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Plasmid-mediated extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase producing Enterobacteriaceae, in particular Escherichia coli and Klebsiella pneumoniae, with potential zoonotic transmission routes, are one of the greatest threats to global health. The aim of this study was to investigate global food products as potential vehicles for ESBL/AmpC-producing bacteria and identify plasmids harboring resistance genes. We sampled 200 food products purchased from Finland capital region during fall 2018. Products originated from 35 countries from six continents and represented four food categories: vegetables (n = 60), fruits and berries (n = 50), meat (n = 60), and seafood (n = 30). Additionally, subsamples (n = 40) were taken from broiler meat. Samples were screened for ESBL/AmpC-producing Enterobacteriaceae and whole genome sequenced to identify resistance and virulence genes and sequence types (STs). To accurately identify plasmids harboring resistance and virulence genes, a hybrid sequence analysis combining long- and short-read sequencing was employed. Sequences were compared to previously published plasmids to identify potential epidemic plasmid types. Altogether, 14 out of 200 samples were positive for ESBL/AmpC-producing E. coli and/or K. pneumoniae. Positive samples were recovered from meat (18%; 11/60) and vegetables (5%; 3/60) but were not found from seafood or fruit. ESBL/AmpC-producing E. coli and/or K. pneumoniae was found in 90% (36/40) of broiler meat subsamples. Whole genome sequencing of selected isolates (n = 21) revealed a wide collection of STs, plasmid replicons, and genes conferring multidrug resistance. bla(CTX-M-15)-producing K. pneumoniae ST307 was identified in vegetable (n = 1) and meat (n = 1) samples. Successful IncFII plasmid type was recovered from vegetable and both IncFII and IncI1-I gamma types from meat samples. Hybrid sequence analysis also revealed chromosomally located beta-lactamase genes in two of the isolates and indicated similarity of food-derived plasmids to other livestock-associated sources and also to plasmids obtained from human clinical samples from various countries, such as IncI type plasmid harboring bla(TEM-52C) from a human urine sample obtained in the Netherlands which was highly similar to a plasmid obtained from broiler meat in this study. Results indicate certain foods contain bacteria with multidrug resistance and pose a possible risk to public health, emphasizing the importance of surveillance and the need for further studies on epidemiology of epidemic plasmids.

Final report of the project "Impact of pests on persistence and spearing of zoonotic bacteria on production farms (PESTANIMAL)" ., FI; PDF; efsafocalpoint@ruokavirasto.fi, {"references":["Andrés-Barranco S et al (2014). Role of wild bird and rodents in the epidemiology of subclinical salmonellosis in finishing pigs.","Backhans A et al (2013). Occurrence of pathogens in wild rodents caught on Swedish pig and chicken farms.","Healing TD ja Greenwood MH. (1991). Frequency of isolation of Campylobacter spp., Yersinia spp. and Salmonella spp. from small mammals from two sites in southern Britain.","Meerburg BG et al (2006). Presence of Salmonella and Campylobacter spp. in wild small mammals on organic farms."]}

This study was conducted by Finnish Food Authority (formerly Finnish Food Safety Authority Evira), Natural Resources Institute Finland Luke and Finnish Environment Institute SYKE in 2015–2018. The aims of the study were to examine how antimicrobial residues and antimicrobial-resistant E. coli bacteria spread via the manure chain from the dairy cows treated with antimicrobials to the farm environment and manure storages. Furthermore, the dissemination routes to the surrounding environment, ecosystems and back to the food chain in Finnish conditions were profiled. The effect of mesophilic anaerobic digestion process on the level of antimicrobial residues and antimicrobial-resistant E. coli was examined and, conversely, the effect of antimicrobial residues on the process as well. Finally, the life-cycle of different manure handling and processing methods, and their potential effects on nutrient recycling, energy production, antimicrobial residues and resistant bacteria were evaluated. Resistant and multi-resistant E. coli spread to the manure storages, although enrichment in their proportion over the manure chain was not detected. Only minor concentrations of antimicrobial residues were measured from the liquid manure. Instead, very high concentrations of antimicrobial residues were measured from the faeces, urine and milk of dairy cows medicated with antimicrobials, which may lead to high local concentrations in (dry) manures composed of them. In Finland, there is no withdrawal period for manure of medicated animals to be used as a fertilizer. Therefore, manures may contain antimicrobial residues, as well as resistant bacteria, which disseminate to the environment when applied to the fields. Animals and plants may be exposed to antimicrobial residues and resistant bacteria in agricultural and natural ecosystems in Finnish conditions as well. Therefore, fertilizer usage of faeces, urine and milk excreted by animals treated with antimicrobials should be subjected to critical assessment. Treating the manure with mesophilic anaerobic digestion may reduce the concentration of antimicrobial residues but does not necessarily destroy them. Furthermore, the process reduced the number of total E. coli bacteria, although it did not have an effect on the proportion of resistant E. coli. Along with energy production and nutrient recycling, different manure handling and processing methods may, however, be suitable for destroying and reducing antimicrobial residues and resistant bacteria from manure, and substantially reduce the emission of antimicrobial residues and resistant bacteria to the environment, as well as to the food chain. The aspect of tackling the spread of antimicrobial resistance is to be considered when manure processing methods are developed in Finland., FI; PDF; johanna.suomi@ruokavirasto.fi

Antimicrobial resistance (AMR) is recognized as one of the most urgent global public health threats facing humanity today. The increasing number of deaths related to bacterial infections untreatable with antibiotics demands new and robust methods for studying and controlling the spread of AMR. Antimicrobial resistance, an ancient phenomenon at its core, has been affected by the discovery and profuse use of antibiotics in different sectors. Antibiotic use has been a key driver behind the successful spread of new resistance determinants in the microbial world. The complex nature behind the epidemiology of AMR stems from versatile transmission mechanisms employed by microorganisms and mobile genetic elements (MGEs), and the interwoven web of potential transmission routes between humans, animals, and the environment. Horizontal transmission of resistance genes via MGEs, especially plasmids, has been essential in the successful dissemination of resistance genes globally. A One Health approach is required to understand the complexity of AMR. Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBL/AmpC) and carbapenemases are among the most critical resistant pathogens globally, as these threaten the effectiveness of commonly used beta- lactam antibiotics used widely in modern medicine. ESBL-producing Enterobacteriaceae, especially Escherichia coli and Klebsiella pneumoniae, are leading causes of both hospital-associated and community-acquired antibiotic-resistant infections. The localization of genes encoding for extended-spectrum beta-lactamases on plasmids enables transfer even between bacteria of different species. Advances in whole genome sequencing (WGS) have aided the discovery of new resistance determinants, made surveillance more accurate and rapid, and offered new powerful tools to interpret the epidemiology and transmission routes of bacteria and MGEs. The aim of this thesis was to study the occurrence and epidemiology of ESBL/AmpC-producing Enterobacteriaceae in different sources in Finland including broiler production, food products, migratory birds, and human clinical samples. Using WGS, bacterial strains were characterized and compared to bacterial strains and plasmids from global databases to discover potential successful plasmids and resistance genes and bacterial sequence types. Furthermore, ESBL-producing E. coli from human clinical isolates from Finland were characterized to assess the similarity between strains of different human and non-human origins and assess potential transmission sources. Poultry and broiler meat have been recognized as a reservoir for ESBL/AmpC- producing E. coli worldwide. In Finland, antibiotics have not been used in broiler production for over a decade, but nevertheless ESBL/AmpC-producing bacteria have been discovered in the production chain. To study the occurrence and transmission routes of ESBL/AmpC-producing E. coli in the broiler production pyramid, samples were taken from different stages of the production pyramid. ESBL/AmpC-producing E. coli was detected in 26.7% of parent level birds, but ESBL/AmpC-producing E. coli was absent from egg surfaces after an incubation period at a hatchery, and E. coli was very rare in hatchlings (2.2%). The findings indicate the transmission routes of these bacteria in the production pyramid are a combination of horizontal and vertical routes, rather than strictly vertical. The global nature of AMR was further studied by sampling migratory birds and imported food products. Fecal samples were collected from barnacle geese (Branta leucopsis) on two occasions in the southern part of Finland. Plasmids considered as internationally successful were recognized from barnacle geese feces and food products. Furthermore, a rare multireplicon plasmid was identified from E. coli from barnacle geese, indicating the adaptive nature of plasmids harboring resistance genes. ESBL/AmpC-producing E. coli was found in 4.5% of the barnacle goose fecal samples, mirroring the limitedly studied prevalence in the asymptomatic human population in Finland. Investigating the occurrence of ESBL/AmpC-producing E. coli and K. pneumoniae in global food products demonstrated raw broiler meat as a potential source for these resistant bacteria. ESBL genes, such as blaCTX-M-15, were recognized in certain food products together with human-associated K. pneumoniae multilocus sequence types, indicating a possible human-related source of transmission. Study of ESBL-producing E. coli isolates from human patients in Finland with WGS identified blaCTX-M-27 as the most common ESBL gene. This demonstrates the spread of globally successful subclade ST131-C1-M27 and supports the notion of a shift in the most dominant CTX-M enzymes in humans. Core genome multilocus sequence typing and comparison of the isolates suggested human-derived ESBL-producing E. coli isolates are distinct from ESBL/AmpC-producing E. coli isolates obtained from animal, food, and environmental sources in Finland. The ever-evolving global health pandemic of AMR demands for a combined effort of different sectors and continuous monitoring, in which WGS has proved to be invaluable. Laajakirjoisia beetalaktamaaseja (ESBL/AmpC) tuottavat Enterobacteriaceae -heimon bakteerit, etenkin Escherichia coli ja Klebsiella pneumoniae, ovat yksi tärkeimmistä antibiooteille vastustuskykyisistä bakteereista, ja näitä havaitaan niin ihmisillä, eläimillä kuin ympäristössäkin. Usein resistenssiä aiheuttava geeni sijaitsee kromosomin ulkopuolisessa DNA-elementissä, plasmidissa, mikä on edesauttanut mikrobilääkeresistenssin leviämistä. Väitöskirjan tavoitteena oli tutkia ESBL/AmpC-entsyymiä tuottavien bakteerien esiintymistä Suomessa eri lähteistä ja verrata bakteerikantojen, plasmidien ja geenien yhtäläisyyksiä kokogenomisekvensoinnin keinoin. Ensimmäisessä osatyössä tutkittiin siipikarjaa, jota on pidetty ESBL/AmpC-entsyymiä tuottavan E. colin reservuaarina maailmanlaajuisesti. Suomessa tuotantopolven broilereita ei ole lääkitty antibiootein yli vuosikymmeneen, mutta tuotantoketjussa on löydetty ESBL/AmpC-entsyymiä tuottavia bakteereja. Hankkeessa tutkituista vanhempaispolven broilereista 26,7 %:lla havaittiin ESBL/AmpC-entsyymiä tuottavaa E. colia, mutta tuotantopolven munien pinnalta haudonta-ajan jälkeen bakteereita ei enää havaittu. Löydökset viittaavat siihen, että ESBL/AmpC-entsyymiä tuottavat bakteerit leviävät tuotantoketjussa useita eri reittejä hyödyntäen, ei pelkästään emolta jälkeläisille. Muuttolintujen mukana mahdollisesti leviävien ESBL/AmpC-entsyymiä tuottavien bakteerien esiintymistä tutkittiin valkoposkihanhista pääkaupunkiseudulla. Näytteistä 4,5 % oli positiivisia ESBL/AmpC-entsyymiä tuottavan E. colin suhteen. Lisäksi löydettiin harvinainen plasmidityyppi, joka osoittaa mahdollisesti uusien resistenssigeeni-plasmidi-yhdistelmien leviämisen olevan mahdollista useita eri reittejä pitkin, yli maarajojen. Tuontielintarvikkeita tutkivassa hankkeessa havaittiin, että etenkin raaka broilerinliha sisälsi ESBL/AmpC-entsyymiä tuottavaa E. colia. Sekä valkoposkihanhista että tuontielintarvikkeista peräisin olevissa näytteissä löydettiin ESBL/AmpC-entsyymiä koodaavia resistenssigeenejä plasmidityypeissä, joita pidetään kansainvälisesti levinneinä. Kliinisistä infektioista eristettyjen ihmisten ESBL-näytteiden yleisin geeni oli blaCTX-M-27, joka on maailmanlaajuisesti lisääntynyt blaCTX-M-15-geenin rinnalla viime vuosina. Kokogenomisekvenssianalyysin perusteella ihmisistä peräisin olevat kannat olivat erillään eläimistä ja elintarvikkeista peräisin olevista kannoista. Väitöskirja tuo uutta tietoa ESBL/AmpC-entsyymiä tuottavien bakteerien esiintyvyydestä ja epidemiologiasta Suomessa, sekä osoittaa kokogenomisekvensoinnin olevan tehokas työkalu mikrobilääkeresistenssin tutkimisessa.

The purpose of the project is to investigate the occurrence of antimicrobial resistance related to use of antimicrobials and biosecurity in the Finnish pork production chain from nursing to slaughter and processing of meat. Additionally, utilising the information about production, management, medication and biosecurity, possible factors related to development and spread of antimicrobial resistance will be identified. Finally, consumers’ exposure to resistant microbes is evaluated. The results will be helpful in improving control measures in order to prevent the emergence and spread of antimicrobial resistance. This poster presents the workflow of the project, when and where the samples are collected. Ten farms’ antimicrobial treatments and indications were used to compile boxplots to visualise differences. Observed resistance features are also presented., FI; en; pdf; efsafocalpoint@evira.fi