animals Article Extended-Spectrum β-Lactam Resistant Klebsiella pneumoniae and Escherichia coli in Wild European Hedgehogs (Erinaceus europeus) Living in Populated Areas Biel Garcias 1 , Laia Aguirre 1 , Chiara Seminati 1 , Nerea Reyes 1 , Alberto Allepuz 1,2 , Elena Obón 3 , Rafael A. Molina-Lopez 3 and Laila Darwich 1,2, * 1 Departament de Sanitat i Anatomia Animal, Universitat Autònoma de Barcelona (UAB), 08193 Cerdanyola del Vallès, Spain; biel.garcias@uab.cat (B.G.); Laia.aguirre@uab.cat (L.A.); Chiara.seminati@uab.cat (C.S.); Nerea.reyes@sanikolas.org (N.R.); Alberto.allepuz@uab.cat (A.A.) 2 IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain 3 Catalan Wildlife Service, Centre de Fauna Salvatge de Torreferrussa, 08130 Santa Perpètua de Mogoda, Spain; Elena.obon@gencat.cat (E.O.); Rafael.molina@gencat.cat (R.A.M.-L.) * Correspondence: Laila.darwich@uab.cat; Tel.: +34-935811046 Simple Summary: The alarming emergence of antimicrobial resistance (AMR) in human and veteri-   nary medicine has activated awareness for monitoring the levels of AMR pollution in the environment and wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban Citation: Garcias, B.; Aguirre, L.; areas in Europe. In this study, the occurrence and distribution of extended-spectrum β-lactam Seminati, C.; Reyes, N.; Allepuz, A.; Obón, E.; Molina-Lopez, R.A.; (ESBL) resistant enterobacteria and AMR genes were assessed in wild European hedgehogs in Darwich, L. Extended-Spectrum Catalonia, NE Spain. The results showed that 36.8% of the animals were detected as carriers of β- β-Lactam Resistant Klebsiella lactamase/carbapenemase resistance genes, with a special occurrence of human nosocomial bacteria pneumoniae and Escherichia coli in such as Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii. In addition, more than half of Wild European Hedgehogs (Erinaceus the enterobacteria presented a multidrug resistance (MDR) phenotype and 31% of the isolates had an europeus) Living in Populated Areas. extended XDR profile. No differences in the spatial distribution of animals with AMR genes were Animals 2021, 11, 2837. https:// observed within the study region. The results of this study suggest that the close contact with human doi.org/10.3390/ani11102837 areas predispose the transmission of AMR genes to wild hedgehogs because they either inhabit and/or feed in an anthropogenic environment. In conclusion, hedgehogs could be good sentinels Academic Editors: Anna Rita Attili, or bioindicators of AMR environmental pollution, especially in highly populated areas with high Alessandro Bellato, Patrizia Nebbia human activity. and Patrizia Robino Abstract: Wildlife has been suggested to be a good sentinel of environmental health because of Received: 8 September 2021 Accepted: 23 September 2021 its close interaction with human populations, domestic animals, and natural ecosystems. The Published: 28 September 2021 alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European Publisher’s Note: MDPI stays neutral hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, with regard to jurisdictional claims in there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of published maps and institutional affil- this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant iations. enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as Copyright: © 2021 by the authors. carriers of β-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically Licensee MDPI, Basel, Switzerland. K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by This article is an open access article E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 distributed under the terms and (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, conditions of the Creative Commons 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an Attribution (CC BY) license (https:// extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study creativecommons.org/licenses/by/ region was shown in the spatial analysis, nor differences in the proportion of positive animals among 4.0/). Animals 2021, 11, 2837. https://doi.org/10.3390/ani11102837 https://www.mdpi.com/journal/animals Animals 2021, 11, 2837 2 of 13 regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach. Keywords: wild European hedgehogs; antimicrobial resistance; ESBL; K. pneumoniae; E. coli 1. Introduction Antimicrobial resistance (AMR) is a problem in human and veterinary medicine worldwide. The increasing prevalence of AMR in clinically important and commensal bacteria in both humans and livestock is attributed largely to selection through the use of antimicrobials [1,2]. However, the emergence of genes conferring resistance to human last-resort antibiotics, such as extended-spectrum β-lactamases (ESBLs), AmpC-type β- lactamases (AmpC), and carbapenem resistance genes, has been reported in the commensal bacteria of wildlife without any history of antimicrobial intake [3,4]. In fact, wild animals do not naturally come into contact with antimicrobials, but commensal bacteria have the potential to act as reservoirs of resistance genes contributing to the development of AMR in pathogens through horizontal transmission [3,5]. Thus, wildlife may be infected with antimicrobial-resistant bacteria (AMRB) or acquire AMR genes from human sources, agricultural facilities, and associated contaminated environments [6,7]. The European hedgehog (Erinaceus europaeus) is a common and widely distributed wild species in Europe. Hedgehogs are small, nocturnal, spiny-coated insectivores that live in a variety of habitats, generally in close contact with humans, but also with livestock in the countryside [8]. In recent years, hedgehog populations are increasingly inhabiting areas with human activity, such as gardens in residential areas or green areas in big cities [9]. Some of the consequences of this close hedgehog−human interaction is that people usually feed these animals and thus increases the risk of human physical contact. Moreover, the presence of hedgehogs in other anthropogenic environments, such as farms or crop fields, also allow for contact with livestock. The prevalence of resistant bacteria in wildlife is linked with human activity [6]. Only a few studies have been conducted in hedgehogs reporting the presence of zoonotic antimi- crobial resistant bacteria or AMR genes, such as β-lactam resistant Enterobacteriaceae in NE Spain [10], tetracycline resistance genes [11], or methicillin-resistant Staphylococcus aureus carrying mecC in Denmark and Sweden [12,13]. However, there is no research on the role of hedgehogs in AMRB dynamics at the interface between human populations, domestic animals, and natural ecosystems. This information is critical for implementing strategies to prevent further development and spread of AMRB between animals and humans. Catalonia (NE Spain) is a densely populated county with a high density of pig fattening farms. Barcelona is Catalonia’s capital, one of Europe’s most densely populated cities. It is estimated that more than five million people live in the metropolitan area of Barcelona and the adjacent suburban areas, which is the largest on the Mediterranean Sea (Figure 1). Thus, the pressure on urban land on the main metropolitan areas of Barcelona is very high. As hedgehogs are a species closely related with the urban life, they could serve as reservoirs, vectors, and bioindicators of resistant bacterial pathogens and genetic determinants of AMR in the environment [6,7,14]. Therefore, the aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria in a wild population of European hedgehogs in Catalonia, NE Spain. Animals 2021, 11, x FOR PEER REVIEW 3 of 14 Animals 2021, 11, 2837 3 of 13 Figure 1. The map shows the human population density of Catalonia. Sampled animals were concentrated in the zones with a higher density. Figure 1. The map shows the human population density of Catalonia. Sampled animals were concentrated in the zones with a higher density. 2. Materials and Methods 2.1. Study Population 2. Materials From 2015 toand Methods 2019, faeces from 114 hedgehogs were collected at the Wildlife Rehabili- tation2.1. Center (WRC) of Study Population Torreferrussa (Catalunya, North-East Iberian Peninsula). Sampling methods and animal handling techniques were in agreement with the Catalan Wildlife From 2015 to 2019, faeces from 114 hedgehogs were collected at the Wildlife Rehabil- Service, which specifies the management protocols and the Ethical Principles according to itation Center (WRC) of Torreferrussa (Catalunya, North-East Iberian Peninsula). Sam- Spanish legislation [15]. pling methods and animal handling techniques were in agreement with the Catalan Wild- Upon admission, a physical exam under inhalant anaesthesia with isofluorane was life Service, which specifies the management protocols and the Ethical Principles accord- performed on the animals and rectal samples were collected using sterile swabs with ing to Spanish legislation [15]. an Amies transport medium (Deltalab, Barcelona, Spain), before any pharmacologic or Upon admission, a physical exam under inhalant anaesthesia with isofluorane was antimicrobial treatment. Data of the origin of the hedgehogs were provided by the wildlife performed rangers who broughton thetheanimals animalsand rectal to the WRC. samples The agewere andcollected sex of theusing animalssterile wereswabs with an recorded Amies transport medium (Deltalab, Barcelona, Spain), before any pharmacologic by veterinary staff of the WRC. The cause of admission was related to the following or anti- microbial treatment. Data of the origin of the hedgehogs were provided categories [16]: orphaned young (27%), fortuitous finds (27%), metabolic/nutritional by the wildlife rangers disease (15%),who brought traumatic the animals injuries to the WRC. (14%), natural The age and disease/incident sex of (13%), andthe animals other were rec- casualties orded by veterinary staff of such as illegal captivity or traps (4%).the WRC. The cause of admission was related to the following categories [16]: orphaned young (27%), fortuitous finds (27%), metabolic/nutritional dis- ease (15%), traumatic 2.2. Microbiological Analysisinjuries (14%), natural disease/incident (13%), and other casualties such as illegal Rectal swabs werecaptivity or traps (4%). transported on an Amies medium to the Veterinary Infectious Diseases Diagnostic Laboratory located at the Autonomous University of Barcelona, Spain. Upon2.2. Microbiological arrival, samples Analysis were processed immediately or were stored at 4 ◦ C until being processedRectal swabs the next day.were transported All rectal on an swabs were Amiescultured directly mediumon toColumbia the Veterinary bloodInfectious agar Diseases (BD GmBh, Diagnostic Munich, Laboratory Germany) located at agar and MacConkey the (Oxoid, Autonomous University Basingstoke, UK),ofnotBarcelona, sup- plemented Spain. and Upon supplemented withwere arrival, samples ceftriaxone (1 mg/L), processed and aerobically immediately incubated or were stored for until at 4 °C at 37 ◦processed 24 h being C. The agarthewithout ceftriaxone next day. supplementation All rectal was used swabs were directly as a quality cultured controlblood on Columbia of theagar sample (BD growth GmBh, to assess if Munich, negative and Germany) growths on the supplemented MacConkey agar were due agar (Oxoid, Basingstoke, to not UK), the ceftriaxone activity or because of a bad quality or degradation of the sample. All supplemented and supplemented with ceftriaxone (1 mg/L), and aerobically incubated for of the colonies 24 hgrown on The at 37 °C. MacConkey agar with agar without ceftriaxone ceftriaxone were re-cultured supplementation wason TSAasagar used in order a quality control Animals 2021, 11, 2837 4 of 13 to conduct bacterial identification, which was performed using conventional biochemical tests (oxidase, catalase, TSI, SIM, urease, citrate, and methyl red) and the API system (bioMérieux, Marcy l’Etoile, France). 2.3. Antimicrobial Susceptibility Test All of the isolates identified from the MacConkey agar with ceftriaxone were stored at −80 ◦ C before antimicrobial susceptibility testing was performed using the disk dif- fusion method [17]. Briefly, one to four colonies were suspended in 5 mL of distilled sterile water to achieve a turbidity of 0.5 in the McFarland scale. The dilution was then seeded onto Mueller–Hinton (Oxoid, Basingstoke, UK) plates. Each isolate was tested for the following antimicrobial groups, using commercial disks: aminopenicillins (ampicillin (BD) and amoxicillin/clavulanic acid (Oxoid)), cephalosporins (ceftiofur (BD), ceftriaxone (BD), and cefquinome (CondaLab)), fluoroquinolones (ciprofloxacin (BD) and enrofloxacin (BD)), aminoglycosides (gentamicin (BD)), tetracyclines (tetracycline (BD)), macrolides (erythromycin (BD)), lincospectin (Oxoid), sulfamethoxazole/trimethoprim (BD), and chlo- ramphenicol (BD). Then, based on the laboratory testing readings, isolates were classified as susceptible, intermediate, or resistant, as previously described [18–20]. For statistical assessments, all isolates that exhibited intermediate resistance were re- classified as resistant. In addition, multidrug resistance (MDR) was defined as resistance to at least one agent in three or more antimicrobial categories; extensive drug resistance (XDR) was defined as resistance to all but two of the tested antimicrobial categories; and finally, pan-drug resistance (PDR) was defined as resistance to all of the categories tested [21]. 2.4. Molecular Characterization of Antimicrobial Resistance Genes DNA was extracted by boiling the bacterial growth from the selective MacConkey Agar supplemented with ceftriaxone (1 mg/L) plates, as previously described [18,22]. Briefly, all the bacterial growths were diluted in 600 µL of sterile distilled water, and 200 µL of the dilution was then transferred to a new tube. Two-hundred microliters of sterile distilled water was added to each tube. The tubes were boiled at 100 ◦ C in a water bath for 10 min, and then centrifuged at 16,000× g for 5 min. After centrifugation, the supernatant was recovered and stored at −80 ◦ C until processing. β-lactamase—blaTEM, blaCTX, blaSHV , blaCMY-1, blaCMY-2 , and blaOXA —and plasmid me- diated colistin—mcr1, mcr2, mcr3, mcr4, and mcr5—genes were assessed using previously described PCR protocols [10]. For all PCR assays, the master mix consisted of: 1 × PCR Buffer, 0.2 mM of each dNTP (Bioline, Livron-sur-Drôme, France), 3 mM of MgCl2, 1 mM of each primer, and 1 U of Taq DNA Polymerase (Bioline, Livron-sur-Drôme, France). A final volume of 2.5 µL of DNA was used in the PCR. In each reaction, positive and negative controls were included. The oligonucleotide sequence of primers is presented in Supplementary Table S1. Briefly, the PCR conditions were homogenized for all of the reactions as follows: 5 min at 94 ◦ C, followed by 25 cycles of 1 min at 94 ◦ C, 1 min of annealing at 55 ◦ C and 1 min of extension at 72 ◦ C, and a final extension step of 7 min at 72 ◦ C. The controls for all genes were provided by the Veterinary Diagnostic Laboratory in Infectious Diseases of the Universitat Autònoma de Barcelona (UAB, Bellaterra, Spain). Sanger DNA sequencing was conducted for blaTEM, blaCTX, blaSHV , and blaOXA PCR products at the Genomic and Bioinformatics Service of the Universitat Autònoma de Barcelona (Spain). Sequences and chromatograms were manually explored to trim bad- quality bases with BioEdit 7.2. Once the assembly of the consensus sequences was con- ducted, both complete and partial sequences were aligned using the Clustal Omega pro- gram, and were finally blasted against the public database (National Center for Biotechnol- ogy Information, NCBI, Bethesda, MD, USA). 2.5. Statistical Analysis All of the statistical analyses were performed with R software, version 3.6.0 [23]. The graphics for representing antimicrobial susceptibility were built using the “AMR” Animals 2021, 11, 2837 5 of 13 package [24]. Generalised lineal models were used to test the association between the presence of AMR genes (considered as the dependent variable) and different variables such as age, sex, month, population, density of the municipality, and cause of admission to WRC (considered as explanatory variables). First, bivariate analysis was used to individually test all the variables, and those with a p-value lower than 0.3 were selected. After that, cor- relation among predictor variables was evaluated by calculating the Spearman correlation coefficient, and variables with a coefficient higher than 0.5 were removed. Non-correlated variables were included in the multivariate analysis and the best model was selected based on the lowest Akaike Information Criteria (AIC) value [25]. The limit of significance of the p-value was set at 0.05. Maps were created using Quantum Gis software, version 3.18 [26]. To evaluate the areas with a higher probability of having animals carriers of AMR genes, cluster analysis was performed with SaTScan version 9.6, using a purely spatial scan analysis with a Bernoulli model [27]. The spatial scan analysis is based in the likelihood ratio statistic based on the number of observed and expected cases in the specific zone and the search for clusters using a variable circular window size to detect spatial clusters in large areas while controlling for the underlying population [27]. The number of Montecarlo simulations was set at 999 and p-values lower than 0.05 were considered statistically significant. 3. Results Sixty seven out of the 114 wild hedgehog fecal samples analyzed (58.3%) had a positive growth on agar supplemented with ceftriaxone. Pure cultures were obtained from most of the animals and 19 out of 67 hedgehogs presented more than one colony, identifying a total of 90 bacterial isolates. Klebsiella spp., principally Klebsiella pneumoniae (29% out of the total bacteria), was the most frequently detected microorganism (42.2%), followed by E. coli (38.9%). Other bacteria such as Citrobacter freundii (5.6%), Serratia spp. (4.4%), Proteus mirabilis (3.3%), Enterobacter cloacae (2.2%), and Shigella spp. (1.1%) were also identified. Regarding the presence of β-lactamase resistance, 36.8% of the animals (42/114) were detected as carriers of AMR genes. Klebsiella spp. (59.6%) was the agent with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The frequency of genes and variants in the studied population was as follows: 19.3% blaCTX-M-15 , 10.5% blaSHV-28 , 9.7% blaCMY-1 , and 8.8% blaCMY-2 (Table 1). In addition, 1.7% of carbapenemases blaOXA-48 was found in isolates of K. pneumoniae and E. coli from two different animals (Table 2). In contrast, no colistin mcr-plasmid-mediated resistance genes were detected in the tested animals. Table 1. Number and frequencies of the samples positive for β-lactamase genes in European hedgehogs. CI, confidence intevals. Number of Animals with Overall Frequency (%) Frequency (%) and CI 95% AMR Gene AMR Gene CI 95% (n = 114) among Postive Animals (n = 42) blaCTX-M-15 22 19.3 (12.1–26.5) 52.4 (37.3–67.5) blaCTX-M-3 1 0.9 (0–2.6) 2.4 (0–7) blaSHV-1 5 4.4 (0.6–8.2) 11.9 (2.1–21.7) blaSHV-11 5 4.4 (0.6–8.2) 11.9 (2.1–21.7) blaSHV-12 3 2.6 (0–5.6) 7.1 (0–14.9) blaSHV-28 12 10.5 (4.9–16.2) 28.6 (14.9–42.2) blaTEM-1b 5 4.4 (0.6–8.2) 11.9 (2.1–21.7) blaCMY-1 11 9.7 (4.2–15.1) 26.2 (12.9–39.5) blaCMY-2 10 8.8 (3.6–14) 23.8 (10.9–36.7) blaOXA-48 2 1.8 (0–4.2) 4.8 (0–11.2) Animals 2021, 11, 2837 6 of 13 Table 2. Antimicrobial resistance genotypes and phenotypes of β-lactamase producing Enterobacteriaceae detected in wild European hedgehogs. Bacterial spp. β-lactam Resistance Genes AMR Phenotyping Escherichia coli CTX-M-15, SHV-28 AMC, E, TET CTX-M-15 CEFQ, E, CRO, XNL CTX-M-15 E CTX-M-15 AMP, CEFQ, ENO, E, LS, SXT, GM, CIP, CRO, C, XNL CTX-M-15 nt CTX-M-15 E, GM CTX-M-15 AMP, CEFQ, ENO, E, TET, CIP, CRO, XNL CTX-M-15 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL SHV-12 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL SHV-11, OXA-48 AMP, AMC, CEFQ, E, CRO, XNL TEM-1b, CMY-2 AMP, AMC, E, SXT, CRO, XNL CMY-2 AMP, ENO, CIP, CRO, XNL CMY-2 AMP, SXT, TET, CRO, XNL CMY-2 AMP, CRO, XNL CMY-2 AMP, ENO, E, LS, TET, XNL CMY-2 AMP, AMC, ENO, E, LS, SXT, TET, CIP, CRO, XNL CMY-2 AMP, AMC, E, CRO, XNL CMY-2 AMP, AMC, E, TET Klebsiella spp. SHV-28, TEM-1b, CMY-2 AMP, AMC, ENO, GM, E, LS, SXT, TET, CIP, C SHV, CMY-1 AMP, ENO, E, LS, SXT, TET, CIP, C CTX-M-15, SHV-1 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL CMY-2 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, C, XNL CTX-M-15, SHV-28, CMY-1 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-11, CMY-1 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-11, CMY-1 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-11 AMP, CEFQ, ENO, E, TET, CIP, CRO, XNL CTX-M-15 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL K. pneumoniae CTX-M-15, CMY-1 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-28, CMY-1 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-28, CMY-1 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL CMY-1, OXA-48 AMP, AMC, ENO, E, LS, SXT, CIP, XNL SHV-28, CMY-1 AMP, AMC, ENO, E, LS, SXT, TET, CIP, XNL CTX-M-15 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-28 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-28 AMP, AMC, CEFQ, ENO, GM, E, LS, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-28 AMP, AMC, E CTX-M-15, SHV-28 AMP, AMC, ENO, E, TET, CRO, XNL CTX-M-15, SHV-28 nt SHV-1 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, C, XNL SHV-1 AMP, AMC, ENO, E, CRO, XNL SHV-1 AMP, AMC, ENO, E, XNL CTX-M-15, SHV-28 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL CTX-M-15, SHV-28, CMY-1, CMY-2 AMP, AMC, CEFQ, ENO, GM, E, SXT, CIP, CRO, XNL CTX-M-15, SHV-1, TEM-1b, CMY-1, CMY-2 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL SHV-28 AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL SHV-12 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL SHV-1, TEM-1b AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL SHV-1 nt K. oxytoca CTX-M-3 AMP, AMC, CEFQ, ENO, E, CIP, CRO, XNL Citrobacter freundii SHV-12, CMY-2 AMP, AMC, CEFQ, ENO, E, LS, SXT, TET, CIP, CRO, XNL TEM-1b, CMY-2 AMP, AMC, CEFQ, ENO, GM, E, TET, CIP, CRO, XNL CMY-2 nt AMP—ampicillin; AMC—amoxicillin + clavulanic acid; CEFQ—cefquinome; ENO—enrofloxacin; GM—gentamicin; E—erythromycin; LS—lincospectin; SXT—trimetoprim + sulphametoxazol; TET, Tetracycline; CIP—ciprofloxacin; CRO—ceftriaxone; XNL—ceftiofur; C—cloramphenicol; nt—not tested. Regarding the AST results, almost 52% (27/52) of the isolates showed an MDR pheno- type and 30.8% had an extended (XDR) profile (Table 2). Interestingly, a high proportion of the bacterial isolates presented more than one AMR gene, especially within the genus Klebsiella, in which 67.7% (21/31) of the isolates presented two or more β-lactamase genes. The most common combination was blaCTX-M-15 and blaSHV-28, which was present in 21.2% CMY-1, CMY-2 SHV-28 AMP, AMC, CEFQ, ENO,GM, E, SXT, TET, CIP, CRO, XNL SHV-12 AMP, AMC, CEFQ, ENO, E, SXT, TET, CIP, CRO, XNL SHV-1, TEM-1b AMP, AMC, CEFQ, ENO, GM, E, SXT, TET, CIP, CRO, XNL SHV-1 nt Animals 2021, 11, 2837 7 of 13 K. oxytoca CTX-M-3 AMP, AMC, CEFQ, ENO, E, CIP, CRO, XNL Citrobacter freundii SHV-12, CMY-2 AMP, AMC, CEFQ, ENO, E, LS, SXT, TET, CIP, CRO, XNL TEM-1b, CMY-2 AMP, AMC, CEFQ, ENO, GM, E, TET, CIP, CRO, XNL (11/52) of the overall isolates, CMY-2 and principally in K. pneumoniae, nt for which 84.6% of the isolates carried AMR AMP—ampicillin; genes, with up AMC—amoxicillin to 4–5 genes + clavulanic acid; in some cases (TableENO—enrofloxacin; CEFQ—cefquinome; 2). AMP—ampicillin; AMC—amoxicillin + clavulanic acid; CEFQ—cefquinome; GM—gentamicin; E—erythromycin; LS—lincospectin; SXT—trimetoprim + sulphametoxazol; ENO— TET, Tetracycline; CIP—ciprofloxacin; CRO—ceftriaxone; XNL—ceftiofur; C—cloramphenicol; nt—not + enrofloxacin; GM—gentamicin; E—erythromycin; LS—lincospectin; SXT—trimetoprim sulphametoxazol; TET, Tetracycline; CIP—ciprofloxacin; CRO—ceftriaxone; XNL—ceftiofur; tested. C—cloramphenicol; nt—not tested. The Theresults results of of the the phenotypic phenotypic antimicrobial antimicrobial susceptibility susceptibility testing testing showed showed high high levels levels of of resistance resistance inin most most ofof the the tested tested antimicrobials antimicrobials (Figure (Figure 2). 2). ItIt is is important important to to note note the the highest levels highest levels of of resistance resistance were were observed observed among among the Klebsiella isolates, the Klebsiella isolates, especially especially for for quinolones, third quinolones, third and andfourth fourthgeneration generationcephalosporines, cephalosporines, trimethoprim/sulphametoxazol, trimethoprim/sulphametoxazol, andtetracyclines. and tetracyclines. Figure 2. Antimicrobial susceptibility phenotype of the different isolates. Interpretation: S—sensitive (in purple); I— Figure 2. Antimicrobial intermediate susceptibility and R—resistant phenotype of the different isolates. Interpretation: S—sensitive (in purple); I—inter- (in yellow). mediate and R—resistant (in yellow). A generalised linear model was created to test the association between the presence A of AMR generalised genes and linear model different was created variables, tocause such as test the association between of hospitalization, thedensity human presenceof of the zone, sex, age, and month of collection of the sample, but no variable was found of AMR genes and different variables, such as cause of hospitalization, human density to the zone, sex, age, be statistically and month significant. As of collection some animalsofwere the sample, carriersbut nomore from variable thanwas onefound to be bacterium, statistically significant. As some animals were carriers from more than one species could not be included as an explanatory variable. So, to evaluate the possiblebacterium, species could association not variables of the be included as an before explained explanatory and thevariable. presenceSo, to evaluate of the the possible most prevalent genus, two specific generalised linear model were created just for animals that were carriers of Klebsiella spp and E. coli. However, again, no variable was found to be statistically significant. Odd ratios and p-values of the models are presented on Supplementary Tables S2 and S3. Regarding animal distribution, at least one animal from the sampled regions was positive for β-lactams resistance genes (Figure 3). association of the variables explained before and the presence of the most prevalent genus, two specific generalised linear model were created just for animals that were carriers of Klebsiella spp and E. coli. However, again, no variable was found to be statistically signifi- cant. Animals 2021, 11, 2837 8 of 13 Odd ratios and p-values of the models are presented on Supplementary Tables S2 and S3. Regarding animal distribution, at least one animal from the sampled regions was positive for β-lactams resistance genes (Figure 3). Figure Figure 3. The map 3. shows The mapthe shows the counties counties wherewhere hedgehogs hedgehogs were were found.Pie found. Pie charts chartsrepresent representthethe occurrence of wild occurrence ofhedge- wild hedgehogs hogs positive for AMR genes from the total number of sampled individuals for the corresponding study areas: green— positive forTarragonès AMR genes (n =from the total number 2); orange—Baix of =sampled Penedès (n individuals 1); azure—Garraf (n = 2);for the corresponding yellow—Alt Penedès (n =study areas: green—Tarragonès 3); brown—Baix Llobre- (n = 2); orange—Baix Penedès (n gat (n = 13); red—Anoia (n = 1);Black—Bages = 1); azure—Garraf (nmint—Vallès (n = 5); = 2); yellow—Alt Occidental Penedès (n = 3); brown—Baix (n = 38); purple—Barcelonès Llobregat (n = 17); blue— (n = 13); red—AnoiaVallès Oriental (n = 17); pink—Maresme (n = 6); beige—Selva (n = 3); grey—Osona (n = 3). Pie charts: purple—positive; (n = 1); Black—Bages (n = 5); mint—Vallès Occidental (n = 38); purple—Barcelonès (n = 17); blue—Vallès Oriental yellow—negative. (n = 17); pink—Maresme (n = 6); beige—Selva (n = 3); grey—Osona (n = 3). Pie charts: purple—positive; yellow—negative. In Figure 4, a map with the location of wild hedgehogs positive for AMR genes is In Figure Most presented. 4, a of map with the the sampled location animals wereof wildin hedgehogs found highly populatedpositive for areas of AMR genes is Catalo- presented. Most nia. The scanof the sampled statistics did not animals detect any were foundcluster significant in highly populated of animals areas carrying AMR of Catalonia. The genes, scan neither were statistics did there not statistically detect any significant differences significant cluster inofthe frequency animals of positive carrying AMR genes, Animals 2021, 11, x FOR PEER REVIEWanimals among the regions. 9 of 14 neither were there statistically significant differences in the frequency of positive animals among the regions. Figure 4. Localization and distribution of the hedgehogs included in the study according to the presence of at least one AMR gene (red star) or their absence (green dot) in each municipality over Figure 4. Localization and distribution of the hedgehogs included in the study according to the presence of at least one the human population density of each zone. AMR gene (red star) or their absence (green dot) in each municipality over the human population density of each zone. 4. Discussion In this study, a high occurrence of β-lactamase resistance genes has been reported in a wild population of European hedgehogs coming from urbanized areas of Catalonia. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild Animals 2021, 11, 2837 9 of 13 4. Discussion In this study, a high occurrence of β-lactamase resistance genes has been reported in a wild population of European hedgehogs coming from urbanized areas of Catalonia. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Sixty seven out of 114 faecal samples (57.8%) had growths in ceftriaxone supplemented agar, but AMR genes were detected in 42 of them (36.8%). Regarding the bacterial isolates, a total of 90 bacterial isolates were identified from those 67 analysed ani- mals, and AMR genes were detected in 52 isolates. Thus, in line with other studies [28,29], we observed that some of the AMRB did not have any of the AMR genes. This could be explained by the possible existence of other mechanisms of resistance such as chromosomal mechanisms that were not included as a target of this study. Interestingly, 36.8% of the animals were detected as carriers of AMR genes, with a special occurrence of human nosocomial bacteria harbouring ESBL, AmpC, and OXA genes. Moreover, Klebsiella pneumoniae was the bacteria with the highest proportion of resistance genes, followed by E. coli and C. freundii. The most frequently detected genes were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), and blaCMY-2 (8.8%). In addition, carbapenemases blaOXA-48 genes were found in two animals. It is interesting to remark that more than half of the enterobacteria presented a MDR phenotype, with an extended profile (XDR) in 30% of the isolates. According to the World Health Organization and the World Organization for Animal Health, third- and fourth-generation cephalosporins are critically important antibiotics to treat infection diseases in humans [30] and animals [31]. However, in the last decades, ac- quired resistance to β-lactams, mainly mediated by extended-spectrum beta-lactamase and AmpC β-lactamases, such as cephalosporinases, has been widely reported worldwide from humans, livestock, companion animals, the environment, and wildlife [32,33]. The results obtained in this study in wild hedgehogs dwelling in highly populated areas of Catalonia corroborate this wide distribution of β-lactam resistance genes. In fact, a high proportion of the bacterial isolates, especially K. pneumoniae, presented two or more β-lactamase genes and a phenotype of resistance to third- and fourth-generation cephalosporins among other compounds. The presence of β-lactamase resistance genes has been historically analysed using E. coli as a marker in livestock [34] and wildlife [35–37]. Regarding wildlife, the presence of ESBL genes has been reported principally in wild birds [38–40], but also in other species such as bats [41], wild boars [29], and fish [42]. The most commonly detected ESBL genes are CTX-M-1, CTX-M-14, and CTX-M-15, which are also found frequently in hospital settings, suggesting a relationship between bacteria from wildlife and health-care facilities [33]. Interestingly, hedgehogs from this study harbouring the CTX-M group were all classified with the CTX-M-15 variant. This variant has been described in the high risk clone ST131 of E. coli, which generates nosocomial issues worldwide [43]. Within the SHV group, we found a variety of variants distributed in all of the regions sampled, with SHV-28 being the most frequent one. The combination of CTX-M-15 with SHV-28 was the most repeated association, found principally in K. pneumoniae isolates. This association, when located in Klebsiella species, has been related with the high risk clone ST15, which is an emerging cause of nosocomial infections [44]. The AmpC β-lactamase genes CMY-1 and CMY-2 were detected in the tested hedge- hogs. As these genes can be either acquired by plasmids or can be chromosomally se- lected [44], it is difficult to attribute the presence of CMY-1 and -2 in these animals as a result of direct environmental contamination. Similarly, the genetic resistance of C. freundii isolates found in three animals is usually originated at a chromosomal level. However, most of the bacteria carrying AmpC genes found in the wild hedgehogs of this study were Klebsiella spp. and E. coli, which usually acquire resistance genes by plasmids [45]. Regarding other families of antibiotics, plasmid mediated colistin resistance genes were also analysed. Colistin is considered a last therapeutic option in human medicine and is widely used in cases of infections caused by ESBL bacteria [46]. However, the coexistence Animals 2021, 11, 2837 10 of 13 of resistance genes to β-lactamase and to colistin has been detected in humans [47], live- stock [48], and wild birds [49]. It has been speculated that colistin use in animal production, and particularly in the pig industry, could be the original source of these genes [22]. In this study, none of the tested animals presented colistin (mcr-1 to -5) resistance genes, but this is an expected finding as most of the tested animals came from urbanized areas with a low risk of contact with residues from pig production. However, the inclusion of a larger sample size with hedgehogs from regions with a high swine population density, such as Osona, in the analysis, could help to improve the odds of detecting these colistin resistance genes. On the other hand, the emergence of resistance to carbapenems—last resort drugs used in hospital settings for the treatment of severe infections caused by β-lactam resistant enterobacteria—has compromised the therapeutic options in human health [50]. Specially, the OXA-48 variant of carbapenemases is becoming highly prevalent in human clinical infections in Europe [51]. This high prevalence in human settings could explain the 1.7% of OXA-48 frequency in the hedgehog populations of urban surroundings. As carbapenems are not allowed in food animals, the increasing presence of carbapenemases genes in live- stock [52], companion animals [53], and wildlife animals [54] is alarming evidence of the anthropogenic environmental contamination. The spatial analysis showed that the frequency and distribution of hedgehogs carrying AMR genes was similar in the study area. However, it is important to mention that most of the animals included in this study came from highly urbanized regions, because there is more chance of them being found and taken to the wildlife centre. In addition, the hedgehog population density has increased in urban areas compared with rural zones (26), so sampling animals from these zones can be more difficult. Finally, as most of the animals came from highly populated regions, it is reasonable to speculate that the close contact with human areas leads to the transmission of AMR genes to wildlife, because they either inhabit and/or feed in an anthropogenic environment [55]. Regarding the AMR phenotype, it is important to remark that, although the AST was only performed on isolates grown on MacConkey agar supplemented with ceftriaxone, in order to focus on ESBLs, some of these strains showed a sensitive phenotype to ceftriaxone after long storage at −80 ◦ C. These long periods of freezing could affect both the viability of some of the strains and the loss of these genes that confer resistance. The high frequency of hedgehogs carrying β-lactamase resistance genes in this study is a cause of concern, because these animals have never been treated with an antibiotic; thus, the presence of AMR genes must be related to exposure to sewage water or soils contaminated with AMR bacteria and/or antimicrobial residues. Sewage treatment plants are not completely efficient at removing all antibiotic residues and AMR bacteria [56], and contaminated water is spread into rivers and seas. Moreover, livestock manure is applied to land and can also contain it [57], enriching the resistome in both soils and earthworms [58]. Earthworms, a key biological component of soil, have been shown to contribute to spread- ing AMR, as they can acquire antibiotics directly from the soil [55]. Considering that most of the hedgehog’s diet is based on earthworms and other invertebrates, hedgehogs are more directly exposed to these antibiotic substances and resistance genes. Hence, the results of this study suggest that hedgehogs are good sentinels of AMR environmental pollution, especially in highly populated areas with high human activity. Furthermore, the presence of wild hedgehogs carrying MDR bacteria as clinical pa- tients at the WRC might compromise the effectiveness of antimicrobial treatments in these animals, and it can also represent a zoonotic risk for staff and the general population who come into contact with them. 5. Conclusions This study reveals a high prevalence of human nosocomial-like bacteria with β- lactamase resistance genes in wild hedgehogs inhabiting highly populated zones in Cat- alonia. It is crucial to raise awareness about the strong interconnection between habitats and compartments induced by multiple exchange routes, which implies that AMR issues Animals 2021, 11, 2837 11 of 13 must be tackled under the One Health approach. Further studies are needed to confirm the relatedness of cephalosporin resistant Enterobacteriaceae strains found in wild hedgehogs as well as those that cause concern in human hospitals at a regional level. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/ani11102837/s1. Table S1: Oligonucleotides used for the detection of ESBL/AmpC and colistin-resistance genes in this study, Table S2: Odd-ratios and p-values of bivariate generalized linear models, Table S3: Odd-ratios and p-values of multivariate generalized linear models. Author Contributions: Conceptualization, R.A.M.-L. and L.D.; methodology, B.G., E.O., R.A.M.- L. and L.D.; software, B.G. and A.A.; validation, L.D.; formal analysis, B.G., L.A., C.S. and N.R.; investigation, L.D.; resources, L.D.; data curation, B.G., L.A., C.S., N.R. and L.D.; writing—original draft preparation, B.G. and L.D.; writing—review and editing, B.G., A.A., E.O., R.A.M.-L. and L.D.; supervision, L.D.; funding acquisition, L.D. All authors have read and agreed to the published version of the manuscript. Funding: Biel Garcias was supported by Departament de Recerca i Universitats de la Generalitat de Catalunya (FI-SDUR 2020). Institutional Review Board Statement: The authors only collected samples. Therefore, the research did not cause any harm or suffering to any animals. Informed Consent Statement: Not applicable. Data Availability Statement: Data will be made available upon reasonable request to the corre- sponding author. 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