Evaluation of the One Health-Ness of 20 Years of Antimicrobial Resistance Surveillance in Norway

We evaluated the One Health-ness (OH-ness) of the surveillance system for antimicrobial resistance (AMR) in Norway by using the recently developed “Evaluation tool for One Health epidemiological surveillance capacities and capabilities” (OH–EpiCap tool). First, we defined the Norwegian AMR surveillance system that we would evaluate. The tool was applied by a group of stakeholders (key persons in the Norwegian AMR surveillance programmes and authors of this paper). The evaluation was performed using a consensus approach. The evaluation resulted in an overall OH-ness score of 68% across all three dimensions included in the tool: Organisation, Operation, and Impact. Suggestions for improvement were only indicated within the areas of internal evaluation and operational costs, whereas most of the indicators included in the tool showed good adherence to the One Health principles. By performing this internal evaluation, we recognized that AMR surveillance in the environment needs to be included in a more systematic and standardized way to improve the OH-ness as defined by the quadripartite organisations. Last but not least, it was beneficial to bring key stakeholders together to conduct the evaluation. It increased a joint perception of the OH-ness of AMR surveillance in Norway and encouraged further collaboration in the future

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.publishedVersio

Screening for antimicrobial- and heavy metal resistant bacteria in copper contaminated areas

A One heath approach is essential to examine the role of the marine environment in the selection and spread of antimicrobial resistance. Norwegian aquaculture used high amounts of antimicrobials during the 80s and 90s, which facilitated widespread resistance among fish pathogens. Moreover, fish farms have since their beginning used copper containing coating as anti-fouling agents on the fish nets, in addition to copper supplements in the fish feed. Consequently, some fish farms have high concentrations of copper in det sediments beneath the pens. Several studies have reported that even sublethal concentrations of heavy metals can facilitate co-selection of antimicrobial resistance and heavy metal resistance. This study applied Vibrio spp. as indicator of antimicrobial- and heavy metal resistance in copper contaminated areas. Six different locations were sampled twice. These represented four different areas in regard of aquaculture activity; Active aquaculture, Non-active aquaculture, Non-aquaculture and Unpolluted. In total, 70 samples, including sediments, seawater, marine fauna and marine algae, were examined for the presence of Vibrio spp. and Escherichia coli. The sediments were examined for heavy metals, where sediments from Active aquaculture areas held exstensively toxic mean concentration with 272.3 mg/kg dry weight (d.w.) copper, significant higher than Non active (24.4 mg/kg d.w.), Non-aquaculture (34.8 mg/kg d.w.) and Unpolluted (2.28 mg/kg d.w.), and moderate toxic mean concentration with 424.6 mg/kg d.w. zinc, also significant higher than Non-active (70.7 mg/kg d.w.), Non-aquaculture (144.6 mg/kg d.w.) and Unpolluted (16.4 mg/kg d.w.). The majority of the retrieved 350 Vibrio splendidus and 72 Vibrio anguillarum isolates were susceptible to all the antimicrobials tested for. Increased minimum inhibition concentrations (MIC) to oxytetracycline and copper were seen for some isolates from Active aquaculture, however only one- to two-fold higher than from Non-active and Non-aquaculture areas. Several antimicrobial- and heavy metal resistance genes were found across the different examined areas, with exception of the nlpE gene encoding a copper sensing lipoprotein inducing efflux pumps, that were only found in one isolate from one location with aquaculture. The majority of the 336 E. coli isolates were susceptible to all antimicrobial tested for. The Active aquaculture areas conferred highest number of E. coli, and most of the resistant E. coli. Many factors, such as weather and topography, can influence the inflow of E. coli from sewage and run-offs from land. No association was found between the detected phenotypic and genotypic resistance traits in V. splendidus and in E. coli in any of the examined areas. Importantly, a higher sample size including more locations is needed to determine whether any possible indicated differences are significant. In conclusion, the high proportion of susceptible isolates found in this project, indicates that the prevalence of selectable resistance mechanisms in the examined marine vibrios from copper contaminated areas are low.publishedVersio

Population dynamics and characteristics of Klebsiella pneumoniae from healthy poultry in Norway

Klebsiella pneumoniae is an important opportunistic pathogen widely studied in relation to human infection and colonization. However, there is a lack of knowledge regarding other niches that K. pneumoniae may inhabit. K. pneumoniae isolated from healthy broiler and turkey flocks in Norway in 2018 have previously been described with regard to population structure, sequence types (STs), and the presence of virulence- and antimicrobial resistance (AMR) genes. In the present study we aimed to evaluate the dynamics of the K. pneumoniae population in poultry over time, with regards to AMR and virulence, and with a special focus on persistence of STs. A total of 391 flocks sampled in 2020 were included in the present study, of which 271 were from broiler flocks and 120 from turkey flocks. Similar to findings from 2018, the occurrence of K. pneumoniae was significantly higher based on culturing in turkey flocks (62.5%) compared to broiler flocks (24.0%). Major STs in 2020 included ST5827 (n = 7), ST37 (n = 7), ST370 (n = 7), ST17 (n = 5), and ST4710 (n = 5). Several STs persisted over time in both host species, including ST35, ST37, ST590, and ST17. This persistence may be due to local re-circulation or reintroduction from parent flocks. Of these five major STs, only ST590 carried AMR genes, indicating that the persistence was not associated with the presence of AMR genes. An ST4710 strain with a hypervirulence-encoding plasmid (p4710; iro5, iuc5) was recovered from turkeys in 2018. The same strain was present in turkeys in 2020, but the plasmid had lost the salmochelin locus. This loss may be attributed to reductive evolution due to the presence of several siderophores within the same isolates. In this study we also characterized a clinical ST4710 isolate from a turkey with airsacculitis. The isolate was closely related to two intestinal ST4710 isolates from healthy turkeys in 2018. These three isolates were sampled within the same location and time frame in 2018, and all carried the full p4710 virulence plasmid. These findings highlight the transmission- and infectious potential of ST4710 in turkeys.publishedVersio

The effect of antimicrobial resistance plasmids carrying BlaCMY-2 on biofilm formation by escherichia coli from the broiler production chain

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.publishedVersio

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.publishedVersio

Technical specifications for the monitoring and reporting of VTEC on animals and food

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Livestock-Associated MRSA CC1 in Norway; Introduction to Pig Farms, Zoonotic Transmission, and Eradication

Farm animals have been identified as an emerging reservoir for transmission of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to humans. The low incidence of MRSA in humans and farm animals in Norway has led to the implementation of a national strategy of surveillance and control of LA-MRSA aiming to prevent livestock becoming a domestic source of MRSA to humans. In 2015, MRSA clonal complex 1 spa-type t177 was identified in nine Norwegian pig herds in two neighboring counties. An outbreak investigation was undertaken, and measures of control through eradication were imposed. We performed a register-based cohort study including pig herds and MRSA-positive persons in Norway between 2008 and 2016 to investigate the livestock-association of MRSA CC1, the transmission of the outbreak strain to humans before and after control measures, and the effect of control measures imposed. Data from the Norwegian Surveillance System of Communicable Diseases were merged with data collected through outbreak investigations for LA-MRSA, the National Registry and the Norwegian Register for Health Personnel. Whole-genome sequencing was performed on isolates from livestock and humans identified through contact tracing, in addition to t177 and t127 isolates diagnosed in persons in the same counties. It is likely that a farm worker introduced MRSA CC1 to a sow farm, and further transmission to eight fattening pig farms through trade of live pigs confirmed the potential for livestock association of this MRSA type. The outbreak strain formed a distinct phylogenetic cluster which in addition to the pig farms included one sheep herd and five exposed persons. None of the investigated isolates from possible cases without direct contact to the MRSA positive farms were phylogenetically related to the outbreak strain. Moreover, isolates of t177 or t127 from healthcare and community-acquired cases were not closely related to the outbreak cluster. Eradication measures imposed were effective in eliminating MRSA t177 from the positive pig holdings, and the outbreak strain was not detected in the national pig population or in persons from these counties after control measures

Antibiotikaresistens – kunnskapshull, utfordringer og aktuelle tiltak

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