ORIGINAL RESEARCH published: 20 January 2021 doi: 10.3389/fmicb.2020.621597 Antimicrobial Resistance in Bacteria Isolated From Cats and Dogs From the Iberian Peninsula Yanli Li 1 , Rubén Fernández 1 , Inma Durán 2 , Rafael A. Molina-López 3 and Laila Darwich 1* 1 Departament de Sanitat i Anatomia Animal, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Departamento Veterinaria de Laboratorios Echevarne, Barcelona, Spain, 3 Catalan Wildlife Service, Centre de Fauna Salvatge de Torreferrussa, Barcelona, Spain Pet animals are assumed to be potential reservoirs in transferring antimicrobial resistance (AMR) to humans due to the extensively applied broad-spectrum antimicrobial agents and their close contact with humans. In this study, microbiological data and antimicrobial susceptibility results of dog (n = 5,086) and cat (n = 789) clinical samples from a private Laboratory of Diagnosis in Barcelona were analyzed. Samples Edited by: came from different counties of the Iberian Peninsula during 2016–2018. In dogs, Marina Spinu, clinical samples were most commonly from otitis, and in cats from wounds, respiratory University of Agricultural Sciences and Veterinary Medicine tract infections and conjunctivitis. In both pet groups, Staphylococcus spp. (31% in of Cluj-Napoca, Romania dogs vs 30% in cats), Streptococcus spp. (19% vs 17%), Pseudomonas spp. (16% Reviewed by: vs 10%), Escherichia coli (8% vs 5.6%), and Enterococcus spp. (5.5% vs 6.8%) were Rosa Del Campo, Ramón y Cajal Institute for Health shown as the most predominant bacteria. However, higher frequencies of P. aeruginosa, Research, Spain P. canis, and S. pseudintermedius were found in dogs, while S. aureus and P. multocida Mehmet Demirci, were more prevalent in cats. The antimicrobial susceptibility testing demonstrated that Beykent University, Turkey Enterococcus spp. and Pseudomonas spp. presented the highest levels of AMR in *Correspondence: Laila Darwich both dogs and cats. Within the Enterobacteriaceae, E. coli showed low levels of laila.darwich@uab.cat AMR compared to Klebsiella, Proteus, or Enterobacter spp. Respiratory tract infections caused by K. pneumoniae presented higher AMR in cats. By contrast, Pasteurella Specialty section: This article was submitted to isolates from the respiratory tract were highly sensitive to all the antimicrobials in cats Antimicrobials, Resistance and dogs. Data from this study could be used to guide empirical antimicrobial selection and Chemotherapy, a section of the journal in companion animal veterinary practices in the Iberian Peninsula. Frontiers in Microbiology Keywords: antimicrobial resistance, bacteria, cats, dogs, Iberian Peninsula Received: 26 October 2020 Accepted: 30 December 2020 Published: 20 January 2021 INTRODUCTION Citation: Li Y, Fernández R, Durán I, The emergence of antimicrobial resistance (AMR) has become a great concern worldwide, Molina-López RA and Darwich L threatening the public healthcare system (Brinkac et al., 2017). Some studies assumed that food (2021) Antimicrobial Resistance in Bacteria Isolated From Cats animals were the main contributors of human AMR by transferring resistant bacteria or genes and Dogs From the Iberian Peninsula. through food chain (Witte, 1998; Fey et al., 2002; Smith et al., 2002; White et al., 2002; Angulo Front. Microbiol. 11:621597. et al., 2009; McEwen and Fedorka-Cray, 2017). However, (Barber et al., 2016) established a doi: 10.3389/fmicb.2020.621597 new analytical model and assumed the non-foodborne transmission of AMR should be equally Frontiers in Microbiology | www.frontiersin.org 1 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs emphasized. Thus, the companion animals, mostly dogs and piperacillin/tazobactam, ampicillin, cephalexin, cephalotin, cats, started to be considered a potential reservoirs of AMR cefazolin, cefuroxime, ceftazidime, cefotaxime, cefovecin, due to their close contact with humans and being extensively cefotaxim, and cefepime), carbapenems (imipenem and treated by broad-spectrum antimicrobial agents (Guardabassi meropenem), and aztreonam; fluoroquinolones (ciprofloxacin, et al., 2004; Lloyd, 2007). If AMR can be transmitted to humans enrofloxacin, and marbofloxacin); aminoglycosides (amikacin, from companion animals, and if multi-drug resistant (MDR) gentamicin, tobramycin, and neomycin); macrolides bacteria exist among the household pets, the risk of antimicrobial (azithromycin and erythromycin); tetracyclines (doxycycline); treatment failure would highly increase in both animals and clindamycin; polymyxin B; trimethoprim/sulphamethoxazole; humans. Thus, understanding the prevalence of AMR among chloramphenicol/florphenicol; fosfomycin; mupiracin; and pets, mainly dogs and cats, is demanded from both veterinary and glycopetides (vancomycin). For Gram negative bacteria, NM44 human medicine perspectives. However, due to the clinical cases MicroScan (Beckman Coulter, Villepinte, France) system are not always entirely recorded and monitored, the available data was performed for all the antimicrobials except for those on pet-related AMR are very minimal. antibiotics authorized for veterinary uses that are not included In this study, we analyzed the clinical microbiological data on in the automatic scan panels (enrofloxacin, pradofloxacin, pet dogs and cats with data collected between 2016 and 2018 in marbofloxacin, doxycycline, cephalexin, and cefovecin). the Iberian Peninsula, and found out the most prevalent bacterial The MicroScan is an automated bacterial identification and infections and AMR profiles among the two companion animals. susceptibility testing system based on microbiology principles of true minimum inhibitory concentration (MIC) testing. Based on the lab readings, isolates were classified as Susceptible, MATERIALS AND METHODS Intermediate or Resistant. For statistical assessments, isolates that exhibited intermediate resistance were re-classified as Data Source and Management resistant. The laboratory has the quality management system Retrospective records of 5,875 microbiological analyses of clinical certificate ISO-9001 since 1998 and the accreditation from specimens from dogs (n = 5,086) and cats (n = 789) between ENAC (National Accreditation Entity) according to criteria 2016 and 2018 were analyzed in the present study. The records included in the ISO/IEC 17025 Standard defined in the Technical were provided by the Veterinary Medicine Department of a Annexes 511/LE1947 for Pharmaceutical Toxicology and large private Laboratory of Diagnosis in Barcelona. The lab Microbiology Testing. records contained information about clinical cases submitted by veterinary clinics covered throughout the Spanish provinces, Portugal, and Andorra (Figure 1). Data were assessed for Statistical Analysis Descriptive and statistical analysis was performed using the duplicates and missing information. Finally, only samples SPSS Advanced Models TM 15.0 (SPSS Inc. 233 South with complete records were analyzed. Repeat samples of the Wacker Drive, 11th Floor Chicago, IL, United States 60.606- same case were not included. The following variables were 6412). The Chi-square (χ2 ) or Fishers Exact tests were extracted from the records: animal species, type/origin of sample, used to compare bacterial spp. and the AMR frequencies county of specimen, bacterial identification, and antimicrobial in both animal groups. Statistical significant was considered susceptibility testing. when p < 0.05. The specimens were classified according to the sample origin as follows: otitis (n = 3,043), wounds (n = 1,142), respiratory tract infections (which included rhinitis, bronchitis, pneumonia, and pleuritic, n = 483), dermatitis (n = 341), RESULTS abscesses (n = 218), conjunctivitis (n = 190), and others (which included reproductive tract infections, musculoskeletal Microbiological Diagnosis of Bacterial infections, arthritis, and osteomyelitis, n = 458). Urine samples Infections were not included in the study. In dogs, most of the samples remitted to the lab were from cases related to otitis (55.3% dogs vs 29% cats, χ2 = 187.2, and Microbiological Analysis and p < 0.05). In cats, samples from wounds (23% cats vs 19% dogs, Antimicrobial Susceptibility Testing χ2 = 6.6, and p = 0.01), respiratory tract infections (24% vs 5.8%, Microbiological identification was performed using the χ2 = 299, and p < 0.05), and conjunctivitis (6% vs 2.8% χ2 = 21.6, MALDITOF mass spectrometeror the API ID system R and p < 0.001) were more frequently remitted (Figure 2). (bioMérieux, Spain). All Gram-positive bacterial isolates Staphylococcus spp. (31–30%), Streptococcus spp. (19–17%) were performed by the antimicrobial susceptibility test and Pseudomonas spp. (16–10%), followed by Escherichia coli using the standard disk diffusion method according to (8.0–5.6%), and Enterococcus spp. (5.5–6.8%), were the most Performance Standards for Antimicrobial Susceptibility predominant bacteria isolated in both dogs and cats (Table 1). Testing for bacteria isolated from animals (M31-A3, CLSI As a differential trait, dogs presented higher frequencies of VET01, 2008) and from humans (M100-S24, CLSI, 2016) for Pseudomonas aeruginosa (92% vs 72%), P. canis (36.7% vs 6.5%), drugs not licensed for veterinary use. The panel included the and S. pseudintermedius (17% vs 4.6%), while S. aureus (6% vs following antimicrobial classes: beta-lactams (amoxicillin- 1.5%) and P. multocida (63% vs 20.4%) were more prevalent in clavulanic acid, oxacillin, cefoxitin, penicillin, piperacillin, cats (Table 1). Frontiers in Microbiology | www.frontiersin.org 2 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs FIGURE 1 | Map of the origin and the number of clinical specimens from dogs and cats in the Iberian Peninsula between 2016 and 2018. The distribution of pathogens for different sample categories (n = 969, n = 132), Enterococcus spp. (n = 281, n = 54), showed that wounds and dermatitis presented similar patterns of Escherichia spp. (n = 405, n = 44), Enterobacter spp. (n = 193, distribution in dogs and cats, with Staphylococcus, Streptococcus, n = 22), Klebsiella spp. (n = 103, n = 23), Pseudomonas spp. Enterococcus, and E. coli identified as the most frequently (n = 825, n = 76), Pasteurella spp. (n = 49, n = 62), and isolated agents (Figure 3). From otitis specimens, infections Corynebacterium spp. (n = 194, n = 22). In addition, for the by Staphylococcus spp. were highly detected in both cats and most relevant gram-negative bacteria species, minimal inhibitory dogs; meanwhile in cats, high frequencies of P. aeruginosa and concentration (CMI) values required to inhibit the growth of 50% E. coli were presented. On the other hand, dogs presented in (MIC50) and 90% (MIC90) of organisms were assessed for some general a larger bacterial diversity in samples from abscess, antimicrobials (Table 2). Interestingly, the Enterobacteriaceae conjunctivitis and respiratory tract infections in comparison to species presented high values of CMI90 for beta-lactams, cats. In this line, cats showed higher percentages of Bordetella ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole spp. and P. multocida infections in conjunctivitis and respiratory in both animal groups. Pseudomonas spp. showed the highest specimens, respectively, (Figure 3). CMI50 values for amoxicillin-clavulanate and cefoxitin (jointly with Enterobacter spp.), for ampicillin (jointly with Klebsiella Antimicrobial Susceptibility Testing spp.), and for cefotaxime and cefuroxime. Finally, Proteus spp. Comparisons of AMR levels between dogs and cats were only isolated from dogs presented a CMI90 value = 8 mg/L to made for bacterial species, which were recorded for more than imipenem, exceeding the resistant breakpoint (Table 2). 20 different strains in the antibiotic sensitivity test. Thus, the Among the Gram-positive bacteria, more than 80% of following species were involved: Staphylococcus spp. (n = 1,572 Enterococcus isolates presented resistance to oxacillin, cefoxitin, isolates from dogs, n = 239 from cats), Streptococcus spp. amikacin, clindamycin, polymyxin B, and fosfomycin from Frontiers in Microbiology | www.frontiersin.org 3 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs FIGURE 2 | Proportion of analyzed samples between cats and dogs according to the type or source of specimens. both dogs and cats (Figure 4). Similar patterns but with and cefuroxime (57.4%, 33.3%). Meanwhile, resistance to lower frequencies were detected for Staphylococcus, Streptococcus, piperacillin, piperacilina/tazobactam, cefotaxime, ciprofloxacin, and Corynebacterium spp., principally in isolates from dog enrofloxacin, marbofloxacin, amikacin, tobramycin, and specimens. Besides, Staphylococcus spp. isolated from dogs trimethoprim/sulfamethoxazole was also found in both pet presented higher levels of AMR to macrolides, tetracycline, groups but in a low proportion of isolates (<20%; Figure 6). trimethoprim/sulfamethoxazole and chloramphenicol compared As regard Pasteurella isolates, they were detected principally to cat isolates. Of note, a significant higher frequency of from respiratory tract, most of the isolates were highly sensitive imipenem and marbofloxacin Corynebacterium resistant isolates to all the antimicrobials in cats and dogs, showing low were found in dog cases (Figure 4). resistance frequencies only to cefuroxime (8.2%) and ampicillin Within the Enterobacteriaceae family, although E. coli was (6.1%) in dogs, and cephalexin (6.5%) and cefovecin (4.8%) in highly isolated from wounds, dermatitis, abscesses, and otitis cats (Figure 6). in both dogs and cats, they presented low levels of AMR (with the exception of ampicillin where 50% of isolates were resistant), in comparison to other members of the family such DISCUSSION as Klebsiella, Proteus, or Enterobacter spp. (Figure 5). More in detail, Enterobacter strains from dog specimens showed a This study provides data of the most frequently isolated bacteria higher level of AMR to β-lactams, imipenem, and mupirocin from cat and dog infections and their associated AMR profiles than cats. K. pneumoniae from cat respiratory tract infections based on a large number of clinical cases (N = 5875) within presented an overall higher resistance to antimicrobials than the Iberian Peninsula. This information can be a guide to from dogs, showing statistical differences for piperacillin and clinicians, especially those working in this region, to make trimethoprim/sulfamethoxazole (Figure 5). rational decisions on the use of antimicrobials, principally when Finally, Pseudomonas spp. presented the highest levels of AMR empirical antimicrobial treatment is recurrent in companion in both dogs and cats, showing between 80 and 97% of resistance animal veterinary medicine. to penicillin and cephalosporin classes, including 3rd GC, 79– Most of the specimens submitted to the lab were from 94% trimethoprim/sulfamethoxazole, 68–85% flophenicol, 55– ears in both cats and dogs, and in cats, a large number of 62% chloramphenicol, and 69–78% fosfomycin. In general, samples were also from respiratory tract infections and wounds. isolates from dogs presented higher levels of resistance than the The distribution of pathogens showed that Staphylococcus, cat isolates (Figure 5). Streptococcus, Pseudomonas, E. coli, and Enterococcus were the Antimicrobial susceptibility in Proteus spp. (n = 205, most frequently isolated agents for different sample categories. n = 5), Serratia spp. (n = 104, n = 14), Acinetobacter spp. In both cats and dogs, Staphylococcus spp. was commonly (n = 61, n = 18), and Bordetella spp. (n = 47, n = 15) was isolated from several sample sources including ears, skin, eyes, mainly done from dog isolates. (Figure 6) Interestingly, more abscesses and wounds. This finding agrees with other studies than 80% of Proteus isolates were resistant to doxycycline conducted in Canada, Sweden, and South Africa (Windahl and polymyxin B. Acinetobacter isolates presented a high et al., 2015; Qekwana et al., 2017; Awosile et al., 2018) which resistance rate to cephalexin (66.1% of dog, 44.4% of cat, and confirms Staphylococcus spp. as an opportunistic pathogen of the p < 0.05), cefovecin (65.0%, 38.9%, and p < 0.05), ampicillin integument and mucosae, causing otitis externa, pyoderma, and (63.8%, 44.4%), amoxicillin (59.0%, 22.2%, and p < 0.05), post-surgical complications in dogs. Frontiers in Microbiology | www.frontiersin.org 4 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs Thirty-one and thirty percent of the studied samples were TABLE 1 | Continued tested positive for Staphylococcus spp., respectively, in dogs and DOGS CATS cats. In dogs, the identified species of Staphylococcus included (N = 5,086) (N = 789) S. pseudintermedius (17.4%), S. intermedius (7%), S. schleiferi n (%) n (%) Proteus 205 (4) 5 (0.6) spp. TABLE 1 | Frequencies of bacterial species identified in dog and cat specimens. P. mirabilis 198 (96.6) P. mirabilis 5 (100) DOGS CATS P. vulgaris 3 (1.5) (N = 5,086) (N = 789) Pseudo 827 (16.3) 76 (9.6) monas spp. n (%) n (%) P. aeruginosa 761 (92) P. aeruginosa 55 (72.4) Acineto 61 (1) 18 (2) P. fluorescens 18 (2.2) P. fluorescens 5 (6.6) bacter spp. Others 34 (4.1) Others 16 (21.1) A. baumannii 22 (36.1) A. lwoffii 8 (44.4) Serratia 104 (2) 14 (1.7) A. lwoffii 14 (23) A. baumannii 2 (11.1) spp. A. haemolyticus 4 (6.6) A. haemolyticus 1 (5.5) S. marcescens 96 (92.3) S. marcescens 12 (85.7) Others 2 (3.3) S. liquefaciens 7 (6.7) S. liquefaciens 2 (14.3) Bordetella 47 (0.9) 15 (1.9) Staphylo 1,581 (31) 239 (30.3) spp. coccus B. bronchiseptica 47 (100) B. bronchiseptica 15 (100) spp. Candida 30 (0.5) 7 (0.9) S. pseudintermedius 275 (17.4) S. aureus 14 (5.9) spp. S. intermedius 109 (6.9) S. epidermidis 12 (5) C. parapsilosis 5 (16.7) C. parapsilosis 3 (42.9) S. schleiferi 30 (1.9) S. felis 12 (5) C. albicans 2 (6.5) C. albicans 2 (28.6) S. aureus 23 (1.5) S. pseudintermedius 11 (4.6) Others 4 (13.3) S. epidermidis 9 (0.6) S. schleiferi 2 (0.8) Coryne 194 (3.8) 22 (2.8) Others 25 (1.6) Others 16 (6.7) bacterium Strepto 972 (19) 132 (16.7) spp. coccus C. amycolatum 7 (3.6) C. amycolatum 2 (9.1) spp. C. auriscanis 5 (2.6) S. canis 23 (2.4) S. canis 2 (1.5) Others 2 (1) S. dysgalacticae 3 (0.3) Entero 84 (1.6) 26 (3.3) S. halichoeri 1 (0.1) bacter spp. E. cloacae 59 (70.2) E. cloacae 22 (84.6) E. aerogenes 13 (15.5) E. aerogenes 3 (11.5) (2%), S. aureus (1.5%), and S. epidermidis (0.6%), which E. gergoviae 8 (9.5) E. gergoviae 1 (3.8) presented a similar prevalence patterns as other studies reported Entero 281 (5.5) 54 (6.8) in South Africa (Qekwana et al., 2017). The lower prevalence of coccus spp. S. aureus compared with S. pseudintermedius was in accordance E. faecalis 92 (32.7) E. faecalis 18 (69.2) with previously published works (Hanselman et al., 2009; E. faecium 8 (2.8) E. avium 2 (7.7) Kawakami et al., 2010; Chanchaithong et al., 2014; Dos Santos E. canintestini 1 (0.4) E. faecium 1 (3.8) et al., 2016). In cats, S. aureus was the most common isolated E. durans 1 (0.4) E. hirae 1 (3.8) specie. The high rate of colonization with S. pseudintermedius Escherichia 405 (8) 44 (5.6) and S. aureus found in dog and cat specimens could represent spp. a public health concern, as has been described in many papers E. coli 400 (98.8) E. coli 42 (95.5) the potential transmission of Staphylococcus spp. from dogs to E. vulneris 4 (1) humans when exposing to carrier or infected dogs (Boost et al., Klebsiella 103 (2) 23 (2.9) 2007; Faires et al., 2009; Frank et al., 2009; Pantosti, 2012; Dos spp. Santos et al., 2016). K. pneumoniae 73 (70.9) K. pneumoniae 17 (73.9) The most common ear pathogens isolated from dogs K. oxytoca 28 (27.2) K. oxytoca 6 (26.1) are coagulase-positive staphylococci (Staphylococcus K. ornithinolytica 1 (1) pseudintermedius) and P. aeruginosa (Cole et al., 1998). By Pasteurella 49 (1) 62 (7.8) contrast, a recent study conducted in France showed that the spp. major causative agents of dog otitis were coagulase-positive P. canis 18 (36.7) P. multocida 39 (62.9) staphylococci, P. aeruginosa, P. mirabilis, and streptococci P. multocida 10 (20.4) P. canis 4 (6.5) (Bourély et al., 2019). In that study, the authors found that P. pneumotropica 3 (6.1) Others 4 (6.5) since 2003 resistance to fluoroquinolones has been decreased in (Continued) both P. aeruginosa and S. pseudintermedius isolates, resulting Frontiers in Microbiology | www.frontiersin.org 5 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs FIGURE 3 | Frequencies of bacterial species according to the origin of infections in dogs and cats. Frontiers in Microbiology | www.frontiersin.org 6 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs for P. aeruginosa, 19.4% of isolates were resistant to both conducted in Canada (Awosile et al., 2018). Enrofloxacin is enrofloxacin and gentamicin (Bourély et al., 2019). In the present commonly used systemically with concurrent topical treatment study, S. pseudintermedius, P. aeruginosa, and E. coli. were in cases of canine otitis caused by P. aeruginosa (Hariharan also frequently isolated from dog otitis specimens, and similar et al., 2006). These results suggest that aminoglycosides percentages of fluoroquinolones and gentamicin resistance were and fluoroquinolones have potential to be used as anti- observed for S. pseudintermedius and P. aeruginosa isolates pseudomonal drugs (Dowling, 1996). Our findings are also (<20%). Meanwhile, the P. aeruginosa isolates showed high consistent with similar retrospective studies from Denmark, levels of resistance to penicillin and cephalosporin classes United States, and Canada (Petersen et al., 2002; Authier et al., (including 3rd GC), trimethoprim/sulfamethoxazole, phenicoles, 2006; Pedersen et al., 2007). and fosfomycin, both in dogs and cats. Pseudomonas spp. Enterococci are MDR from both intrinsic and acquired were intrinsically resistant to beta-lactams, combinations with features. Specifically, Enterococcus spp. are naturally resistant β-lactamase inhibitors, chloramphenicol, erythromycin, and to clindamycin, as well as to penicillin G and cephalothin, trimethoprim/sulfamethoxazole. In this study, high proportions giving them a characteristic of AMR profile (Prescott et al., of Pseudomonas were susceptible to the aminoglycosides 2002; Delgado et al., 2007). Enterococci isolates of this study (>95%). As well, the frequency of enrofloxacin resistance was were principally isolated from wounds and dermatitis of low (27% in dogs and 20% in cats) compared to other studies companion animal specimens. More than 80% of Enterococcus TABLE 2 | Minimal Inhibitory Concentrations (MIC, mg/L) values in Gram-negative bacteria isolated from dogs and cats. DOG SPECIMENS Acinetobacter spp. Pseudomonas spp. Escherichia spp. Klebsiella spp. Enterobacter spp. Proteus spp. MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 AMC 4 >32 >32 >32 4 >32 4 >32 >32 >32 <2 16 AMK <2 16 >2 16 <2 <8 <2 16 <2 <8 <2 4 AMP 16 >32 >32 >32 16 >32 >32 >32 16 >32 <2 >32 CAZ 4 16 4 8 <1 16 <1 16 <1 >64 <1 <1 CIP <0.25 >4 <0.25 >4 <0.25 >4 <0.25 >4 <0.25 >4 <0.25 >4 CTX 8 32 16 >64 <1 8 <1 >64 <1 >64 <1 4 CXM 32 >64 >64 >64 4 >64 4 >64 4 >64 <1 16 FOX >64 >64 >64 >64 <4 >64 >8 >64 >64 >64 <4 16 GEN <1 8 <1 8 <1 >16 <1 >16 <1 8 <1 >16 IPM <0.25 1 2 2 <0.25 <0.5 <0.25 <0.25 <0.5 2 2 8 SXT <20 >320 160 >320 <20 >320 <20 >320 <20 >320 <20 >320 TZP 8 16 8 32 <4 8 <4 >128 <8 >128 <4 <4 CAT SPECIMENS Acinetobacter spp. Pseudomonas spp. Escherichia spp. Klebsiella spp. Enterobacter spp. Proteus spp. MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 AMC 4 16 >32 >32 4 >32 16 >32 >32 >32 8 8 AMK <2 <8 <2 16 <2 8 <2 16 <2 16 <2 <2 AMP 4 >32 >32 >32 >16 >32 >32 >32 >32 >32 >32 >32 CAZ 4 4 16 >64 <1 >16 <1 >16 <1 >64 <1 <1 CIP <0.25 <0.5 <0.5 >4 <0.25 >4 >2 >4 <0.25 >4 <0.25 2 CTX 8 16 16 >64 <1 >64 <1 >64 <1 >64 <1 <1 CXM 16 >64 >64 >64 4 >64 >16 >64 16 >64 <1 4 FOX >64 >64 >64 >64 >4 8 >64 >64 <4 16 GEN <1 <2 <1 8 <1 <2 <1 >16 <1 8 <1 >16 IPM <0.25 <1 2 2 <0.25 <1 <0.25 <1 <0.25 <1 – – SXT <20 <20 >320 >320 <20 >320 >320 >320 <20 >320 >320 >320 TZP <8 16 8 >128 <4 64 16 >128 <8 >64 <4 <4 AMC, Amoxicillin-clavulanic; AMK, amikacin; AMP, ampicillin; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; CXM, cefuroxime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; SXT, trimethoprim/sulphamethoxazole; and TZP, piperacillin/tazobactam. CLSI (M100-S24): AMC ≥ 32/16, AMK ≥ 64, and AMP ≥ 16; CAZ ≥ 16, CIP ≥ 1, CTX ≥ 4, CXM ≥ 32, FOX ≥ 32, GEN ≥ 16, IPM ≥ 4, SXT ≥ 4/76, and TZP ≥ 128/4. CLSI (VET01): AMC ≥ 1, AMP > 8, AMK ≥ 16, CAZ ≥ 16, and GEN ≥ 8. Frontiers in Microbiology | www.frontiersin.org 7 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs FIGURE 4 | Comparison of antimicrobial resistance frequencies in Gram negative bacteria isolated from dogs and cats. Statistical significance was calculated by Chi-square (χ2 ) or Fishers Exact tests, *p < 0.05. AMC, Amoxicillin-clavulanic; AMP, ampicillin; FOX, cefoxitin; LEX, cephalexin; CFZ, cefazolin; CEF, cephalotin; CXM, cefuroxime; CAZ, ceftazidime; CTX, cefotaxime; CVN, cefovecin; FEP, cefepime; IPM, imipenem; MEM, meropenem; CIP, ciprofloxacin; ENR, enrofloxacin; MFX, marbofloxacin; PRA, pradofloxacin; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; DOX, doxycycline; FOF, Fosfomycin; NIT, nitrofurantoin; and SXT, trimethoprim/sulfamethoxazole. isolates showed resistance to cephalosporins, clindamycin and 2007; Awosile et al., 2018). Nevertheless, our isolates from polymyxin B, and more than 50% of them were also resistant dogs presented the highest resistance percentage for amikacin to aminoglycosides. These results are consistent with findings and neomycin (>50%); this finding could compromise the from Canada, United States, Portugal (Delgado et al., 2007; bactericidal activity of therapies holding aminoglycosides for Jackson et al., 2009; Awosile et al., 2018), and Spain (unpublished the empirical treatment of streptococcal infections in dogs of data) where enterococcal isolates from urinary infections had the studied region. similar levels of resistance to cephalosporins, clindamycin, and Among the Enterobacteriaceae family, E. coli and Proteus spp. polymyxin B, but high levels of susceptibility to penicillin, were highly isolated from wounds, dermatitis, abscesses ampicillin, and amoxicillin-clavulanate. Thus, oral ampicillin and otitis in dog specimens in this study. The reduced or amoxicillin which is commonly prescribed as a first line susceptibility patterns of these bacterial species was found treatment for empirical therapy in enterococcal infections could to cephalosporins (1st generation, 30% for cephalexin) and be appropriate for the studied region. Nevertheless, the increased to ampicillin (50%). Proteus isolates presented resistance to AMR to gentamicin observed in this study could compromise the doxycycline and polymyxin B (>80%) as well. Ampicillin effectivity of combined therapies with ampicillin or amoxicillin was used in the susceptibility test to predict activity of (Arias et al., 2010). amoxicillin (Weese et al., 2019), and is a good first-line In this study, Streptococcus spp. were highly susceptible option for the treatment of sporadic bacterial cystitis to several antimicrobials, including penicillin, ampicillin, associated to E. coli in cats and dogs (Weese et al., 2011, amoxicillin-clavulanate, trimethoprim/sulfamethoxazole, 2019). The use of this antimicrobial for empirical treatment fluoroquinolones, and allowing for several likely effective of E. coli infections should be with caution due to the choices for empirical therapy. Similar susceptibility pattern rapid development of resistance caused by beta-lactamase of Streptococcus spp. has also been reported (Pedersen et al., production (Boehmer et al., 2018). Nonetheless, our results Frontiers in Microbiology | www.frontiersin.org 8 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs FIGURE 5 | Comparison of antimicrobial resistance frequencies in Gram positive bacteria isolated from dogs and cats. Statistical significance was calculated Chi-square (χ2 ) or Fishers Exact tests, *p < 0.05. AMC, Amoxicillin-clavulanic; AMP, ampicillin; LEX, cephalexin; CFZ, cefazolin; CXM, cefuroxime; CTX, cefotaxime; CVN, cefovecin; IPM, imipenem; MEM, meropenem; CIP, ciprofloxacin; ENR, enrofloxacin; MFX, marbofloxacin; PRA, pradofloxacin; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; DOX, doxycycline; ERY, erythromycin; FOF, fosfomycin; NIT, nitrofurantoin; SXT, trimethoprim/sulfamethoxazole; and VAN, vancomycin. support than other antimicrobials, also effective against E. coli 2018). Clinically, doxycycline and amoxicillin-clavulanate are and Proteus spp., such as amoxicillin-clavulanate, amikacin, often used for the treatment of Pasteurella infections (Lappin and gentamicin could be included as empirical selection et al., 2017). Since most of the isolates were highly sensitive to (Awosile et al., 2018). antimicrobials including fluoroquinolones, aminoglycosides, and In the present study, E. coli strains isolated from dogs trimethoprim/sulfamethoxazole which are reasoned to be used and cats showed low levels of AMR (with the exception for the treatment of Pasteurella infections in cats and dogs. of ampicillin) in comparison with other members within The antimicrobial options for empirical therapy can be the same family, i.e., Klebsiella, Proteus, or Enterobacter spp. compromised in companion animals (Prescott et al., 2002; Accordingly, Enterobacter strains from dog specimens showed Jung et al., 2020) basically due to: (1) the increased incidence higher levels of AMR for β-lactams, imipenem and mupirocin in the last years of antimicrobial-resistant bacteria such as compared to cats. Moreover, K. pneumoniae from respiratory MDR Enterococcus spp., Enterobacter spp., P. aeruginosa tract infections in cats presented in general higher resistance to and K. pneumoniae, and (2) the extended AMR to other antimicrobials than dog specimens, mainly for piperacillin, and antimicrobial families (i.e., aminoglycosides, fluoroquinolones, trimethoprim/sulfamethoxazole. and carbapenems). Of note, the results obtained from pets of this On the other hand, high susceptibility to many antimicrobials study are similar to those reported in human hospitals in Spain has been observed for Pasteurella isolates from respiratory tract (ESTUDIO EPINE-EPPS, 2017). The most prevalent bacterial of cats and dogs. This is consistent with findings in other reports species found in human nosocomial and community infections (Pedersen et al., 2007; Kroemer et al., 2014; Awosile et al., are E. coli (19.5%), S. aureus (9%) and P. aeruginosa (8%), Frontiers in Microbiology | www.frontiersin.org 9 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs FIGURE 6 | Comparison of antimicrobial resistance frequencies in other bacteria spp. isolated from dogs and less representative from cats. Statistical significance was calculated by Chi-square (χ2) or Fishers Exact tests, *p < 0.05. AMC, Amoxicillin-clavulanic; FOX, cefoxitin; PIP, piperacillin; TZP, piperacillin/tazobactam; AMP, ampicillin; LEX, cephalexin; CXM, cefuroxime; CAZ, ceftazidime; CVN, cefovecin; CTX, cefotaxime; CPD, cefpodoxime; IPM, imipenem; CIP, ciprofloxacin; ENR, enrofloxacin; MFX, marbofloxacin; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; N, Neomycin; DOX, doxycycline; PB, polymyxin B, SXT, trimethoprim/sulfamethoxazole; FFC, florfenicol, and CHL, chloramphenicol. followed by K. pneumoniae (6.3%), Enterococcus spp. (5.8%), Some limitations have to be considered in the present P. mirabilis (3.2%), and Enterobacter spp. (2.2%). Moreover, study. Firstly, data on clinical history and antimicrobial CMI90 results of Enterobacteriaceae isolated from dogs and usage were not available. Secondly, some cases might have cats of this study presented values for amoxicillin-clavulanate been treated empirically prior to culture and susceptibility >32–16 mg/L, ceftazidime = 8 mg/L, cefotaxime = 4 mg/L, testing. Thirdly, the use of laboratory data may represent a cefuroxime > 64 mg/L, cefoxitin > 32 mg/L, and bias toward resistance, since cultures from complicated cases piperacillin/tazobactam = 16–4 mg/L, which have been associated tend to be requested more often than uncomplicated cases. with a BLEE phenotype in E. coli, K. pneumoniae, and E. cloacae Finally, isolates that exhibited intermediate resistance were from human isolates (Canton, 2010). Finally, the presence of classified as resistant, this could have biased the results to Proteus isolates from dogs with imipenem CMI90 > 4 mg/L is some extent toward overestimating the resistance levels among highly suspicious for carbapenemasa production. To prevent the the tested strains. selection of BLEEs and carbapenem- resistance profiles in both Despite these limitations, the results of this study provides human and animal medicine, is very important to implement information on susceptibility patterns in major cat and dog the One Health approach, and monitor the resistance patterns bacterial isolates from the Iberian Peninsula. These results of these pathogenic bacteria in companion animals (ESTUDIO show Staphylococcus spp., Streptococcus spp., Pseudomonas EPINE-EPPS, 2017; Nigg et al., 2019; Jung et al., 2020). spp., E. coli, and Enterococcus spp. as the most predominant Frontiers in Microbiology | www.frontiersin.org 10 January 2021 | Volume 11 | Article 621597 Li et al. Antimicrobial Resistance in Cats Dogs bacteria in cats and dogs, and with the highest levels of ETHICS STATEMENT AMR in Enterococcus spp. and Pseudomonas spp. Within the Enterobacteriaceae, E. coli presented low levels of AMR Authors declare no Institutional Animal Care and Use compared to Klebsiella, Proteus or Enterobacter spp. Since dogs Committee (IACUC) or other approval declaration was needed. and cats are supposed to act as reservoirs of AMR genes that may transfer to humans, data from this study combined with clinical judgment can be used as a guide for rationalizing antimicrobial AUTHOR CONTRIBUTIONS treatment of companion animals, at least in the Iberian Peninsula. Finally, optimizing antimicrobial use in the vet clinics will benefit YL and LD contributed to the analysis and the interpretation of to limit the selection and spreading of resistant bacteria not only the data and the writing of the manuscript. ID contributed to among our pets but also among the human population. data collection. RF and RM-L contributed to data analysis. LD supervised the work. All authors contributed to the article and approved the submitted version. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included ACKNOWLEDGMENTS in the article/supplementary material, further inquiries can be directed to the corresponding author/s. We gratefully thank the Echevarne Lab for data extraction. REFERENCES Delgado, M., Neto, I., Correia, J. H. D., and Pomba, C. (2007). Antimicrobial resistance and evaluation of susceptibility testing among pathogenic Angulo, F. J., Johnson, K. R., Tauxe, R. V., and Cohen, M. L. (2009). Origins and enterococci isolated from dogs and cats. Int. J. Antimicrob. Agents 30, consequences of antimicrobial-resistant nontyphoidal Salmonella: implications 98–100.doi: 10.1016/j.ijantimicag.2007.03.007 for the use of fluoroquinolones in food animals. Microb. 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Chemother. 60, 775–781.doi: 10.1093/jac/ absence of any commercial or financial relationships that could be construed as a dkm269 potential conflict of interest. Petersen, A. D., Walker, R. D., Bowman, M. M., Schott, H. C., and Rosser, E. J. (2002). Frequency of isolation and antimicrobial susceptibility patterns of Copyright © 2021 Li, Fernández, Durán, Molina-López and Darwich. This is an Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine open-access article distributed under the terms of the Creative Commons Attribution skin and ear samples over a 6-year period (1992-1997). J. Am. Anim. Hosp. License (CC BY). The use, distribution or reproduction in other forums is permitted, Assoc. 38, 407–413.doi: 10.5326/0380407 provided the original author(s) and the copyright owner(s) are credited and that the Prescott, J. F., Brad Hanna, W. J., Reid-Smith, R., and Drost, K. (2002). original publication in this journal is cited, in accordance with accepted academic Antimicrobial drug use and resistance in dogs. Can. Vet. J. 43, 107–116. doi: practice. No use, distribution or reproduction is permitted which does not comply 10.1111/j.1751-0813.2002.tb11003.x with these terms. Frontiers in Microbiology | www.frontiersin.org 12 January 2021 | Volume 11 | Article 621597