Combating antimicrobial resistance in the Norwegian food production chain

The NoResist project seeks to obtain knowledge that can be used to prevent, reduce or inhibit antimicrobial resistance in the Norwegian food production chain, with the prime focus on poultry. In 2016, all Norwegian broiler farms were sampled during May-October for the presence of extended-spectrum cephalosporin resistant (ESCR) bacteria, of which approx. 10% of the flocks were positive. A previous positive flock in the same house was a risk factor. Some isolates harbored an ESCR-encoding gene not previously detected in Norwegian broiler production. Studies indicate that both clonal dissemination of ESCR E. coli and horizontal transfer of ESCR-plasmids have contributed to the dissemination of this gene. Both plasmids and E. coli isolates were genetically related to plasmids and E. coli from broiler production in other European countries, indicating a common source. We tested a hypothesis that the excess levels of Zn and Cu in chicken feed additives increase transfer of resistance plasmids from ESC-producing E. coli. However, results from extensive laboratory experiments showed that Zn and Cu on the contrary reduced the conjugation frequency. A down-regulation of conjugational genes in response to Zn and Cu resulted in the decreased conjugation frequency. Health-related consequences for humans exposed to ESC-producing E. coli through chicken meat are addressed with special attention to urinary tract infections. ESCR E. coli isolates from retail chicken during 2012-16 in the Norwegian monitoring program (NORM-VET) were whole genome sequenced and analysed for the presence of virulence-associated and antimicrobial resistance genes. A selected group was analysed for uropathogen-associated virulence traits in laboratory experiments. The results indicate that the pathogenic potential of ESCR E. coli from the Norwegian poultry reservoir is limited. However, it is important to maintain monitoring programs to manage eventual emergence of potential pathogenic variants in the Norwegian broiler production. A specific variant of ESCR E. coli from Norwegian broiler production, ST38, has been compared with isolates of the same sequence type from infected and healthy humans. Comparative genomic analysis with whole genome sequence data showed that the broiler isolates group into a distinct sub-cluster, different from the human isolates. Close genetic relationship to ST38 isolates from Swedish and Icelandic broiler production has been demonstrated, indicating a common origin for these isolates. Collection, identification and antibiotic resistance characterisation of Pseudomonas isolates from raw chicken meat spanning a time period of isolation of 26 years have been performed. Resistance testing and whole genome sequence analyses indicated relatively stable prevalence and levels of resistance in the study period. Pseudomonas has numerous intrinsic multidrug resistance genes but transferable antibiotic resistance genes seemed not prevalent. Klebsiella pneumoniae is a major human pathogen and a central contributor to the global spread of antibiotic resistance, but knowledge about this bacterium in animal and environmental reservoirs is limited. By using selective culturing methods, Klebsiella spp was isolated from faecal samples of healthy turkeys and broilers, all identified as K. pneumoniae. There were more findings of K. pneumoniae from turkey than from chicken. The occurrence of antibiotic resistance was low. The isolates were generally good biofilm producers. Biofilm formation contributes to bacterial reservoirs in production environments. Only two of three disinfectants commonly used in broiler production showed a satisfactory effect on quinolone resistant E. coli (QREC) in young biofilms in laboratory tests, whereas none were effective on old biofilms- QREC from broiler production were better biofilm formers than ESCR E. coli. Interestingly, QREC strains displayed reduced biofilm forming abilities after acquisition of plasmids carrying an ESCR gene, indicating a biofilm regulating role of these resistance plasmids. We have detected a strong association between vancomycin resistant enterococci (VRE) and narasin resistance. This suggests that feed containing narasin to be an important factor in promoting persistence of VRE in the Norwegian broiler production. After the Norwegian broiler production stopped using narasin as a feed additive in 2016, a significant reduction in narasin-resistant bacteria in broilers has been observed, and VRE has fallen below the detection limit. We have cloned and performed the first characterization of the narasin resistance mechanism, and named it NarAB. NarAB confers resistance to certain other polyether ionophores, but does not give resistance to any of the clinically used antibiotics tested.

Prosjektet har fremskaffet viktig kunnskap om utvikling, forekomst og spredning av antibiotikaresistens i matproduksjonskjeden for slaktekylling. Denne kunnskapen kan brukes av næring og forvaltning i arbeidet med å identifisere risiko, forbygge og redusere slik resistens på nasjonalt nivå, noe som også vil komme konsumentene til gode. Kunnskapen vil også være nyttig for internasjonale aktører, spesielt fordi det gunstige nivået på norsk antibiotikabruk og resistens gjør det mulig å studere resistens fra et "lavresistens-perspektiv" som ikke kan gjøres så mange andre steder. Den nye kunnskapen om sammenheng mellom antibiotikaresistens og narasin, som er vanlig å bruke som fôrtilsetning i andre land, vil være av stor internasjonal interesse. Prosjektet har styrket samarbeid og kunnskapsutveksling mellom partnerne, økt den faglige kompetansen, bidratt til kompetanseutvikling for to PostDoc-stipendiater, samt forskerutdannelse for to master-, en forskerlinje- og 16 bachelor-studenter.

Antimicrobials revolutionized medicine in the 20th century, but the development of antimicrobial resistance in bacteria is becoming an increasing threat to global public health. There are numerous routes by which resistant bacteria and resistance genes are spread to and from humans, one being the food chain. The NoResist project will seek to obtain knowledge on persistence and spread of antimicrobial resistance in the Norwegian food production chain which can be used to prevent, reduce or inhibit such resistance. The prime focus of the project will be on the poultry production chain, which currently is associated with the most serious challenges of antimicrobial resistant bacteria, i.e. extended spectrum betalactamase (ESBL) producing bacteria and quinolone resistant bacteria. These antimicrobials are defined by WHO to be among the critically important antimicrobial agents to be reserved for treatment of severe infections in humans. The project will: 1) Identify occurrence and possible reservoirs of antimicrobial resistance in animals, food products and various production environments, e.g. in biofilms. 2) Identify risk factors of development and spread of antimicrobial resistance. 3) Study the dissemination of antimicrobial resistance throughout the production chain from production animals via animal products to the human consumer. 4) Study mechanisms and drivers of transfer of resistance genes between bacteria in various compartments production chain 5) Identify the effect of anticoccidial feed additives on antimicrobial resistance development and dissemination in bacteria. 6) Based on the results obtained, identify factors which can prevent or inhibit spread of resistance. Norwegian and international institutions with complementing competence within antimicrobial resistance, food safety and human health, will cooperate with the industry to ensure both scientific quality and relevance of the results obtained in the NoResist project.