Science of the Total Environment 830 (2022) 154814 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Occurrence of multidrug resistant Gram-negative bacteria and resistance genes in semi-aquatic wildlife - Trachemys scripta, Neovison vison and Lutra lutra - as sentinels of environmental health Tilaye Shibbiru Mengistu a, Biel Garcias b, Gabriela Castellanos b, Chiara Seminati b, ⁎ Rafael A. Molina-López c, Laila Darwich b, a School of Veterinary Medicine, Wolaita Sodo University, Ethiopia b Department Sanitat i Anatomia Animals, Veterinary Faculty, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, CP 08193, Spain c Wildlife Rehabilitation Center of Torreferrusa, Barcelona, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • Pond sliders (25.3%), American minks (21%) and Eurasian otters (14.5%) had AMR genes. • The highest frequency of AMR was ob- served against β-lactams. • ESBL/AmpC were found in highly- populated areas and in high livestock den- sity areas. • Semi-aquatic wild animals are good senti- nels of AMR environmental contamina- tion • One Health Approach is urgently needed to reduce the impact of anthropogenic actions. A R T I C L E I N F O A B S T R A C T Article history: Emergence of antimicrobial resistance (AMR) in bacterial pathogens has been recognized as a major public health con- Received 8 November 2021 cern worldwide. In the present study, antimicrobial resistant Gram-negative bacteria (AMRGNB) and AMR genes were Received in revised form 20 March 2022 assessed in semi-aquatic wild animals from a highly populated and intensive farming region of Spain, Catalonia. Clo- Accepted 21 March 2022 acal/rectal swab samples were collected from 241 animals coming from invasive species Trachemys scripta (n = 91) Available online 24 March 2022 and Neovison vison (n = 131), and endangered-protected species Lutra lutra (n = 19). Accordingly, 133 (55.2%) iso- Editor: Rafael Mateo Soria lates were identified as AMRGNB. Escherichia coli and Pseudomonas fluorescens were among the bacteria most fre- quently isolated in all animal species, but other nosocomial agents such as Klebsiella pneumoniae, Salmonella spp. or Keywords: Citrobacter freundii, were also prevalent. The phenotypic susceptibility testing showed the highest resistance to β- Antimicrobial resistance lactams (91%). Molecular analysis showed 25.3% of turtles (15.4% ESBL/Ampc genes), 21% of Eurasian otters ESBL/AmpC (10.5% ESBL/Ampc genes) and 14.5% of American minks (8.4% ESBL/Ampc genes) were positive to AMR genes. Trachemys scripta The genotyping frequency was tetM (20.6%), blaCMY-2 (13%), ermB (6.1%), blaCMY-1 (4.6%), blaCTX-M-15 (3.1%) Neovison vison and mcr-4 (0.8%). Turtles had a larger prevalence of AMRGNB and AMR genes than mustelids, but American mink car- Lutra lutra ried mcr-4 colistin-resistance gene. Moreover, cluster analysis of AMR gene distribution revealed that an ESBL/AmpC Semi-aquatic wildlife cluster in a highly populated area comprising big metropolitan regions, and another tetM/emrB cluster in an expended area with highly intensive livestock production. Although the mcr-4 positive case was not included in those clusters, ⁎ Corresponding author. E-mail addresses: tilayeshiberu@gmail.com (T.S. Mengistu), biel.garcias@uab.cat (B. Garcias), gabriela.nicole.casmor@gmail.com (G. Castellanos), chiara.seminati@uab.cat (C. Seminati), rafael.molina@gencat.cat (R.A. Molina-López), laila.darwich@uab.cat (L. Darwich). http://dx.doi.org/10.1016/j.scitotenv.2022.154814 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 that case was found in a county with a high pig farm density. In conclusion, semi-aquatic wild animals are a good sen- tinel for environmental contamination with AMRGNB and AMR genes. Therefore, One Health Approach is urgently needed in highly populated regions, and with intensive livestock production like Catalonia. 1. Introduction delimited in the east by Mediterranean Sea, in the north by France and in the west and south by autonomous communities of Aragon and Valencia, Antibiotics have been used to treat a wide range of bacterial infections in respectively. Catalonia has an estimated population over 7.8 million with humans (Zinner, 2007) and animals (da Cunha et al., 2019). They have also a population density of 242.3 inhabitants per km2 (Statistical Institute of been used as growth promoters to increase food production in animals (da Catalonia, 2020). Regarding livestock production, Catalonia is a region Cunha et al., 2019; Van et al., 2020). β-Lactams are among the most clinically with a high density of livestock farms, mainly pig farms, leading the important antibiotics in both human and veterinary medicine (Li et al., 2007), Spanish pig production with 21.41 million pigs slaughtered in the first being third-generation cephalosporins the most commonly used antimicro- 11 months of 2020 (Pig Process, 2021). bial drugs in humans and animals (Hasman et al., 2005). Ceftriaxone is used to treat invasive Gram-negative bacterial infections in humans, and 2.2. Study design ceftiofur in food producing animals (Heider et al., 2009). However, intensive use of antibiotics in human and veterinary medicine has resulted in increased A cross-sectional study was conducted on semi-aquatic wild animals prevalence of antimicrobial resistant bacteria (AMRB), opportunistic infec- from invasive species such as Neovison vison (American mink) and tions in hospital settings, and global spread of resistant bacteria (Durão Trachemys scripta (Pond slider), and protected species such as Lutra lutra et al., 2018; Pitout and Laupland, 2008). Emergence of antimicrobial resis- (Eurasian otter). tance (AMR) in most common bacterial pathogens has been recognized as a The American minks and the Pond sliders are categorized as invasive major public health concern worldwide. Resistant bacteria have emerged in species in Catalonia, and they were captured and euthanized as part of hospital as well as community settings suggesting the presence of reservoirs the regular population management plan for the species, according to the of AMRB outside the hospital (Munita and Arias, 2016). national legislation (Spanish RD 630/2013, August 2nd, regulation of the Emergence and spread of multidrug resistant bacteria, specifically the ex- Spanish Catalogue of invasive exotic species). On the other hand, Eurasian tended spectrum β-lactamases (ESBLs) producing strains, are often responsi- otter is a protected species, thus, a rehabilitation program is always imple- ble for the failure of antibiotic treatment in hospital settings. ESBLs are mented. For this study, the Wildlife Rehabilitation Center (WRC) of rapidly evolving group of β-lactamases which share the ability to hydrolyze Torreferrusa received two kind of animals: live animals (all turtles and penicillins, first-, second-, and third-generation cephalosporins, and aztreo- most of the minks and otters) and dead animals (some minks and few ot- nam (but not the cephamycins or carbapenems) yet are inhibited by ters). In the case of live animals, samples were always taken on arrival to clavulanic acid (Ghafourian et al., 2014; Paterson and Bonomo, 2005; the WRC before being euthanized (for invasive species) or before receiving Rawat and Nair, 2010). ESBLs are frequently plasmid encoded and can be ex- any pharmacologic or antimicrobial treatment (for protected species). In changed readily among bacterial populations horizontally. Plasmids responsi- the case of dead animals, fresh corpses (less than 24 h postmortem) found ble for ESBL production frequently carry genes encoding resistance to other in the field (otters) or captured and euthanized animals by licensed staff drug classes such as aminoglycosides (Paterson and Bonomo, 2005). in the field (minks), were submitted at the WRC and frozen at −18 °C Studies conducted in wild birds and mammalian species showed that these until the necropsy and sample collection. animals can serve as reservoirs and potential vectors for the spread of resistant The most common causes of admission for Eurasian otter to the center bacteria and antimicrobial resistance genes (Carroll et al., 2015; Darwich were anthropological origins ranging from direct persecution (gunshot or et al., 2019). Similar studies conducted in reptiles such as green sea turtles poisoning) to involuntary human-induced threats (collisions with vehicles (Ahasan et al., 2017), loggerhead sea turtles (Blasi et al., 2020), Galapagos tor- or fences). toises (Claudin et al., 2019), European pond turtle (Ruzauskas et al., 2016), For logistic purposes, the sampling was centralized at the WRC of and sea turtle (Zavala-norzagaray et al., 2015) confirmed that these animals Torreferrusa (Barcelona) according to the direction of the Catalan Wildlife harbored antimicrobial resistant bacteria regardless of their health status. In Service - Forestal Catalana, SA (Generalitat de Catalunya Government), addition, there are few studies conducted in American minks that reported during a period comprised from May 2018 to May 2021. Thus, sampling AMR mostly in pathogenic bacteria isolated from clinical cases in Denmark methods and handling protocols used for semi-aquatic wild animals were (Pedersen et al., 2009; Nikolaisen et al., 2017) or from a commercial mink in agreement with the management protocols, and Ethical Principles of farm in United States (Agga et al., 2021). However, to the best of our knowl- the Spanish legislation (Spanish R.D.1201/2005 of the Ministry of edge, no information has been published yet in wild mustelids. Presidency of Spain, 2005). In Catalonia (NE Spain), although there are some studies conducted on multidrug resistant bacteria (Molina-Lopez et al., 2015; Vidal et al., 2017) 2.3. Sample collection and processing and antimicrobial resistance genes in wildlife (Darwich et al., 2019), there are limited data regarding the role of semi-aquatic wild animals as Cloacal or rectal swab samples were obtained using sterile swabs from sentinels of environmental health. For this reason, in this study, we estimate all live and dead animals admitted at the WRC. Swab samples were the occurrence of AMRGNB in wild semi-aquatic animals, principally from transported on Amies transport medium (deltalab, Spain) to the Veterinary invasive wild species such as Pond slider and American mink, and from Eur- Infectious Diseases Diagnostic Laboratory located at Autonomous Univer- asian otters. Moreover, antimicrobial susceptibility phenotyping profile of sity of Barcelona, Spain. Upon arrival, samples were processed immediately isolates and genotyping of β-lactams, erythromycin and tetracycline- or stored at 4 °C until being processed on the next day. resistance genes were determined, as well as the geographical distribution of AMRGNB and genes in these wild animals in Catalonia, NE Spain. 2.4. Microbiological analysis 2. Material and methods All samples were analyzed for microbiological identification of Entero- bacteriaceae, and some non-Enterobacteriaceae Gram-negative bacteria. Clo- 2.1. Study area acal or rectal swabs were streaked on MacConkey agar (Oxoid, UK) and MacConkey agar supplemented with ceftriaxone (1 mg/L), and incubated The present study was conducted in Catalonia, an autonomous commu- at 37 °C for 24 h (Garcias et al., 2021). Bacterial cultures on MacConkey nity located in the NE Spain, from January 2018 to July 2021. Catalonia is agar were used as a positive control for their corresponding culture on 2 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 ceftriaxone supplemented plates. Bacterial growth was examined for colony finally blasted against the public database (National Center for Biotechnol- morphology, and ability of the bacteria to ferment lactose in the ogy Information, NCBI, Bethesda, MD, USA). MacConkey agar. Based on their lactose fermenting ability, bacteria were classified as lactose fermenters, and non-lactose fermenters. Single colonies 2.7. Statistical analysis were subculture on MacConkey agar and incubated at 37 °C during 24 h for further biochemical identification by API® biochemical test strips All data obtained during the study were recorded on Microsoft® excel (bioMérieux SA, Marcy l'Etoile, France) or by VITEK 2 systems (bioMerieux, spread sheet. Recorded data included: animal's identification (including Spain) in case of not reaching very good levels of identification with the date of admission, species, location), isolated bacteria, phenotypic suscepti- API® (Darwich et al., 2019). bility profile, and antimicrobial resistance genes. Descriptive statistics was Oxidase negative bacteria were analyzed by a panel of six conventional performed under 95% confidence interval and using SPSS to describe fre- biochemical tests including triple sugar iron, methyl red, citrate, sulfur- quency of occurrence of resistant bacteria, phenotypic antimicrobial sus- indole-motility, urease, and phenylalanine deaminase tests. Additionally, ceptibility, and genotypic antimicrobial resistance genes. Chi-square or API® 20 E biochemical test strips were used for identification of oxidase Fisher exact test was used for comparison between proportions when ap- negative isolates when the conventional biochemical tests failed to suffi- propriate. Statistical significance (unadjusted p-value) was set at 0.05. ciently identify the bacteria. On the other hand, oxidase positive isolates were analyzed by API® 20 NE biochemical test strips or VITEK 2 2.8. Geographical distribution of resistant bacteria and antimicrobial resistance (bioMerieux, Spain) systems (Darwich et al., 2019). genes After identifying the bacteria isolated from the ceftriaxone supple- mented MacConkey medium, phenotypic antimicrobial susceptibility test- Geographic Information System (GIS) was applied to investigate the dis- ing, and genotypic characterization of resistance genes were done. In tribution of antimicrobial resistance genes detected in bacteria isolated addition, all isolates were stored in glycerol/brain heart infusion solution from semi-aquatic wildlife in Catalonia, Spain. The location of each animal (30/70 v/v) at −80 °C for future use. was retrieved from database at the Wildlife Rehabilitation Center (WRC). Maps were created using Quantum GIS software, version 3.18 (“QGIS 2.5. Phenotypic antimicrobial susceptibility testing Development Team. QGIS geographic information system. Open Source Geospatial Foundation Project,” 2020). To evaluate area with higher prob- A Kirby-Bauer disk diffusion susceptibility test protocol (Hudzicki, ability of having animal carriers of AMR genes, cluster analysis was per- 2012) was used to determine phenotypic antimicrobial susceptibility of iso- formed with SaTScan™ version 9.6, using a purely spatial scan analysis lates against a panel of 13 different antimicrobials. Each isolate was tested with a Bernoulli model. The spatial scan analysis is based in the likelihood for the following antimicrobial classes using commercial disks: ratio statistic based on the number of observed and expected cases in the aminopenicillins (ampicillin and amoxicillin/clavulanic acid), cephalospo- specific zone and search for clusters using a variable circular window size rins (ceftiofur and ceftriaxone), fluoroquinolones (ciprofloxacin and to detect spatial clusters in large areas while controlling for the underlying enrofloxacin), aminoglycosides (gentamicin), tetracyclines (tetracycline), population (Kulldorff, 2007). The number of Montecarlo simulations was macrolides (erythromycin), lincosamides (lincomycin/spectinomycin), set at 999 and values of p-value lower than 0.05 were consider as statisti- sulphonamides (sulphamethoxazole/trimethoprim), amphenicols (chlor- cally significant. amphenicol) and, carbapenems (imipenem). The susceptibility of bacteria to each antimicrobial agent was interpreted as Susceptible (S), Intermediate 3. Results (I), and Resistant (R) based on the breakpoints provided by Clinical & Lab- oratory Standards Institute, and the European Committee on Antimicrobial 3.1. Study population Susceptibility Testing. CLSI veterinary breakpoints were preferably used (CLSI, 2018). If not available, CLSI human (CLSI, 2020), EUCAST or CA- The study included 241 wild animals admitted at the WRC during 2018 SFM veterinary breakpoints as previously published by Vidal et al. (2020). to 2021: Trachemys scripta (n = 91), Neovison vison (n = 131) and Lutra lutra (n = 19). 2.6. Genotypic characterization of antimicrobial resistance genes The sampled animals were found in rivers distributed throughout the Catalan territory; turtles were taken from highly populated areas whereas PCR was performed, as previously reported, to detect presence of most mustelids came from less urbanized and rural areas (Fig. 1). common antimicrobial resistance genes coding for ESBL/AmpC – blaCTX, blaTEM, blaCMY-1, blaCMY-2, blaOXA, colistin resistance – mcr-1 and 3.2. Microbiological analysis mcr-4 (Darwich et al., 2019), erythromycin resistance – ermB, and tetracy- cline resistance – tetM (Jacob et al., 2008). DNA was extracted from bacte- All samples presented bacterial growth in the MacConkey control plates rial culture by boiling. Briefly, bacterial growth from the MacConkey agar and 133 out of 241 (50.2%) animals tested isolates had a positive growth in plates were suspended in tube containing 400 μL distilled water and boiled the MacConkey agar supplemented with ceftriaxone. In particular, positive in water bath at 100 °C for 10 min. Then, all tubes were centrifuged at samples came from 80.2% (73/91) Trachemys scripta, 38.2% (50/131) 13,000 rpm for 5 min. After centrifugation, the supernatant from each Neovison vison and 52.6% (10/19) Lutra lutra (Table 1). For each positive tube was recovered into a new tube and stored at −20 °C until further anal- sample or animal, one isolate from pure culture or majoritarian growth ysis. A PCR mix was prepared as previously described elsewhere (Vidal was further studied. Therefore, 133 bacterial isolates were subjected to an- et al., 2020). The positive controls for all genes were provided by the Veter- timicrobial resistance phenotyping, and 131 isolates (two samples were inary Infectious Diseases Diagnostic Laboratory of Autonomous University lost) to genotyping analysis. of Barcelona (Bellaterra, Spain). Analysis of amplified PCR products was The bacterial prevalence from the overall population showed that 63% performed in 1.5% agarose gel by electrophoresis. and 37% of isolates were Enterobacteriaceae and non-Enterobacteriaceae, re- Sanger DNA sequencing was conducted for verification of blaCTX posi- spectively. Within the Enterobacteriaceae, Escherichia coli was the most rep- tive PCR products at the Genomic and Bioinformatics Service of the Auton- resentative bacteria and Pseudomonas fluorescens was the most common omous University of Barcelona (Spain). Sequences and chromatograms non-enterobacteria (Table 2). Behind E. coli (42%), K. pneumoniae (17%) were manually explored to trim bad-quality bases with BioEdit 7.2. Once and Salmonella (8%) were highly prevalent enterobacteria in Neovison the assembly of the consensus sequences was conducted, both complete vison, whereas in Lutra lutra, Hafnia alvei (10%) and Serratia marcescens and partial sequences were aligned using the Clustal Omega program, and (10%) were the principal ones (Fig. 2). In turtles, E. coli (31%) was followed 3 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 Fig. 1. The map shows the human population density, counties and principal rivers of Catalonia. The localization of the sampled animals is represented in circles (American minks and Eurasian otters) and triangles (Pond sliders). Black filled figures represent positive individuals to AMR genes whereas white figures represent negative ones. by Morganella morganii (11%) and Citrobacter freundii (9%) among other presented AMR genes and 11.2% of them were ESBL/AmpC genes Enterobacteriaceae such as Salmonella (7%) or Klebsiella spp. (Fig. 3). (Table 1). Pond sliders showed higher prevalence of ESBL/AmpC genes than mustelids, 15.4% versus 9.4% respectively (p = 0.08). 3.3. Phenotypic antimicrobial susceptibility testing of isolates The gene frequency results showed tetM (20.6%, 27/131) as the most frequent gene detected followed by blaCMY-2 (13.0%), ermB (6.1%), Out of 133 bacterial isolates of different species that were subjected to blaCMY-1 (4.6%), blaCTX-M-15 (3.1%) and mcr-4 (0.8%). None of the iso- antimicrobial susceptibility testing by disk diffusion against a panel of 13 lates was found positive to carbapenem resistance genes (OXA-48). One iso- antibiotics of different categories, highest resistance was seen against late of Pseudomonas fluorescens from an American mink was seen to harbor macrolides (99%), and β-lactams (91%). Varying degrees of resistance three different resistance genes, blaCMY-1, ermB, and tetM and fifteen iso- were also seen against lincosamides (39%), amphenicols (31%), lates (11.5%) were found to carry two different resistance genes. tetracyclines (30%), quinolones (27%), sulphonamides (26%), and amino- Bacterial isolates found positive for AMR genes were also analyzed for glycosides (16%). None of the isolates showed resistance to imipenem their corresponding phenotypic antimicrobial susceptibility profile against (Fig. 4). non-β-lactams (Table 3). E. coli was the most common enterobacteria iso- lated from all animal groups, showing blaCTX-M-15, blaCMY-2 or tetM 3.4. Molecular characterization of antimicrobial resistance genes of isolates genes. Citrobacter positive to blaCMY-2 was also found in American minks and Pond sliders. K. pneumoniae expressing blaCMY-1 and tetM genes was Molecular analysis of 131 bacterial isolates for the detection of AMR also isolated from Pond sliders. It is important to remark the presence of Sal- genes showed that 35.1% (46/131) of the bacteria isolated from monella positive to blaCMY-2 and K. pneumoniae positive to colistin mcr-4 in Ceftriaxone-MacConkey selective medium possess at least one antimicro- American minks (Table 3). Finally, P. fluorescens was the most frequent non- bial resistance gene. That represents 19.2% of the total population enterobacteria found in both mustelids and turtles presenting two or three Table 1 Frequency of bacterial isolation on ceftriaxone supplemented MacConkey agar (CRO) and AMR genes from semi-aquatic wildlife in Catalonia, Spain. Animal spp Total sample Positive animals CRO growth Prevalence (CI95%) Prevalence (CI95%) Prevalence (CI95%) CRO growth AMR genesa ESBL/AmpC genesa Trachemys scripta 91 73 80.2 (72–88.4) 25.3 (16.4–34.2) 15.4 (8.0–22.8) Neovison vison 131 50 38.2 (29.8–46.5) 14.5 (8.5–20.5) 8.4 (3.7–13.2) Lutra lutra 19 10 52.6 (30.2–75.1) 21.1 (2.7–39.4) 10.5 (0–24.3) Total 241 133 55.2 (48.9–61.5) 19.2 (14.1–24.1) 11.2 (7.2–15.2) a N = 131 (2 samples were not recovered for the analysis). 4 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 Table 2 different AMR genes, blaCTX-M-15 and tetM among them. Turtles addition- Species of animals versus frequency of bacterial isolation from semi-aquatic wildlife ally presented other non-enterobacteria such as Burkholderia and in Catalonia, Spain (n = 133). Aeromonas with AMR genes (Table 3). Species of Bacteria Number (%) of animals isolates 3.5. Geographic information system analysis of antimicrobial resistance genes Family Species Trachemys scripta Enterobacteriaceae Escherichia coli 14 (19%) (n = 73) (n = 45) Morganella morganii 5 (6.8%) Geographic distribution of all AMR genes of our interest was investi- Citrobacter freundii 4 (5.5%) gated based on the location of animals from which samples were collected. Citrobacter braakii 3 (4.1%) For this purpose, out of 241 animals, the county (zones) of 211 animals was Hafnia alvei 3 (4.1%) retrieved from database at the WRC; however, the location of 30 animals Klebsiella oxytoca 3 (4.1%) was missing. Accordingly, wild animals originated from 23 different Salmonella spp. 3 (4.1%) Klebsiella pneumoniae 2 (2.7%) counties in Catalonia were included in this analysis. Enterobacter cloacae 2 (2.7%) The geographical distribution of the AMR genes was analyzed at county Enterobacter amnigenus 1 (1.4%) level by using QGIS 3.16 (Hannover). Animals harboring bacteria with at Proteus vulgaris 1 (1.4%) least one AMR gene were considered positive. In some counties such as, Rahnella aquatilis 1 (1.4%) Serratia fonticula 1 (1.4%) Baix Camp, Maresme, and Ribera d'Ebre (indicated in full red circle in the Serratia marcescens 1 (1.4%) map), only one animal sample was obtained, and the sample was detected Shigella spp. 1 (1.4%) positive for AMR genes (Fig. 5). There was no statistically significant asso- Non-Enterobacteriaceae Pseudomonas fluorescens 10 (13.7%) ciation between the species of animals from which the bacteria were recov- (n = 28) Aeromonas hydrophila 8 (11%) ered and occurrence of AMR genes in bacteria (χ2 = 4.139; p = 0.124). Burkholderia cepacia 4 (5.5%) Pseudomonas putida 3 (4.1%) Furthermore, the geographic distributions of ESBL/AmpC, and colistin re- Pseudomonas aeruginosa 2 (2.7%) sistance genes (mcr-1 and mcr-4), and erythromycin (ermB) and tetracycline Ralstonia pictettii 1 (1.4%) (tetM) resistance genes were analyzed separately by using the same GIS Neovison vison Enterobacteriaceae Escherichia coli 15 (30%) software. Interestingly, one isolate from Osona, one of the counties with (n = 50) (n = 35) Klebsiella pneumoniae 6 (12%) Salmonella spp. 3 (6%) the highest density of pig farms, was found positive for mcr-4. Shigella spp. 2 (4%) Cluster analysis using a purely spatial scan analysis to evaluate areas Hafnia alvei 2 (4%) with higher probability of having animal carriers of AMR genes revealed Morganella morganii 2 (4%) that significant high-risk cluster of animals harboring a tetracycline resis- Proteus vulgaris 2 (4%) tance gene in the zones of Tarragona and Lleida (Relative Risk = 4.95, p- Citrobacter braakii 1 (2%) Citrobacter freundii 1 (2%) value = 0.006). Moreover, another high-risk cluster of individuals carrying Providencia stuartii 1 (2%) ESBL/AmpC genes, which was at limit of significance, was detected in the Non-Enterobacteriaceae Pseudomonas fluorescens 13 (26%) zones of Tarragona and the metropolitan area of Barcelona (RR = 3.74, (n = 15) Ralstonia pictettii 1 (2%) p-value = 0.07). These results are shown on Fig. 6. Bordetella bronchiseptica 1 (2%) Lutra lutra Enterobacteriaceae Escherichia coli 2 (20%) (n = 10) (n = 4) Hafnia alvei 1 (10%) 4. Discussion Serratia marcescens 1 (10%) Non-Enterobacteriaceae Pseudomonas fluorescens 5 (50%) In the present study, different species of semi-aquatic wild animals such (n = 6) Mannheimia haemolytica 1 (10%) as Pond sliders, American minks, and Eurasian otters, were examined as Fig. 2. Frequency of Enterobacteriaceae and Non-Enterobacteriaceae isolated from cloacal samples of Neovison vison in Catalonia, Spain. 5 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 Fig. 3. Frequency of Enterobacteriaceae and Non-Enterobacteriaceae isolated from cloacal samples of Trachemys scripta in Catalonia, Spain. reservoirs of AMRGNB in Catalonia, NE Spain. A wide range of antimicro- Moreover, non-Enterobacteriaceae such as Pseudomonas fluorescens were bial resistant bacterial species were isolated with E. coli leading the also isolated in the present study. This result was in line with the finding of Enterobacteriaceae group and P. fluorescens as the most representative non- Dias et al., who reported resistant P. fluorescens isolated from faecal samples Enterobacteriaceae species. Other nosocomial enterobacteria such as of different species of wild animals (Dias et al., 2018). Although Citrobacter freundii, and Klebsiella pneumoniae were also frequently isolated. P. fluorescens is one of the less virulent members of Pseudomonadaceae E. coli was the most common AMR pathogen described in wildlife from family, this agent has been implicated as a causative agent of infection in different parts of the world. A study conducted in Poland showed that resis- humans, such as pseudobacteraemia, especially in immunocompromised tant E. coli were recovered from wild animals such as red deer, roe deer, fal- individuals (Pappas et al., 2006; Pratishtha et al., 2021). low deer, European bison, and wild boar (Wasyl et al., 2018). Similarly, a The present study also determined AMR profiles, and types of resistance study conducted in Singapore on wild birds and rodents also reported the in bacterial isolates recovered from the study animals. Highest proportion presence of antimicrobial resistant E. coli, with 80.8% of these isolates of antimicrobial resistance to β-lactams (91%) was found. Varying degrees were resistant to at least one antimicrobial tested (Ong et al., 2020). Tardón of resistance were also seen to lincosamides, amphenicols, tetracyclines, et al., also identified antimicrobial resistant Enterobacteriaceae in fractures quinolones, sulphonamides, and aminoglycosides. These results were in of wild birds from wildlife rehabilitation centers in Spain (Tardón et al., agreement with the findings of Nikolaisen et al., who reported AMR in 2021). Despite the fact that E. coli is a common normal microbiota in the most pathogenic bacteria from mink in Denmark, particularly E. coli large intestine (Katouli, 2010), the increase of resistant strains causing in- (Nikolaisen et al., 2017). In recent years, due to increased number of bacte- fection in animals and humans, such as urinary tract infections (Darwich rial isolates carrying ESBL genes, the clinical use of carbapenems has in- et al., 2021a), may difficult empirical treatment of patients thereby posing creased. As a result, the overuse of carbapenems has led to increased a public health concern worldwide (Olorunmola et al., 2013). number of clinical bacterial isolates producing carbapenemases (Elshamy Fig. 4. Occurrence of phenotypic antimicrobial resistance profile of bacteria from semi-aquatic wildlife in Catalonia, Spain. 6 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 Table 3 Anthropogenic activities such as use of antimicrobials in agriculture and Antimicrobial resistance genes and phenotypic resistance to non-β-lactams in bacte- aquaculture, other nonhuman applications of antimicrobials, and waste dis- rial species from semi-aquatic wildlife in Catalonia, Spain. posal play an important role to create major environmental reserves of re- Animal spp. Bacterial spp. AMR genes Resistance phenotype to sistance, virulence genes and the organism that harbor them (Davies and non-β-lactams Davies, 2010). Poorly treated wastes contaminated with antimicrobials Trachemys Enterobacteriaceae from humans and livestock are often assumed to be the main sources of scripta Escherichia coli blaCMY-2 CIP, ENO, GM, TE, LS AMR in wildlife (Arnold et al., 2016). Waste water is a meeting place for an- blaCMY-2 XNL timicrobials, antimicrobial resistance genes, and bacteria from different blaCMY-2, ENO, GM, TE, LS tetM sources (Karkman et al., 2018). In this study, the most frequently detected blaCMY-2, CIP, ENO, SXT, TE, LS, C gene was tetM (20.6%) followed by blaCMY-2 (13.0%), ermB (6.1%), tetM blaCMY-1 (4.6%), blaCTX-M-15 (3.1%) and mcr-4 (0.8%). These genes Klebsiella pneumoniae blaCMY-1, – have been also reported in enterobacteria from wild birds and mammals tetM in Spain, suggesting that are quite frequent in the environment (Darwich Citrobacter braakii blaCMY-2, – tetM et al., 2019, 2021b; Garcias et al., 2021). blaCMY-2 LS Other AMR genes such as tetM or ermB detected in this study, have been Hafnia alvei blaCMY-2 – also reported in bacterial isolates obtained from humans, domestic and wild Citrobacter freundii blaCMY-2 LS animals elsewhere in the world (Bryan et al., 2004). Erythromycin resis- blaCMY-2 ENO tance gene – ermB and tetracycline resistance genes – tetM are among tetM ENO, SXT, C Serratia marcescens tetM GM, LS plasmid-mediated AMR genes that confer resistance to erythromycin, and Serratia fonticula tetM – tetracycline, respectively. Both antimicrobial agents are widely used in Non-Enterobacteriaceae human and animal clinical practices (Bryan et al., 2004; Dönhöfer et al., Pseudomonas blaCTX-M-15, LS 2012; Villedieu et al., 2004).The high prevalence of tetM and ermB resis- fluorescens tetM tetM CIP, ENO, GM, SXT, TE, LS tance genes found in semi-aquatic wildlife could be consequence of the en- Pseudomonas putida blaCMY-1, ENO, SXT, C vironmental pollution caused by faecal resistome in livestock, especially in tetM slaughter pigs from countries with a high antimicrobial usage patterns blaCMY-1 ENO, SXT, C (Munk et al., 2018). blaCMY-1, ENO, SXT, LS Mobile colistin resistance (mcr) genes are plasmid-mediated resistance blaCMY-2 Burkholderia cepacia ermB ENO, SXT, TE, C genes recently discovered in humans and food animals. The mcr genes are tetM CIP, ENO, SXT, TE, LS, C known to confer resistance to colistin sulphate – one of the last effective Aeromonas hydrophila tetM GM, SXT, TE, LS drugs for the treatment of multidrug resistant Gram-negative bacteria infec- tetM TE, LS tions (Sun et al., 2018). Colistin is included within the reserve group of Ralstonia pictettii tetM CIP, SXT, TE, LS, C Neovison Enterobacteriaceae drugs according to the Aware list of the World Health Organization vison Escherichia coli blaCTX-M-15 GM, TE, LS (WHO, 2021), which means that it is considered a “last-resort” drug to be blaCMY-2 – used when all alternatives have failed. This antibiotic was widely used in blaCMY-2 LS veterinary medicine as preventive mass-medication against colibacillosis blaCMY-2, CIP, GM, LS in piglets (Rhouma et al., 2016). However, nowadays there is a worldwide tetM tetM – trend to limit colistin usage in animal production due to significant public Citrobacter braakii blaCMY-2 LS health concern related to emergence and transmission of resistance genes Citrobacter freundii blaCMY-2 LS (Sun et al., 2018).The mcr-1 and mcr-4 were the most frequent colistin- Salmonella spp. blaCMY-2 – resistance genes reported in Enterobacteriaceae in pigs from Spain (Sevilla Klebsiella pneumoniae mcr-4 – tetM – et al., 2021). The presence of mcr-4 in Klebsiella pneumoniae from Neovison Shigella spp. ermB, tetM – vison in the present study could be due because Osona is one of the counties ermB, tetM – in Catalonia with the highest pig density and previous abuse of colistin sul- Proteus vulgaris tetM – phate in pig production as a preventive for controlling post-weaning diar- Non-Enterobacteriaceae rheas could happen. In fact, from 2013 to 2015, Spain was the European Pseudomonas blaCTX-M-15, ENO, GM, SXT, TE, LS fluorescens tetM country with the highest sales of polymyxins for food-producing animals blaCMY-1, – (EMA (European Medicine Agency), 2017), and with a significant preva- ermB, tetM lence of colistin-resistance genes, principally mcr-4 (Aguirre et al., 2020). blaCMY-1, – The Spanish Agency for Medicines and Health Products established a tetM ermB C drastic reduction (97%) in the use of colistin in swine from 2015 to 2018 ermB, tetM C in Spain, with a positive impact in the decrease in colistin resistance ermB, tetM – genes (Aguirre et al., 2020). In this study, we have detected the presence Lutra lutra Enterobacteriaceae of mcr-4 in a Neovison vison, confirming that these genes are still present Escherichia coli ermB – in the environment in counties with a high pig production. The presence Non-Enterobacteriaceae Pseudomonas blaCTX-M-15, CIP, ENO, GM, SXT, TE, LS, of these colistin resistant genes in natural waters can suggest that the reduc- fluorescens tetM C tion in the use of this antimicrobial is not enough to prevent gene selection blaCMY-2 – in live pigs or that river contamination come from stable mcr genes in old TetM C non-treated purines. C, chloramphenicol; CIP, ciprofloxacin; ENO, enrofloxacin; GM, gentamicin; LS, lin- In this study, turtles had a larger prevalence of AMRGNB and AMR comycin/spectinomycin; SXT, sulphamethoxazole/trimethoprim; TE, tetracyclin; genes than mustelids, and colistin-resistant genes were only detected in XNL, ceftiofur. the mustelid group. Different behaviour, feeding habits and ecological niche could explain the difference among these species. The Pond sliders, and Aboshanab, 2020). Although carbapenem resistance has been detected and American minks are invasive and generalist species that have spread in bacteria isolated from wild mammals and wild birds in Spain (Darwich throughout much of Europe and currently occupies the Iberian Peninsula et al., 2019), none of the isolates from semi-aquatic wildlife of the present because of their environmental release as exotic pets or their multiple es- study showed resistance to carbapenems. capes from mink farms, respectively (Bonesi and Palazon, 2007). 7 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 Fig. 5. Geographical distribution of antimicrobial resistance genes detected in bacteria isolated from semi-aquatic wildlife in Catalonia, Spain. Circles represent the propor- tion of positive animals for each county (red = positive cases; pink = negative cases). Fig. 6. Spatial cluster analysis performed with SatScan. Red circles represent high-risk clusters. Red points are locations situated into the clusters and black points are locations out of the cluster. 8 T.S. Mengistu et al. Science of the Total Environment 830 (2022) 154814 On the other hand, American minks and Eurasian otters are always been Resources, L.D.; Data curation, T.S.M., B.G., R.A.M.-L., and L.D.; Writing linked to clean and crystalline waters and their feeding habits may differ de- —original draft preparation, T.S.M., B.G. and L.D.; Writing—review and pending on the habitat. In rivers and streams, the American minks are more editing, T.S.M., B.G., G.C., C.S., R.A.M.-L., and L.D.; Supervision, L.D.; generalists and feeds on frogs, small mammals (rabbits, rodents and Funding acquisition, L.D. All of the authors have read and agreed to the shrews), fish and waterfowl. Whereas otters are basically feeding upon published version of the manuscript. fish, crustaceans and amphibians (Romero and Guitián, 2017). As regards the pond sliders, they prefer ponds, swamps, or slow-flowing portions of Declaration of competing interest rivers and estuaries and they are omnivores, feeding on a wide variety of plants, insects, fish, crayfish, freshwater sponges, snails, slugs, small The authors declare that they have no known competing financial inter- amphibians, and other reptiles (Bonin et al., 2006). Thus, the colonization ests or personal relationships that could have appeared to influence the of these invasive species could be consequence of the water pollution work reported in this paper. with AMRGNB, and AMR genes but also due to the consumption of aquatic/terrestrial contaminated preys (Fu et al., 2017; Liu et al., 2013). Acknowledgements The distribution of β-lactams-, erythromycin-, and tetracycline- resistance genes were homogeneous in all the regions of the study, with Tilaye Shibbiru Mengistu was supported by a scholarship from Erasmus the exception of two clusters. The first one was found for ESBL/AmpC Mundus Joint Master's Degree in Infectious Diseases and One Health Pro- genes, in a highly populated area comprising big metropolitan regions of gram. Biel Garcias was supported by Departamento de Recerca i Universitat Barcelona and Tarragona with large human hospitals and also receiving de la Generalitat de Catalunya (FI-SDUR 2020). Gabriela Castellanos was the river-mouths of rivers coming from northern areas with a large pig student of the Master's degree in Zoonosis and One Health. production. Thus, Pond sliders sampled from streams, flood plains, the river-mouths of main rivers of those highly urbanized areas of Catalonia References presented higher burden of AMR bacteria and AMR genes from both human and animal origin. The other cluster was observed for tetM/emrB Agga, G.E., Silva, P.J., Martin, R.S., 2021. 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