Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates

Environmental Pollution 291 (2021) 118136 Available online 10 September 2021 0269-7491/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates ☆ Rita Tinoco Torres a , * , M´ onica V. Cunha b , c , D´ ebora Araujo d , e , Helena Ferreira d , f , Carlos Fonseca a , g , Josman Dantas Palmeira a a Departamento de Biologia & CESAM, Universidade de Aveiro, Campus de Santiago, 3810-193, Aveiro, Portugal b Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciˆ encias, Universidade de Lisboa, 1749-016, Lisbon, Portugal c Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciˆ encias, Universidade de Lisboa, 1749-016, Lisbon, Portugal d Faculty of Engineering of University of Porto, Porto, Portugal e UCIBIO - Applied Molecular Biosciences Unit, REQUIMTE – University of Porto, Porto, Portugal f Microbiology, Biological Sciences Department, Faculty of Pharmacy of University of Porto, Porto, Portugal g ForestWISE - Collaborative Laboratory for Integrated Forest & Fire Management, Quinta de Prados, 5001-801, Vila Real, Portugal ARTICLE INFO Keywords: mcr-1 Colistin Antimicrobial resistance genes (ARG) Wildlife Environmental reservoirs ABSTRACT The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a “last resort” antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n =2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces. 1. Introduction Antimicrobial resistant bacteria (AMR) and antimicrobial resistance genes (ARG) are a multifaceted problem that poses a worldwide threat to human, animal and environmental health (Woolhouse and Farrar, 2014). Antimicrobials are essential for the treatment of bacterial infections in humans and animals but AMR has become an obstacle to the treatment of infectious diseases, posing a significant threat to public health (Laxminarayan et al., 2013). The emergence of multidrug-resistant (MDR) Gram-negative bacteria in clinical and nosocomial settings is a growing problem worldwide (Li et al., 2006). Colistin, a “last resort” drug for the treatment of severe infections caused by MDR Gram-negative bacteria and one of the single effective drugs for treating life-threatening bacterial infections (Li et al., 2006), has been listed as a “Highest Priority Critically Important Antimicrobial“ by WHO (WHO, 2019). Additionally, and worrisome, apart from its use in hospital settings, colistin has been historically used in veterinary medicine, either as a therapeutic option or as a feed growth promoter in livestock. ☆ This paper has been recommended for acceptance by Sarah Harmon. * Corresponding author. CESAM and Department of Biology, University of Aveiro, Campus de Santiago, 3810-193, Aveiro, Portugal. E-mail address: [email protected] (R.T. Torres). Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/envpol https://doi.org/10.1016/j.envpol.2021.118136 Received 1 July 2021; Received in revised form 2 September 2021; Accepted 6 September 2021 Environmental Pollution 291 (2021) 118136 2 Examples include some Asian countries (e.g., India, Japan, and Vietnam) (see Kempf et al., 2016), the U.S.A., and Brazil, where colistin is widely used (Fernandes et al., 2016; Palmeira et al., 2018a). Due to the inevitable risk of underlying emerging resistance, the use of colistin in food-producing animals has been globally limited or banned (Sun et al., 2018). Acquired resistance to colistin has been linked to chromosomal mutations but recently it has been related with plasmid-mediated mechanisms (Liu et al., 2016). A plasmid-mediated colistin resistance gene, mcr-1 (mobile colistin resistance 1), was firstly reported in southern China in Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolates from human and animal origin, that had been recovered between 2011 and 2014 (Liu et al., 2016), flashing lights on our (in) ability to treat MDR infections. Plasmid-mediated colistin resistance genes pose additional public and environmental risk, as resistance genes can easily, and rapidly, spread via horizontal transfer. In fact, since Liu and colleagues (Liu et al., 2016), mcr-1 genes have been recovered from livestock (Dantas Palmeira and Ferreira, 2020; Duggett et al., 2017; Zhang et al., 2019), food products (Luo et al., 2017; Monte et al., 2017), the wider environment (Fernandes et al., 2017) and humans worldwide (Yin et al., 2017). These findings confirm that mcr-1 is already disseminated globally (Sun et al., 2018), highlighting the complexity of reservoirs and transmission pathways. The environment is a sink of many anthropogenic sources of contamination (Huijbers et al., 2015) but it can also harbour antibiotic resistance genes regardless of the contact with xenobiotics arising from human activities. So, the wider environment is considered a reservoir of resistant microorganisms and their genes (Allen et al., 2011a). The increasing incidence of AMR in humans and livestock has been linked to the emergence of AMR in wildlife (Jones et al., 2008; Wellington et al., 2013), putting under the spotlight environmental reservoirs of antibiotic resistance (Mills and Lee, 2019). Recently, growing evidence suggests that the wildlife compartment could provide important insights into AMR emergence and persistence (Allen et al., 2011b; Torres et al., 2020a,b; Wellington et al., 2013) and various authors suggest wildlife as biological indicators of environmental pollution caused by xenobiotics related to antimicrobial resistance (Furness et al., 2017; Radhouani et al., 2012; Torres et al., 2020a,b). However, the role of this wild setting in the dissemination and transmission pathways of colistin resistant genes is understudied. Until now, only a few studies have reported this resistance determinant in bacteria from wildlife species from different countries (Xiaomin et al., 2020). For example, mcr-1 has been detected in gulls from South America (Liakopoulos et al., 2016) and Europe (Ahlstrom et al., 2019; Ruzauskas and Vaskeviciute, 2016), Barbary macaques from Africa (Bachiri et al., 2018), birds from Asia (Mohsin et al., 2016) and Europe (Tarabai et al., 2018) and recently in the endangered P` ere David’s Deer in China (Lu et al., 2020), indicating that the dissemination of mcr-1 has gone wild (Liakopoulos et al., 2016) and most importantly, that wildlife can contribute to the spread of resistance. While birds have been widely surveyed for mcr-1 bacteria due to their long-distance migratory movements and exposure to anthropogenic sources (e.g., landfills, wastewater from urban areas), wild ungulates have been neglected but this taxonomic group surely deserves attention, as they are known to carry AMR and ARG in their gut microbiota, including some highly virulent strains (Dias et al., 2019; Navarro-Gonzalez et al., 2018; Torres et al., 2020a,b). These are perfect model species due to their ubiquity, considerably large home ranges, unlikeliness of being treated with antibiotics and habitat overlapping with livestock and humans, connecting anthropogenic and natural areas. Also, they are emerging as a source of foodborne diseases in humans due to the consumption of game meat, placing them in direct contact with humans. Some species, e.g., wild boar (Sus scrofa) have been suggested as a sentinel of AMR bacteria and genes due to their wide range (Torres et al., 2020a,b). They have also increased in number and distribution all over Europe, inhabiting from natural to non-natural environments. As a consequence of this expansion, its wide distribution and link between natural and humanized environments, this species occupies a central place as reservoir and vector of several zoonotic pathogens (Fredriksson-Ahomaa, 2019). If generalist species such as these carry mcr-positive bacteria, they will certainly play a central role in AMR and ARG life cycles. Therefore, research on the dissemination of mcr-1-positive E. coli from widely distributed ungulates is of great significance but, to the best of our knowledge, the distribution of mcr-1 in wild ungulates remains to be investigated, with the exception of P` ere David’s deer in China (Lu et al., 2020). To fill this caveat, this study investigated the presence of colistinresistant bacteria and their genes among the commensal microbiota of widely distributed ungulates, which through excretion into the environment could pose a potential risk to humans, livestock and other wildlife species. Our results are discussed under the One Health umbrella, stressing the need for ongoing wildlife surveillance for early identification of colistin resistance threats. 2. Materials and methods 2.1. Sampling and bacterial isolation Sampling of wild ungulates was completed during two hunting seasons (October to February, 2018/2019 and 2019/2020), from 36 different hunting grounds, located in 15 of the 18 districts of continental Portugal (Fig. 1). A total of 181 faecal samples were opportunistically collected from legally hunted animals (n =88 from wild boar, Sus scrofa; n =62 from red deer, Cervus elaphus; n =29 from fallow deer, Dama dama and n =2 from mouflon, Ovis orientalis musimon) within 1–3 h after Fig. 1. Location of the sampling locations (black dots) in Portugal. R.T. Torres et al. Environmental Pollution 291 (2021) 118136 3 death. No animals were sacrificed for the purposes of this study. None of the authors was responsible for the death of any animals. All applicable institutional and/or national/international guidelines for the care and use of animals have been followed. The faecal samples were collected directly from the rectus of the animals with sterilized material and subjected to refrigeration conditions during transportation to the laboratory, where they were stored at −20 ◦C until microbiological analysis in the following days. Faecal samples were thawed, submitted to enrichment with Tryptic Soy Broth (TSB, Liofilchem – Italy) and incubated without agitation at 37 ◦C for24 h. To select resistantEnterobacteriaceae isolates, 100 μ L of bacterial suspensions grown in the enrichment broth were inoculated onto the surface of MacConkey agar (Liofilchem – Italy) supplemented with ampicillin (AMP, 100 μ g/mL, Sigma-Aldrich - Germany), cefotaxime (CTX, 1 μ g/mL, Labesfal - Portugal), meropenem (MRP, 0,5 μ g/mL, Sigma-Aldrich - Germany), ciprofloxacin (CIP, 1 μ g/mL, Acros Organics – United Kingdom) or tetracycline (TET, 100 μ g/mL, Sigma-Aldrich - Germany), followed by incubation at 37 ◦C during 24 h. One lactose-fermenter colony of each plate was streaked into the same antibiotic-selection plate, at 37 ◦C/ 24H, for isolation and resistance confirmation purposes, as described previously (Palmeira et al., 2020a). 2.2. Antimicrobial susceptibility-testing and identification All selected isolates were submitted to the disk-diffusion method to evaluate their antimicrobial resistance profile. The methodology was carried out according to EUCAST guidelines (2021), Clinical and Laboratory Standard Institute (CLSI, 2017) standards used for ceftiofur (EFT), enrofloxacin (ENR), TET and gentamycin (GEN), using antibiotic disks (Oxoid – United Kingdom) for the following antibiotics: AMP (10 μ g), ampicillin plus clavulanic acid (AMC, 30 μ g), cefoxitin (FOX, 30 μ g), CTX (5 μ g), ceftazidime (CAZ, 10 μ g), EFT (30 μ g), cefepime (FEP, 30 μ g), aztreonam (ATM, 30 μ g), MRP (10 μ g), CIP (5 μ g), ENR (5 μ g), TET (30 μ g), tigecycline (TGC, 15 μ g), GEN (10 μ g), sulfamethoxazole plus trimethoprim (SXT, 25 μ g), chloramphenicol (CHL, 30 μ g), fosfomycin (FOT, 200 μ g) and nitrofurantoin (FUR, 100 μ g). For colistin, minimal inhibitory concentration (MIC) was determined by the broth microdilution method in accordance with EUCAST standards for clinical breakpoints and ECOFFs analysis (EUCAST, 2021). Multidrug-resistant profiles (MDR) were determined according to the criteria of Magiorakos et al. (2012), considering the MDR profile when isolates show antibiotic-resistant profile to 3 or more antibiotics categories. The E. coli isolates were presumptively identified with CHROMagar Orientation (CHROMagar – France) and confirmed by biochemical tests or PCR. 2.3. Plasmid-mediated colistin resistance and accessory resistance genes Screening for mcr-1 to 5 genes was performed by PCR according to standard methodology and applied to all selected isolates of Enterobacteriaceae (Rebelo et al., 2018). Amplicons were sequenced by sanger sequencing (Eurofins genomics – Germany) was performed to confirm the mcr genes. Resistance genes for accessory resistance of mcr-positives isolates to beta-lactams (bla TEM , bla SHV and bla OXA ), fluoroquinolones (qnrA, qnrB, qnrS and aac6′Ib-cr), aminoglycosides (aac3′-II, aac3′-IV, ant2′′, strA and strB), tetracycline (tetA and tetB), sulfamethoxazole (sul1, sul2 and sul3), trimethoprim (dfrA1, dfrA12 and dfrA17) and chloramphenicol (cmlA1, catA and floR) were screened by PCR (Table S1). 2.4. Characterization of Escherichia coli phylotypes, pathogenic potential and plasmid replicon typing For mcr-positive E. coli isolates, a standard protocol was performed to characterize the phylogenetic group (A, B1, B2, C, D, E and F) (Clermont et al., 2013). To determine their pathogenic potential, we evaluated if mcr-positive E. coli belonged to one of seven major pathotypes of E. coli, using an established methodology through a 12 genes multiplex PCR (Müller et al., 2007); additionally 5 multiplex PCR were done to screen for 30 different E. coli virulence factors (Johnson and Stell, 2000). For clonality evaluation, pulsed-field gel electrophoresis (PFGE) was performed according to PulseNet USA standard protocol, through a CHIF-DR III system (BIO-RAD - U.S.A.) using 10 s as initial time and 40 s as final time for 21 h of running, after digestion of immobilized DNA with XbaI restriction enzyme (Bioron - Germany) (Palmeira et al., 2020b). MLST was performed following the Achtman scheme (https://pubml st.org/bigsdb?db=pubmlst_mlst_seqdef) for mcr-1-positive isolates by sanger sequencing (Eurofins genomics – Germany) the amplicons of 7 housekeeping genes and the sequencing type (ST) determined according to their allelic profile. Plasmid content of mcr-positive isolates was determined using standard protocols based on replicon typing (Inc), through five multiplex and three simplex PCR assays (Carattoli et al., 2005). All PCR assays were performed using SuperHot Master Mix (Bioron - Germany) and the primers used are listed in table S1. 3. Results A total of 151 antibiotic resistant Enterobacteriaceae were selected from 83 (45.8%) of the sampled animals. Regarding the antibiotic selection distribution, the majority of resistant isolates were selected in AMP (67/151) and TET (61/151), followed by CTX (14/151) and CIP (9/151), no isolates was selected in MEM. The resistance profile of Enterobacteriaceae isolates (Table 1) was diversified, with ampicillin (71.5%), tetracycline (63.6%) and sulfamethoxazole plus trimethoprim (23.8%) being the less effective antimicrobials in vitro (Table 1). The mcr screening evidenced that four (2.6%) of the antibiotic resistant isolates harbour mcr-1 gene, being all E. coli (Table 2). The four mcr-1-positive E. coli were selected from different fallow deer hunted in the same hunting ground, highlighting the possibility of circulation of resistant-E. coli determinant among these animals and/or exchange of this mobile resistance across their commensal microbiota. All mcr-1positive E. coli show MIC of 2 μ g/mL, according to these results the isolates are susceptible to colistin. Regarding the plasmid content of mcrpositive isolates, the plasmids IncP (4/4), IncFIB (3/4), IncK (3/4) and IncY (2/4) were detected in more than one isolate. All mcr-1-positive isolates showed accessory resistance, with three in vitro resistance patterns: AMP/TET (2/4), CIP/ENR (1/4) and AMP/ TET/CIP/GEN/SXT/CHL (1/4); one isolate (U147) was classified as MDR since it is resistant to antimicrobials from six antibiotic classes. Seven ARG were identified, namely bla TEM (3/4), tetA (1/4), tetB (2/4), Table 1 Resistance profile of 151 Enterobacteriaceae isolated in wild ungulates from Portugal. Antibiotic Disk Concentration ( μ g) Resistant Isolates No. % Ampicillin 10 108 71.5% Amoxicillin +Clav. Ac. 30 10 6.6% Cefoxitin 30 14 9.3% Cefotaxime 5 11 7.3% Ceftazidime 10 7 4.6% Ceftiofur 30 8 5.3% Cefepime 30 4 2.6% Aztreonam 30 8 5.3% Meropenem 10 0 0.0% Ciprofloxacin 5 17 11.3% Enrofloxacin 5 9 6.0% Gentamycin 10 7 4.6% Tetracycline 30 96 63.6% Tigecycline 15 0 0.0% Fosfomycin 200 7 4.6% Sulfam. +Trimethoprim 25 36 23.8% Chloramphenicol 30 16 10.6% Nitrofurantoin 100 1 0.7% R.T. Torres et al. Environmental Pollution 291 (2021) 118136 4 sul2, (1/4), sul3 (1/4), dfrA1 (1/4), dfrA12 (1/4), cmlA1 (1/4) and floR (1/4) that confers resistance to beta-lactams, tetracyclines, sulphonamides, trimethoprim and chloramphenicol, respectively. About the E. coli phylogenetic profile, all mcr-1-positive isolates belonged to B1 group, which are related with commensal E. coli in mammals’ gut (Carlos et al., 2010). No E. coli isolates exhibited a pathotype profile accordingly to the used protocol, but showed presence of virulence factors fimH (4/4), traT (1/4) and fuyA (1/4). The clonality evaluation by PFGE (Figure S1) showed that two E. coli (isolates U54 and U144) from two individuals shared high similarity, suggesting the circulation of clonal commensal bacteria among animals from the same habitat. Regarding the sequencing type of the mcr-1-positive isolates, the results confirm the relation of U54 and U144 that share the ST345. The isolate 147 belongs to the ST533, highlighting the link between wildlife and livestock. The ST155 is the sequencing type of U54 isolates, giving this isolates the status of high-risk clone, once this E. coli clone is internationally recognized due their global distribution, pathogenicity skills and capacity of acquired and spread of AMR (Mathers et al., 2015). 4. Discussion The global dissemination of mobile colistin resistance genes (mcr) across widely distributed species, and their environment, is alarming and represents a global threat to public and environmental health. At present, there is a dearth of studies screening and/or reporting bacteria carrying mcr-1 from wildlife, since the bulk has been focused on the human and veterinary settings. Our results show that it is imperative to expand the surveillance to wildlife species, particularly those who represent major epidemiological links between natural and humanized environments, such as wild ungulates. In the present study, opportunistic fresh samples were collected on a nationwide framework, taking advantage of a network of collaboration with hunter federations. Diverse environmental scenarios were monitored, from natural to humanized settings, and several ungulate species were screened. In the present study, the mcr-1 gene was detected in four E. coli isolated from fresh faeces collected from hunted fallow deer in Portugal. These four animals were hunted in the same hunting ground, suggesting that this mobile resistance genetic element (or the resistant-E. coli harbouring it) circulates among these animals and their environment. Furthermore, clonality of E. coli from different fallow deer was confirmed, thus corroborating the former hypothesis. The environmental context was probably determinant in the clonal spread of mcr-1 bacteria amongst these ungulates, but possibly also because these animals have large range areas, due to their daily movement features, highlighting the role of wild ungulates as epidemiological links of antimicrobial resistance at the interface of natural-humanized environments (Tiedje et al., 2019; Tymensen et al., 2018). Apparently, the mcr-1–producing E. coli reported here was confined to a single geographical area, calling for the urgent implementation of a wide-range surveillance and monitoring network aimed at other important emerging resistance genes in wildlife reservoirs, to better quantify the risk to human and environmental health. To the best of our knowledge, this is the first report of mcr-1 gene from ungulates, both in Portugal and Europe. For instance, in a national screening of 660 wild ungulates (red deer, roe deer, fallow deer, European bison and wild boar) in Poland, no mcr gene was found (Wasyl et al., 2018). So far, besides our study, the single report of mcr-1 occurrence in wild ungulates was published in 2020, in China, and it involved the P` ere David’s deer (Elaphurus davidianus) (Lu et al., 2020). Our results raise several concerns as they clearly indicate that resistance towards drugs from antimicrobial classes of the highest priority in human medicine, such as the oxyimino-beta-lactam (cefotaxime, ceftazidime, cefepime, aztreonam) and fluoroquinolones (ciprofloxacin), are widespread in the wider environment. The high rates (83/ 151.45.8%) of resistant-bacteria among commensal microbiota of these wild ungulates confirm their role of simultaneously reservoirs and dispersers of resistant bacteria, including for an extended range of antibiotic classes (beta-lactams, including 3rd and 4th generation cephalosporins, fluoroquinolones, tetracycline, fosfomycin, aminoglycosides, sulphonamides, chloramphenicol and nitrofuran), even when they have little contact with humans and livestock. The occurrence of mcr-1-positive isolates with resistance to other antibiotic classes has been frequently reported, most commonly when this gene shares its location with other ARG including those found in mcr-1-positive E. coli (Don` a et al., 2017). The diversity of ARG detected in mcr-1-positive E. coli isolates confirms their role as potential sources of resistance determinants and their relation with mobile genetic elements such as plasmids (Chen et al., 2020).). It is relevant to note the presence of virulence factors in these commensal B1 mcr-1-positive E. coli, since these genes provide virulence attributes that increase bacterial adaptability to act as pathogenic agents (Johnson and Stell, 2000). The combination of antibiotic resistance and virulence in these bacterial isolates sets up a dangerous threat to human and animal health and highlights the importance of wildlife as source of pathogenic microorganisms (Dias et al., 2019). Interestingly, all mcr-1-positiveisolates displayed low MIC values (2 μ g/mL) to colistin that places these E. coli in the wild-type population, accordingly to ECOFF interpretation (EUCAST, 2021). The presence of mcr-1 is normally related with high prevalence of colistin resistance (Kieffer et al., 2017), but some workers have reported the present of mcr-1 in colistin-susceptible isolates (Palmeira et al., 2018b). The description of wild-type E. coli carrying mcr-1 can be related to gene expression profiles that are affected by plasmid types, bacterial host and antibiotic pressure (Zhang et al., 2019). The spread of mcr-1 spread is deeply connected to the dynamics of plasmid mobility (Li et al., 2010). This gene has been related with a large range of Inc types of plasmids, including IncP, FIB, K and Y, all present in mcr-1-positive E. coli. The presence of mcr-1-related plasmids reassert the theory that mcr-1 spread reached the wild animals in a context of pressure over the natural environment exerted by human activities (Don` a et al., 2017; dos Santos et al., 2020; Li et al., 2010; Swift et al., Table 2 Characterization of mcr-1-positive Escherichia coli from wild ungulates in Portugal. Ungulate species Sex Age* Isolate ID Resistance gene Colintin MIC Accessory resistance Accessory resistance genes MDR profile Plasmid content (Inc) Phylogroup Sequencing Type Virulence factors Fallow deer M J U54 mcr-1 2 μ g/mL AMP, TET bla TEM , tetB – FIB, Y, P B1 ST345 fimH Fallow deer F J U140 mcr-1 2 μ g/mL CIP, ENR – I1, P, K B1 ST155 fimH Fallow deer M A U144 mcr-1 2 μ g/mL AMP, TET bla TEM , tetB – FIB, Y, P, K B1 ST345 fimH Fallow deer M A U157 mcr-1 2 μ g/mL AMP, CIP, TET, GEN, SXT, CHL tetA, sul2, sul3, dfrA1, dfrA12, cmlA1, floR MDR FIB, P, T, K B1 ST533 fimH, traT, fuyA *J – juvenile, A - adult. R.T. Torres et al. Environmental Pollution 291 (2021) 118136 5 2019). E. coli is still the most frequent gram-negative bacterium harbouring the mcr gene, a finding consistent with the study of Ahmed et al. (2019). Worryingly, we also found plasmid-mediated colistin resistance (mcr-1) in fallow deer. Interestingly, the mcr-1 positive isolates were recovered from 4 different fallow deer that shared the same habitat and inhabited a touristic fenced hunting ground on southeast of central Portugal, with supposedly limited contact with human and livestock. Most touristic hunting grounds on this region, where food and water are scarce, particularly in the summer, use artificial supplementary feeding and watering. Even though supplementary feeding is mostly with no commercial feed additives, the feeding and watering sites are aggregation points, which has been associated with increased risk of infectious disease transmission, e.g., wild boar and red deer for tuberculosis (Reis et al., 2020). It is difficult to infer the origin of mcr-1 genes in the ecosystem but some potential sources of could be hypothesized. Being a touristic hunting ground means that the turnover of different people in this area is very high, thus creating the opportunity for animal exposure to xenobiotic disturbance and to alien bacteria. The zoonotic potential of mcr-1-carrying bacteria has been suggested and discussed by many studies (Ahlstrom et al., 2019; Liakopoulos et al., 2016; Lu et al., 2020; Ruzauskas and Vaskeviciute, 2016). One study showed that the mcr-1-positive plasmid harboured by wildlife is IncI2 plasmid that is consistent with plasmid documented from human bloodstream infection in Denmark (Liakopoulos et al., 2016), further supporting that the mcr-1 detected in wildlife might have come from humans or food animals. Few studies have explored the transmission of mcr-1 between humans and wildlife, therefore, more studies encompassing different ecological niches are needed to identify the reservoirs and transmission routes of mcr-1. Wild ungulates are one of the most extensively distributed mammals across the world, colonizing and occupying a variety of environments, exhibiting high tolerance to human disturbance, while also exploring anthropogenic food available in these environments. They are also carriers of several zoonotic pathogens (Fredriksson-Ahomaa, 2019) and also AMR bacteria (Dias et al., 2019; Torres et al., 2020). Due to their feeding ecology, their link to natural and humanized areas, wild ungulates have a high potential of harbouring ARG and particularly mcr-positive genes, contributing to their environmental dissemination. Previous studies in Portugal showed that mcr-1 genes are disseminated in humans (Teixeira et al., 2020), livestock (pigs, Kieffer et al., 2017), intensive rabbits farming (Freitas-Silva et al., 2018) and the wider environment (yellow-legged gulls Ahlstrom et al., 2019), retail meat (Figueiredo et al., 2016) and also fresh vegetables (Manageiro et al., 2020). Portugal has been ranked, in 2015, the 7th country with the highest sales of antimicrobials for veterinary use in Europe and the 5th in the consumption of polymyxins, where colistin is widely used in veterinary medicine (EMA, 2018). Based on these numbers and on their ecology, we hypothesise that fallow deer may have been exposed to environmental pollution and anthropogenic factors (e.g., human waste, sewage treatment plant effluents, and manure), which may have induced colistin resistance and acquisition of resistance determinants. Dissemination via third parties such as birds cannot be discarded as various studies have suggested that birds might be considered an important vector of mcr-positive genes (Ruzauskas and Vaskeviciute, 2016; Ahmed et al., 2019). 5. Conclusions Here, we reported the first case of colistin–resistant mcr-1 genes in E. coli isolates from wild ungulates in Portugal. This is the first description of mcr genes in Europe, highlighting their global dissemination in wild mammals, even in species with low contact with humans. Detection of a plasmid-mediated gene in the context of resistance to a last-line drug in ungulates is worrisome, posing a possible threat to public and animal health. However continued surveillance in these species may also be an opportunity since wild ungulates may be bioindicators for clinically important antibiotic-resistant pathogens that can seriously affect human communities related with these ecosystems. Our results emphasize the importance of continued surveillance of E. coli and other commensal bacteria with transferred colistin resistance mediated by mcr genes. The environmental dissemination of critical priority pathogens is a serious threat to planetary health. Author statement R.T.T: Conceptualization, Writing, Supervision, Funding acquisition; Project administration; M.V.C.: Writing – review & editing; D.A.: Methodology; H.F.: Resources; C.F.: Resources; J.D.P.: Methodology, Conceptualization, Writing – review and editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was funded by the project EcoARUn: POCI-01-0145FEDER-030310funded by FEDER, through COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds (OE), through FCT/MCTES. R. T. Torres is funded by national funds (OE), through FCT–Fundacao para a Ciˆ encia e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19. Thanks are due to FCT/MCTES for the financial support to CESAM (UID/AMB/50017/2019), through national funds. Strategic funding to cE3c (UIDB/00329/20192020), BioISI (UIDB/04046/2020) and UCIBIO (UIDP/04378/2020 and UIDB/ 04378/2020) from FCT is also acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.envpol.2021.118136. References Ahlstrom, C.A., Ramey, A.M., Woksepp, H., Bonnedahl, J., 2019. Early emergence of mcr-1-positive Enterobacteriaceae in gulls from Spain and Portugal. Environ. Microbiol. Rep. 11, 669–671. https://doi.org/10.1111/1758-2229.12779. Ahmed, Z.S., Elshafiee, E.A., Khalefa, H.S., Kadry, M., Hamza, D.A., 2019. Evidence of colistin resistance genes (mcr-1 and mcr-2) in wild birds and its public health implication in Egypt. Antimicrob. Resist. Infect. Contr. 8, 1–8. https://doi.org/ 10.1186/s13756-019-0657-5. 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