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

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.publishedVersio

The Microplastic-Antibiotic Resistance Connection

Microplastic pollution is a big and rapidly growing environmental problem. Although the direct effects of microplastic pollution are increasingly studied, the indirect effects are hardly investigated, especially in the context of spreading of disease and antibiotic resistance genes, posing an apparent hazard for human health. Microplastic particles provide a hydrophobic surface that provides substrate for attachment of microorganisms and readily supports formation of microbial biofilms. Pathogenic bacteria such as fish pathogens Aeromonas spp., Vibrio spp., and opportunistic human pathogens like Escherichia coli are present in these biofilms. Moreover, some of these pathogens are shown to be multidrug resistant. The presence of microplastics is known to enhance horizontal gene transfer in bacteria and thus, may contribute to dissemination of antibiotic resistance. Microplastics can also adsorb toxic chemicals like antibiotics and heavy metals, which are known to select for antibiotic resistance. Microplastics may, thus, serve as vectors for transport of pathogens and antibiotic resistance genes in the aquatic environment. In this book chapter, we provide background information on microplastic biofouling (“plastisphere concept”), discuss the relationship between microplastic and antibiotic resistance, and identify knowledge gaps and directions for future research.publishedVersio

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 β-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.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

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

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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.publishedVersio

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

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).publishedVersio

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.publishedVersio

Monitoring programme for veterinary control on seafood products imported to Norway from third countries – Results from 2019: In accordance with Commission Regulation (EC) No 136/2004, Annex II, Part 1

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Co-localization of clinically relevant antibiotic- and heavy metal resistance genes on plasmids in Klebsiella pneumoniae from marine bivalves

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with antibiotic resistance and present in a wide range of environments, including marine habitats. However, little is known about the development, persistence, and spread of antibiotic resistance in such environments. This study aimed to obtain the complete genome sequences of antibiotic-resistantKlebsiella pneumoniae isolated from marine bivalves in order to determine the genetic context of antibiotic- and heavy metal resistance genes in these isolates. Five antibiotic-resistant Klebsiella pneumoniae isolates, of which four also carried heavy metal resistance genes, were selected for complete genome sequencing using the Illumina MiSeq platform and the Oxford Nanopore Technologies GridION device. Conjugation experiments were conducted to examine the transfer potential of selected plasmids. The average length of the complete genomes was 5.48 Mbp with a mean chromosome size of 5.27 Mbp. Seven plasmids were detected in the antibiotic-resistant isolates. Three IncFIB, one IncFIB/IncFII, and one IncFIB/IncHIB plasmid, respectively, carried antibiotic resistance genes such as qnrS1, aph(6)-Id and aph(3′)-Ia, aadA1, and aadA2. Four of these plasmids also carried genes encoding resistance to copper (pco), silver (sil), and arsenic (ars). One plasmid carrying tet(D) and blaSHV-1 as well as pco, sil, and ars genes was transferred to Escherichia coli by conjugation. We show the co-occurrence of antibiotic- and heavy metal resistance genes on a conjugative IncFIB plasmid from K. pneumoniae from marine bivalves. Our study highlights the importance of the marine environment and seafood as a possible dissemination route for antimicrobial resistance and provides insights into the potential for co-selection of antibiotic resistance genes by heavy metals