Listeria monocytogenes in salmonid slaughter facilities - Screening program for the Norwegian Food Safety Authority

Listeria monocytogenes is a bacterium of special concern in the seafood industry beacuse it possesses several properties adapted for the food production environment and may cause severe disease in humans. The Institute of Marine Research has on behalf of the Norwegian Food Safety Authority conducted a screening program for L. monocytogens in salmonid slaughter facilities. In total, 358 samples from 60 slaugher facilities (49 slaughtering plants and 11 slaughtering vessels) were examined. Samples were collected from the production environment (n=108), from the surface of fish entering the facilities (n=47), and from the surface of fish (n=59) and raw material (n=144) at end point at the examined facility. None of the samples from the slaughtering vessels were positive for L. monocytogenes, whereas 22 positive samples were detected in nine different slaugtering plants. In five of these plants, several positive samples were found. Six of the slaughtering plants had positive samples at the end of the production line, where a higher prevalence was found when swabbing the fish skin and gills compared to the examined raw matierial. All the raw material samples contained low numbers of L. monocytogenes below the quatification limit (<10 CFU/g). However, this study found that L. monocytogenes can be present in both fish and the production environment, and that in some cases L. monocytogenes will be present in fish ready for furhter processing in the salmonid value chain.publishedVersio

Resistance profiles and diversity of β-lactamases in Escherichia coli strains isolated from city-scale sewage surveillance in Bergen, Norway mimic clinical prevalence

The aim of this study was to examine antibiotic resistance profiles and diversity of β-lactamases in Escherichia coli present within the population and the potential spread of resistant E. coli into the receiving environment using city-scale sewage surveillance. In E. coli isolates from ECC plates without antibiotics from ten influent samples (n = 300), highest resistance was observed against ampicillin (16.6%), sulfamethoxazole (9.7%) and trimethoprim (9.0%), while in effluent samples (n = 262) it was against sulfamethoxazole (11.8%), ampicillin (11.5%) and tetracycline (8.8%). All isolates (n = 123) obtained on cefotaxime-containing plates were multidrug-resistant. Several clinically important antibiotic resistance genes (ARGs) were detected in 46 E. coli isolates subjected to whole-genome sequencing, including carbapenemases like NDM-6, VIM-1 and OXA-48-variant, as well as tigecycline resistance gene tet(X4). CTX-M-15 was the most prevalent (42.9%) extended-spectrum β-lactamase among cefotaxime-resistant isolates, followed by CTX-M-27 (31.4%) and CTX-M-14 (17.1%), resembling clinical prevalence in Norway. Most of the sequenced isolates carried other clinically relevant ARGs, such as dfrA17, sul1, sul2, tet(A), aph(6)-Id, aph(3’’)-Ib and aadA5. Sixteen different sequence types (STs) were identified, including ST131 (39.1%), ST38 (10.9%) and ST69 (8.7%). One E. coli isolate belonging to novel ST (ST11874) carried multiple virulence factors including genotoxin, salmochelin, aerobactin and yersiniabactin, suggesting that this isolate has potential to cause health concerns in future. Our study reveals presence of clinically relevant ARGs like blaNDM-6 and tet(X4) in pathogenic strains, which have so far not been reported from the clinics in Norway. Our study may thus, provide a framework for population-based surveillance of antibiotic resistance.publishedVersio

Tilsynsprogrammet for skjell 2011 - fremmedstoffer (tungmetaller og organiske miljøgifter i skjell og tungmetaller i snegler) og mikroorganismer.

I den mikrobiologiske delen av tilsynsprogrammet for skjell, ble det i 2011 tatt ut i alt 384 prøver fordelt gjennom hele året. Av disse var 312 prøver av blåskjell (Mytilus edulis), 40 av kamskjell (Pecten maximus), 21 av østers (Ostrea edulis), fire av oskjell (Modiolus modiolus) og syv av strandsnegl (Littorina littorea). Prøvene ble sendt til Nasjonalt institutt for ernærings- og sjømatforskning (NIFES) i henhold til instruks utarbeidet av Mattilsynet, Seksjon for fisk og sjømat. Ved NIFES ble alle prøvene analysert for E. coli og til sammen 369 av prøvene ble analysert for enterokokker. Til sammen 38 av prøvene ble også analysert med hensyn på Salmonella. Antall E. coli ble bestemt ved en flerrørs fortynningsmetodikk (MPN) i henhold til EUs referansemetode (Donovans metode, ISO ), mens enterokokker ble bestemt ved hjelp av NMKL metode nr. 68, 5. utgave 2011: Enterococcus, bestemmelse i næringsmidler. Prøvene ble analysert med hensyn på forekomst av Salmonella ved hjelp av en automatisert immunologisk metodikk (ELFA, Vidas Salmonella), kombinert med konvensjonell dyrkning og verifikasjon for eventuelle positive prøver. Innholdet av E. coli var under 230/100 g i 323 (84 %) av de 384 prøvene som ble undersøkt, og kom dermed under EUs grenseverdi for klassifisering av dyrkingslokaliteter til såkalte A-områder. De øvrige 61 prøvene (16 %) hadde innhold av E. coli over 230/100 g. Høyeste avleste konsentrasjoner av E. coli for blåskjell var på over /100 g, for kamskjell 750 /100 g, for østers 3 500/100 g, for oskjell 20 /100 g og for strandsnegl 2 400/100 g. Av prøvene som ble undersøkt med tanke på E. coli, var 49 fra sluttprodukter, og fordelte seg med 31 av kamskjell, 12 av blåskjell, fire av oskjell og to av strandsnegl. Av disse hadde en prøve E. coli i konsentrasjoner over 230/100g. Dette var en prøve av blåskjell tatt ut i Sogn og Fjordane, der påvist mengde var /100g. Enterokokker kunne påvises i fem av de 369 undersøkte prøvene. Konsentrasjonene for fire av de positive prøvene var lik påvisningsgrensen, som for denne parameteren er 100 enterokokker/g skjellmat, mens én prøve hadde 200 enterokokker/g skjellmat. Alle positive prøver var av blåskjell. Det ble ikke funnet Salmonella-bakterier i noen av de 38 undersøkte prøvene fra tilsynsprogrammet. I tillegg til prøver innsendt av Mattilsynet, som en del av dette overvåkingsprogrammet, ble det også gjennomført mikrobiologisk analyse av 96 prøver innsendt av næringen. Innholdet av E. coli var under 230/100 g i 80 av disse prøvene (83 %). De øvrige 16 prøvene (17 %) hadde innhold av E. coli over 230/100 g. Høyeste avleste konsentrasjoner av E. coli for blåskjell var på /100 g og for hjerteskjell 9 100/100 g. Enterokokker kunne påvises i en av 18 undersøkte prøver. Prøven var av blåskjell og konsentrasjonen var lik påvisningsgrensen på 100 enterokokker/g skjellmat. Det ble ikke funnet Salmonella-bakterier i noen av de 5 undersøkte prøvene innsendt av næringen.Tilsynsprogrammet for skjell 2011 - fremmedstoffer (tungmetaller og organiske miljøgifter i skjell og tungmetaller i snegler) og mikroorganismer.publishedVersio

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.publishedVersio

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 β-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 β-lactamase (ESBL)-producing K. pneumoniae that may be transmitted through the food chain.publishedVersio

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

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Kartlegging av antibiotikaresistente bakteriar i marine skjel

På oppdrag for Miljødirektoratet, har HI undersøkt førekomst av antibiotikaresistente bakteriar i marine skjel samla inn langs Norskekysten i perioden frå juni til august 2017. Det vart undersøkt 26 parti med skjel frå i alt 18 lokalitetar med ulik grad av menneskeleg innverknad. Totalt 252 bakterieisolat frå desse prøvane vart undersøkte for fenotypisk antibiotikaresistens, samt resistens mot eit utval tungmetall med kjende resistensdrivande eigenskapar (koppar, sink og kadmium). Bakterieisolata som vart dyrka fram utan antibiotika tilsett i vekstmediet og seinare undersøkt for resistens, gav innsyn i den generelle førekomsten av resistenseigenskaper både hos bakteriar som høyrer heime i havet og hos bakteriar tilført frå andre kjelder. Det vart og dyrka fram bakterieisolat med medier tilsett antibiotika for å fremje vekst av antibiotikaresistente bakteriar og for å betre kunne undersøkje koplinga mellom antibiotikaresistens og tungmetallresistens.publishedVersio

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.publishedVersio

Microbiological aspects of fish handling and processing in the Norwegian pelagic sector

Most global marine fish resources seem to have reached their limit of sustainable exploitation, whereas the demand for high quality fish products is still increasing. The fish sector needs to comply with the growing market by optimising the utilization of fish that have been harvested already, as approx. 10 - 25% of the total global wild-captured fish are rejected from the market. Microorganisms play an important role in product quality reduction, and an increased knowledge of the bacteria associated with fish, is needed. The overall aim of the work included in this thesis was to describe the bacteria associated with pelagic fish, to better utilise the fish and ensure quality and safety. The indigenous bacterial biota of gills, skin and intestinal content from Atlantic mackerel (Scomber scombrus), as well as in the intestinal content of blue whiting (Micromesistius poutassou), were described by cultivation and PCRDGGE methods. These samples were dominated by Gram-negative bacteria. The frequently occurring parasitic nematode larva of the genera Anisakis enters the fish through the intestine. The bacterial biota of these larvae was therefore expected to reflect the bacterial biota found in the fish intestine, and the shared bacterial genera comprised of Pseudoalteromonas, Psychrobacter, Photobacterium, and Pseudomonas. The latter two are known to cause spoilage in fresh fish. During capture, landing and processing, the fish become exposed to extrinsic factors in the production environment that might affect the bacterial biota and possibly the quality of the fish. A spot sampling programme was performed at fishing vessels and fish processing factories over a ten years period (2005-2014). The microbiological conditions of fish, surfaces of equipment and water samples was analysed by quality, hygiene and safety parameters. Samples were assessed according to a proposed assessment scheme, and good hygiene and safety conditions were found at 19 of 41 samplings. Contamination by the human pathogen Listeria monocytogenes was detected in fish from three samplings, and on equipment at nine samplings. One sampling on board a fishing vessel showed contamination by E. coli in the refrigerated sea water (RSW) tanks and on the fish, and this is of concern, since any contamination that occur early in the production chain might follow the fish throughout the process. The effect of pumping of fish on board vessels was examined by comparing samples of Atlantic mackerel collected from the purse seine and the RSW tanks, 12 hour post capture. During the pumping intestinal content is forced out of the fish, contaminating the outer surfaces such as gills and skin of the surrounding fish. This is shown by the increased bacterial load on Iron Agar Lyngby (IAL) found in gill- and skin samples of fish collected from the RSW tank. Additionally, the genera Vibrio and Oceanisphaera were only identified in intestine samples from fish collected from the purse seine, but in all samples from fish collected from the RSW tank. The muscle-invading Anisakis larvae were suspected to carry bacteria into the assumed sterile fish muscle. The presence of bacteria in larvae excised from fish muscle, was confirmed by Gram-stained histology sections. Samples from blue whiting muscle, with and without Anisakis larvae, were cultured and showed that the parasite acts as a contaminating vector, increasing the bacterial load in the fish muscle. However, these bacteria are not likely to be spread within the fish muscle unless the fish is processed in a way that disrupt the capsule and the parasite, as for instance during fish mincing. A follow-up study included a 15-days long storage trial with fish mince inoculated with an Anisakis homogenate. The study gave surprisingly positive results, as the heterotropic plate count (HPC) were lower and the shelf-life was prolonged when the fish mince was added the Anisakis-homogenate. These results show that fish that are infected with parasite larvae, should not necessarily be excluded as a food resource

Effect of Anisakis simplex (sl) larvae on the spoilage rate and shelf-life of fish mince products under laboratory conditions

Wild caught marine fish are commonly infected with anisakid nematodes lodging in the intestinal linings or in the fish muscle. One of the most commonly found nematode parasites in marine fish is Anisakis simplex. During production of mince from the muscle of wild caught Anisakis-infected fish, the larvae would be disrupted during mince production. Any bacteria within or on the surface of such larvae are during the mincing process evenly distributed throughout the mince, and could thus possibly affect the spoilage rate of the final products. To explore if or how any bacteria associated with muscle-invading Anisakis larvae may affect the spoilage rate of fish mince, a controlled storage trial was conducted. Fillets of farmed Atlantic cod (Gadus morhua), exclusively fed on dried and heat-treated compound feed and hence expectably free from Anisakis larvae, were aseptically collected and homogenised. Fish mince aliquots were added different volumes of Anisakis homogenate based on larvae which were freshly sampled from the visceral cavity of NE Atlantic blue whiting (Micromesistius poutassou). The volumes of added parasite homogenate (parasite(+)-samples) reflected different infection intensities from 15 (low) to 50 (high) larvae per 100 g fish fillet, representing an actual Anisakis intensity range in the flesh of blue whiting. The samples were kept at 4 °C for 15 days and subjected to microbiological, sensory and chemical evaluation at 3 days intervals. Upon visual examination and plate count measurements (PC) on Iron Agar Lyngby (IAL), the samples without any parasite additives (no[parasite]) spoiled differently and more rapidly than any of the parasite(+)-samples. However, H2S-producing bacteria were only recorded in the latter samples, which were also the only ones that showed increased levels of the spoilage indicator substance trimethylamine (TMA). Moreover, the parasite(+)-samples changed their sensory characteristics at a later stage compared to the no[parasite]-samples. Although some cultures of H2S-producing bacteria were found on IAL, molecular identification by PCR-DGGE of the actual bacteria was not conclusive. Psychrobacter sp. which has no or only little spoilage activity, was identified in all samples until trial day 9, but was probably outgrown by the stronger spoilers Pseudomonas fluorescence/fragi and Photobacterium phosphoreum. Thus, and somewhat unexpected, our findings indicate that – under the present trial conditions – fish mince contaminated with bacteria which originate from Anisakis larvae, spoiled less rapidly than samples without any parasite-related bacteria present. Moreover, the shelf-life of fish mince was apparently not reduced by the presence of bacteria transferred to the mince by Anisakis larvae