The effect of enzymatic and viability dye treatment in combination with long-range PCR on assessing Tulane virus infectivity.

Human norovirus (HuNoV) is regularly involved in food-borne infections. To detect infectious HuNoV in food, RT-qPCR remains state of the art but also amplifies non-infectious virus. The present study combines pre-treatments, RNase and propidium monoazide, with three molecular analyses, including long-range PCR, to predominantly detect infectious Tulane virus (TuV), a culturable HuNoV surrogate. TuV was exposed to inactivating conditions to assess which molecular method most closely approximates the reduction in infectious virus determined by cell culture (TCID50). After thermal treatments (56 °C/5 min, 70 °C/5 min, 72 °C/20 min), TCID50 reductions of 0.3, 4.4 and 5.9 log10 were observed. UV exposure (40/100/1000 mJ/cm2) resulted in 1.1, 2.5 and 5.9 log10 reductions. Chlorine (45/100 mg/L for 1 h) reduced infectious TuV by 2.0 and 3.0 log10. After thermal inactivation standard RT-qPCR, especially with pre-treatments, showed the smallest deviation from TCID50. On average, RT-qPCR with pre-treatments deviated by 1.1-1.3 log10 from TCID50. For UV light, long-range PCR was closest to TCID50 results. Long-range reductions deviated from TCID50 by ≤0.1 log10 for mild and medium UV-conditions. However, long-range analyses often resulted in qPCR non-detects. At higher UV doses, RT-qPCR with pre-treatments differed by ≤1.0 log10 from TCID50. After chlorination the molecular methods repeatedly deviated from TCID50 by >1.0 log10, Overall, each method needs to be further optimized for the individual types of inactivation treatment.

Genomic insights into Enterococcus faecium isolates from marine bivalves highlight One Health concerns and healthcare linkages.

Enterococci, especially Enterococcus faecium, are one of today's leading causes of multidrug-resistant infections in hospital settings. The marine environment may harbour enterococci, but its role as an evolutionary niche and as a vector for the spread of enterococci is sparsely investigated. Hence, by applying enterococci in bivalves as a sentinel tool, this study aimed to describe the prevalence of enterocooci along the Norwegian coast and in addition the phylogeny of E. faecium in particular. Enterococci in batch samples of marine bivalves, harvested from 86 different locations, were quantitatively examined by a culture-dependent most probable number (MPN) method. Isolates were identified by MALDI-TOF-MS prior to antimicrobial susceptibility testing by broth microdilution. In-detail analyses of a representative selection of E. faecium isolates (n=148) were done by Illumina whole-genome sequencing, and assembled genomes were compared to closed E. faecium genomes in the public databases and to genomes from commensal and clinical isolates from Norway. Diversity among E. faecium within the same batch sample of bivalves was also explored. Enterococci were detected in 287 of the 471 examined bivalve samples, but in low concentrations with a median value of <18 MPN /100 g. From positive samples, 479 isolates of enterococci were identified belonging to ten different species, where E. faecium (n=247), Enterococcus hirae (n=114) and Enterococcus faecalis (n=66) were most frequently found. Resistance towards one or more antimicrobial agents was observed in 197 isolates (41 %), none of the isolates showed acquired resistance to vancomycin or linezolid. Phylogenetic analyses revealed high diversity among the E. faecium isolates and showed that the marine niche is dominated by strains from the non-clinical setting belonging to clade A2 (n=85) and B (E. lactis) (n=60). Only three isolates belonged to the hospital-associated clade A1 (ST80 and ST117). Two of these clustered with one isolate from a hospitalized patient and one from a non-hospitalized person. This study demonstrated a high prevalence, but low concentrations of enterococci in bivalves, and low levels of antimicrobial resistance. E. faecium genomes showed high population diversity and that very few E. faecium isolates in bivalves may have arisen from the human healthcare system. A systematic surveillance of target micro-organisms applying methods examining multiple isolates from the same bivalve sample provides important data to assess the enterococcal phylogeny, antimicrobial resistance and the level of faecal pollution in the marine environment.

From tide to table: A whole-year, coastal-wide surveillance of antimicrobial resistance in Escherichia coli from marine bivalves.

This work is the first of its kind to report a whole-year and coastal-wide surveillance of antimicrobial resistance (AMR) of Escherichia coli with samples from the EU imposed Norwegian surveillance programme for marine bivalves. In total, 390 bivalve samples collected from January to December in 2016 at 59 different harvest locations, were examined. The occurrence of resistant E. coli in relation to the concentration of E. coli was also analysed. From each sample with E. coli (n = 261), one isolate was susceptibility tested against a panel of 14 antimicrobials from ten classes. The occurrence of resistance to at least one antimicrobial was 8.4 %. Resistance to tetracycline was most commonly detected (5.7 %), followed by resistance to ampicillin (4.6 %) and sulfamethoxazole (3.1 %). The occurrence of extended spectrum cephalosporin (ESC)-resistant E. coli, quinolone-resistant E. coli (QREC) and carbapenem-resistant Enterobacteriaceae (CRE) were detected through selective screening in 3.3 %, 12.8 % and none of the samples, respectively. Among the ESC-resistant E. coli, the blaCTX-M-15 gene was detected in nine isolates, where two isolates also carried the blaCMY-42 gene, followed by blaCTX-M-3 in two and blaCTX-M-1 in one. One isolate was resistant to ESC due to the n.-42C>T mutation in the AmpC gene. Only the presence of QREC clustered significantly (p < 0.013) in space including nine harvest locations. An increased risk (OR 9.4) of detecting ESC-resistant E. coli or QREC was found for samples with E. coli concentrations above the threshold of Class A for direct distribution to the market (i.e. 230 E. coli/100 g). However, five of the ESC-resistant E. coli and 26 of the QREC positive samples, had levels of E. coli below the threshold, thus from areas cleared for sale. Among the 17 ESC-resistant E. coli subjected to whole genome sequencing, two originated from two samples of great scallops and two samples of flat oysters, which are often consumed raw or lightly processed. One of these isolates belonged to the high-risk clone sequence type 131 and carried a plasmid born senB gene encoding the Shigella enterotoxin 2 (ShET2) attributed to cause watery diarrhoea in infections caused by Enteroinvasive E. coli (EIEC). Thus, our study shows that there is a potential risk for transmission of resistant and pathogenic E. coli to the consumers from these products.

Co-localization of clinically relevant antibiotic- and heavy metal resistance genes on plasmids in Klebsiella pneumoniae from marine bivalves.

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with antibiotic resistance and present in a wide range of environments, including marine habitats. However, little is known about the development, persistence, and spread of antibiotic resistance in such environments. This study aimed to obtain the complete genome sequences of antibiotic-resistant K. pneumoniae isolated from marine bivalves in order to determine the genetic context of antibiotic- and heavy metal resistance genes in these isolates. Five antibiotic-resistant K. pneumoniae isolates, of which four also carried heavy metal resistance genes, were selected for complete genome sequencing using the Illumina MiSeq platform and the Oxford Nanopore Technologies GridION device. Conjugation experiments were conducted to examine the transfer potential of selected plasmids. The average length of the complete genomes was 5.48 Mbp with a mean chromosome size of 5.27 Mbp. Seven plasmids were detected in the antibiotic-resistant isolates. Three IncFIB, one IncFIB/IncFII, and one IncFIB/IncHIB plasmid, respectively, carried antibiotic resistance genes such as qnrS1, aph(6)-Id and aph(3')-Ia, aadA1, and aadA2. Four of these plasmids also carried genes encoding resistance to copper (pco), silver (sil), and arsenic (ars). One plasmid carrying tet(D) and blaSHV-1 as well as pco, sil, and ars genes was transferred to Escherichia coli by conjugation. We show the co-occurrence of antibiotic- and heavy metal resistance genes on a conjugative IncFIB plasmid from K. pneumoniae from marine bivalves. Our study highlights the importance of the marine environment and seafood as a possible dissemination route for antimicrobial resistance and provides insights into the potential for co-selection of antibiotic resistance genes by heavy metals.

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.

Temperature Dependent Depuration of Norovirus GII and Tulane Virus from Oysters (Crassostrea gigas).

Raw oysters are considered a culinary delicacy but are frequently the culprit in food-borne norovirus (NoV) infections. As commercial depuration procedures are currently unable to efficiently eliminate NoV from oysters, an optimisation of the process should be considered. This study addresses the ability of elevated water temperatures to enhance the elimination of NoV and Tulane virus (TuV) from Pacific oysters (Crassostrea gigas). Both viruses were experimentally bioaccumulated in oysters, which were thereafter depurated at 12 °C and 17 °C for 4 weeks. Infectious TuV and viral RNA were monitored weekly for 28 days by TCID50 and (PMAxx-) RT-qPCR, respectively. TuV RNA was more persistent than NoV and decreased by < 0.5 log10 after 14 days, while NoV reductions were already > 1.0 log10 at this time. For RT-qPCR there was no detectable benefit of elevated water temperatures or PMAxx for either virus (p > 0.05). TuV TCID50 decreased steadily, and reductions were significantly different between the two temperatures (p < 0.001). This was most evident on days 14 and 21 when reductions at 17 °C were 1.3-1.7 log10 higher than at 12 °C. After 3 weeks, reductions > 3.0 log10 were observed at 17 °C, while at 12 °C reductions did not exceed 1.9 log10. The length of depuration also had an influence on virus numbers. TuV reductions increased from < 1.0 log10 after seven days to > 4.0 log10 after 4 weeks. This implies that an extension of the depuration period to more than seven days, possibly in combination with elevated water temperatures, may be beneficial for the inactivation and removal of viral pathogens.

Seawater from Bergen harbor is a reservoir of conjugative multidrug-resistance plasmids carrying genes for virulence.

Aquatic environments play important roles in the dissemination of clinically-relevant antibiotic resistance genes (ARGs) and pathogens. Limited knowledge exists about the prevalence of clinically-relevant acquired resistance genes in the marine environment, especially in Norway. The aim of the current study was to investigate the presence of and characterize self-transmissible resistance plasmids from Bergen harbor seawater, with exogenous-plasmid capture, using a green fluorescent protein (GFP)-tagged Escherichia coli strain as a recipient. We obtained transconjugants resistant against ampicillin and cefotaxime from four of the 13 samples processed. Nine transconjugants, selected on the basis of antibiotic sensitivity patterns, were sequenced, using Illumina MiSeq and Oxford Nanopore MinION platforms. Ten different plasmids (ranging from 35 kb to 136 kb) belonging to incompatibility groups IncFII/IncFIB/Col156, IncFII, IncI1 and IncB/O/K/Z were detected among these transconjugants. Plasmid p1A1 (IncFII/IncFIB/Col156, 135.7 kb) carried resistance genes blaTEM-1, dfrA17, sul1, sul2, tet(A), mph(A), aadA5, aph(3″)-Ib and aph(6)-Id, conferring resistance against six different classes of antibiotics. Plasmid p1A4 carried blaCTX-M-55, lnu(F), aadA17 and aac(3)-IId. Cephalosporinase blaCMY-2 was detected on plasmids captured from an area impacted by wastewater from a local marine aquarium. Along with ARGs, some plasmids also carried virulence factors, such as enterotoxins, adhesion factors and siderophores. Our study demonstrates the presence of clinically-important multidrug-resistance conjugative plasmids in seawater from Bergen harbor, which have the potential to be transferred to human microbiota. The results highlight the need for surveillance of antibiotic resistance in the environment, as suggested by the World Health Organization, especially in low prevalence settings like Norway.

Sewage-based surveillance shows presence of Klebsiella pneumoniae resistant against last resort antibiotics in the population in Bergen, Norway.

The aim of this study was to understand the prevalence of antibiotic resistance in Klebsiella pneumoniae present in the population in Bergen city, Norway using city-scale sewage-based surveillance, as well as the potential spread of K. pneumoniae into the marine environment through treated sewage. From a total of 30 sewage samples collected from five different sewage treatment plants (STPs), 563 presumptive K. pneumoniae isolates were obtained on Simmons Citrate Agar with myo-Inositol (SCAI) plates, and 44 presumptive K. pneumoniae isolates on SCAI plates with cefotaxime. Colistin resistance was observed in 35 isolates, while cefotaxime resistance and tigecycline resistance was observed in only five isolates each, out of 563 presumptive K. pneumoniae isolates. All 44 isolates obtained on cefotaxime-containing plates were multidrug-resistant, with 25% (n = 11) showing resistance against tigecycline. Clinically important acquired antibiotic resistance genes (ARGs), like blaCTX-M-14, blaCTX-M-15, qnrS1, aac(3)-IIe, tet(A), and sul1, were detected in several sequenced Klebsiella spp. isolates (n = 53). All sequenced colistin-resistant isolates (n = 13) had a mutation in the mgrB gene with nucleotide substitution at position C88T creating a premature stop codon. All sequenced tigecycline-resistant isolates (n = 4) harbored a Tet(A) variant with 22 amino acid (aa) substitutions compared to the reference protein. The sequenced K. pneumoniae isolates (n = 44) belonged to 22 different sequence types (STs) with ST730 (29.5%) as most prevalent, followed by pathogenic ST307 (11.4%). Virulence factors, including aerobactin (iutA), enterobactin (entABCDEFS and fepABCDG), salmochelin (iro), and yersiniabactin (ybt) were detected in several sequenced K. pneumoniae isolates, suggesting pathogenicity potential. Heavy metal resistance genes were common in sequenced K. pneumoniae isolates (n = 44) with silver (silABCEFPRS) and copper (pcoABDRS) resistance genes present in 79.5% of the isolates. Sewage-based surveillance can be a useful tool for understanding antibiotic resistance in pathogens present within a population and to provide up-to date information on the current resistance situation. Our study presents a framework for population-based surveillance of resistance in K. pneumoniae.

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.

Insights into the genetic diversity, antibiotic resistance and pathogenic potential of Klebsiella pneumoniae from the Norwegian marine environment using whole-genome analysis.

Klebsiella pneumoniae (Kp) can cause hospital- and community acquired infections. Although, Kp is widespread in the environment, very little is known about the genetic diversity and pathogenicity of Kp from the marine environment. The aim of our study was to understand the genetic diversity, resistome and pathogenic potential of 87 Kp isolates from the Norwegian marine environment, using whole-genome sequencing. We identified 50 sequence types, including globally disseminated sequence types associated with multidrug resistance or hypervirulence. Ten isolates carried the yersiniabactin loci. Acquired antibiotic resistance genes were identified in six Kp isolates. Heavy metal resistance genes were widespread among the isolates, with 71% carrying genes encoding resistance to copper, silver, arsenic, nickel and/or mercury. Co-occurrence of antibiotic resistance genes and heavy metal resistance genes was seen in five Kp isolates. Phylogenetic analysis revealed a close genetic relationship between Kp 2016-1200 ST25 isolated from blue mussels (Mytilus edulis) and a clinical isolate reported in Germany. To the best of our knowledge, this study provides the first comprehensive account of genetic diversity among Kp from the marine environment. Our study reveals high diversity of Kp in the Norwegian marine environment and seafood, including globally disseminated pathogenic sequence types carrying clinically relevant antibiotic resistance genes and virulence factors, as well as several heavy metal resistance genes.

Microbiological Food Safety of Seaweeds.

The use of seaweeds in the human diet has a long history in Asia and has now been increasing also in the western world. Concurrent with this trend, there is a corresponding increase in cultivation and harvesting for commercial production. Edible seaweed is a heterogenous product category including species within the green, red, and brown macroalgae. Moreover, the species are utilized on their own or in combinatorial food products, eaten fresh or processed by a variety of technologies. The present review summarizes available literature with respect to microbiological food safety and quality of seaweed food products, including processing and other factors controlling these parameters, and emerging trends to improve on the safety, utilization, quality, and storability of seaweeds. The over- or misuse of antimicrobials and the concurrent development of antimicrobial resistance (AMR) in bacteria is a current worldwide health concern. The role of seaweeds in the development of AMR and the spread of antimicrobial resistance genes is an underexplored field of research and is discussed in that context. Legislation and guidelines relevant to edible seaweed are also discussed.

Draft Genome Sequence of Hafnia paralvei Strain VBC_1714, Isolated from Frozen Cod Fillet Imported from Russia to Norway.

Hafnia spp. have the potential to cause opportunistic infections in humans and animals. This announcement describes the draft genome sequence of an H2S-positive Hafnia paralvei strain that was isolated as a presumptive Salmonella sp. from a frozen cod fillet.

Draft Genome Sequence of Multidrug-Resistant Pseudomonas protegens Strain 11HC2, Isolated from Marine Plastic Collected from the West Coast of Norway.

We report the draft genome sequence of multidrug-resistant Pseudomonas protegens strain 11HC2, isolated from polypropylene collected from the water column near a beach in Øygarden, Norway. The genome sequence is 6,861,219 bp long, with a G+C content of 63.4%. Strain 11HC2 is resistant to cefotaxime, ampicillin, trimethoprim, and chloramphenicol.

Antibiotic Sensitivity Screening of Klebsiella spp. and Raoultella spp. Isolated from Marine Bivalve Molluscs Reveal Presence of CTX-M-Producing K. pneumoniae.

Klebsiella spp. are a major cause of both nosocomial and community acquired infections, with K. pneumoniae being responsible for most human infections. Although Klebsiella spp. are present in a variety of environments, their distribution in the sea and the associated antibiotic resistance is largely unknown. In order to examine prevalence of K. pneumoniae and related species in the marine environment, we sampled 476 batches of marine bivalve molluscs collected along the Norwegian coast. From these samples, K. pneumoniae (n = 78), K. oxytoca (n = 41), K. variicola (n = 33), K. aerogenes (n = 1), Raoultella ornithinolytica (n = 38) and R. planticola (n = 13) were isolated. The number of positive samples increased with higher levels of faecal contamination. We found low prevalence of acquired resistance in all isolates, with seven K. pneumoniae isolates showing resistance to more than one antibiotic class. The complete genome sequence of cefotaxime-resistant K. pneumoniae sensu stricto isolate 2016-1400 was obtained using Oxford Nanopore and Illumina MiSeq based sequencing. The 2016-1400 genome had two contigs, one chromosome of 5,088,943 bp and one plasmid of 191,744 bp and belonged to ST1035. The ß-lactamase genes blaCTX-M-3 and blaTEM-1, as well as the heavy metal resistance genes pco, ars and sil were carried on a plasmid highly similar to one found in K. pneumoniae strain C17KP0055 from South-Korea recovered from a blood stream infection. The present study demonstrates that K. pneumoniae are prevalent in the coastal marine environment and that bivalve molluscs may act as a potential reservoir of extended spectrum ß-lactamase (ESBL)-producing K. pneumoniae that may be transmitted through the food chain.

Vibrios from the Norwegian marine environment: Characterization of associated antibiotic resistance and virulence genes.

A total of 116 Vibrio isolates comprising V. alginolyticus (n = 53), V. metschnikovii (n = 38), V. anguillarum (n = 21), V. antiquarius (n = 2), and V. fujianensis (n = 2) were obtained from seawater, fish, or bivalve molluscs from temperate Oceanic and Polar Oceanic area around Norway. Antibiotic sensitivity testing revealed resistance or reduced susceptibility to ampicillin (74%), oxolinic acid (33%), imipenem (21%), aztreonam (19%), and tobramycin (17%). Whole-genome sequence analysis of eighteen drug-resistant isolates revealed the presence of genes like ß-lactamases, chloramphenicol-acetyltransferases, and genes conferring tetracycline and quinolone resistance. The strains also carried virulence genes like hlyA, tlh, rtxA to D and aceA, E and F. The genes for cholerae toxin (ctx), thermostable direct hemolysin (tdh), or zonula occludens toxin (zot) were not detected in any of the isolates. The present study shows low prevalence of multidrug resistance and absence of virulence genes of high global concern among environmental vibrios in Norway. However, in the light of climate change, and projected rising sea surface temperatures, even in the cold temperate areas, there is a need for frequent monitoring of resistance and virulence in vibrios to be prepared for future public health challenges.

Pharmacokinetic Data Show That Oxolinic Acid and Flumequine Are Absorbed and Excreted Rapidly From Plasma and Tissues of Lumpfish.

This study examined the uptake, tissue distribution and elimination of the antibacterial agents oxolinic acid and flumequine in lumpfish (Cyclopterus lumpus L.) by use of LC-MS/MS following a single oral administration of 25 mg/kg fish given in feed. Lumpfish are increasingly used as cleaner fish for removal of sea lice on commercially farmed salmon. The production of lumpfish is successful, but there are challenges with bacterial infections and the number of antibacterial treatments has increased in recent years. As the lumpfish is a novel species to farming, there is a need for pharmacokinetic data and establishment of protocols for efficient antibacterial treatment. The current study describes the pharmacokinetic properties of oxolinic acid and flumequine in lumpfish. Absorption of oxolinic acid was moderate and was characterized by a calculated peak plasma concentration (Cmax) of 2.12 µg/ml after 10.3 h (Tmax) and an elimination half-life (t1/2ß) of 21 h. Area under curve (AUC) and AUC from 0 to 24 h (AUC0-24h) were calculated to be 60.9 and 34.0 h µg/ml, respectively. For flumequine, plasma Cmax was found to be 2.77 µg/ml after 7.7 h (Tmax) with t1/2ß of 22 h. The area under the curve (AUC) and AUC from 0 to 24 h (AUC0-24) were calculated as 104.3 and 50.3 h µg/ml, respectively. Corresponding Cmax values in muscle, liver, and head-kidney for oxolinic acid were 4.01, 3.04, and, 4.68 µg/g, respectively and Tmax of 11.1, 9.2, and 10.0 h, respectively. For flumequine, Cmax values of 4.16, 4.01, and 7.48 µg/g were obtained in muscle, liver, and head kidney, respectively, with corresponding Tmax values of 10.2, 10.3, and 6.0 h. Antimicrobial susceptibility values as determined by minimum inhibitory concentration (MIC) analyses against 28 isolates of Aeromonas salmonicida isolated from diseased lumpfish ranged from 0.06 to 15 µg/ml for oxolinic acid and 0.024 to 6.25 µg/ml for flumequine. Bimodal distributions in susceptibility to both oxolinic acid and flumequine were observed. The combination of pharmacokinetic properties and MIC data make possible calculation of efficient treatment doses, which are needed to improve the welfare of lumpfish and minimize development of antibiotic resistant bacteria.

Isolation and characterisation of Shiga toxin-producing Escherichia coli from Norwegian bivalves.

Only a few studies concerning Shiga toxin-producing E. coli (STEC) detection in bivalves and their harvesting areas have been reported, and to the best of our knowledge there are no outbreaks associated with STEC from bivalves described. The aim of the present study was to investigate the occurrence of STEC in Norwegian bivalves, and to characterize potential STEC isolated from the samples. A total of 269 samples of bivalves were screened for the presence of stx and eae genes, and markers for the serogroups O26, O103, O111, O145 and O157 by using ISO TS 13136 (2012). The screening returned 19 samples that were positive for stx and eae, and attempts of isolation of STEC were made from these samples. Presumptive STEC were obtained from three samples, and three isolates (one from each sample) were subjected to whole-genome-sequencing (WGS). The WGS revealed that one of the isolates did not carry the stx genes, while the other two were identified as stx2i positive E. coli O9:H19 and stx2g positive E. coli O96:H19. Neither of the two STEC isolates were positive for virulence markers such as eae and ehx. The results suggest that the occurrence of STEC in Norwegian bivalves is low.

Assessment of food quality and microbial safety of brown macroalgae (Alaria esculenta and Saccharina latissima).

BACKGROUND: There has been a rapid increase in the number of seaweed farms in the Western world, and it is crucial for these companies and their customers to have standardized methods for quality assessment and optimization. The aim of this study was to adapt known methods for food-quality determination for the analysis of seaweed quality, including color, texture, and microbiology, and to discuss optimal heat treatments for the popular macroalgae Saccharina latissima and Alaria esculenta. RESULTS: The development of an attractive, green color during heating was highly specific to species, freezing history, and part of the thallus. Resilience and thermostability were also species dependent. Low microbial numbers (1-3 log cfu/g) for total aerobic count, psychrotrophic bacteria, and spore-forming bacteria were found, but Bacillus spp. were isolated. No enterococci, coliforms, pathogenic vibrios, or Listeria monocytogenes were detected. CONCLUSION: The methods employed were able to describe clearly the physical and microbial qualities of A. esculenta and S. latissima, and quality changes during processing. Based on the results, optimal cooking for a minimum of 15 min at 95 °C was suggested for S. latissima. Fresh and frozen A. esculenta showed the greenest color after heating for 5-9 s at a high temperature (> 85 °C). If a higher heat load is needed to achieve safe and stable food products, using fresh and not frozen A. esculenta is highly recommended, as fresh specimens remain green even after 15 min at 95 °C. © 2018 Society of Chemical Industry.

A comparative study of quality and safety of Atlantic cod (Gadus morhua) fillets during cold storage, as affected by different thawing methods of pre-rigor frozen headed and gutted fish.

BACKGROUND: The catch of marine whitefish is typically seasonal, whereas the land-based processing industry has a need for all-year stable supply of raw materials. This challenge can be met by applying fish frozen at sea. When using frozen fish, the methods employed for thawing may influence the safety and quality of the final product. This study aimed to investigate the applicability of novel thawing strategies in order to provide an all-year supply of high-quality and safe cod products. RESULTS: Comparative investigations of quality and safety factors after thawing in water, with and without air circulation, and contact thawing were performed. The parameters included water-holding capacity, thawing loss, drip loss, cooking yield, sensory evaluation and microbiological analyses (including total volatile bases nitrogen). Water thawing with air circulation provided faster thawing than water thawing without air circulation and contact thawing. For all three methods, the quality of the thawed fish was acceptable and the shelf life of the fillets during chilled storage was between 10 and 14 days post-filleting. CONCLUSION: The results show that controlled freezing of cod, followed by appropriate thawing, may provide the processing industry with an all-year delivery of raw materials, without compromising quality and safety of the final product. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The species accuracy of the Most Probable Number (MPN) European Union reference method for enumeration of Escherichia coli in marine bivalves.

Continuous European Union programmes with specified methods for enumeration of Escherichia coli in bivalves for human consumption are currently running. The objective of this research was to examine the species accuracy of the five times three tube Most Probable Number (MPN) EU reference method used for detection of E. coli in marine bivalves. Among 549 samples of bivalves harvested from Norwegian localities during 2014 and 2015, a total number of 200 bacterial isolates were prepared from randomly selected culture-positive bivalves. These presumptive E. coli isolates were characterized biochemically by the Analytical Profile Index (API) 20E, as well as by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). The majority of isolates (90%) were identified as E. coli, by both API 20E and MALDI-TOF MS. Ten isolates (5%) were identified as Klebsiella pneumoniae, while one isolate was identified as K. oxytoca by both methods, whereas three isolates were identified as Acinetobacter baumannii, Citrobacter braakii, and Enterobacter cloacae, respectively. The identification of the remaining six isolates were not in compliance between the two methods.