Polyether ionophore resistance in a one health perspective.

Antimicrobial resistance is a major threat to human health and must be approached from a One Health perspective. Use of antimicrobials in animal husbandry can lead to dissemination and persistence of resistance in human pathogens. Polyether ionophores (PIs) have antimicrobial activities and are among the most extensively used feed additives for major production animals. Recent discoveries of genetically encoded PI resistance mechanisms and co-localization of resistance mechanisms against PIs and antimicrobials used in human medicine on transferrable plasmids, have raised concerns that use of PIs as feed additives bear potential risks for human health. This review summarizes the current knowledge on PI resistance and discusses the potential consequences of PI-usage as feed additives in a One Health perspective.

Detection of Klebsiella pneumoniae in healthy poultry: Insights and perspectives from culturing and metagenomics.

Previously, Klebsiella pneumoniae was found to occur more frequently in healthy turkey flocks than in healthy broiler flocks in Norway. This study aimed to investigate whether this higher occurrence could be attributed to a greater abundance of K. pneumoniae in turkey flocks. We compared culturing, qPCR, and shotgun metagenomic sequencing for the detection and quantification of K. pneumoniae. Using qPCR, we found that 20.7% of broiler flock samples and 63.9% of turkey flock samples were positive for K. pneumoniae. Culturing revealed a significantly higher abundance of K. pneumoniae in turkey flocks compared to broiler flocks. However, metagenomic analysis showed no difference in the relative abundance of Klebsiella spp. between broiler and turkey flocks, and no correlation between the results of culturing and metagenomic quantification. Interestingly, the differential abundance of K. quasipneumoniae was significantly different between the two hosts. Our results indicate that Klebsiella spp. are present in both turkey and broiler flocks at relatively low levels but with a higher abundance in turkey flocks. Our findings also suggest that shotgun metagenomic studies targeting low-abundance taxa such as Klebsiella have poor sensitivity when comparing groups, indicating that reliance on results from metagenomic analysis without experimental validation should be done with caution.

Estimation of epidemiological cut-off values for eight antibiotics used for treatment of bovine mastitis caused by Streptococcus uberis and Streptococcus dysgalactiae subsp. dysgalactiae.

Interpretive criteria for antimicrobial susceptibility testing are lacking for most antimicrobials used for bovine streptococcal mastitis. The objectives of this study were to determine (tentative) epidemiological cut-off ((T)ECOFF) values for clinically relevant antibiotics used for treatment of bovine mastitis, and to estimate the proportion of acquired resistance (non-wild-types) in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus uberis. A total of 255 S. uberis and 231 S. dysgalactiae subsp. dysgalactiae isolates were obtained in Denmark and Norway from bovine mastitis. The isolates were tested for susceptibility to 10 antibiotics using broth microdilution. In accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standard operating procedure, additional published MIC distributions were included for the estimation of ECOFFs for cloxacillin, cephapirin, lincomycin and tylosin, and TECOFFs for amoxicillin, benzylpenicillin, cephapirin and oxytetracycline. The proportion of non-wild-type (NWT) isolates for the beta-lactams was significantly higher in the Danish S. uberis (45-55%) compared to the Norwegian isolates (10-13%). For oxytetracycline, the proportion of NWT was significantly higher in the Danish isolates, both for S. uberis (28% vs. 3%) and S. dysgalactiae (22% vs. 0%). A bridging study testing in parallel MICs in a subset of isolates (n = 83) with the CLSI-specified and the EUCAST-specified broths showed excellent correlation between the MICs obtained with the two methods. The new ECOFFs and TECOFFs proposed in this study can be used for surveillance of antimicrobial resistance, and - for antimicrobials licensed for streptococcal bovine mastitis - as surrogate clinical breakpoints for predicting their clinical efficacy for this indication.

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.

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.

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.

Pilot testing the EARS-Vet surveillance network for antibiotic resistance in bacterial pathogens from animals in the EU/EEA.

Introduction: As part of the EU Joint Action on Antimicrobial Resistance (AMR) and Healthcare-Associated Infections, an initiative has been launched to build the European AMR Surveillance network in veterinary medicine (EARS-Vet). So far, activities included mapping national systems for AMR surveillance in animal bacterial pathogens, and defining the EARS-Vet objectives, scope, and standards. Drawing on these milestones, this study aimed to pilot test EARS-Vet surveillance, namely to (i) assess available data, (ii) perform cross-country analyses, and (iii) identify potential challenges and develop recommendations to improve future data collection and analysis. Methods: Eleven partners from nine EU/EEA countries participated and shared available data for the period 2016-2020, representing a total of 140,110 bacterial isolates and 1,302,389 entries (isolate-antibiotic agent combinations). Results: Collected data were highly diverse and fragmented. Using a standardized approach and interpretation with epidemiological cut-offs, we were able to jointly analyze AMR trends of 53 combinations of animal host-bacteria-antibiotic categories of interest to EARS-Vet. This work demonstrated substantial variations of resistance levels, both among and within countries (e.g., between animal host species). Discussion: Key issues at this stage include the lack of harmonization of antimicrobial susceptibility testing methods used in European surveillance systems and veterinary diagnostic laboratories, the absence of interpretation criteria for many bacteria-antibiotic combinations of interest, and the lack of data from a lot of EU/EEA countries where little or even surveillance currently exists. Still, this pilot study provides a proof-of-concept of what EARS-Vet can achieve. Results form an important basis to shape future systematic data collection and analysis.

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.

Review and Analysis of National Monitoring Systems for Antimicrobial Resistance in Animal Bacterial Pathogens in Europe: A Basis for the Development of the European Antimicrobial Resistance Surveillance Network in Veterinary Medicine (EARS-Vet).

The monitoring of antimicrobial resistance (AMR) in bacterial pathogens of animals is not currently coordinated at European level. To fill this gap, experts of the European Union Joint Action on Antimicrobial Resistance and Healthcare Associated Infections (EU-JAMRAI) recommended building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet). In this study, we (i) identified national monitoring systems for AMR in bacterial pathogens of animals (both companion and food-producing) among 27 countries affiliated to EU-JAMRAI, (ii) described their structures and operations, and (iii) analyzed their respective strengths, weaknesses, opportunities and threats (SWOT). Twelve countries reported having at least one national monitoring system in place, representing an opportunity to launch EARS-Vet, but highlighting important gaps in AMR data generation in Europe. In total, 15 national monitoring systems from 11 countries were described and analyzed. They displayed diverse structures and operations, but most of them shared common weaknesses (e.g., data management and representativeness) and common threats (e.g., economic vulnerability and data access), which could be addressed collectively under EARS-Vet. This work generated useful information to countries planning to build or improve their system, by learning from others' experience. It also enabled to advance on a pragmatic harmonization strategy: EARS-Vet shall follow the European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards, collect quantitative data and interpret AMR data using epidemiological cut-off values.

Defining the scope of the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet): a bottom-up and One Health approach.

BACKGROUND: Building the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) was proposed to strengthen the European One Health antimicrobial resistance (AMR) surveillance approach. OBJECTIVES: To define the combinations of animal species/production types/age categories/bacterial species/specimens/antimicrobials to be monitored in EARS-Vet. METHODS: The EARS-Vet scope was defined by consensus between 26 European experts. Decisions were guided by a survey of the combinations that are relevant and feasible to monitor in diseased animals in 13 European countries (bottom-up approach). Experts also considered the One Health approach and the need for EARS-Vet to complement existing European AMR monitoring systems coordinated by the ECDC and the European Food Safety Authority (EFSA). RESULTS: EARS-Vet plans to monitor AMR in six animal species [cattle, swine, chickens (broilers and laying hens), turkeys, cats and dogs], for 11 bacterial species (Escherichia coli, Klebsiella pneumoniae, Mannheimia haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus hyicus, Streptococcus uberis, Streptococcus dysgalactiae and Streptococcus suis). Relevant antimicrobials for their treatment were selected (e.g. tetracyclines) and complemented with antimicrobials of more specific public health interest (e.g. carbapenems). Molecular data detecting the presence of ESBLs, AmpC cephalosporinases and methicillin resistance shall be collected too. CONCLUSIONS: A preliminary EARS-Vet scope was defined, with the potential to fill important AMR monitoring gaps in the animal sector in Europe. It should be reviewed and expanded as the epidemiology of AMR changes, more countries participate and national monitoring capacities improve.

Longitudinal Sampling Reveals Persistence of and Genetic Diversity in Extended-Spectrum Cephalosporin-Resistant Escherichia coli From Norwegian Broiler Production.

There are knowledge gaps concerning dynamics of extended-spectrum cephalosporin (ESC)-resistant Escherichia coli and their plasmids in broiler production and the persistence of strains on broiler farms. Thus, we aimed at characterising ESC-resistant Escherichia coli collected from all flocks reared on 10 different farms during a six-months sampling period. All isolates (n = 43) were subjected to whole-genome sequencing, and a subset of isolates (n = 7) were also sequenced using oxford nanopore technology and subsequent hybrid assembly in order to do in-depth characterisation of the ESC resistance plasmids. The 43 isolates belonged to 11 different sequence types, and three different ESC resistance gene/plasmid combinations were present, namely, IncK2/bla CMY-2 (n = 29), IncI1/bla CMY-2 (n = 6) and IncI1/bla CTX-M-1 (n = 8). ESC-resistant E. coli of different STs and with different ESC resistance gene/plasmid combinations could be present on the same farm, while a single ST and ESC resistance gene/plasmid displaying zero or few SNP differences were present on other farms. In-depth characterisation of IncK2/bla CMY-2 plasmids revealed that at least two distinct variants circulate in the broiler production. These plasmids showed close homology to previously published plasmids from other countries. Our longitudinal study show that ESC-resistant E. coli belong to a multitude of different STs and that different ESC resistance genes and plasmids occur. However, there is also indication of persistence of both ESC-resistant E. coli strains and IncK2/bla CMY-2 plasmids on farms. Further studies are warranted to determine the dynamics of strains, plasmids and ESC resistance genes within single broiler flocks.

Corrigendum: bla CTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].

Corrigendum: bla CTX-M-1 /IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].

Building the European Antimicrobial Resistance Surveillance network in veterinary medicine (EARS-Vet).

Antimicrobial resistance (AMR) should be tackled through a One Health approach, as stated in the World Health Organization Global Action Plan on AMR. We describe the landscape of AMR surveillance in the European Union/European Economic Area (EU/EEA) and underline a gap regarding veterinary medicine. Current AMR surveillance efforts are of limited help to veterinary practitioners and policymakers seeking to improve antimicrobial stewardship in animal health. We propose to establish the European Antimicrobial Resistance Surveillance network in Veterinary medicine (EARS-Vet) to report on the AMR situation, follow AMR trends and detect emerging AMR in selected bacterial pathogens of animals. This information could be useful to advise policymakers, explore efficacy of interventions, support antimicrobial stewardship initiatives, (re-)evaluate marketing authorisations of antimicrobials, generate epidemiological cut-off values, assess risk of zoonotic AMR transmission and evaluate the burden of AMR in animal health. EARS-Vet could be integrated with other AMR monitoring systems in the animal and medical sectors to ensure a One Health approach. Herein, we present a strategy to establish EARS-Vet as a network of national surveillance systems and highlight challenges of data harmonisation and bias. Strong political commitment at national and EU/EEA levels is required for the success of EARS-Vet.

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.

Actinobacillus pleuropneumoniae Eradication with Enrofloxacin May Lead to Dissemination and Long-Term Persistence of Quinolone Resistant Escherichia coli in Pig Herds.

Norway has a favourable situation with regard to health status and antimicrobial usage in the pig production sector. However, one of the major disease-causing agents in the commercial pig population is Actinobacillus pleuropneumoniae (APP). In some herds, APP eradication has been performed by using enrofloxacin in combination with a partial herd depopulation. The aim of this study was to investigate the long-term effects of a single treatment event with enrofloxacin on the occurrence of quinolone resistant Escherichia coli (QREC). The study was designed as a retrospective case/control study, where the herds were selected based on treatment history. Faecal samples were taken from sows, gilts, fattening pigs and weaners for all herds where available. A semi-quantitative culturing method was used to identify the relative quantity of QREC in the faecal samples. A significant difference in overall occurrence and relative quantity of QREC was identified between the case and control herds, as well as between each animal age group within the case/control groups. The results indicate that a single treatment event with enrofloxacin significantly increased the occurrence of QREC in the herd, even years after treatment and with no subsequent exposure to quinolones.

The prevalence and genomic context of Shiga toxin 2a genes in E. coli found in cattle.

Shiga toxin-producing Escherichia coli (STEC) that cause severe disease predominantly carry the toxin gene variant stx2a. However, the role of Shiga toxin in the ruminant reservoirs of this zoonotic pathogen is poorly understood and strains that cause severe disease in humans (HUSEC) likely constitute a small and atypical subset of the overall STEC flora. The aim of this study was to investigate the presence of stx2a in samples from cattle and to isolate and characterize stx2a-positive E. coli. In nationwide surveys in Sweden and Norway samples were collected from individual cattle or from cattle herds, respectively. Samples were tested for Shiga toxin genes by real-time PCR and amplicon sequencing and stx2a-positive isolates were whole genome sequenced. Among faecal samples from Sweden, stx1 was detected in 37%, stx2 in 53% and stx2a in 5% and in skin (ear) samples in 64%, 79% and 2% respectively. In Norway, 79% of the herds were positive for stx1, 93% for stx2 and 17% for stx2a. Based on amplicon sequencing the most common stx2 types in samples from Swedish cattle were stx2a and stx2d. Multilocus sequence typing (MLST) of 39 stx2a-positive isolates collected from both countries revealed substantial diversity with 19 different sequence types. Only a few classical LEE-positive strains similar to HUSEC were found among the stx2a-positive isolates, notably a single O121:H19 and an O26:H11. Lineages known to include LEE-negative HUSEC were also recovered including, such as O113:H21 (sequence type ST-223), O130:H11 (ST-297), and O101:H33 (ST-330). We conclude that E. coli encoding stx2a in cattle are ranging from strains similar to HUSEC to unknown STEC variants. Comparison of isolates from human HUS cases to related STEC from the ruminant reservoirs can help identify combinations of virulence attributes necessary to cause HUS, as well as provide a better understanding of the routes of infection for rare and emerging pathogenic STEC.

Comparative Genome Analyses of Wild Type- and Quinolone Resistant Escherichia coli Indicate Dissemination of QREC in the Norwegian Broiler Breeding Pyramid.

Quinolones are important antimicrobials for both humans and animals, and resistance toward these compounds is a serious threat to public health. In Norway, quinolone resistant E. coli (QREC) have been detected at low levels in a high proportion of broiler flocks, even without the use of quinolones in rearing of broilers. Due to the pyramidal structure of broiler breeding, QREC isolates may be disseminated from grandparent animals down through the pyramid. However, quinolone resistance can also develop in wild type E. coli through specific chromosomal mutations, and by horizontal acquisition of plasmid-mediated quinolone resistance genes. The goal of this study was to determine whether QREC is disseminated through the broiler breeding pyramid or developed locally at some stage in the broiler production chain. For this purpose, we whole genome sequenced wild type- and QREC isolates from broiler and parent flocks that had been isolated in the Norwegian monitoring program for antimicrobial resistance in feed, food and animals (NORM-VET) between 2006 and 2017, from 22 different production sites. The sequencing data was used for typing of the isolates, phylogenetic analysis and identification of relevant resistance mechanisms. Highly similar QREC isolates were identified within major sequence types from multiple production sites, suggesting dissemination of QREC isolates in the broiler production chain. The occurrence of potential resistance development among the WT E. coli was low, indicating that this may be a rare phenomenon in the Norwegian broiler production. The results indicate that the majority of the observed QREC at the bottom of the broiler production pyramid originates from parent or grandparent animals. These results highlight the importance of surveillance at all levels of the broiler production pyramid and of implementation of proper biosecurity measures to control dissemination of QREC.

Biofilm forming properties of quinolone resistant Escherichia coli from the broiler production chain and their dynamics in mixed biofilms.

BACKGROUND: Quinolone resistant Escherichia coli (QREC) have been found in samples from Norwegian broiler chicken, despite quinolones not being administered to poultry in Norway. Biofilm production may be one factor contributing to the observed persistence in the broiler production chain. In the present study, 158 QREC strains from chicken caecal and retail meat samples were screened for biofilm production in microtiter plates, biofilm morphotype on Congo Red (CR) agar plates and phylotype by multiplex PCR. Furthermore, the dynamics in mixed biofilms with strains of different morphotypes were studied on glass slides and on CR agar plates. RESULTS: All strains but one produced biofilm in microtiter plates and/or on CR agar plates at room temperature. There were no differences between strains from chicken caecum and chicken retail meat in the mean amount of biofilm produced in microtiter plates. Furthermore, no differences in biofilm production were observed between phylotypes. However, significant differences in biofilm production were found between biofilm morphotypes. The morphotype RDAR (red dry and rough, which has both curli and cellulose in the matrix, was displayed by 70% of the strains. Mean biofilm production by these strains were significantly higher than by strains with the morphotypes PDAR (pink dry and rough) with only cellulose or BDAR (brown dry and rough) with only curli. Interestingly, the two latter morphotypes produced biofilms with the morphotype RDAR when grown together. None of the strains achieved significantly higher numbers of colony forming units (cfu) in mixed biofilms than in single strain biofilms on glass slides. CONCLUSIONS: The results indicate that QREC can form biofilm reservoirs on both inert and organic surfaces in production environments, as well as on meat. This may contribute to persistence and dissemination of the strains. Strains with both curli and cellulose in the biofilm matrix were significantly better biofilm formers than strains lacking one of these components. However, strains with only one of the components could compensate for this by producing mixed biofilms with strains having the other component, and thereby most likely enhance their probabilities of persistence in the production environment.

blaCTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

Escherichia coli carrying blaCTX-M-1 mediating resistance to extended-spectrum cephalosporins was recently described as a new genotype in Norwegian broiler production. The aim of this study was to characterize these isolates (n = 31) in order to determine whether the emergence of the genotype was caused by clonal expansion or horizontal dissemination of blaCTX-M-1-carrying plasmids. All included isolates were subjected to whole genome sequencing. Plasmid transferability was determined by conjugation, and plasmid replicons in the transconjugants were described using PCR-based replicon typing. Plasmid sizes were determined using S1 nuclease digestion. Plasmids in a subset of strains were reconstructed and compared to plasmids from broiler production in other European countries. The isolates belonged to nine different sequence types (STs), with the largest group being ST57 (n = 12). The vast majority of blaCTX-M-1-carrying plasmids were conjugative. All transconjugants were positive for the IncI1-Iγ replicon, and several also harbored the IncFIB replicon. Highly similar plasmids were present in different E. coli STs. Additionally, high similarity to previously published plasmids was detected. A reconstructed plasmid from an ST57 isolate harbored both IncI1-Iγ and IncFIB replicons and was considered to be co-integrated. The presence of one large plasmid was confirmed by S1 nuclease digestion. Our results show that dissemination of blaCTX-M-1 in Norwegian broiler production is due to both clonal expansion and horizontal transfer of plasmids carrying blaCTX-M-1. The blaCTX-M-1/IncI1-Iγ plasmids grouped into two main lineages, namely clonal complex (CC)-3 and CC-7. The genetic diversity at both strain and plasmid level indicates multiple introductions to Norway. We also show that the blaCTX-M-1 plasmids circulating in Norwegian broiler production are highly similar to plasmids previously described in other countries.