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

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.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

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24−28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 μg ml−1), gentamicin (≤4 μg ml−1), oxytetracycline (≤0.25 μg ml−1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 μg ml−1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cutoff values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.publishedVersio