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-resistant K. 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 K. 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.

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 cut-off 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.

Insights into the genetic diversity, antibiotic resistance and pathogenic potential of Klebsiella pneumoniae from the Norwegian marine environment using whole-genome analysis.

Klebsiella pneumoniae (Kp) can cause hospital- and community acquired infections. Although, Kp is widespread in the environment, very little is known about the genetic diversity and pathogenicity of Kp from the marine environment. The aim of our study was to understand the genetic diversity, resistome and pathogenic potential of 87 Kp isolates from the Norwegian marine environment, using whole-genome sequencing. We identified 50 sequence types, including globally disseminated sequence types associated with multidrug resistance or hypervirulence. Ten isolates carried the yersiniabactin loci. Acquired antibiotic resistance genes were identified in six Kp isolates. Heavy metal resistance genes were widespread among the isolates, with 71% carrying genes encoding resistance to copper, silver, arsenic, nickel and/or mercury. Co-occurrence of antibiotic resistance genes and heavy metal resistance genes was seen in five Kp isolates. Phylogenetic analysis revealed a close genetic relationship between Kp 2016-1200 ST25 isolated from blue mussels (Mytilus edulis) and a clinical isolate reported in Germany. To the best of our knowledge, this study provides the first comprehensive account of genetic diversity among Kp from the marine environment. Our study reveals high diversity of Kp in the Norwegian marine environment and seafood, including globally disseminated pathogenic sequence types carrying clinically relevant antibiotic resistance genes and virulence factors, as well as several heavy metal resistance genes.

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 ß-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 ß-lactamase (ESBL)-producing K. pneumoniae that may be transmitted through the food chain.

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.