animals Article Antimicrobial Resistance in Bacteria Isolated from Exotic Pets: The Situation in the Iberian Peninsula Eleonora Muñoz-Ibarra 1 , Rafael A. Molina-López 2 , Inma Durán 3 , Biel Garcias 1 , Marga Martín 1 and Laila Darwich 1, * 1 Department Sanitat i Anatomia Animals, Veterinary Faculty, Universitat Autònoma de Barcelona (UAB), 08193 Cerdanyola del Vallès, Spain; munozeleonora@gmail.com (E.M.-I.); biel.garcias@uab.cat (B.G.); marga.martin@uab.cat (M.M.) 2 Catalan Wildlife Service, Centre de Fauna Salvatge de Torreferrussa, 08130 Santa Perpètua de Mogoda, Spain; rafael.molina@gencat.cat 3 Departamento de Veterinaria de Laboratorio Echevarne, 08037 Barcelona, Spain; idduran@laboratorioechevarne.com * Correspondence: laila.darwich@uab.cat Simple Summary: Antimicrobial resistance in exotic pets has not been widely studied. The close contact of this type of animal with the human population increases the risk of untreatable bacterial infections, which represent a veterinary and human public health challenge. We analyze the database of microbiological diagnoses and the bacterial susceptibility to antimicrobials in exotic pets from the Iberian Peninsula. We found that the most prevalent bacteria in birds and mammals were Staphylococcus spp., while in reptiles, they were the Pseudomonas spp. In addition, Pseudomonas showed the highest levels of resistance among the three animal groups, and on the other hand, the multidrug resistance level was significant in Enterobacterales. Most of the bacteria we found have zoonotic importance. The prevalent bacteria are resistant to antimicrobials that have been described as critical for human use, implying that the threat of antimicrobial resistance extends not only to Citation: Muñoz-Ibarra, E.; domestic and companion animals but also to humans due to the potential transmission of resistant Molina-López, R.A.; Durán, I.; Garcias, B.; Martín, M.; Darwich, L. genes. Once seen from the lens of the One-Health paradigm, these findings are concerning, as they Antimicrobial Resistance in Bacteria highlight the risk of spreading antibiotic-resistant genes between different individuals and their Isolated from Exotic Pets: The environments. In order to prevent antibiotic resistance, we encourage the development of joint work Situation in the Iberian Peninsula. between animal and human health specialists. Animals 2022, 12, 1912. https:// doi.org/10.3390/ani12151912 Abstract: Literature related to antimicrobial resistant (AMR) bacteria in exotic pets is minimal, being Academic Editor: Mandy Paterson essential to report objective data on this topic, which represents a therapeutic challenge for veterinary medicine and public health. Between 2016 and 2020, laboratory records of 3156 exotic pet specimens’ Received: 9 June 2022 microbiological diagnoses and antibiotic susceptibility testing (AST) results were examined. The Accepted: 25 July 2022 samples were classified into three animal classes: birds (n = 412), mammalia (n = 2399), and reptilian Published: 27 July 2022 (n = 345). The most prevalent bacteria in birds and mammals were Staphylococcus spp. (15% and 16%), Publisher’s Note: MDPI stays neutral while in reptiles they were Pseudomonas spp. (23%). Pseudomonas was the genus with the highest with regard to jurisdictional claims in levels of AMR in all animal groups, followed by Enterococcus spp. By contrast, Gram-positive cocci published maps and institutional affil- and Pasteurella spp. were the most sensitive bacteria. Moreover, in reptiles, Stenotrophomonas spp., iations. Morganella spp., and Acinetobacter spp. presented high levels of AMR. Multidrug-resistant (MDR) bacteria were isolates from reptiles (21%), birds (17%), and mammals (15%). The Enterobacterales had the highest MDR levels: S. marcescens (94.4%), C. freundii (50%), M. morganii (47.4%), K. pneumoniae (46.6%), E. cloacae (44%), and E. coli (38.3%). The prevalence of MDR P. aeruginosa strains was 8%, Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. detecting one isolate with an XDR profile. Regarding antimicrobial use, many antibiotics described This article is an open access article as critically important for human use had significant AMR prevalence in bacteria isolated from exotic distributed under the terms and pets. Under the One-Health approach, these results are alarming and of public health concern since conditions of the Creative Commons potential transmission of AMR bacteria and genes can occur from exotic pets to their owners in Attribution (CC BY) license (https:// both senses. For this reason, the collaboration between veterinarians and public health professionals creativecommons.org/licenses/by/ is crucial. 4.0/). Animals 2022, 12, 1912. https://doi.org/10.3390/ani12151912 https://www.mdpi.com/journal/animals Animals 2022, 12, 1912 2 of 24 Keywords: exotic pets; antimicrobial resistance; birds; mammals; reptiles; bacteria 1. Introduction There is increasing worry about the development of antibiotic resistance due to the numerous instances of bacterial infections that have affected humans and animals and may be fatal or exceedingly challenging to treat. Several studies have shown that the shift of resistant bacteria or genes through pets to humans is highly probable since pets play a role as a potential reservoir of AMR [1–8]. Although it is important to note that spillover transmission from people to animals is feasible, research on this situation is limited. Some worldwide studies have evidenced the transmission of diseases from exotic pets to their owners, handlers, or other animal species [2,3,9] but most of the monitoring programs are focused on antimicrobial use in livestock animals, and recommendations and policies that control or rule antibiotics stewardship in exotic pets are lacking [10]. In Spain, there are few studies conducted on exotic pets showing the potential risk of human infections by zoonotic bacteria such as Salmonella spp. in turtles [11,12] or respiratory infectious diseases in guinea pigs and chinchillas [13]. Although these studies have shown concern for investigating bacterial infections in exotic pets, there is a lack of published literature about the antimicrobial sensitivity profiles of bacterial infections in exotic pets against common antibiotics used in veterinary and human medicine. This information is critical since vet clinicians have no guidelines for selecting the best empirical antimicrobial treatment in terms of the risk of selection and dissemination of antibiotic resistance in exotic pets and its repercussion on the health of people and other domestic animals. The present study analyses the clinical microbiological data on exotic pets collected between 2016 and 2020 by a large Diagnostic Laboratory in Spain to determine the most prevalent bacterial infections and AMR profiles among different exotic pet classes. The final objective is to provide evidence of the risk of zoonotic transmission of AMR bacteria between exotic pets and their owners in order to optimize treatment and preventive measures in veterinary practices. 2. Materials and Methods 2.1. Database Source and Management The database used for this study comprises recorded data of the microbiological outcomes from clinical samples that contain information about clinical cases submitted by veterinary clinics throughout Spain and Portugal (Figure 1). The records were provided by the Veterinary Medicine Department of a large private laboratory of diagnosis in Barcelona (Spain). The lab has held the quality management system certificate ISO-9001 since 1998 and the accreditation from ENAC (National Accreditation Entity) according to criteria included in the ISO/IEC 17025 Standard defined in the Technical Annexes 511/LE1947 for Pharmaceutical Toxicology and Microbiology Testing. The first step was to preclean the database, excluding the results without animal species information, type of sample, or antibiogram results. Likewise, the data were screened for samples collected in duplicate and those that were found to be repeated. The following variables were extracted from the records: animal species, type/origin of the sample, geographic origin of the specimen, bacterial identification, and antimicrobial susceptibility testing. Then, the animals were segregated following the categorization of animals depending on their purpose, behavior, or habitat, as mentioned in the Legislative Decree 2/15 April 2008 (Anon, 2011). Animals 2022, 12, Animals 2022, 12, 1912 x FOR PEER REVIEW 3 3of of 24 24 Figure 1. Distribution of clinical samples by geographical origin in Spain and Portugal analyzed Figure 1. Distribution of clinical samples by geographical origin in Spain and Portugal analyzed between 2016 and 2020. between 2016 and 2020. The first 2.2. Study stepand Design wasAnimal to preclean Samplesthe database, excluding the results without animal spe- cies information, A total of 3986 microbiologicalantibiogram type of sample, or analyses from results. Likewise, clinical specimens the taken data were screened between 2016 for samples collected in duplicate and those that were found to be repeated. and 2020 comprised the final sample size. The sample was classified according to the exotic The following variables pet class as were extracted follows: birdsfrom (n = the 591 records: samples), animal mammalsspecies, (n =type/origin of the 3003), reptiles (n =sample, 392). geo- graphic origin ofdata In addition, the specimen, were grouped bacterial identification, into the and antimicrobial following categories: susceptibility respiratory (nasal and testing. Then, the animals were segregated following the categorization bronchioalveolar samples); skin/external mucous (skin, turtle shell, ocular, otic, and other of animals de- pending on their purpose, behavior, or habitat, as mentioned in the Legislative mucous); digestive (liver, gastrointestinal tract, and cloaca); urinary (urine and cloaca); Decree 2/2008 of 15 April(bones, musculoskeletal (Anon,joints, 2011).and muscles); lymphoreticular (lymph nodes, coelomic cav- ity, and glands); embryo/egg/eggshell (fetus, yolk sacs, and amniotic liquid); reproductive 2.2. Studyand (vaginal Design and penis Animal Samples secretions, and gonads); and circulatory (pericardium, heart, or blood). A total of 3986 microbiological analyses from clinical specimens taken between 2016 2.3. Microbiological and 2020 comprised Diagnosis the finalTechniques andThe sample size. Antimicrobial sample was Susceptibility Testing classified according to the ex- Microbiological otic pet class as follows:identification wassamples), birds (n = 591 performed using the(nMALDI-TOF mammals mass = 3003), reptiles (nspectrom- = 392). eter or the API Rdata In addition, ID were system (bioMérieux, grouped into theMadrid, following Spain), as previously categories: respiratorydescribed (nasal andby Darwich and others bronchioalveolar [1] andskin/external samples); Li and others [6]. All(skin, mucous Gram-positive turtle shell,bacterial isolates ocular, otic, were and other performeddigestive mucous); by the antimicrobial susceptibility (liver, gastrointestinal tract,test and(AST) using cloaca); the standard urinary (urine anddisk diffu- cloaca); sion method according to Performance Standards for Antimicrobial musculoskeletal (bones, joints, and muscles); lymphoreticular (lymph nodes, coelomic Susceptibility Test- ing for bacteria isolated from animals (M31-A3, CLSI VET01, 2008) and cavity, and glands); embryo/egg/eggshell (fetus, yolk sacs, and amniotic liquid); repro- humans (M100- S24, CLSI, ductive 2016) and (vaginal for monitoring resistant penis secretions, and microorganisms as a potential gonads); and circulatory risk to public (pericardium, heart, health. or blood). The panel included 45 antimicrobials corresponding to 8 classes or categories and 7 single drug classes and their respective disc concentration: β-lactams (amoxicillin (AMO 2.3. 30 µg), AMO Microbiological + clavulanic Diagnosis acidand Techniques (AMC/30 µg), oxacillin Antimicrobial (OXA/1 Susceptibility µg), cefoxitin Testing (CXI/30 µg), penicillin (PEN/10 U), piperacillin (PIP/110 µg), piperacillin/tazobactam Microbiological identification was performed using the MALDI-TOF mass spectrom- (PIT/110 µg), ampicillin (AMP/10 µg), cephalexin (CLE/30 µg), cephalothin (CET/30 µg), eter or the API R ID system (bioMérieux, Madrid, Spain), as previously described by Dar- cefazolin (CZO/30 µg), cefuroxime (CUR/30 µg), ceftazidime (CTZ/30 µg), cefotaxime wich and others [1] and Li and others [6]. All Gram-positive bacterial isolates were per- (CTA/30 µg), cefovecin (CVN/30 µg), and cefepime (CEP/30 µg)), imipenem (IMI/10 µg), formed by the antimicrobial susceptibility test (AST) using the standard disk diffusion meropenem (MER/10 µg), and aztreonam (AZT/30 µg); fluoroquinolones (ciprofloxacin method according to Performance Standards for Antimicrobial Susceptibility Testing for (CIP/5 µg), enrofloxacin (ENR/5 µg), nalidixic acid (NAL/30 µg)); aminoglycosides bacteria isolated from animals (M31-A3, CLSI VET01, 2008) and humans (M100-S24, CLSI, Animals 2022, 12, 1912 4 of 24 (amikacin (AMI/30 µg), gentamicin (GEN/10 µg), tobramycin (TOB/10 µg), neomycin (NEO/30 µg), kanamycin (KAN/30 µg)); macrolides (azithromycin (AZI/15 µg), ery- thromycin (ERY/5 µg)); tetracyclines (tetracycline (TET/30 µg), doxycycline (DOX/30 µg)); lincosamides (clindamycin (CLI/2 µg), lincomycin (LCM/2 µg)); polymyxins (polymyxin B (PMB/300 µg)); trimethoprim/sulfamethoxazole (TRS/25 µg); phenicols (chloram- phenicol (CHL/10 µg), florfenicol (FLO/30 µg)); fosfomycin (FOS/50 µg); mupirocin (MUP/200 µg); metronidazole (MET/5 µg); glycopeptides (vancomycin (VAN/30 µg)); fusidic acid (FUS/10 µg); nitrofurantoin (NIT/300 µg), and rifampicin (RIF/5 µg). For Gram-negative bacteria, NM44 MicroScan (Beckman Coulter, Villepinte, France) system testing was performed for all the antimicrobials except for those authorized for vet- erinary uses that are not included in the automatic scan panels (enrofloxacin, pradofloxacin, marbofloxacin, doxycycline, cephalexin, and cefovecin) [6]. Additionally, quality control for the AST was performed using internal controls in each automatic panel of NM44 MicroScan (Beckman Coulter, Villepinte, France). In the case of manual antibiograms, McFarland standards were used as a reference, previously confirmed by a Densicheck (bioMérieux, Madrid, Spain). Isolates were classified as susceptible, intermediate, or resistant according to the results obtained from the lab records. In addition, multidrug resistance (MDR) was defined as resistance to at least one agent in ≥3 antimicrobial categories; extensive drug resistance (XDR) as resistance to all but two of the tested antimicrobial categories; and finally, pan-drug (PDR) as resistance to all the categories tested [14]. These classifications and definitions were also used in the MDR analysis. 2.4. Statistical Analysis The statistical analysis was performed using R version 4.1.0 (R v 4.1.0 (R Core Team, Vienna, Austria), 2021) [15], applying the novel AMR package [16]. The statistical unit is bacterial culture results, which were considered individually for the analysis. Bacterial names were first manually and then automatically classified by the AMR package, the taxonomy of microorganisms was extracted from the Catalogue of Life database and the List of Prokaryotic names with Standing in Nomenclature; the interpretation of disk diffusion values were based on the CLSI and EUCAST guidelines available between 2011 and 2020, which are included in this package as well as antibiotic and AMR analysis. Inconsistencies in the classification were manually reviewed. For statistical assess- ments, the package performed an accurate analysis avoiding the false susceptibility or bias susceptibility frequency of microorganisms, reporting the AST, regardless of the in- trinsic resistance or taking out the repeated cases. The pack of functions worked with Chi-square (X2 ) test function to compare the animals’ classes, bacteria species, and the AMR frequencies; in this regard, the statistical significance was considered when p < 0.05. 3. Results 3.1. Microbiological Results in Exotic Pets The results of the microbiological cultures were performed once the database was cleaned from duplicated results and the fungus cultures were removed. Consequently, the final data of the microbiological testing were 3156 samples that presented a positive diagnosis confirmation of a pure or majoritarian bacterial infection (Table 1). Table 1. Number of samples according to the microbiological testing results. Class % Negatives (N) % Positives (N) Sum * Birds 26.6 (149) 73.4 (412) 561 Mammals 18.3 (539) 81.7 (2399) 2938 Reptiles 7.8 (29) 92.2 (345) 374 Total 18.5 (717) 81.5 (3156) 3873 * Fungi and yeasts were not considered. Animals 2022, 12, 1912 5 of 24 From the microbiological results, birds (n = 412) represented 13%, mammals (n = 2399) represented 76%, and reptiles (n = 345) 11% of the total sample (Table 2). Within the birds, psittacines represented 85.2% of the avian class, with a particular frequency of Agapornis spp. (lovebirds) with 20%. The mammal class was principally represented by lagomorphs (73.2%) and rodents (22%), with Oryctolagus cuniculus (European rabbit) and the Cavia porcellus (guinea pig) being the principal species with 56% and 11% of the mammals, respectively. Finally, Trachemys scripta (red-eared slider turtle) represented 25% among reptiles, followed by 14% of Python regius (royal python). Table 2. Number and frequency of exotic pet cases analyzed by animal class and years studied from the database. Class 2016 2017 2018 2019 2020 Total (%) Birds 53 73 62 121 103 412 (13) Columbiformes 3 1 4 (1) Galliformes 5 7 10 15 16 53 (12.8) Passeriformes 1 1 1 1 4 (1) Psittaciformes 47 65 52 102 85 351 (85.2) Mammals 265 342 434 602 756 2399 (76) Carnivora 20 22 20 22 16 100 (4.1) Eulipotyphla 2 1 1 3 7(0.3) Lagomorpha 200 235 297 454 571 1757 (73.2) Rodentia 43 85 116 125 166 535 (22.3) Reptiles 60 44 55 96 90 345 (11) Squamata 27 14 22 41 34 138 (40) Testudines 33 30 33 55 56 207 (60) Total 378 459 551 819 949 3156 (100) According to the sample’s origin, skin/external mucous samples were the most fre- quent ones, followed by the respiratory specimens. Digestive problems were more common in birds and reptiles, while urinary infections were most important in mammals. Detailed information about the sample origin is depicted in the Appendix A Figure A1. The most prevalent bacterial genus was Staphylococcus spp. (14.9%) and Pseudomonas spp. (14.3%), followed by Streptococcus spp. (9%) and Enterobacteria (Klebsiella spp. 6.6%, Enterobacter spp. 6.4%, and Escherichia spp. 6.3%) (Table 3). Within the birds and mammals groups, Staphylococcus spp. was the predominant agent, representing 16% of the cases. In reptiles, Pseudomonas spp. was the most frequent one representing 23% of the cases (Table 3). In birds and mammals groups, a similar distribution of the bacterial agents was found for the different system categories (Figure 2a,b). In general, Pseudomonas spp. was the most important agent found in most categories, except in the urinary and musculoskeletal systems, where the most prevalent bacteria were E. coli and Staphylococcus spp., and the lymphoreticular system, with Enterococcus spp. as the principal agent. Staphylococcus spp. and Enterobacteria such as E. coli, Klebsiella spp., and Enterobacter spp. were also frequently isolated from digestive, respiratory, and skin/external mucous systems. Animals 2022, 12, 1912 6 of 24 Table 3. Frequencies of bacterial species identified in each animal class. Isolations Per Animal Class (% 1 ) Microbiological Results Birds Mammals Reptiles Total Population Acinetobacter spp. 2 16 (3.9) 96 (4) 12 (3.5) 124 (3.9) A. baumannii 3 (0.7) 12 (0.5) 4 (1.1) A. Iwoffii 5 (1.2) 17 (0.7) 1 (0.3) Aeromonas spp. 2 5 (1.2) 18 (0.7) 34 (9.8) 57 (1.8) A. hydrophila 2 (0.4) 14 (0.6) 17 (4.9) A. veronii 1 (0.2) 0 6 (1.7) Bordetella spp. 2 1 (0.2) 164 (6.8) 7 (2) 172 (5.4) B. bronchiseptica 1 (0.2) 155 (6.5) 5 (1.4) Citrobacter spp. 2 5 (1.2) 20 (0.8) 20 (5.8) 45 (1.4) C. freundii 4 (1) 10 (0.4) 14 (4) Enterobacter spp. 2 35 (8.5) 152 (6.3) 14 (4) 201 (6.4) E. cloacae 27 (6.5) 120 (5) 11 (3.2) Enterococcus spp. 2 33 (8) 103 (4.3) 18 (5.2) 154 (4.9) E. faecalis 14 (3.4) 56 (2.3) 8 (2.3) Escherichia spp. 2 55 (13.3) 128 (5.3) 15 (4.3) 198 (6.3) E. coli 54 (13.1) 118 (4.9) 15 (4.3) Klebsiella spp. 2 46 (11.1) 146 (6.1) 16 (4.6) 208 (6.6) K. pneumoniae 30 (7.3) 98 (4.1) 3 (0.9) K. oxytoca 12 (2.9) 37 (1.5) 12 (3.5) Morganella spp. 2 0 6 (0.2) 14 (4) 20 (0.6) M. morganii 0 5 (0.2) 14 (4) Pasteurella spp. 2 2 (0.4) 198 (8.3) 1 (0.3) 201 (6.4) P. multocida 2 (0.4) 144 (6) 1 (0.3) Pseudomonas spp. 2 57 (13.8) 315 (13.1) 80 (23.2) 452 (14.3) P. aeruginosa 44 (10.7) 217 (9) 62 (18) P. fluorescens 0 16 (0.7) 0 Staphylococcus spp. 2 69 (16.7) 389 (16.2) 11 (3.2) 469 (14.9) S. aureus 11 (2.7) 122 (5.1) 1 (0.3) S. epidermidis 8 (1.9) 25 (1) 0 S. pseudintermedius 0 20 (0.8) 0 S. sciuri 7 (1.7) 9 (0.4) 1 (0.3) S. xylosus 2 (0.4) 37 (1.5) 2 (0.6) Stenotrophomonas spp. 2 4 (1) 18 (0.8) 16 (4.6) 38 (1.2) S. maltophilia 4 (1) 18 (0.8) 16 (4.6) Streptococcus spp. 2 30 (7.3) 236 (9.8) 17 (4.9) 283 (9) S. intermedius 0 26 (1.1) 0 Other spp. 54 (13.1) 410 (17.1) 70 (20.3) 534 (16.9) Total 412 (100) 2399 (100) 345 (100) 3156 (100) 1 Percentage related to the total number of samples per animal group and the total population. 2 All species are included. Chi-square (X2) test applied to all the results showed no significant differences (p-value > 0.001). In reptiles, Pseudomonas spp. was also the most common agent isolated from almost all the system categories, except for the lymphoreticular system, where the principal bacteria were Aeromonas spp. and Citrobacter spp. These two former bacterial species were also found in other systems (digestive, respiratory, and skin/external mucous systems). In contrast to birds and mammals, reptiles presented a high prevalence of Enterococcus spp. in urinary infections (Figure 2c). Animals 2022, 12, x FOR PEER REVIEW 7 of 24 and the lymphoreticular system, with Enterococcus spp. as the principal agent. Staphylo- Animals 2022, 12, 1912 7 of 24 coccus spp. and Enterobacteria such as E. coli, Klebsiella spp., and Enterobacter spp. were also frequently isolated from digestive, respiratory, and skin/external mucous systems. a. Bird 100% Stenotrophomonas spp. 90% Aeromonas spp. 80% Citrobacter spp. 70% Acinetobacter spp. 60% Corynebacterium spp. 50% Enterobacter spp. Enterococcus spp. 40% Escherichia spp. 30% Klebsiella spp. 20% Pseudomonas spp. 10% Staphylococcus spp. 0% Streptococcus spp. Others b. Mammals 100% Acinetobacter spp. 90% Aeromonas spp. 80% Citrobacter spp. 70% Corynebacterium spp. 60% Enterobacter spp. 50% Enterococcus spp. 40% 30% Escherichia spp. 20% Klebsiella spp. 10% Pseudomonas spp. 0% Staphylococcus spp. Stenotrophomonas spp. Streptococcus spp. Others Figure 2. Cont. Animals 2022,12, Animals 2022, 12,1912 x FOR PEER REVIEW 8 of8 24 of 24 c. Reptiles 100% Acinetobacter spp. 90% Aeromonas spp. 80% 70% Citrobacter spp. 60% Enterobacter spp. 50% Enterococcus spp. 40% Escherichia spp. 30% 20% Klebsiella spp. 10% Neisseria spp. 0% Pseudomonas spp. Staphylococcus spp. Stenotrophomonas spp. Streptococcus spp. Others Figure2.2. Distribution Figure Distribution ofof bacterial bacterial genus genusaccording accordingtotothe thesample sampleorigin originand andthe animal the animalclass: birds class: (a), mammals (b), and reptiles (c). Others include Achromobacter spp., Agrobacterium birds (a), mammals (b), and reptiles (c). Others include Achromobacter spp., Agrobacterium spp., spp., Avibacte- rium spp., Bacillus spp., Bordetella spp., Brevibacterium spp., Burkholderia spp., Chryseobacterium spp., Avibacterium spp., Bacillus spp., Bordetella spp., Brevibacterium spp., Burkholderia spp., Chryseobacterium Corynebacterium spp. (in reptiles), Cronobacter spp., Delftia spp., Elizabethkingia spp., Empedobacter spp., Corynebacterium spp. (in reptiles), Cronobacter spp., Delftia spp., Elizabethkingia spp., Empedobacter spp., Haemophilus spp., Kocuria spp., Kosakonia spp., Leclercia spp., Lelliottia spp., Leuconostoc spp., spp., Haemophilus spp., Kocuria spp., Kosakonia spp., Leclercia spp., Lelliottia spp., Leuconostoc spp., Ligilactobacillus spp., Lysinibacillus spp., Mammaliicoccus spp., Microbacterium spp., Moraxella spp., Ligilactobacillus Myroides spp., spp., Lysinibacillus Neisseria spp., Mammaliicoccus spp. (in birds and mammals), spp., Microbacterium Ochrobactrum spp., Moraxella spp., Pantoea spp., spp., Pasteurella Myroides spp., Neisseria spp. (in birds and mammals), Ochrobactrum spp., Pantoea spp., Pasteurella spp., Peptostreptococcus spp., Pluralibacter spp., Proteus spp., Providencia spp., Raoultella spp., spp., Rothia Peptostreptococcus spp., Salmonella spp., Pluralibacter spp.,Serratia spp., spp., Proteusspp., Vagococcus Providencia spp.,Vibrio spp., spp., and Raoultella Weissella spp., Rothia spp., spp. Salmonella spp., Serratia spp., Vagococcus spp., Vibrio spp., and Weissella spp. In reptiles, Pseudomonas spp. was also the most common agent isolated from almost 3.2. Prevalence of Antimicrobial-Resistant Bacteria all the system categories, except for the lymphoreticular system, where the principal bac- teriaThe frequencies were Aeromonas ofspp. AMRandamong birds, mammals, Citrobacter spp. Theseand tworeptiles’ former classes bacterialforspecies the most were frequent bacterial genus are in the Appendix A and shown in Figures 2, 3 and A1. Overall, also found in other systems (digestive, respiratory, and skin/external mucous systems). In Pseudomonas was the genus with the highest levels of AMR, whereas Gram-positive contrast to birds and mammals, reptiles presented a high prevalence of Enterococcus spp. cocci (Staphylococcus, Streptococcus, or Aeromonas spp.) and Pasteurella spp. were the most in urinary infections (Figure 2c). sensitive bacteria in all animal classes. Moreover, in birds and mammals, Enterobacter spp. and Klebsiella spp. also presented high levels of AMR to a large number of antimicrobials, 3.2. Prevalence of Antimicrobial-Resistant Bacteria followed by Enterococcus spp. In reptiles, Stenotrophomonas presented the highest resistance levelsThe in a frequencies of AMR global assessment, amongby followed birds, othermammals, and reptiles’ highly resistant classes genus such for the most as Morganella frequent spp. bacterial genus and Acinetobacter spp. are in the Finally, appendix AMR and in E. coli shown was in Figures intermediate, 2–4. Overall, normally Pseudo- lower levels monas than was other the genus members with of the the highest levels Enterobacterales of AMR, order such aswhereas Gram-positive Enterobacter, cocci (Staph- Klebsiella, Citrobacter, or Serratia spp. ylococcus, Streptococcus, or Aeromonas spp.) and Pasteurella spp. were the most sensitive bacteria in all animal classes. Moreover, in birds and mammals, Enterobacter spp. and Klebsiella spp. also presented high levels of AMR to a large number of antimicrobials, fol- lowed by Enterococcus spp. In reptiles, Stenotrophomonas presented the highest resistance levels in a global assessment, followed by other highly resistant genus such as Morganella spp. and Acinetobacter spp. Finally, AMR in E. coli was intermediate, normally lower levels than other members of the Enterobacterales order such as Enterobacter, Klebsiella, Citrobac- ter, or Serratia spp. Animals 2022, 12, x FOR PEER REVIEW 9 of 24 Animals 2022, 12, 1912 9 of 24 Escherichia coli 100 % cases reported 80 60 40 20 0 % R - Bird % R - Mammalia % R - Reptilia Klebsiella pneumoniae 100 % cases reported 80 60 40 20 0 % R - Bird % R - Mammalia % R - Reptilia Enterobacter cloacae 100 % cases reported 80 60 40 20 0 % R - Bird % R - Mammalia % R - Reptilia Figure 3. Cont. Animals 2022, 12, x FOR PEER REVIEW 10 of 24 Animals 2022, 12, 1912 10 of 24 Serratia marcescens 100 % cases reported 80 60 40 20 0 % R - Bird % R - Mammalia % R - Reptilia Citrobacter freundii 100 % cases reported 80 60 40 20 0 % R - Mammalia % R - Reptilia Morganella morganii 100 % cases reported 80 60 40 20 0 % R - Mammalia % R - Reptilia Figure 3. Cont. Animals 2022, 12, x FOR PEER REVIEW 11 of 24 Animals 2022, 12, 1912 11 of 24 Pseudomonas aeruginosa 100 % cases reported 80 60 40 20 0 % R - Bird % R - Mammalia % R - Reptilia Enterococcus faecalis 100 % cases reported 80 60 40 20 0 % R - Bird % R - Mammalia Staphylococcus aureus 100 80 % cases reported 60 40 20 0 % R - Bird % R - Mammalia Figure 3. Comparison of MDR patterns (percentage of resistance) of different bacterial spp. for each animal class. Animals 2022, 12, 1912 12 of 24 3.3. Frequency of Multidrug Resistance Profiles The study of antimicrobial susceptibility patterns showed that 97% of the bacteria were resistant to at least one agent of the total antimicrobial categories: 99.5% in birds, 96% in mammals, and 98.5% in reptiles. Moreover, 16% of the microorganisms presented a MDR pattern. No cases of PDR were observed. The percentage of strains with higher MDR levels found in reptiles was 21%, in birds it was 17%, and in mammals 15%. Enterobacterales pre- sented the most significant frequencies of MDR, remarking the prevalence of S. marcescens (94.4%), C. freundii (50%), M. morganii (47.4%), K. pneumoniae (46.6%), E. cloacae (44%), and E. coli (38.3%). Gram-positive cocci showed MDR frequencies of 24.6% for S. aureus and 15.4% for E. faecalis. Regarding P. aeruginosa, 8% of the isolates presented MDR patterns and one of the isolates showed an XDR profile. In birds, most bacterial species showed high resistance to macrolides, fusidic acid, lincosamides, nitroimidazoles, and aminopenicillins. Additionally, E. cloacae, S. marcescens, and P. aeruginosa were highly resistant to 1st and 2nd generation cephalosporins, amoxicillin, clavulanic acid, and nitrofurans. For P. aeruginosa, this resistant pattern was also expanded to tetracyclines, chloramphenicol, and trimethoprim-sulfonamides (Figure 3). In mammals, all bacterial species, with the exception of S. aureus, were highly resis- tant to fusidic acid, aminopenicillins (except for E. coli), nitroimidazoles and macrolides (except for C. freundii), and lincosamides (except for P. aeruginosa). Moreover, E. cloacae, S. marcescens, and P. aeruginosa were also resistant to 1st and 2nd generation cephalosporins and aminopenicillins β-lactamase inhibitors, and S. marcescens and P. aeruginosa expanded their resistance to 4th generation fluoroquinolones, chloramphenicol, and tetracyclines (Figure 3). In the reptiles, all bacteria were highly resistant to fusidic acid, lincosamides, and macrolides. Furthermore, except for E. coli, all the bacteria showed high resistance to 1st and 2nd generation cephalosporins. In addition, most enterobacteria and P. aeruginosa demonstrated to have high resistance to penicillins, aminopenicillins, aminopenicillins β-lactamase inhibitors, and tetracyclines. P. aeruginosa also showed extended AMR to chloramphenicol and trimethoprim/sulfonamides (Figure 3). 4. Discussion This work reports novel data about AMR in bacteria isolated from exotic pets in the Iberian Peninsula. These results provide helpful information for veterinarian clinicians and the scientific community since AMR in exotic pets can represent both a serious animal and public health concern. In this study, the most prevalent bacteria in birds and mammals were Staphylococcus spp. (16%), while in reptiles they were Pseudomonas spp. (23%). The AST results showed Pseudomonas presented the highest AMR levels in birds and mammals, detecting one isolate with an XDR profile, while in reptiles, it was found to be Stenotrophomonas spp., Morganella spp., and Acinetobacter spp. Moreover, the highest levels of MDR were observed in Enterobacterales: S. marcescens (94.4%), C. freundii (50%), M. morganii (47.4%), K. pneumoniae (46.6%), E. cloacae (44%), and E. coli (38.3%). Regarding antimicrobial use, many antibiotics described as critically important for human use had significant AMR prevalence in bacteria isolated from exotic pets. Thus, it is essential to deal with the burden of AMR, select the appropriate therapy, and research effective antibiotic stewardship in these animals. According to the antibiotic categorization for prudent and responsible use in animals, made by AMEG [17], the antibiotic classes that clinical veterinarians could use to combat specific bacterial infections are those included in the C -caution- or D -prudence- cate- gories authorized for vet use as a first-line treatment options. Among these categories can be found the 1st generation of cephalosporins, aminoglycosides, chloramphenicol, penicillin/β-lactam inhibitor, tetracyclines, and trimethoprim/sulfonamides. On the other hand, the antibiotics included in the A -avoid- or B -restrict- categories are the 3rd and 4th generation of cephalosporins; and 2nd and 3rd generation of fluoroquinolones, polymyxins, Animals 2022, 12, 1912 13 of 24 carbapenems, which are not authorized in veterinary medicine, cannot be used unless there is no other option, or shall always be used based on AST results. The results of AMR bacteria obtained in exotic pets agree with data reported on dogs and cats in Spain [1,6]. The AST demonstrated that Pseudomonas spp. and Enterococcus spp. presented the highest levels of AMR in both dogs and cats and in exotic pets. Moreover, within the Enterobacterales, E. coli showed low levels of AMR compared with K. pneumoniae, E. cloacae, S. marcescens, C. freundii, and M. morganii. Even so, in these common pets, Pasteurella isolates were highly sensitive to all antimicrobials tested. In exotic pets, the most sensitive bacteria were also Pasteurella spp., followed by Staphylococcus spp. and Streptococcus spp. Furthermore, many bacterial isolates observed in this study have been described as important zoonotic bacteria in human infectious diseases [18–20]. On the other hand, the prevalence of MDR profile in overall exotic pet cases was 16%, higher than that reported in urinary tract infections (UTI) in dogs and cats (8%) [1]. Analyzing individual cases, E. coli, K. pneumoniae, and S. aureus from exotic pets presented lower resistance patterns than the equivalent isolates in dogs and cats, whereas E. cloacae, S. marcescens, and P. aeruginosa presented higher resistance profiles [1]. The results of AST in exotic pets reveal that P. aeruginosa was the bacteria with the largest frequency of AMR in all animal categories, followed by E. cloacae and S. marcescens in mammals, which commonly require the use of antimicrobials as a last resort for human medicine. P. aeruginosa is an opportunistic pathogen that causes nosocomial infections, pulmonary infections in cystic fibrosis patients, disseminated infections in immunocom- promised humans [21], and severe infections in domestic and companion animals [1,6]. P. aeruginosa is also one of the most critical AMR priority pathogens, according to the World Health Organization [22]. There is an increase in the circulation of strains resistant to several antibiotic classes, including carbapenems [23]. Carbapenems are last-resort antibiotics used to treat serious infections by MDR bacteria, including P. aeruginosa. In our study, some P. aeruginosa avian strains presented an XDR profile, intrinsically resistant to beta-lactams and combinations with b-lactamase inhibitors, chloramphenicol, erythromycin, and trimetho- prim/sulfamethoxazole. Furthermore, Pseudomonas spp., principally in birds and mammals, were also resistant to the aminoglycosides (around 60%), carbapenems (50% approx.), and second-generation fluoroquinolones (50% approx.). By contrast, approximately 70% of the isolates in mammals were susceptible to lincomycin, polymyxins, and third-generation fluoroquinolones. In comparison to pseudomonal infections in canines and felines of the Iberian Peninsula, exotic pets presented higher frequencies of AMR for enrofloxacin and aminoglycosides than dogs and cats [6]. These results represent a serious health concern since the circulation of these MDR P. aeruginosa strains is increasing among exotic pets, highlighting its global spread. Another related Gram-negative oxidase-positive bacterium often seen as a co-organism along with P. aeruginosa is Stenotrophomonas spp., detected in this study as MDR strains in the respiratory tract of reptiles. Stenotrophomonas can cause opportunistic infections in humans owing to biofilm formation and antibiotic resistance. Occasionally, this bacterium has been involved in sepsis and severe lung infections in immunocompromised patients [24]. Regarding Enterobacterales, Morganella morganii was the enterobacteria with the largest resistance frequencies in birds and reptiles. In mammals, E. cloacae, S. marcescens, and K. pneumoniae presented MDR profiles, making them difficult to combat with conventional antimicrobials for veterinary use. M. morganii causes urinary infection and, to a lesser extent, other gynecological-related infections or contaminating surgical wounds. Occasion- ally, it has been related to septic arthritis, especially in elderly patients with long-standing diseases [25]. On the other hand, in this study, K. pneumoniae only presented low resistance to carbapenems, which are reserved for critical use in human medicine; considering other antimicrobials authorized for vet medicine, the best options were aminoglycosides and chloramphenicol, with around 50% of the isolates presenting resistance to these drugs. An increasing problem for doctors globally is K. pneumoniae, as it is a MDR infection and is considered a zoonotic agent of great relevance to both animal and human health [26,27]. Animals 2022, 12, 1912 14 of 24 Studies on bacterial resistance in animals, food, and the environment have revealed that multidrug-resistant and extended-spectrum β-lactamase (ESBL) strains are also resistant to carbapenems and β-lactam group drugs. For instance, samples of soft tissue, respiratory tract, genital tract, urinary tract infections, wounds, and feces from domestic and com- panion animals have contained MDR and ESBL carbapenemase-producing strains of this bacteria [6,27,28]. As a consequence, it is imperative to improve the monitoring of MDR bacteria and prevent their evolution, such as K. pneumoniae and P. aeruginosa in animals, i.e., minimizing antibiotic exposure in veterinary by the use of microbiological diagnostic techniques for effective treatments. It is equally critical to monitor for and adopt hygiene methods in order to reduce the spread of pathogens in practice. From a zoonotic point of view, the high levels of AMR to critically important antibiotics in human medicine found in exotic pets are of great concern since potential transmission of resistant genes from pets to humans or other animals can occur, considering that the predominant bacteria in this study are among the six pathogens directly attributed to human deaths due to AMR; likewise, they are significantly present in the urban microbiome with resistant genes [29,30]. Regarding antimicrobial use, many of the antibiotics described by the WHO as critically important for human use had significant AMR prevalence in bacteria isolated from exotic pets. Furthermore, under the AWaRe classification by the WHO [31], the principal bacteria found in this study present clear resistant patterns to different antibiotics used in human medicine (See Appendix A Table A1). It is necessary to highlight the susceptible bacterial patterns recommended by this study and its comparison with those allowed for animal use; the therapies shaped from this interpretation should be in contrast with the individual animal case and, as much as possible, with AST. Additionally, performing the susceptibility test with the antibiotics for veterinary medicine might support decision-making by veterinarians, prudent antibiotic use, and the reduction in the use of antimicrobials sensitive to human medicine. From the One Health standpoint, it is critical that veterinarians and physicians work together to optimize, rationalize, and prudently use antimicrobial therapies in domestic, companion, and exotic animals and humans since most bacterial pathogens and their resistance mechanisms can be shared between animals and humans. Examples include adopting interrelated education on zoonotic infections and ownership implications in health, as well as exchanging data sources, experiences, and discussing clinical cases. The results of this study provide objective data on the microbiological results (AMR bacteria and AST profiles) obtained in exotic pets of the Iberian Peninsula. On the one hand, these data can be useful for vet clinicians to apply empirical therapy in exceptional situations where the severity of the disease requires immediate antimicrobial treatment, with no time for AST analysis. On the other hand, this situation of AMR bacteria found in exotic pets to critically important antibiotics in human medicine is significant and a public health concern since potential transmission of resistant genes from exotic pets to humans or other animals can occur in both senses. These findings are concerning in terms of a One-Health approach since they indicate the likelihood of resistance genes spreading through animals, the environment, and humans. For this reason, the collaboration between veterinary and public health professionals to combat AMR is essential. Author Contributions: E.M.-I. and L.D. conceptualized, analyzed, investigated, and wrote the drafts of the manuscript. Additionally, E.M.-I. worked in the software, data curation, and visualization. All the authors, E.M.-I., R.A.M.-L., I.D., B.G., M.M., and L.D. were involved in the validation, reviewing, and editing; I.D. had substantial participation in the data resourcing. Supervision, funding acquisition, and project administration were conducted by L.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. BG was supported by Departament de Recerca i Universitats de la Generalitat de Catalunya (FI-SDUR 2020). Institutional Animals 2022, 12, 1912 Review Board Statement: Not applicable. 15 of 24 Informed Consent Statement: Not applicable. Data Availability Statement: Theresearch Funding: This data that support received the findings no external of this funding. BG study arebylimited was supported available Departament de Recerca from the corresponding author de i Universitats upon reasonable la Generalitat request. (FI-SDUR de Catalunya Restrictions 2020).apply to the availability of these data, which were used under Institutional license Review BoardforStatement: this study. Not applicable. Acknowledgments: EMI wasConsent Informed a student of the Not Statement: Master’s Degree in Zoonosis and One Health of the applicable. University Autonomous of Barcelona (UAB). Data Availability Statement: The data that support the findings of this study are limited available from the corresponding author upon reasonable request. Restrictions apply to the availability of Conflicts of Interest: The authors declare that they have no known competing financial interests or these data, which were used under license for this study. personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments: EMI was a student of the Master’s Degree in Zoonosis and One Health of the University Autonomous of Barcelona (UAB). Appendix A Conflicts of 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. Percentage of samples for animal class versus sample origin Appendix A 100% 90% Respiratory 80% Skin/Shell/External mucous 70% Digestive 60% Urinary 50% Muscle-skeletal 40% Lympho-reticular 30% Embryo/Egg/Egg shell 20% Reproductive 10% Circulatory 0% Bird Mammalia Reptilia Figure A1. Distribution of samples according to the animal class and the sample origin, following the grouped categories. Figure A1. Distribution of samples according to the animal class and the sample origin, following the grouped categories. Animals 2022, 12, x FOR PEER REVIEW 16 o Animals 2022, 12, 1912 16 of 24 Staphylococcus spp. Enterococcus spp. Pseudomonas spp. RIF MER TIC MET FOS NAL LCM VAN COL CMX PEN CEP CHL FLO AZT VAN AMC IMI FLO MER AMO TRS TOB AMP MER CTZ PIT CLE PIT RIF NEO GEN IMI FOS PMB TRS CUR AMI MET ENR GEN CTA ENR CIP AMI DOX MET CEP TET OXA IMI CIP FLO DOX GEN TRS CET CMX FOS ENR CHL DOX CTR NIT TET TET TOB AMC CIP AMI NEO OXA NEO AMP AMC CTA CTA CLI CLI AMO TOB CHL CLE AZI VAN ERY ERY RIF CUR PEN PEN OXA AMP NIT CTZ PIT LMC CEP NIT ERY AMO AZI CUR TIC PMB CTR CTZ NAL CMX PMB CTR CLI NAL COL CLE COL CET CET AZT AZT AZI 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R Escherichia spp. Klebsiella spp. Enterobacter spp. TIC PMB MER PMB MER FLO MER IMI PIT IMI AMI IMI CET PIT PMB CEP ENR FOS PIT GEN TET GEN CTZ TRS TOB OXA TOB FOS TOB NEO AMI TRS CHL CTZ DOX DOX OXA CTA ENR FLO CLE CIP CLE CHL GEN CTA CIP AMI ENR CUR OXA CUR TET CMX FLO DOX MET FOS TRS CLE AMC CHL AMC CMX AMC VAN CEP NEO RIF NEO CMX PEN VAN CIP NIT TIC TET NAL RIF AMO LMC PEN AMP ERY NIT MET NAL CUR NIT MET CTZ VAN LMC CTA RIF ERY CLI PEN CLI CET LMC CET CEP ERY AZI AZI CLI AMP AMP AZI AMO AMO 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R Figure A2. Proportion Figure A2.of susceptible Proportion (S), susceptible—increased of susceptible exposure (I), (S), susceptible—increased and resistance exposure (R) (I), and resistance (R) terial bacterial strains strains in bird in bird class. class. Animals 2022, 12, x FOR PEER REVIEW 17 Animals 2022, 12, 1912 17 of 24 Staphylococcus spp. Streptococcus spp. Pseudomonas spp. LCM MER NAT VAN IMI AZT CMX CTR MER CHL AMC TIC FLO PIT PMB RIF VAN PIT TRS RIF IMI MER FLO TOB FOS CUR CEP CUR CTA CTZ NEO CHL GEN CTA CIP AMI CEP CLE ENR CLE GEN CIP CET TRS FLO IMI AMP OXA CTR DOX TRS AMI PEN DOX ENR ENR CMX AMC CEP TET DOX ERY NIT GEN NIT CTA OXA CLI AMC CIP AMO TOB CHL OXA TET MET AZI CLI FOS CET ERY TET AMP AZI FOS NEO PIT MET AMO PEN TIC VAN AMP LCM RIF MET CMX PEN AMO TOB LCM TIC PMB ERY NIT NEO CUR CTZ NAT CTR PMB CTZ CLI NAT COL CLE COL AZT CET AZT AMI AZI 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R Escherichia spp. Klebsiella spp. Enterobacter spp. MER CTR MER FOS MER AZT CET IMI IMI AZT PMB PMB IMI AMI PIT FLO FOS NEO PIT PIT FLO CLE NAT CMX AMI FLO GEN CUR CHL CHL CTA DOX TOB PMB NEO AMI AMC CEP NAT GEN AMC ENR CMX CTZ DOX ENR TOB TET TOB CHL CEP CIP CEP TIC GEN DOX CIP TRS NEO TRS TET ENR FOS CTZ TRS NIT CUR TIC OXA CTA TET CLE OXA OXA MET CLE CMX AMC AZT CIP CET AMP NAT NIT AMO AMO MET CUR AMP VAN VAN NIT TIC RIF MET RIF PEN VAN PEN LCM RIF LCM ERY PEN ERY CTZ LCM CLI CTA ERY AZI CLI CLI AMP CET AZI AMO AZI 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R Figure A3. Cont. Animals 2022, 12, x FOR PEER REVIEW 1 Animals 2022, 12, x FOR PEER REVIEW 18 Animals 2022, 12, 1912 18 of 24 Pasteurellaspp. Pasteurella spp. Bordetellaspp. Bordetella spp. NEO PMB NEONAT PMBNAT NATLCM NATCTR LCM CTR COL CTZ CTZ COLCEP CTR CTR CEP AZT CEP CEP AZT MER AZT AZT MER IMI DOX IMI FLO DOX TRS FLO PIT TRS MER PIT CHL MERTOB CHL TET TOB AMC TET GEN AMC IMI GEN CIP IMI AMI CIP AMI AMI PIT AMITRS PIT CLE TRS CMX CLE ENR CMXTOB ENR GEN TOB AMC GENPEN AMC TIC PEN CUR TIC DOX CURCTA DOXCTZ CTA CHL CTZ ENR CHL ENR NEO AZI AZI NEOOXA AMO AMO OXAAMP ERY ERY AMPCTA TET TET CTA AMO AMP AMO FOS AMP CIP FOSNIT CIP FLO NIT MET FLO PMB METVAN PMB TIC VAN RIF TIC OXA RIF PEN OXA CLI PEN LCM CLI FOS LCM ERY FOS CMX ERY CUR CMX CET CUR CLI CET MET CLI CLE MET VAN CLECET VAN NIT CETAZI NIT AZI 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R %S %I %R Figure A3. Proportion of susceptible (S), susceptible—increased exposure (I), and resistance (R Figure A3. Figure A3. Proportion ofProportion susceptibleof(S), susceptible (S), susceptible—increased susceptible—increased exposure exposure (I), (I), and resistance and resistance (R) (R terial strains in mammals. bacterial strainsterial strains in mammals. in mammals. Stenotrophomonas Stenotrophomonas Pseudomonasspp. Pseudomonas spp. Aeromonasspp. Aeromonas spp. IMI spp. spp. PMB IMI MER PMB PMB PMB NAL MERPMB FLO NAL FLO FLO PMBTOB CMX FLO CMX CTR TOB GEN DOX CTR CLI GENCTZ DOX ENR CLI CEP CTZ AZT ENR CHL CEP AZT AZT PIT CHL TRS AZT PIT OXA TRS ENR CIP ENR OXA CEP CIP TRS CTZ TRS CEPFLO CTZ CTZ GEN CTZ FLO ENR CIP GENNIT ENR TIC CIP DOX NIT VAN TIC MET DOX CMX VAN TOB MET CIP CMX TOB CHL CIP AMI TIC CHL TIC TOB AMI NAL TET TOB TET MER NALCOL RIF MER RIF AZI COL DOX PIT AZI AMI DOXCMX PIT PEN AMI TET CMX TRS PEN OXA TET PEN TRS AMC OXA NEO PEN AMCCTA NEO OXA MET OXA CTANIT MET CUR MER CUR NIT FOS MER CLE LCM CLE FOSTET IMI LCMIMI TET CHL IMI PIT IMI FOS CHL AMO PIT CTA FOS ERY AMOAMP CTA ERY GEN AMPNEO CUR GEN CUR VAN NEOVAN CTR VAN CTR NEO VAN RIF CTA NEO CTA FOS RIF PEN CLI FOS ERY PEN LCM CLI CLE ERY NIT LCM ERY CLE CET NIT MET ERY CUR CET AZT MET LCM CURCTR AZT AZI LCM CTR CLI AZI CET AMP CET CLI CLE AMP AMP AMO AMP CLECET AMO AMO AMI AMO CETAZI AMC AMI AMC AZI AMC AMC 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R %S %I %R %S %I %R %S %I %R Figure A4. Cont. Animals 2022, 12, x FOR PEER REVIEW 1 Animals 2022, 12, 1912 19 of 24 Enterococcus spp. Streptococcus spp. Citrobacter spp. RIF VAN TRS FLO TRS TOB IMI PIT PMB VAN OXA PIT MER MER IMI MER FOS CHL FOS IMI DOX CET FOS AMC CEP FLO PIT TET CMX AMP AMC CHL AMO DOX RIF ENR PEN TRS FLO GEN CHL AMI TET GEN NIT CIP AMO ENR TET ENR CTZ DOX PEN CIP MET NEO GEN CLI CTZ MET CUR CTA CUR CIP ERY CUR AMP NEO CLE ERY CLI AMC CMX CTA VAN CLE OXA CTA TIC TOB TOB RIF CLE TIC AMI PEN PMB PMB NIT NIT NAL NEO LCM LCM NAL ERY CTR LCM CLI COL CTZ CET CET CTR AZI CEP COL AZT AZT AMP AZI AMI AMO 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R Klebsiella spp. Escherichia spp. Morganella spp. PMB TRS NEO OXA TIC GEN NEO PMB CMX MER AZT PIT IMI TOB MET FOS PIT FLO MER PIT ENR CHL GEN CMX TRS CIP CEP CIP ENR ENR CTZ TOB IMI DOX TRS FOS GEN AMI AMI TET TET VAN IMI AMC TET CIP RIF DOX AMC PMB CTZ TOB PEN FLO MER NIT CUR AMI MET CTA CHL LCM CHL FLO CLE CMX ERY AMP CLE DOX NIT AMO CUR VAN FOS CTZ TIC CTA CTR RIF CUR CTA PEN NIT COL VAN CLI LCM CLE ERY RIF CET CLI PEN CEP CET LCM AZI AZI ERY AMP AMP CLI AMO AMO AZI AMC 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% 0% 25% 50% 75% 100% %S %I %R %S %I %R %S %I %R Figure A4. Cont. Animals 2022, 12, 1912 Animals 2022, 12, x FOR PEER REVIEW 20 of 24 2 Antibiotics abbreviations: AMC, amoxicillin-clavulanic acid; Acinetobacter spp. AMI, amikacin; AMO, amoxicillin; AMP, ampicillin; AZI, azithromycin; AZT, aztreonam; CEP, cefepime; CET, cepha- TRS PMB lothin; CHL, chloramphenicol; CIP, ciprofloxacin; CLE, IMI cephalexin; CLI, clindamycin; CMX, cotrimoxazole; COL, col- PIT MER istin; CTA, cefotaxime; CTR, ceftriaxone; CTZ, ceftazidime; CIP CTZ CUR, cefuroxime; DOX, doxycycline; ENR, enrofloxacin; ERY, ENR CHL erythromycin; FLO, florfenicol; FOS, fosfomycin; GEN, gen- TOB CMX tamicin; IMI, imipenem; LCM, lincomycin; MER, meropenem; GEN AMI MET, metronidazole; NAL, nalidixic acid; NEO, neomycin; CTA NIT, nitrofurantoin; OXA, oxacillin; PEN, penicillin; PIT, pip- AMP AMC eracillin/tazobactam; PMB, polymyxin B; RIF, rifampicin; VAN TET TET, tetracycline; TIC, ticarcillin; TOB, tobramycin; TRS, tri- RIF PEN methoprim/sulfamethoxazole; VAN, vancomycin. NIT LCM FOS ERY DOX CUR CTR CLI CLE CET CEP AZT AZI AMO 0% 25% 50% 75% 100% %S %I %R Figure A4. Proportion Figure A4.of susceptible Proportion of(S), susceptible—increased susceptible exposure (I),exposure (S), susceptible—increased and resistance (I), and(R) resistance ( bacterial strains terial in reptiles. strains in reptiles. Table A1.resistance Table A1. Antimicrobial Antimicrobial of theresistance principal of the principal bacteria isolatedbacteria from theisolated from the three animal three animal classes and their AWaRe classification: Access (green), Watch (yellow), and their AWaRe classification: Access (green), Watch (yellow), Reserve (red). Reserve (red). a. Birds a. Birds E. cloacae E. faecalis E. coli K. pneumoniae P. aeruginosa S. au E. cloacae E. faecalis E. coli K. pneumoniae P. aeruginosa S. aureus CFR CFR CLE CLE CFR AM CFR CFR CLE CLE CLE CLE AMI CFR AMO AMI CLE GE CLE CLE CET AMI CET AMO AMP-S CLE AMP GEN CET AM CET CET CFZ AMP-S CFZ AMO AMP CLI CET CFZ AMO AM CFZ CFZ AMI AMO AMI AMP CLI AMC CFZ AMI AMP CL AMI AMI AMP-S AMP GEN CHL AMC DOX AMI GEN CLI DO AMO AMO CLI TET AMP-S TE AMP-S GEN CHL DOX GEN DOX AMP AMP AMC CUR AMO CT AMO AMO CLI TET AMP-S TET CLI CHL DOX CTA AMP NE AMP AMP AMC CUR AMO CTZ AMC CLI TET CTZ CHL TO CLI CHL OXA DOX AMC CTA FOX AMP TOB NEO CLI FA AMC CLI DOX TET OXA CTZ CUR CHL FA TOB AMC AZ OXA AMC TRS FOX DOX CTA TOB LMCCLI DOX FA CL DOX OXA TET CUR TET CTZ FA AZI AMC TET AZI ER TRS DOX FOX CTA FOX TOB LMC CLR DOX FOX CLR PI CUR CUR FA ERY CUR TI TET TET CTZ AZI TET ERY CTZ CDN LMC JO CFM CO FOX FOX TOB CLR FOX PIT TOB CFM AZI PIT CEF PM CUR CUR FA ERY CUR TIC FA CTA CLR TIC CTA MI LMC CPD ERY CPD Animals 2022, 12, 1912 21 of 24 Table A1. Cont. a. Birds E. cloacae E. faecalis E. coli K. pneumoniae P. aeruginosa S. aureus CTZ CDN LMC JO CFM COL TOB CFM AZI PIT CEF PMB FA CTA CLR TIC CTA MIN LMC CPD ERY CPD AZI CTZ JO CTR CLR CTR PIT KAN ERY KAN MIN NEO JO NEO TOB SPT ETP IMI IMI FA MER AZI FA CLR LMC ERY AZI JO CLR PIT ERY COL JO PIT PMB MIN TGC b. Mammals B. bronchis E. cloacae E. coli K. pneumoniae P. multocida P. aeruginosa S. aureus CFR CFR CET CET AMI CFR CFR CLE CLE GEN AMI GEN CLE CLE CET CFZ AMP-S GEN AMO CET CET CFZ AMI AMO AMP-S AMP CFZ CFZ AMI GEN AMP AMO CHL AMI AMI GEN AMP-S CHL AMP CLI GEN GEN AMP-S AMO CLI CHL NIT AMP-S AMP-S AMO AMP AMC CLI CDN AMO AMO AMP CLI OXA AMC LMC AMP AMP CLI AMC MET OXA ERY CHL CLI AMC OXA NIT MET JO CLI AMC OXA MET DOX DOX PMB AMC OXA MET NIT TRS TRS OXA MET DOX DOX TET TET MET NIT TRS TRS CUR FOX DOX DOX FOX TET CDN CUR TRS TRS CUR FOX CFM CDN TET TET Animals 2022, 12, 1912 22 of 24 Table A1. Cont. b. Mammals B. bronchis E. cloacae E. coli K. pneumoniae P. multocida P. aeruginosa S. aureus CPD CUR CEF CFM FOX FOX CTR CDN CTR CEF CUR CUR KAN CFM KAN CPD CDN CDN CIP CEF TOB CTR CTA CEF MER CTA MER KAN CEP CTA AZI CPD LMC SPT CPD CEP CLR CTR AZI CIP CTR CPD ERY KAN CLR MER KAN CTR LVX SPT ERY LMC NEO KAN JO TOB LVX MXF TOB NEO PIT CIP JO AZI ETP SPT TIC MER PIT CLR CIP MER MIN LMC TIC ERY MER FA AZI MIN LVX FA LMC CLR JO AZI MXF ERY PIT CLR ERY LVX TIC ERY LVX JO NOR LVX JO PIT PMB JO TIC TIC MIN PIT NOR MIN TIC COL PMB PMB MIN MIN TGC c. Reptiles A. hydrophyla C. freundii E. cloacae E. coli M. morganii P. aeruginosa S. maltophilia CLE CFR CFR GEN CFR CFR CFR GEN CLE CLE AMC CLE CLE CLE AMP-S CFZ CFZ OXA CFZ CFZ CFZ AMO AMI AMI CEF AMI AMI AMI AMP AMO GEN CEP GEN GEN GEN CHL AMP AMP-S NEO AMO AMP-S AMP-S DOX CHL AMO LMC AMP AMO AMO FOX AMC AMP MXF CHL AMP AMP CUR OXA CHL AZI AMC CHL CHL CEP FOX AMC CLR OXA CLI AMC MER CUR OXA ERY DOX AMC OXA AZI CEF NIT JO TRS OXA TRS LMC DOX CUR DOX TET MXF FOX CEF TRS FOX Animals 2022, 12, 1912 23 of 24 Table A1. Cont. c. Reptiles A. hydrophyla C. freundii E. cloacae E. coli M. morganii P. aeruginosa S. maltophilia AZI CUR KAN TET CUR CLR CFM NEO FOX CDN ERY LMC CIP CDN CFM JO MXF MER CFM CEF MIN AZI LMC CEF CEP CLR MXF CEP CTR ERY AZI CTR SPT JO CLR SPT TOB TIC ERY TOB ETP PMB JO CIP CIP TGC COL IMI IMI PMB LMC MER MIN MXF FA TGC AZI LMC CLR MXF ERY AZI JO CLR MIN ERY TGC JO PIT TIC AMI, Amikacin; AMO, Amoxicillin; MC, Amoxicillin/clavulanic-acid; AMP, Ampicillin; AMP-S, Ampi- cillin/sulbact.; CFR, Cefadroxil; CFZ, Cefazolin; CLE, Cephalexin; CET, Cephalotin; CHL, Chloramphenicol; CLI, Clindamycin; DOX, Doxycycline; GEN, Gentamicin; MET, Metronidazole; NIT, Nitrofurantoin; OXA, Oxacillin; TET, Tetracycline; TRS, Trimethoprim/Sulfamoxole; AZI, Azithromycin; CDN, Cefditoren-pivoxil; CEP, Cefepime; CFM, Cefixime; CEF, Cefodizime; CTA, Cefotaxime; FOX, Cefoxitin; CPD, Cefpodoxime; CTZ, Ceftazidime; CTR, Ceftriaxone; CUR, Cefuroxime; CIP, Ciprofloxacin; CLR, Clarithromycin; ETP, Ertapenem; ERY, Erythromycin; FA, Fusidic-acid; IMI, Imipenem; JO, Josamycin; KAN, Kanamycin; LVX, Levofloxacin; LMC, Lincomycin; MER, Meropenem; MXF, Moxifloxacin; NEO, Neomycin; NOR, Norfloxacin; PIT, Piperacillin/tazobactam; SPT, Streptomycin; TIC, Ticarcillin; TOB, Tobramycin; COL, Colistin; MIN, Minocycline; PMB, Polymyxin-B; TGC, Tigecycline. 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