced into sushi through both raw vegetables and fish. The results highlight the importance of high quality ingredients and proper temperature control to ensure stable quality and safety of these food products.

Rapid reagentless quantification of alginate biosynthesis in Pseudomonas fluorescens bacteria mutants using FT-IR spectroscopy coupled to multivariate partial least squares regression.

Alginate is an important medical and commercial product and currently is isolated from seaweeds. Certain microorganisms also produce alginate and these polymers have the potential to replace seaweed alginates in some applications, mainly because such production will allow much better and more reproducible control of critical qualitative polymer properties. The research conducted here presents the development of a new approach to this problem by analysing a transposon insertion mutant library constructed in an alginate-producing derivative of the Pseudomonas fluorescens strain SBW25. The procedure is based on the non-destructive and reagent-free method of Fourier transform infrared (FT-IR) spectroscopy which is used to generate a complex biochemical infrared fingerprint of the medium after bacterial growth. First, we investigate the potential differences caused by the growth media fructose and glycerol on the bacterial phenotype and alginate synthesis in 193 selected P. fluorescens mutants and show that clear phenotypic differences are observed in the infrared fingerprints. In order to quantify the level of the alginate we also report the construction and interpretation of multivariate partial least squares regression models which were able to quantify alginate levels successfully with typical normalized root-mean-square error in predictions of only approximately 14%. We have demonstrated that this high-throughput approach can be implemented in alginate screens and we believe that this FT-IR spectroscopic methodology, when combined with the most appropriate chemometrics, could easily be modified for the quantification of other valuable microbial products and play a valuable screening role for synthetic biology.