Multidrug-resistant Enterococcus faecium strains enter the Norwegian marine environment through treated sewage.

This study aimed to understand the antibiotic resistance prevalence among Enterococcus spp. from raw and treated sewage in Bergen city, Norway. In total, 517 Enterococcus spp. isolates were obtained from raw and treated sewage from five sewage treatment plants (STPs) over three sampling occasions, with Enterococcus faecium as the most prevalent (n = 492) species. E. faecium strains (n = 307) obtained from the influent samples, showed the highest resistance against quinupristin/dalfopristin (67.8%). We observed reduced susceptibility to erythromycin (30.6%) and tetracycline (6.2%) in these strains. E. faecium strains (n = 185) obtained from the effluent samples showed highest resistance against quinupristin/dalfopristin (68.1%) and reduced susceptibility to erythromycin (24.9%) and tetracycline (8.6%). We did not detect resistance against last-resort antibiotics, such as linezolid, vancomycin, and tigecycline in any of the strains. Multidrug-resistant (MDR) E. faecium strains were detected in both influent (2.3%) and effluent (2.2%) samples. Whole genome sequencing of the Enterococcus spp. strains (n = 25) showed the presence of several antibiotic resistance genes, conferring resistance against aminoglycosides, tetracyclines, and macrolides, as well as several virulence genes and plasmid replicons. Two sequenced MDR strains from the effluents belonged to the hospital-associated clonal complex 17 and carried multiple virulence genes. Our study demonstrates that clinically relevant MDR Enterococcus spp. strains are entering the marine environment through treated sewage.

Complete sequence of a new conjugative multidrug-resistance encoding IncFII/IncFIA/IncFIB plasmid carrying NDM-6 metallo-ß-lactamase from pathogenic Escherichia coli sequence type 167 isolated from sewage in Norway.

OBJECTIVES: The aim of the current study was to determine the genomic map of resistance genes and to understand the potential for mobility of a new NDM-6-carrying plasmid from a pathogenic Escherichia coli strain. A complete and closed genome sequence of the E. coli strain was obtained by applying a combination of short-read Illumina and long-read Nanopore-based sequencing. METHODS: Isolation of E. coli was performed, using ECC CHROMagar™, and antibiotic sensitivity patterns were determined, using Sensititre™ EUVSEC3 plates. Whole-genome sequencing was performed, using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed, using filter-mating and a green fluorescent protein (GFP)-tagged E. coli strain. RESULTS: Two carbapenem-resistant E. coli strains were isolated from sewage. These strains (2-331 and 2-333) belonged to sequence type (ST) 167 and carried an NDM-6 carbapenemase. The complete genome of strain 2-331 (GenBank accession no.: CP110117-22.1) was assembled into six contigs, representing a complete circular chromosome of 4 947 178 bp and five plasmids, ranging from 143 596 bp to 1549 bp. Plasmid p2-331_1 (∼144 kb), belonging to the IncFII/IncFIA/IncFIB group, carried multiple antibiotic resistance genes, including mph(A), mrx(A), blaTEM-1, rmtB1, blaNDM-6, ble, sul1, qacEΔ1, aadAΔ, dfrA12, and tet(A). Plasmid p2-331_1 was transferred from strain 2-331, via conjugation, conferring resistance against eight different classes of antibiotics to a GFP-tagged E. coli strain. CONCLUSIONS: Our study showed the emergence of a new conjugative plasmid-carrying NDM-6 carbapenemase from pathogenic E. coli ST167 for the first time in Norway. The importance of population-based sewage-surveillance for understanding the antimicrobial resistance situation within the community is highlighted.

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.

Resistance profiles and diversity of ß-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 ß-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 ß-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.

First report of mobile tigecycline resistance gene tet(X4)-harbouring multidrug-resistant Escherichia coli from wastewater in Norway.

OBJECTIVES: The mobile tigecycline resistance gene tet(X4), conferring resistance to all tetracyclines, is largely reported from China, however the global spread of such a novel resistance mechanism is a concern for preserving the efficacy of these last-resort antibiotics. The aim of our study was to determine the genetic basis of resistance in a tigecycline-resistant Escherichia coli strain (2-326) isolated from sewage in Bergen, Norway, using whole-genome sequencing (WGS). METHODS: WGS was carried out using Illumina MiSeq-based sequencing. In vitro conjugation assays were performed to determine the potential of isolate 2-326 to transfer tigecycline resistance to other strains. RESULTS: Escherichia coli isolate 2-326 belongs to pathogenic sequence type 167 (ST167) and carries several clinically important antibiotic resistance genes including tet(X4), blaCTX-M-14, dfrA12, sul2, qnrS1 as well as several aminoglycoside resistance genes. Tigecycline resistance along with resistance to tetracycline, sulfamethoxazole, chloramphenicol and azithromycin was transferred to green fluorescent protein (GFP)-encoding E. coli strain CV601-GFP by conjugation. CONCLUSION: To the best of our knowledge, this is the first report of E. coli carrying mobile tet(X4) gene from Norway. Our study demonstrates the ongoing spread of new mechanisms of resistance against last-resort antibiotics and the need for surveillance of such resistance factors in the population in order to mitigate their spread.

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.

Nanopore sequencing reveals genomic map of CTX-M-type extended-spectrum ß-lactamases carried by Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway.

BACKGROUND: Environmental surveillance of antibiotic resistance can contribute towards better understanding and management of human and environmental health. This study applied a combination of long-read Oxford Nanopore MinION and short-read Illumina MiSeq-based sequencing to obtain closed complete genome sequences of two CTX-M-producing multidrug-resistant Escherichia coli strains isolated from blue mussels (Mytilus edulis) in Norway, in order to understand the potential for mobility of the detected antibiotic resistance genes (ARGs). RESULTS: The complete genome sequence of strain 631 (E. coli sequence type 38) was assembled into a circular chromosome of 5.19 Mb and five plasmids (between 98 kb and 5 kb). The majority of ARGs cluster in close proximity to each other on the chromosome within two separate multidrug-resistance determining regions (MDRs), each flanked by IS26 transposases. MDR-1 carries blaTEM-1, tmrB, aac(3)-IId, aadA5, mph(A), mrx, sul1, qacEΔ1 and dfrA17; while MDR-2 harbors aph(3″)-Ib, aph(6)-Id, blaTEM-1, catA1, tet(D) and sul2. Four identical chromosomal copies of blaCTX-M-14 are located outside these regions, flanked by ISEc9 transposases. Strain 1500 (E. coli sequence type 191) exhibited a circular chromosome of 4.73 Mb and two plasmids (91 kb and 4 kb). The 91 kb conjugative plasmid belonging to IncI1 group carries blaCTX-M-15 and blaTEM-1 genes. CONCLUSION: This study confirms the efficacy of combining Nanopore long-read and Illumina short-read sequencing for determining complete bacterial genome sequences, enabling detection and characterization of clinically important ARGs in the marine environment in Norway, with potential for further dissemination. It also highlights the need for environmental surveillance of antibiotic resistance in low prevalence settings like Norway.

Morphological and molecular identification of a new Kudoa thyrsites isolate in Mediterranean silver scabbardfish Lepidopus caudatus.

Myxozoans of the genus Kudoa (Myxosporea, Multivalvulida) infect marine and estuarine fish species worldwide. Some Kudoa species are of concern to the seafood industry since they may generate macroscopic cysts in the fish host's musculature, or cause post mortem myoliquefaction, commonly known as 'soft flesh'. One of the economically most important species is K. thyrsites, a myoliquefactive myxosporean parasite that occurs in many wild and cultured marine fish species worldwide. Here we identified a K. thyrsites isolate as the causative agent of myoliquefaction in silver scabbardfish Lepidopus caudatus from the Alboran Sea (western Mediterranean Sea). For comparative and validation purposes, the morphological and molecular characteristics of the isolate were compared with fresh spores of a K. thyrsites isolate infecting Atlantic mackerel Scomber scombrus from the Norwegian Sea. Myxospores of both isolates shared a stellate appearance and contained 4 unequal pyriform polar capsules (1 large, 1 small and 2 intermediate). These morphological traits were consistent with all other previously described K. thyrsites isolates. Moreover, the small subunit rDNA sequences of the Mediterranean and Norwegian Sea isolates revealed 100% similarity, and matched 100% with K. thyrsites isolates previously recorded in myoliquefactive Atlantic mackerel from the North Sea and off southern England. The findings suggest that K. thyrsites is the primary cause of myoliquefaction in silver scabbardfish from the Alboran Sea. This report represents the first morphological and molecular characterization of K. thyrsites in the Mediterranean Sea. A set of new allometric characters is proposed as additional descriptors for more accurate and specific description of kudoid myxospores.

Marine Bivalve Mollusks As Possible Indicators of Multidrug-Resistant Escherichia coli and Other Species of the Enterobacteriaceae Family.

The mechanisms for the development and spread of antibacterial resistance (ABR) in bacteria residing in environmental compartments, including the marine environment, are far from understood. The objective of this study was to examine the ABR rates in Escherichia coli and other Enterobacteriaceae isolates obtained from marine bivalve mollusks collected along the Norwegian coast during a period from October 2014 to November 2015. A total of 549 bivalve samples were examined by a five times three tube most probable number method for enumeration of E. coli in bivalves resulting in 199 isolates from the positive samples. These isolates were identified by biochemical reactions and matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, showing that 90% were E. coli, while the remaining were species within the genera Klebsiella, Citrobacter, and Enterobacter. All 199 isolates recovered were susceptibility tested following the European Committee on Antimicrobial Susceptibility Testing disk diffusion method. In total, 75 of 199 (38%) isolates showed resistance to at least one antibacterial agent, while multidrug-resistance were seen in 9 (5%) isolates. One isolate conferred resistance toward 15 antibacterial agents. Among the 75 resistant isolates, resistance toward extended-spectrum penicillins (83%), aminoglycosides (16%), trimethoprim (13%), sulfonamides (11%), tetracyclines (8%), third-generation cephalosporins (7%), amphenicols (5%), nitrofurans (5%), and quinolones (5%), were observed. Whole-genome sequencing on a selection of 10 E. coli isolates identified the genes responsible for resistance, including blaCTX-M genes. To indicate the potential for horizontal gene transfer, conjugation experiments were performed on the same selected isolates. Conjugative transfer of resistance was observed for six of the 10 E. coli isolates. In order to compare E. coli isolates from bivalves with clinical strains, multiple-locus variable number tandem repeats analysis (MLVA) was applied on a selection of 30 resistant E. coli isolates. The MLVA-profiles were associated with community-acquired E. coli strains causing bacteremia. Our study indicates that bivalves represent an important tool for monitoring antibacterial resistant E. coli and other members of the Enterobacteriaceae family in the coastal environment.