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.

Tigecycline-resistant Klebsiella pneumoniae strains from sewage in Norway carry heavy-metal resistance genes encoding conjugative plasmids.

OBJECTIVES: Tigecycline is a last-resort antibiotic used for treatment of infections with carbapenem-resistant Klebsiella pneumoniae. The aim of the study was to understand the genetic mechanism of resistance and the genetic context of resistance genes in two tigecycline-resistant K. pneumoniae strains isolated from sewage in Bergen, Norway. METHODS: Complete genome sequencing of the two strains was accomplished using a combination of short-read Illumina MiSeq-based and long-read Oxford Nanopore MinION-based sequencing. Conjugation experiments were performed using filter mating and a green fluorescent protein (GFP)-tagged Escherichia coli strain. RESULTS: The complete genome sequences of strain K6-320.1 and strain K7-325 were assembled into two contigs for each strain, one contig representing the complete circular chromosomes of 5 223 440 bp (K6-320.1) and 5 263 092 bp (K7-325), respectively, and the other representing plasmids with sizes of 276 509 bp (pK6-320.1) and 246 731 bp (pK7-325). Strain K6-320.1 belonged to sequence type (ST)869, whereas strain K7-325 belonged to the pathogenic ST307. Both plasmids belonged to the IncFIB(K)/IncFII(K) group and carried several antibiotic resistance genes (ARGs), including tet(A) and blaCTX-M. Both plasmids (pK6-320.1 and pK7-325) were transferred to a GFP-tagged E. coli strain, leading to the acquisition of resistance against multiple classes of antibiotics. Several heavy-metal resistance genes (HMRGs) encoding resistance against silver (sil), copper (pco), and arsenic (ars) were also present on both plasmids. CONCLUSIONS: Our study demonstrates the presence of multidrug-resistant K. pneumoniae strains carrying conjugative plasmids encoding both ARGs and HMRGs that have potential for persistence in the environment and human microbiota.

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.

Genomic characterisation of multidrug-resistant Bacillus toyonensis strain 4HC1 isolated from marine plastic in Norway.

OBJECTIVES: Bacillus toyonensis is widespread in nature. Multidrug-resistant B. toyonensis strain 4HC1 was isolated from polyethylene submerged in the water column near a beach in Øygarden, Norway. We analysed the whole genome sequence of strain 4HC1 in order to understand the genetic basis of the observed phenotypic antibiotic resistance. METHODS: Whole-genome sequencing of B. toyonensis strain 4HC1 was performed on Illumina MiSeq platform using 2 × 300 bp chemistry. The genome sequence was assembled using SPAdes v.3.13.0 and was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). RESULTS: The draft genome of strain 4HC1 is 6 156 259 bp (133 contigs) in size with a GC content of 34.95%. The genome comprises 6089 protein-coding genes, 86 tRNAs and 24 rRNAs. Strain 4HC1 is resistant to cefotaxime, trimethoprim and ampicillin and carries various antibiotic resistance genes (ARGs), including several ß-lactamases, aminoglycoside 6-adenylyltransferase, a TetM family tetracycline resistance gene, two different tetracycline efflux pumps, and a bleomycin resistance gene. Several virulence genes including genes involved in immune evasion, iron acquisition and toxins were also detected in strain 4HC1. CONCLUSION: The draft genome sequence of B. toyonensis strain 4HC1 released here shows the presence of various ARGs and virulence genes in a multidrug-resistant strain isolated from marine plastic.

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.

Marine Plastics from Norwegian West Coast Carry Potentially Virulent Fish Pathogens and Opportunistic Human Pathogens Harboring New Variants of Antibiotic Resistance Genes.

To our best knowledge this is the first study characterizing fish pathogens isolated from marine plastics from the West coast of Norway for their potential for pathogenicity using whole genome sequencing. Marine plastic polymers identified as polyethylene, polyethylene/ethylene vinyl acetate copolymer and polypropylene, yielded a total of 37 bacterial isolates dominated by Pseudomonas spp. (70%). Six isolates representing either fish pathogens or opportunistic human pathogens were selected for whole genome sequencing (WGS). These included four isolates belonging to Aeromonas spp., one Acinetobacter beijerinckii isolate and one Morganella morganii isolate. Three Aeromonas salmonicida isolates were potentially virulent and carried virulence factors involved in attachment, type II and type VI secretion systems as well as toxins such as aerA/act, ahh1, ast, hlyA, rtxA and toxA. A. salmonicida and Acinetobacter beijerinckii carried new variants of antibiotic resistance genes (ARGs) such as ß-lactamases and chloramphenicol acetyltransferase (catB), whereas Morganella morganii carried several clinically relevant ARGs. Our study shows that marine plastics carry not only potentially virulent fish pathogens but also multidrug resistant opportunistic human pathogens like M. morganii and may serve as vectors for transport of these pathogens in the marine environment.