Although Enterobacterales are natural inhabitants of the intestinal tract of mammals, some genus and species can cause infections of the respiratory and urinary systems, skin, ear, and soft tissue of human and non-human hosts (Zogg et al., 2018). In this regard, Escherichia coli and Klebsiella pneumoniae are leading causes of healthcare-associated infections worldwide (Pesesky et al., 2015), with carbapenem- and broad-spectrum cephalosporin-resistant lineages being categorized as critical priority pathogens by the World Health Organization (Tacconelli et al., 2018). Certainly, and even more worrying is the fact that extended-spectrum β-lactamase (ESβL)-producing strains are no longer restricted to hospital locations but also represents a serious problem involving pets, wildlife, and environmental and food safety (Lopes et al., 2021; Salgado-Caxito et al., 2021).

CTX-M enzymes have become the most prevalent type of ESβLs globally (Cantón & Coque, 2006; Pitout and Laupland, 2008). It is remarkable that the first report on the emergence of a CTX-M enzyme was in 1988, from a laboratory dog used in β-lactams research in Japan (Matsumoto et al., 1988), whereas E. coli producing bla _CTX-M-1-type enzyme was first described in a healthy dog in Portugal. Since then, a significant occurrence of CTX-M-type ESβL-producing Enterobacterales has been documented in healthy and diseased dogs and cats from Asian, European and South American countries (Salgado-Caxito et al., 2021).

From a public health perspective, the rapid appearance of resistant bacterial populations among dogs and cats, and the close contact between household pets and people have favored the transmission of antibiotic-resistant bacteria from companion animals to humans (Damborg et al., 2016; Kawamura et al., 2017; Salgado-Caxito et al., 2021; Sellera et al., 2021). Transfer of resistant bacteria between humans and their dogs has been well documented (Albrechtova et al., 2012), as was illustrated by the identification of the same E. coli clone from a urinary tract infection in a dog, and from its household members (Johnson et al., 2008), although the direction of transfer is often difficult to prove (Pomba et al., 2017). In addition, the intensive use of antimicrobials in animals can be an important factor in the development of antimicrobial-resistant microorganisms (Caprioli et al., 2000; Umber and Bender, 2009; Marshall and Levy, 2011; Seiffert et al., 2013; Samanta et al., 2015). In this sense, companion animals might act as source of human contamination but may also be contaminated by human bacteria (Okubo et al., 2014; Fernandes et al., 2018; Melo et al., 2018). Furthermore, the role of companion animals as a source of AMR has, so far, been neglected (Ewers et al., 2012).

In South America, multidrug-resistant Enterobacterales are a major concern as the region exhibits some of the higher rates of antimicrobial resistance worldwide (Bonelli et al., 2014). The first report of ESβL in this region in companion animals was published in 2008 from E. coli isolates obtained from fecal samples of dogs and cats in Chile (Moreno et al., 2008). In that context, nosocomial infections caused by ESβL producing Enterobacterales have increased in the region more than others, since 2005 (Guzmán-Blanco et al., 2014). Several factors such living in crowded conditions, malnutrition, ineffective healthcare systems, deficient drug supply chain, massive use of antimicrobials in livestock and agriculture linked to lack of financial resources might be related to the greater prevalence of ESβLs in countries with lower economic resources (Villegas et al., 2008). In this study, we report the occurrence and genomic data of ESβL-producing E. coli and K. pneumoniae strains in dogs from Imbabura, Ecuador.

Forty-tree cephalosporin-resistant Gram-negative bacteria were isolated from 23 healthy dogs and 16 sick dogs ( Supplementary Tables 1, 2 ). From the latter, eight dogs presented with gastrointestinal complications, four with metabolic syndrome, two with dermatological disease, one with respiratory problems, and another with cerebrovascular accident. Based on confirmation of ESβL phenotype, four bacterial isolates exhibiting a MDR profile (Magiorakos et al., 2012) were sequenced: i) E. coli strain ECU3_SQ178 (GenBank accession number: JACWHI000000000.1) isolated from a 6-month-old healthy female dog mixed breed, with no previous treatments reported. This strain presented resistance to ceftazidime, cefotaxime (MIC > 32 µg/mL), ceftriaxone, cefepime, aztreonam, nalidixic-acid, enrofloxacin, ciprofloxacin, and chloramphenicol, being susceptible to amoxicillin-clavulanic acid, cefoxitin, ertapenem, meropenem, imipenem, gentamicin, amikacin, trimethoprim-sulfamethoxazole ( Supplementary Table 2 ). In this regard, WGS analysis predicted the presence of genes associated with resistance to β-lactams (bla _CTX-M-55-like), phenicols (floR), tetracyclines (tetA), sulphonamides (sul2), aminoglycosides [aph(3”)-Ib, aph(6)-Id], fosfomycin (fosA3), and quinolones (gyrA-D87N and parC-S80I point mutations, marA). On the other hand, genes conferring tolerance to heavy metals [arsenic (arsBCR) and tellurium (tehAB)], herbicide [glyphosate (phnCDFGHIJKLMNOP)], biocides and disinfectants (mdtEFKN, emrDK, acrAEF and tolC) were also predicted ( Table 1 ); ii) E. coli strain EE12_SQ154 (GenBank accession number: JACWHK000000000.1) isolated from a 4-years-old female Yorkshire terrier dog with a history of physical decline, cerebrovascular accident and shock. It was not reported by the private veterinary clinic the treatment received prior to the sample collection. Antimicrobial susceptibility testing revealed resistance to ceftazidime, cefotaxime (MIC > 32 µg/mL), ceftriaxone, cefepime, aztreonam, nalidixic-acid, enrofloxacin, ciprofloxacin, trimethoprim-sulfamethoxazole, chloramphenicol, and gentamicin, and susceptibility to amoxicillin-clavulanic acid, cefoxitin, ertapenem, meropenem, imipenem and amikacin ( Supplementary Table 2 ). The antimicrobial resistome included genes conferring resistance to β-lactams (bla _CTX-M-65), aminoglycosides (aadA1, aadA2b), fosfomycin (fosA3), phenicol (cmlA1), sulphonamides (sul3), trimethoprim (dfrA1), quinolones (marA), heavy metals [arsenic (arsBR), tellurium (tehAB) and mercury (merR)], herbicide [glyphosate (phnJ)], biocides and disinfectants (mdtEFK, emrDK, acrAEF, tolC, qacF and mvrC) ( Table 1 ); iii) K. pneumoniae strain ECU12_SQ166 (GenBank accession number: JACWHJ000000000.1), isolated from a 12-year-old male English Shepherd dog admitted to a private veterinary clinic with signs of diarrhea, melena, vomiting, septicemia, and chronic kidney failure leading to death. Based on the anamnesis and initial physical examination, fluid therapy was established, as a stabilization measure (lactated ringer solution), and a not specified β-lactam antibiotic was administered. The strain exhibited resistance to amoxicillin-clavulanic acid, ceftazidime, cefotaxime (MIC > 32 µg/mL), ceftriaxone, cefepime, aztreonam, ceftiofur, trimethoprim-sulfamethoxazole, nalidixic-acid, enrofloxacin, ciprofloxacin, and gentamicin, being susceptible to cefoxitin, ertapenem, meropenem, imipenem, and amikacin ( Supplementary Table 2 ). The resistome analysis predicted resistance genes to β-lactams (bla _CTX-M-27, bla _SHV-27), fosfomycin (fosA6), trimethoprim (dfrA27), rifampicin (arr-3), sulfonamides (sul1, sul3), aminoglycosides [aac(3)IV, aac(6′)-Ib-cr, aadA1, aadA16, aadA2b, aph(4)-la], macrolides (mphA), quinolones [aac(6’)-Ib-cr, oqxA, oqxB, qnrB52], phenicols (cmlA), tetracyclines (tetD), silver (silABCEFRS) and ammonium quaternary compounds (qacF) ( Table 2 ); (iv) K. pneumoniae strain EE25K_SQ190 (GenBank accession number: JACWHL000000000.1) isolated from an 8-year-old male German shepherd dog, presenting with discomfort, anorexia and foreign body gingivitis. After clinical examination, the foreign body was removed and a combination of amoxicillin/clavulanic acid plus a non-steroidal anti-inflammatory was prescribed. Antimicrobial susceptibility testing revealed resistance to cefotaxime (MIC > 32 µg/mL), ceftriaxone, cefepime, nalidixic-acid, enrofloxacin, ciprofloxacin, and trimethoprim-sulfamethoxazole. This strain showed to be susceptible to amoxicillin-clavulanic acid, ceftazidime, cefoxitin, aztreonam, ertapenem, meropenem, imipenem, amikacin and gentamicin ( Supplementary Table 2 ). Resistome encompass genes resistant to β-lactams (bla _CTX-M-14, bla _SHV-33, bla _LAP2), fosfomycin (fosA6), trimethoprim (dfrA1), quinolones (oqxA, oqxB, qnrS1), and sulphonamides (sul1), silver (silABCEFRS), arsenic (arsBCDR) and chlorhexidine (smvR) ( Table 2 ).

While CTX-M-55- and CTX-M-65-positive E. coli strains belonged to ST10 and ST162, K. pneumoniae producing CTX-M-27 and CTX-M-14 ESβLs belonged to ST661 and ST35, respectively. E. coli ST10 and ST162 have been previously associated with human infections (Coelho et al., 2011; Chen et al., 2014), being further identified in hospital sewage (Zhao et al., 2017), bovines (Umpiérrez et al., 2017), birds (Fuentes-Castillo et al., 2020), and dogs (Yasugi et al., 2021). The bla _CTX-M-55 gene has been widely identified globally in E. coli isolates from various animal species (Kiratisin et al., 2007; Zhang et al., 2014; Birgy et al., 2018; Lupo et al., 2018). The remarkable prevalence of this gene, accompanied by a high propensity for horizontal gene transfer has facilitated its rapid and wide spread (Yang et al., 2023). In Ecuador bla _CTX-M-55 has been the most prevalent allele of the bla _CTX-M family in E. coli from poultry settings, followed by bla _CTX-M-65 and bla _CTX-M-2 (Ortega-Paredes et al., 2020a). On the other hand, according to Enterobase (https://enterobase.warwick.ac.uk/), ST10 has been identified in dogs from Germany, United States of America (USA), United Kingdom, South Korea, Canada and New Zealand, whereas in Ecuador ST10 has been identified in humans, wild animals, and environmental samples; confirming the One Health importance of this global lineage in this country. In fact, phylogenomic analysis showed that strain ECU3_SQ178 (CTX-M-55/ST10) clustered (95-105 SNP differences) with food and human E. coli strains of ST10 isolated in 2016, in Australia and Cambodia, respectively, whereas CTX-M-65-positive E. coli ST162 (strain EE12_SQ154) showed ubiquity, being clustered (207-265 SNP differences) with other four drug-resistant E. coli strains of ST162 isolated from livestock (USA, 2016), poultry (USA, 2020), human (Australia, 2014) and companion animal (USA, 2007) ( Figure 1 , Supplementary Table 3 ). Moreover, data retrieved from Enterobase confirm occurrence of this E. coli clone in companion animals from Germany, USA, and Canada. Interestingly, this is the first report of E. coli ST162 found in companion animal, in South America.

In the case of K. pneumoniae ST661 and ST35 clones, they have been previously isolated from nosocomial pneumonia in humans (Zhao et al., 2019), rectal swabs from pigs and fecal human samples (Leangapichart et al., 2021). Moreover, ST661 has been recovered from aquatic environments (Furlan et al., 2020), hospitalized patients (Piazza et al., 2019), being recently reported as responsible for outbreaks in Europe (Martin et al., 2017); whereas ST35 has been identified among ESβL-producing K. pneumoniae strains in hospital settings (Marcade et al., 2013; Frenk et al., 2020), being lately recognized as a multidrug-resistant clone with worldwide distribution (Shen et al., 2020).

For CTX-M-27-positive K. pneumoniae ST661 (ECU12_SQ166), phylogenomic analysis revealed relationship (201-216 SNP differences) with human strains identified in Italy, in 2013 and 2017, respectively ( Figure 2 , Supplementary Table 3 ). Strikingly, all the three isolates within the clade carried an IncFIB-type plasmid. Moreover, ECU12_SQ166 and the human strain isolated in 2017 exhibited an identical MDR profile, sharing bla _SHV-27, sul1 and mph(A) resistance genes. In brief, K. pneumoniae ST661 is other global clone identified in Italy, China, England, Brazil, Tunisia, Thailand, Uruguay, Mexico and Taiwan (Yan et al., 2015; Ku et al., 2017; Martin et al., 2017; Patil et al., 2019; Piazza et al., 2019; Sghaier et al., 2019; Furlan et al., 2020; Hassen et al., 2020; Ludden et al., 2020; Leangapichart et al., 2021; Papa-Ezdra et al., 2021; Toledano-Tableros et al., 2021).

In companion animals, ESβL production among Klebsiella isolates has been associated with CTX-M-14 and CTX-M-15 variants (Harada et al., 2016). In this study, CTX-M-14-positive K. pneumoniae EE25K_SQ190 belonged to ST35. Although this clone has been previously identified in China, Romania, Yemen, Israel, France, Spain and Thailand (Marcade et al., 2013; Cubero et al., 2016; Alsharapy et al., 2020; Frenk et al., 2020; Kong et al., 2020; Shen et al., 2020; Surleac et al., 2020; Zhong et al., 2020; Leangapichart et al., 2021), phylogenomic analysis clustered (353-354 SNP differences) EE25K_SQ190 with a human clone identified in Turkey in 2013 and 2014 ( Figure 2 , Supplementary Table 3 ).

Although, in Ecuador, occurrence of E. coli producing ESβL has been reported in pets, chicken, humans, food, vegetables, broiler farms, and river water samples, in Quito (Vinueza-Burgos et al., 2016; Chiluisa-Guacho et al., 2018; Ortega-Paredes et al., 2018; Ortega-Paredes et al., 2019; Vinueza-Burgos et al., 2019; Zurita et al., 2019; Ortega-Paredes et al., 2020a; Ortega-Paredes et al., 2020b; Zurita et al., 2020), and in other cities such as Guayaquil (Soria Segarra et al., 2018), Esmeraldas (Hedman et al., 2019), Loja (Delgado et al., 2016), and Cuenca (Zurita et al., 2013); as well as in the provinces of Tungurahua and Cotopaxi (Sánchez-Salazar et al., 2020), genomic data are scarce. Specifically, while CTX-M-55 and CTX-M-65-producing E. coli have been previously reported in dogs in central Ecuador, and in Quito (Ortega-Paredes et al., 2019; Albán et al., 2020; Salinas et al., 2021), CTX-M-producing K. pneumoniae have been isolated from human hosts in Cuenca (Nordberg et al., 2013), Quito, Guayaquil, and Azogues (Zurita et al., 2013), so far.

In summary, we report genomic data of global One Health-associated clones of CTX-M-55 and CTX-M-65-producing E. coli, and CTX-M-14 and CTX-M-15-producing K. pneumoniae in dogs from the province of Imbabura, in Ecuador. The implementation of a national epidemiological surveillance program is necessary to establish future strategies to control the dissemination of antibiotic-resistant priority pathogens using a One Health approach.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

The animal studies were approved by MSc. Elena Dorothea Balarezo Cisneros President of the Ethics Committee for Research Processes Yachay Tech University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

FAG-Z: Conceptualization, Formal analysis, Project administration, Supervision, Writing – original draft, Writing – review & editing. JT: Formal analysis, Writing – review & editing. JV-B: Formal analysis, Methodology, Writing – original draft, Writing – review & editing. MV-B: Investigation, Methodology, Writing – review & editing. AC-A: Methodology, Writing – review & editing. FE: Formal analysis, Methodology, Software, Writing – review & editing. QM: Formal analysis, Methodology, Writing – review & editing. BF: Methodology, Writing – review & editing. ES: Formal analysis, Methodology, Software, Writing – review & editing. JGMP: Formal analysis, Methodology, Validation, Writing – review & editing. MJ: Investigation, Writing – review & editing. NL: Conceptualization, resources, Formal analysis, Writing – original draft, Writing – review & editing.