Extended-Spectrum Beta-Lactamase-producing Enterobacteriaceae through the lens of One Health in the Whole Genome Sequencing Era

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

Plasmids conferring resistance to extended-spectrum beta-lactamases including a rare IncN+IncR multireplicon carrying blaCTX-M-1 in Escherichia coli recovered from migrating barnacle geese (Branta leucopsis) [version 1; peer review: 2 approved]

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Whole-Genome Sequencing of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli From Human Infections in Finland Revealed Isolates Belonging to Internationally Successful ST131-C1-M27 Subclade but Distinct From Non-human Sources

Antimicrobial resistance (AMR) is a growing concern in public health, particularly for the clinically relevant extended-spectrum beta-lactamase (ESBL) and AmpC-producing Enterobacteriaceae. Studies describing ESBL-producing Escherichia coli clinical samples from Finland to the genomic level and investigation of possible zoonotic transmission routes are scarce. This study characterizes ESBL-producing E. coli from clinical samples in Finland using whole genome sequencing (WGS). Comparison is made between animal, food, and environmental sources in Finland to gain insight into potential zoonotic transmission routes and to recognize successful AMR genes, bacterial sequence types (STs), and plasmids. ESBL-producing E. coli isolates (n = 30) obtained from the Eastern Finland healthcare district between 2018 and 2020 underwent WGS and were compared to sequences from non-human and healthy human sources (n = 67) isolated in Finland between 2012 and 2018. A majority of the clinical isolates belonged to ST131 (n = 21; 70%), of which 19 represented O25:H4 and fimH30 allele, and 2 O16:H5 and fimH41 allele. Multidrug resistance was common, and the most common bla gene identified was bla(CTX-M-27) (n = 14; 47%) followed by bla(CTX-M-15) (n = 10; 33%). bla(CTX-M-27) was identified in 13 out of 21 isolates representing ST131, with 12 isolates belonging to a recently discovered international E. coli ST131 C1-M27 subclade. Isolates were found to be genetically distinct from non-human sources with core genome multilocus sequence typing based analysis. Most isolates (n = 26; 87%) possessed multiple replicons, with IncF family plasmids appearing in 27 (90%) and IncI1 in 5 (17%) isolates. IncF[F1:A2:B20] replicon was identified in 11, and IncF[F-:A2:B20] in 4 isolates. The results indicate the ST131-C1-M27 clade gaining prevalence in Europe and provide further evidence of the concerning spread of this globally successful pathogenic clonal group. This study is the first to describe ESBL-producing E. coli in human infections with WGS in Finland and provides important information on global level of the spread of ESBL-producing E. coli belonging to the C1-M27 subclade. The results will help guide public health actions and guide future research.Peer reviewe

Characterisation of Listeria monocytogenes Isolates from Hunted Game and Game Meat from Finland

Listeria monocytogenes is an important foodborne zoonotic bacterium. It is a heterogeneous species that can be classified into lineages, serogroups, clonal complexes, and sequence types. Only scarce information exists on the properties of L. monocytogenes from game and game meat. We characterised 75 L. monocytogenes isolates from various game sources found in Finland between 2012 and 2020. The genetic diversity, presence of virulence and antimicrobial genes were studied with whole genome sequencing. Most (89%) of the isolates belonged to phylogenetic lineage (Lin) II and serogroup (SG) IIa. SGs IVb (8%) and IIb (3%) of Lin I were sporadically identified. In total, 18 clonal complexes and 21 sequence types (STs) were obtained. The most frequent STs were ST451 (21%), ST585 (12%) and ST37 (11%) found in different sample types between 2012 and 2020. We observed 10 clusters, formed by closely related isolates with 0–10 allelic differences. Most (79%) of the virulence genes were found in all of the L. monocytogenes isolates. Only fosX and lin were found out of 46 antimicrobial resistance genes. Our results demonstrate that potentially virulent and antimicrobial-sensitive L. monocytogenes isolates associated with human listeriosis are commonly found in hunted game and game meat in Finland

Zoonotic bacterial pathogens in slaughter turkeys

Peer reviewe

Plasmid-Borne and Chromosomal ESBL/AmpC Genes in Escherichia coli and Klebsiella pneumoniae in Global Food Products

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.Peer reviewe

Low-level colonization of methicillin-resistant Staphylococcus aureus in pigs is maintained by slowly evolving, closely related strains in Finnish pig farms

Over the past two decades, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has become widely prevalent in pig production in Europe. The carriage status of LA-MRSA is known to vary among individual pigs, but bacterial load in pigs has rarely been studied. We assessed the quantity of LA-MRSA in nasal and skin samples of pigs and investigated the genetic diversity of the strains together with sequenced strains from national surveillance and pathology samples from the Finnish Food Authority. On two farms with assumed MRSA-positive status, farm 1 and farm 2, 10 healthy pigs were sampled three times during 2 weeks from the nares and skin (study A). On farm 1, 54 additional pigs were sampled and from confirmed MRSA-positive animals, 10 were randomly selected and transported to a clean, controlled environment for further sampling (study B). From the samples taken on farms 1 and 2 and in the controlled environment, MRSA was isolated both by direct plating and enrichment on selective media. spa types, multilocus sequence types, staphylococcal cassette chromosome mec types, resistance and virulence genes were determined. Core genome multilocus sequence typing (cgMLST) analysis was performed, including the sequences deriving from the surveillance/pathology samples from the Finnish Food Authority.Peer reviewe

Nordic Vets against AMR—An Initiative to Share and Promote Good Practices in the Nordic–Baltic Region

In the Nordic countries, antimicrobial use in animals and the prevalence of antimicrobial resistance are among the lowest in Europe. The network “Nordic Vets Against AMR” organized a meeting in 2021, with key actors including representatives from universities, veterinary authorities and veterinary organizations in Finland, Norway and Sweden. This paper reflects the most important discussions on education, research, policy and future perspectives, including the experiences of these countries. It concludes that Nordic veterinarians are well placed to lead the way in the fight against antimicrobial resistance and that the sharing of experiences can support colleagues in other countries. Veterinary education must go hand in hand with research activities and continuously updated guidelines and legislation. There is also a need for postgraduate training on antimicrobial resistance and prudent antimicrobial use. The veterinary profession must, by any means necessary, protect the efficiency of antimicrobials for the sake of animal health, animal welfare and productivity, as well as public health. While restrictive use of antimicrobials is crucial, the ability of veterinarians to use this medical tool is also important for the sake of animal welfare and global food security

Wastewater surveillance of antibiotic-resistant bacterial pathogens : A systematic review

Publisher Copyright: 2022, Tiwari, Kurittu, Al-Mustapha, Heljanko, Johansson, Thakali, Mishra, Lehto, Lipponen, Oikarinen, Pitkänen, WastPan Study Group and Heikinheimo.Infectious diseases caused by antibiotic-resistant bacterial (ARB) pathogens are a serious threat to human and animal health. The active surveillance of ARB using an integrated one-health approach can help to reduce the emergence and spread of ARB, reduce the associated economic impact, and guide antimicrobial stewardship programs. Wastewater surveillance (WWS) of ARB provides composite samples for a total population, with easy access to the mixed community microbiome. This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remain poorly understood especially in relation to clinical evidence in sewershed communities. Here, we systematically searched the literature to identify studies that have compared findings from WWS of ARB and antibiotic resistance genes (ARG) with clinical evidence in parallel, thereby evaluating how likely WWS of ARB and ARG can relate to the clinical cases in communities. Initially, 2,235 articles were obtained using the primary search keywords, and 1,219 articles remained after de-duplication. Among these, 35 articles fulfilled the search criteria, and an additional 13 relevant articles were included by searching references in the primary literature. Among the 48 included papers, 34 studies used a culture-based method, followed by 11 metagenomics, and three PCR-based methods. A total of 28 out of 48 included studies were conducted at the single sewershed level, eight studies involved several countries, seven studies were conducted at national or regional scales, and five at hospital levels. Our review revealed that the performance of WWS of ARB pathogens has been evaluated more frequently for Escherichia coli, Enterococcus spp., and other members of the family Enterobacteriaceae, but has not been uniformly tested for all ARB pathogens. Many wastewater-based ARB studies comparing the findings with clinical evidence were conducted to evaluate the public health risk but not to relate with clinical evidence and to evaluate the performance of WWS of ARB. Indeed, relating WWS of ARB with clinical evidence in a sewershed is not straightforward, as the source of ARB in wastewater cannot be only from symptomatic human individuals but can also be from asymptomatic carriers as well as from animal sources. Further, the varying fates of each bacterial species and ARG within the sewerage make the aim of connecting WWS of ARB with clinical evidence more complicated. Therefore, future studies evaluating the performance of many AMR pathogens and their genes for WWS one by one can make the process simpler and the interpretation of results easier.Peer reviewe