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.

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.

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.

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.

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.

An Outbreak of Norovirus Infection from Shellfish Soup Due to Unforeseen Insufficient Heating During Preparation.

Norovirus causes large outbreaks involving all age groups and are considered the most common cause of infectious foodborne diseases worldwide. The aim of this study was to describe a norovirus outbreak connected to insufficient heat treatment during preparation of a shellfish soup in serving portions, during a company Christmas celebration in Norway, December 2013. A questionnaire sent to the employees, showed that 67 % (n = 43) of the celebration participants, reported gastrointestinal symptoms including stomach pain, vomiting, diarrhoea and light fever in the period between 24 and 48 h post celebration. Several dishes were served, including shellfish soup made with carpet shell clams (Tapes rhomboides) in porcelain cups. Consuming this soup, was the only significant risk factor for infection. Norovirus GI and GII were detected in the remaining raw shellfish. To mimic the time and temperature obtained during bivalve soup preparation, raw chopped shellfish tissue and raw cepa onion were added in porcelain cups tempered to 20 °C. To each of these cups, boiling soup base was added. The temperature in the shellfish tissue was continuously recorded, and showed a maximum of 49 °C in the period between 3 and 7 min after adding the boiling soup base. After 1 h the temperature was 30 °C. This time and temperature combination was obviously not sufficient for inactivation of norovirus present in the shellfish tissue. In conclusion, the heat-absorbing capacity of cold ingredients, utensils and table wear porcelain should not be underestimated during food production. Consumers who want to avoid eating raw shellfish, should not assume that the shellfish tissue in preparation as described in our study is adequately heat treated.

Microbiological water examination during laboratory courses generates new knowledge for students, scientists and the government.

Contaminated water is globally the main vehicle for microbial pathogens in most regions. Teaching future microbiologist and employees in the food industry on the importance of hygienically satisfactory water, microbiological analyses and how to ensure good water quality and safety is highly relevant. This paper presents a complete experimental design for water analyses as a tool to teach students the methods and other key elements in microbiology, including food safety, environmental dissemination and survival of microorganisms, laboratory practices, water legislation and critical evaluation of results. All results from the last 10 classes (2006-2015) in a university course on seafood microbiology have been compiled and are presented here. Questionnaires used with former students reveal that the laboratory course is highly appreciated, and that many students remember important aspects of the water analyses, even after several years. The questionnaire results were consistent with our perception that some students find calculation of dilutions difficult to comprehend.

Microbiological assessment along the fish production chain of the Norwegian pelagic fisheries sector--Results from a spot sampling programme.

Microbes play an important role in the degradation of fish products, thus better knowledge of the microbiological conditions throughout the fish production chain may help to optimise product quality and resource utilisation. This paper presents the results of a ten-year spot sampling programme (2005-2014) of the commercially most important pelagic fish species harvested in Norway. Fish-, surface-, and storage water samples were collected from fishing vessels and processing factories. Totally 1,181 samples were assessed with respect to microbiological quality, hygiene and food safety. We introduce a quality and safety assessment scheme for fresh pelagic fish recommending limits for heterotrophic plate counts (HPC), thermos tolerant coliforms, enterococci and Listeria monocytogenes. According to the scheme, in 25 of 41 samplings, sub-optimal conditions were found with respect to quality, whereas in 21 and 9 samplings, samples were not in compliance concerning hygiene and food safety, respectively. The present study has revealed that the quality of pelagic fish can be optimised by improving the hygiene conditions at some critical points at an early phase of the production chain. Thus, the proposed assessment scheme may provide a useful tool for the industry to optimise quality and maintain consumer safety of pelagic fishery products.

Characterisation of the microbiota of Atlantic mackerel (Scomber scombrus).

In this study the microbiota of Atlantic mackerel (Scomber scombrus) collected by a commercial purse seiner was examined. Fish were collected directly from the purse seine and from the Refrigerated Sea Water (RSW) transport tank after loading. The culturable microbiota and Specific Spoilage Bacteria (SSB) were quantified on Iron Agar Lyngby (IAL) and identified using commercially available Biochemical API® kits on pure cultured isolates. These kits showed to be sub-optimal in characterising the isolates, since only half of the strains were identified. The same isolates were also identified by a nucleic acid based PCR-DGGE approach, and only half of the sequences gave the same results as the API®. Characterisation by PCR-DGGE was also performed on bacterial DNA from IAL plates (bulk cell samples) and on samples where the bacterial DNA was extracted directly from fish material without any cultivation (direct DNA samples). The microbiota of Atlantic mackerel was dominated by members of the Gram-negative genera as Psychrobacter sp., Proteus sp., Photobacterium sp., Vibrio sp., Shewanella sp., Synechococcus sp., Oceanisphaerae sp., Bizonia sp., Pseudoalteromonas sp., and members of Flavobacteriaceae. Gram-positive bacteria in the genera Vagococcus sp., Bacillus sp., Mycobacterium sp., Staphylococcus sp., Mycoplasma sp. and Clostridia sp. were also found. Examination by PCR-DGGE and sequencing of the bulk cell pellet after cultivation on IAL, gave a higher number of taxa as compared to extraction and examination of bacterial DNA from fish materials without prior cultivation. This shows the benefit of combining both culture dependent and culture independent methods, when studying the microbiota of marine fish. Several Vibrio spp. were found only in gut samples collected from the purse seine, but in all samples including the skin and the gills collected from the RSW tank, indicating microbial contamination by faecal bacteria from the fish under these transport conditions.