Comparative genomics of quinolone-resistant Escherichia coli from broilers and humans in Norway.

BACKGROUND: The usage of fluoroquinolones in Norwegian livestock production is very low, including in broiler production. Historically, quinolone-resistant Escherichia coli (QREC) isolated from Norwegian production animals rarely occur. However, with the introduction of a selective screening method for QREC in the Norwegian monitoring programme for antimicrobial resistance in the veterinary sector in 2014; 89.5% of broiler caecal samples and 70.7% of broiler meat samples were positive. This triggered the concern if there could be possible links between broiler and human reservoirs of QREC. We are addressing this by characterizing genomes of QREC from humans (healthy carriers and patients) and broiler isolates (meat and caecum). RESULTS: The most frequent mechanism for quinolone resistance in both broiler and human E. coli isolates were mutations in the chromosomally located gyrA and parC genes, although plasmid mediated quinolone resistance (PMQR) was also identified. There was some relatedness of the isolates within human and broiler groups, but little between these two groups. Further, some overlap was seen for isolates with the same sequence type isolated from broiler and humans, but overall, the SNP distance was high. CONCLUSION: Based on data from this study, QREC from broiler makes a limited contribution to the incidence of QREC in humans in Norway.

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.

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.

Escherichia coli multilocus sequence type 38 from humans and broiler production represent distinct monophyletic groups.

Escherichia coli belonging to multilocus sequence type 38 (ST38) is a well-known cause of extra-intestinal infections in humans, and are frequently associated with resistance to extended-spectrum cephalosporins (ESCs). Resistance to carbapenems, mediated by blaOXA-genes has also been reported in this ST. Recently, the European Centre for Disease Prevention and Control (ECDC) released a rapid risk assessment on the increased detection of OXA-244 producing E. coli ST38 in humans, requesting further knowledge to determine the source. ST38 is also one of the most common STs among ESC-resistant E. coli from broiler production. Our aim was to investigate the genetic characteristics and relationship between E. coli ST38 from broiler production and humans, and to investigate if there has been a potential spillover between these sources. A total of 288 E. coli ST38 genomes isolated from humans in Europe (collected 2009-2019) and from Nordic broiler production (collected 2011-2014) were analyzed. The results showed distinct monophyletic clades associated to humans and broiler production. Furthermore, there were differences in the ESC resistance genes present in E. coli ST38 from the two sources. The blaOXA-244 gene was not present in E. coli from broiler production. Our results show that ST38 from humans and broiler production belong to well-separated clades, and suggest that the increased detection of OXA-244-producing E. coli ST38 in humans is not associated with spillover from broiler production.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Closed Genome Sequences of Providencia alcalifaciens Isolates from Dogs.

Eight Providencia alcalifaciens isolates from eight different dogs in Norway with acute hemorrhagic diarrhea were sequenced. Based on Illumina and Oxford Nanopore Technologies sequencing, all of the genomes were complete and closed after hybrid assembly.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae.

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

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.].

Complete Genome Sequences of 12 Quinolone-Resistant Escherichia coli Strains Containing qnrS1 Based on Hybrid Assemblies.

In total, 12 quinolone-resistant Escherichia coli (QREC) strains containing qnrS1 were submitted to long-read sequencing using a FLO-MIN106 flow cell on a MinION device. The long reads were assembled with short reads (Illumina) and analyzed using the MOB-suite pipeline. Six of these QREC genome sequences were closed after hybrid assembly.

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.

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.

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.

NarAB Is an ABC-Type Transporter That Confers Resistance to the Polyether Ionophores Narasin, Salinomycin, and Maduramicin, but Not Monensin.

Polyether ionophores are antimicrobial compounds used as feed additives in poultry feed to control diseases caused by coccidia. In addition to the anticoccidial activity of these compounds, polyether ionophores also contain antibacterial properties. Resistance to the polyether ionophore narasin was recently shown to exist on mobile plasmids in Enterococcus faecium and the resistance mechanism was suggested to be associated with a two-gene operon encoding an ABC-type transporter. In this study we demonstrate that the genes encoding the putative narasin resistance mechanism confers reduced susceptibility to the polyether ionophores narasin, salinomycin and maduramicin, but not to monensin and suggest that this resistance mechanism should be referred to as NarAB. Importantly, NarAB does not affect the susceptibility of E. faecium to any of the tested antimicrobial compounds that are used in clinical medicine. However, we show that conjugation in the presence of certain polyether ionophores increases the number of vancomycin resistant E. faecium suggesting that narasin and certain other polyether ionophores can contribute to the persistence of VRE in poultry populations.

Dissemination of Quinolone-Resistant Escherichia coli in the Norwegian Broiler and Pig Production Chains and Possible Persistence in the Broiler Production Environment.

In Norway, the use of quinolones in livestock populations is very low, and prophylactic use is prohibited. Despite this, quinolone-resistant Escherichia coli (QREC) isolates are present at low levels in several animal species. The source of these QREC isolates is unknown. The aim of this study was to characterize and compare QREC isolates from different animal species to identify putative factors that may promote the occurrence of QREC. A total of 280 QREC isolates, from broilers, pigs, red foxes, and wild birds, were whole-genome sequenced and analyzed. Well-known chromosomal and plasmid-mediated resistance mechanisms were identified. In addition, mutations in marR, marA, and rpoB causing novel amino acid substitutions in their respective proteins were detected. Phylogenetic analyses were used to determine the relationships between the isolates. Quinolone resistance mechanism patterns appeared to follow sequence type groups. Similar QREC isolates with similar resistance mechanism patterns were detected from the samples, and further phylogenetic analysis indicated close evolutionary relationships between specific isolates from different sources. This suggests the dissemination of highly similar QREC isolates between animal species and also the persistence of QREC strains within the broiler production chain. This highlights the importance of both control measures at the top of the production chain as well as biosecurity measures to avoid the further dissemination and persistence of QREC in these environments.IMPORTANCE Since antimicrobial usage is low in Norwegian animal husbandry, Norway is an ideal country to study antimicrobial resistance in the absence of selective pressure from antimicrobial usage. In particular, the usage of quinolones is very low, which makes it possible to investigate the spread and development of quinolone resistance in natural environments. Comparison of quinolone-resistant E. coli (QREC) isolates from livestock and wild animals in light of this low quinolone usage provides new insights into the development and dissemination of QREC in both natural and production environments. With this information, preventive measures may be taken to prevent further dissemination within Norwegian livestock and between other animals, thus maintaining the favorable situation in Norway.

Significant reduction of vancomycin resistant E. faecium in the Norwegian broiler population coincided with measures taken by the broiler industry to reduce antimicrobial resistant bacteria.

Vancomycin resistant enterococci (VRE) belong to the most common causes of nosocomial infections worldwide. It has been reported that use of the glycopeptide growth promoter avoparcin selected for a significant livestock-reservoir of VRE in many European countries, including Norway. However, although avoparcin was banned as a feed-additive in 1995, VRE have for unknown reasons consistently been reported in samples from Norwegian broilers. When avoparcin was banned, broiler-feed was supplemented with the polyether ionophore narasin in order to control the diseases coccidiosis and the frequent sequela necrotic enteritis. A potential link between transferrable vancomycin resistance and reduced susceptibility to narasin was recently reported. The use of narasin as a feed additive was abolished by the Norwegian broiler industry in 2016 and since then, broilers have been reared without in-feed antibacterial supplements. In this study, we demonstrate that all VRE isolates from Norwegian broilers collected in 2006-2014 displayed reduced susceptibility to narasin. Surveillance data collected two years after the narasin abolishment show a significant reduction in VRE, below the detection limit of the surveillance method, and a concurrent marked reduction in Enterococcus faecium with reduced susceptibility to narasin. The significant decline of E. faecium with reduced susceptibility to these antimicrobial compounds also coincided with an increased focus on cleaning and disinfection between broiler flocks. Furthermore, data from a controlled in vivo experiment using Ross 308 broilers indicate that the proportion of E. faecium with reduced susceptibility to narasin was heavily reduced in broilers fed a narasin-free diet compared to a diet supplemented with narasin. Our results are consistent with that the abolishment of this feed additive, possibly in combination with the increased focus on cleaning and disinfection, has had a substantial impact on the occurrence of VRE in the Norwegian broiler population.

Occurrence of and risk factors for extended-spectrum cephalosporin-resistant Enterobacteriaceae determined by sampling of all Norwegian broiler flocks during a six month period.

All broiler flocks reared and slaughtered in Norway from May-October 2016 (n = 2110) were screened for the presence of extended-spectrum cephalosporin (ESC) -resistant Enterobacteriaceae. Furthermore, we investigated possible risk factors for occurrence of such bacteria in broiler flocks. The odds of a flock being positive for ESC-resistant Enterobacteriaceae increased if the previous flock in the same house was positive, and if the flock was reared during September-October. However, we cannot exclude seasonal fluctuations in occurrence of ESC-resistant Enterobacteriaceae during the months November to April. The overall occurrence of ESC-resistant Enterobacteriaceae was 10.4%, and primarily linked to the presence of blaCMY (82.6%) in positive isolates. We describe the first findings of Escherichia coli with blaCTX-M-1, Klebsiella pneumoniae with both blaCTX-M-15 and blaSHV-12, and K. pneumoniae with blaCMY isolated from Norwegian broiler production. This study gives us a unique overview and estimate of the true occurrence of ESC-resistant Enterobacteriaceae in Norwegian broilers over a six-month period. To the best of our knowledge, this is the most comprehensive study performed on the occurrence of ESC-resistant Enterobacteriaceae in a broiler population.