Antimicrobial Resistance Genes and Diversity of Clones among Faecal ESBL-Producing Escherichia coli Isolated from Healthy and Sick Dogs Living in Portugal

The purpose of this study was to analyse the prevalence and genetic characteristics of ESBL and acquired-AmpC (qAmpC)-producing Escherichia coli isolates from healthy and sick dogs in Portugal. Three hundred and sixty-one faecal samples from sick and healthy dogs were seeded on MacConkey agar supplemented with cefotaxime (2 µg/mL) for cefotaxime-resistant (CTXR) E. coli recovery. Antimicrobial susceptibility testing for 15 antibiotics was performed and the ESBL-phenotype of the E. coli isolates was screened. Detection of antimicrobial resistance and virulence genes, and molecular typing of the isolates (phylogroups, multilocus-sequence-typing, and specific-ST131) were performed by PCR (and sequencing when required). CTXR E. coli isolates were obtained in 51/361 faecal samples analysed (14.1%), originating from 36/234 sick dogs and 15/127 healthy dogs. Forty-seven ESBL-producing E. coli isolates were recovered from 32 sick (13.7%) and 15 healthy animals (11.8%). Different variants of blaCTX-M genes were detected among 45/47 ESBL-producers: blaCTX-M-15 (n = 26), blaCTX-M-1 (n = 10), blaCTX-M-32 (n = 3), blaCTX-M-55 (n = 3), blaCTX-M-14 (n = 2), and blaCTX-M-variant (n = 1); one ESBL-positive isolate co-produced CTX-M-15 and CMY-2 enzymes. Moreover, two additional CTXR ESBL-negative E. coli isolates were CMY-2-producers (qAmpC). Ten different sequence types were identified (ST/phylogenetic-group/β-lactamase): ST131/B2/CTX-M-15, ST617/A/CTX-M-55, ST3078/B1/CTX-M-32, ST542/A/CTX-M-14, ST57/D/CTX-M-1, ST12/B2/CTX-M-15, ST6448/B1/CTX-M-15 + CMY-2, ST5766/A/CTX-M-32, ST115/D/CMY-2 and a new-ST/D/CMY-2. Five variants of CTX-M enzymes (CTX-M-15 and CTX-M-1 predominant) and eight different clonal complexes were detected from canine ESBL-producing E. coli isolates. Although at a lower rate, CMY-2 β-lactamase was also found. Dogs remain frequent carriers of ESBL and/or qAmpC-producing E. coli with a potential zoonotic role.


Introduction
Antimicrobial resistance has become a major challenge for public health worldwide. The selective pressure, which results from the long-term use of antibiotics, allowed bacterial species to be resistant to these agents. It has been believed that this resistance is reaching alarming levels, considering that resistance rates have risen extremely, during the last two decades [1,2].
Escherichia coli, a Gram-negative bacterium belonging to the Enterobacteriaceae family, is a common member of the intestinal microbiota of humans and companion animals [3,4]. However, this opportunistic pathogen can cause intestinal and extra-intestinal diseases. It may contribute, in many cases, to antimicrobial resistance dissemination. Recently, the World Health Organization [5] published a global priority list of antibiotic-resistant bacteria, where third-generation cephalosporin-and/or carbapenem-resistant Enterobacteriaceae, including E. coli, were included in the Priority 1 group. It is important to note that firstgeneration cephalosporins and amoxicillin + clavulanic acid are among the most prescribed drugs for dogs [3,4,6].
During recent years, the emergence and rapid dissemination of Enterobacteriaceae carrying genes encoding the extended-spectrum-β-lactamases (ESBLs), acquired AmpC β-lactamases (qAmpC), or carbapenemases are considered of great concern [4,7]. One of the most important mechanisms is the plasmid-mediated production of extendedspectrum β-lactamases (ESBLs), which can hydrolyse broad-spectrum cephalosporins (such as cefotaxime). The horizontal gene transfer (HGT) among bacteria is driven by plasmids [8,9], which play an important role in the transference of antibiotic-resistance genes among bacteria, contributing to the spread of multidrug resistance (MDR), and limiting therapeutic options [10]. ESBLs of the CTX-M-type and the qAmpC CMY-2 are increasingly being reported in bacteria worldwide, while livestock or companion animals are potential sources, leading to the spread of β-lactam-resistant bacteria in humans [11,12].
The close proximity between dogs and their owners increases the possibility of transmitting resistant bacteria [13,14]. According to Dupouy et al. [6], dogs could transmit MDR bacteria due to their close contact with humans, the high consumption of β-lactams in small animal veterinary practice, and also the frequent occurrence of ESBL/qAmpCproducing E. coli. The occurrence of ESBL-producing E. coli has been widely reported in both healthy companion animals [12,15] and diseased ones [1,[16][17][18][19]. International high-risk clones of E. coli are frequently detected worldwide, not only in human infections but also in those of companion animals [2,3,17]. Over the past 5 years, the presence of ESBL/qAmpC genes in Enterobacteriaceae strains from faeces of dogs in Europe has been reported in several studies [6,12,13,20], including Portugal [21,22]. However, knowledge about the clonality of ESBL/qAmpC-producing isolates and the potential zoonotic reservoir of human-associated STs is not well documented. Moreover, there is still a lack of data about their prevalence in sick and healthy dogs, simultaneously. In this study, we aim at characterizing the prevalence and diversity of ESBL-and qAmpC-producing E. coli faecal isolates from healthy and sick dogs in Portugal, as well as determining their genetic lineages and phylogenetic groups.

Animals and Sampling
A total of 361 faecal samples were recovered from 127 healthy and 234 hospitalized dogs from different cities in Portugal. All samples were collected between April andAugust 2017 (one sample/animal) using standardized procedures [23].
The hospitalized dogs came from 7 different veterinary hospitals or clinic centers; the healthy dogs came from a local kennel located in Vila Real (n = 31) and from local houses (n = 96). The seven hospitals/clinic centers were located in different centers of the Portuguese territory: Bragança (1 hospital, n = 29 dogs), Vila Real (4 hospitals, n = 62), Aveiro (1 hospital, n = 58), Leiria (1 hospital, n = 17), and Lisbon (1 hospital, n = 68) ( Figure S1). It is important to note that faecal samples from unhealthy dogs were collected from the ordinary population of animals hospitalized in hospitals or veterinary clinics, not endangering their health, or causing harm or pain. In the same line, faecal samples from healthy animals were also recovered by their owners. All of them were analysed with the owner's permission or with kennel collaboration. The faecal samples were dispatched immediately to the Microbiology Laboratory of the University of Trás-os-Montes and Alto-Douro (UTAD).

E. coli Isolation
From each faecal sample, a small portion of 2 g was diluted in Brain Heart Infusion (BHI, Condalab, Spain) and incubated in aerobic conditions for 24 h at 37 • C. After that, samples were seeded on MacConkey agar (Becton, Dickinson and Company Sparks, Le Pont de Claix, France) supplemented with cefotaxime (2 µg/mL) and incubated for 24 h at 37 • C. Colonies showing E. coli morphology were recovered (one colony per sample) and identified by a classical biochemical method named IMViC (Indol, Methyl-red, Voges-Proskauer, and Citrate).
The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry method (MALDI-TOF MS, MALDI Biotyper®from Bruker Daltonik, Bremen, Germany) was applied in this study to confirm bacterial species identification. E. coli isolates were kept at −80 • C and were further characterized.

DNA Extraction and Quantification
Genomic DNA from cefotaxime-resistant (CTX R ) isolates were extracted using the boiled method [25]. In order to quantify the nucleic acid concentration and the level of purity, the absorbance readings were taken at 260 and 280 nm (Spectrophotometer ND-100, Nanodrop, Thermo Fisher Scientific, Waltham, MA USA).

Antibiotic Resistance and Virulence Genes Detection
The genetic basis of resistance was investigated using PCR methods and subsequent sequencing of the obtained amplicons (specific genes). Negative and positive controls of the University of La Rioja were used in this work. Moreover, the data regarding PCR conditions for each primer (Sigma-Aldrich, Madrid, Spain) as well as the size of the obtained amplicons that were sequenced are illustrated in detail in Table S1.

Multilocus Sequence Typing and Phylogroup Typing of E. coli Isolates
Multilocus sequence typing (MLST), by the analysis of seven housekeeping genes (fumC, adk, purA, icd, recA, mdh, and gyrB), was carried out for thirteen representative E. coli isolates (based on the antimicrobial resistance phenotype) according to the protocol described on PubMLST (Public databases for molecular typing and microbial genome diversity) website [34]. The allele combination was determined after sequencing of the seven genes, and the sequence type (ST) and clonal complex (CC) were identified.
Phylogenetic classification of all E. coli isolates was performed according to the presence of chuA, yjaA, and TSPE4.C2 genes [35].

Statistical Analyses
All statistical analyses were performed using the JMP Statistics software (v7.0, SAS Institute). The Pearson's Chi-square and Fisher's exact tests were performed to understand and identify the associations between the origin of strain (healthy or sick dog) and antibiotic resistance (antibiotic and gene). In this line, we consider two categorical variables: the sick or healthy animal, and the resistance for each antibiotic/gene. A p-value < 0.05 was established as indicating statistical significance [36].
Forty-seven ESBL-producing E. coli isolates were detected among the 51 CTX R isolates, recovered from 32 sick and 15 healthy dogs (frequencies of 13.7% and 11.8%, respectively). The phenotypes of antibiotic resistance for these ESBL-producing isolates are shown in Table 1 and the rates of antibiotic resistance of these isolates depending on their origin (sick or healthy dogs) are represented in Figure 1. No statistical difference could be established between the origin of the strain (healthy or sick dog) and the resistance to different antibiotics (p > 0.05) ( Figure 1).
The two remaining ESBL-positive isolates were revealed negative to all ESBL genes under study. Furthermore, a bla TEM gene was detected in eight bla CTX-M -producing isolates. On the other hand, six ESBL-positive isolates showed cefoxitin-resistance (FOX R ), and the bla CMY-2 gene was detected in one CTX-M-15-producing isolate obtained from a sick dog; the others ESBL-positive-FOX R isolates were negative for bla CMY-2 and bla DHA genes by PCR. Among the ESBL-positive isolates, resistance to tetracycline was mediated by the tetA (24 isolates) and/or tetB genes (Table 1).   (4), purA (5), icd (25), gyrB (2), recA (2), and mdh (5).

Discussion
Regarding the Portuguese situation, the prevalence of ESBL-producing E. coli isolates in healthy dogs obtained in this work is similar to previous studies performed in dogs and cats [12,22,23] in the South and the North of Portugal. Worldwide, this prevalence was lower than the ones obtained with faecal samples of healthy dogs in Germany, Brazil, or China (24-29%) [15,37,38], but it is similar to the results of previous studies performed in Tunisia and France (12.7-17%) [11,39]. These differences could be explained by differences in the epidemiology of ESBL genes among different countries, considering the year in which the studies were performed, but we cannot discard methodological effects in the different studies.
Five types of CTX-M ESBLs were detected, indicating a high diversity of CTX-M genes (mainly blaCTX-M-15 gene) among the CTX R E. coli isolates; these results are in accordance with a previous study done in Portugal on healthy dogs [12]. This blaCTX-M-15 gene was also the most frequently detected in E. coli isolated from dogs in different countries [3,15,40]. The CTX-M-1-and CTX-M-15-encoding genes were also detected among E. coli canine isolates in Italy [41] and Denmark [13], which are in agreement with our data. The same variants of CTX-M genes were observed in a recent study conducted on healthy humans in Spain [42]. Moreover, during the last few years, new variants are becoming more common, in particular CTX-M-55 [3], especially from companion animals in Asian countries [43].
In the past, the blaCTX-M-15 gene was mainly associated with strains of human origin while blaCTX-M-1 was the major CTX-M sub-type among livestock and companion animal isolates in Europe [15,41]. Actually, this close correspondence is no longer so obvious, and our results confirm these data. A further study should be implemented to determine the ESBL gene in the two uncharacterized ESBL-producing isolates.
In this study, the CMY-2 gene was the qAmpC β-lactamase type found among two CTX R -ESBL-negative isolates and one ESBL-producing isolate, and it has been previously reported among E. coli strains from healthy and sick pets worldwide [20,23,39,44]. The detection of tetA and/or tetB genes in most of our tetracycline-resistant isolates seem to be similar to the results obtained by Costa et al. [45] from dogs, in Northern Portugal.
In this work, the most common phylogenetic groups among our isolates were B1 and A, these being the phylogroups more associated with commensal E. coli both in humans and in dogs, as well as in other animals [11,13]. On the other hand, isolates belonging to Two of the four CTX R and ESBL-negative isolates were CMY-2-producers (qAmpC type), and they were recovered from a healthy and a sick dog (one each) ( Table 2). We could not detect the mechanisms of CTX R in the two remaining ESBL-negative isolates. None of the CTX R E. coli isolates carried the mcr-1 gene (related to colistin resistance).
Moreover, other β-lactamases genes such as bla VEB , bla NDM, bla OXA-48, and bla VIM were tested by PCR/sequencing but all isolates were revealed to be negative. Furthermore, the stx 1,2 genes related to Shiga toxin-producing E. coli (STEC) were not detected among our isolates.

Discussion
Regarding the Portuguese situation, the prevalence of ESBL-producing E. coli isolates in healthy dogs obtained in this work is similar to previous studies performed in dogs and cats [12,22,23] in the South and the North of Portugal. Worldwide, this prevalence was lower than the ones obtained with faecal samples of healthy dogs in Germany, Brazil, or China (24-29%) [15,37,38], but it is similar to the results of previous studies performed in Tunisia and France (12.7-17%) [11,39]. These differences could be explained by differences in the epidemiology of ESBL genes among different countries, considering the year in which the studies were performed, but we cannot discard methodological effects in the different studies.
Five types of CTX-M ESBLs were detected, indicating a high diversity of CTX-M genes (mainly bla CTX-M-15 gene) among the CTX R E. coli isolates; these results are in accordance with a previous study done in Portugal on healthy dogs [12]. This bla CTX-M-15 gene was also the most frequently detected in E. coli isolated from dogs in different countries [3,15,40]. The CTX-M-1-and CTX-M-15-encoding genes were also detected among E. coli canine isolates in Italy [41] and Denmark [13], which are in agreement with our data. The same variants of CTX-M genes were observed in a recent study conducted on healthy humans in Spain [42]. Moreover, during the last few years, new variants are becoming more common, in particular CTX-M-55 [3], especially from companion animals in Asian countries [43].
In the past, the bla CTX-M-15 gene was mainly associated with strains of human origin while bla CTX-M-1 was the major CTX-M sub-type among livestock and companion animal isolates in Europe [15,41]. Actually, this close correspondence is no longer so obvious, and our results confirm these data. A further study should be implemented to determine the ESBL gene in the two uncharacterized ESBL-producing isolates.
In this study, the CMY-2 gene was the qAmpC β-lactamase type found among two CTX R -ESBL-negative isolates and one ESBL-producing isolate, and it has been previously reported among E. coli strains from healthy and sick pets worldwide [20,23,39,44]. The detection of tetA and/or tetB genes in most of our tetracycline-resistant isolates seem to be similar to the results obtained by Costa et al. [45] from dogs, in Northern Portugal.
In this work, the most common phylogenetic groups among our isolates were B 1 and A, these being the phylogroups more associated with commensal E. coli both in humans and in dogs, as well as in other animals [11,13]. On the other hand, isolates belonging to phylogroup B 2 and D are more likely to be recovered from extra-intestinal infections of companion animals [4]. An interesting study related to 78 dogs that visited a veterinary hospital in Northern Portugal (either for a normal checkout or in case of disease) revealed the prevalence of E. coli isolates of groups A (n = 19), D (n = 9), and B 1 (n = 7) [46], similar to our observation. So, the carriage of ESBL/qAmpC producing E. coli of these phylogroups in the gastrointestinal tract suggests a potential reservoir of MDR ESBL-producing bacteria in dogs.
Regarding the MLST results, the pandemic virulent E. coli ST131-B2 clone was detected among two isolates of sick dogs tested in this study. It is important to note that this clone was widely detected in pets [47,48], including in sick dogs in Portugal [17,49].
On the other hand, we detected one E. coli strain, ST57/D/CTX-M-1, that was recently detected in Portugal (associated with CMY-2 gene) in a dog with a UTI from a Lisbon hospital [17]. Similarly, the same lineage was identified in a faecal isolate from a healthy dog in Mexico, characterized as CMY-2/ST57/D) [50].
The frequency of the ST6448 lineage, which was observed in two sick dogs in this study, is considered an infrequent clone in humans and companion animals. This lineage was also found among a vulture faecal sample from Canary Islands [51]. To our knowledge, there is only one previous report related to the detection of this clone in humans, which was recently reported in healthy children from Sweden [52].
Additionally, our data indicate the presence of E. coli ST12/B2/CTX-M-15, which should be considered an agent of high clinical relevance for humans and animals. Furthermore, the ST12 lineage (associated with CMY-2) was identified in healthy dogs from Spain [6], Brazil [2], and France [11]. Furthermore, this lineage was found among isolates from children with a febrile UTI in France [53] and in healthy humans in Spain [42]. These findings highlight the dissemination of ST12 lineage and its presence in animal and human' isolates.
To our knowledge, the ST617 lineage (clonal complex ST10) was identified for the first time in pets from Portugal in this study. CTX-M-15-producing E. coli isolates of sequence type ST617/phylogroup A have been reported in sick dogs in France [40] and in hospitalized patients in Tunisia [54,55]. Similarly, Rocha-Gracia et al. [50] identified the same lineage among a faecal isolate from healthy dogs in Mexico (ST617/A/CTX-M-15). According to a recent study, Gauthier, et al. [56] found this lineage in four isolates from dogs in France harbouring carbapenemase genes. Furthermore, this clone was widely disseminated.
The ST542 lineage detected in one of the healthy dogs is not commonly reported; however, this clone was found in a farmworker from Germany [57] and in a pig in Australia [58]. On the other hand, an ST115/CMY-2 isolate (found in a sick dog from the Vouga clinic) was previously reported among chickens and human patients in Germany [47].
We also detected a ST5766/A/CTX-M-32 isolate in a healthy dog; this clone is unusual, and it was previously reported in broilers' osteomyelitis in Brazil [59]. To our knowledge, this is the first report of the ST5766 clone among pets, and the first detection in Europe. In this study, we also found an E. coli isolate, ST3078/B1/CTX-M-32, recovered from a healthy dog from a kennel. To our knowledge, the only unique previous study related to the ST3078 lineage was found in wastewater in Eastern France [60]. This suggests that the environment likely plays a role in the spread of ESBL-producing E. coli isolates in the community, associated with a One Health approach (human-animals-environment). Importantly, a new combination of alleles was found in an isolate of a healthy dog, rendering a new ST.
The use of β-lactams in the clinical practice of veterinary medicine may be considered one of the reasons for the high incidence of ESBL-producers worldwide. Thus, pets can be a significant source of ESBL/qAmpC-producing E. coli isolates. Considering the prevalence of ESBLs (notably the large reservoir in dogs of E. coli isolates with genes encoding CTX-M-15 and CTX-M-1, or CMY-2 β-lactamases), there is a serious and plausible risk of future acquisition of these resistant genes by their owners.

Conclusions
Antimicrobial resistance can make infections difficult to treat, which represents a global public health problem, due to the negative consequences for human health. This study shows that healthy and sick dogs are frequent carriers of faecal ESBL-producing E. coli strains, harbouring different variants of bla CTX-M genes (mostly bla CTX-M-15 and bla CTX-M-1 ), and presenting a high genetic MLST diversity (including the ST131/B2 lineage). Although at a lower rate, the bla CMY-2 gene was also found. This fact suggests the implication of mobile genetic elements in the dissemination of this relevant mechanism of resistance. This underlies the complexity of the antimicrobial resistance of bacteria occurring in dogs and the possible interspecies transmission between humans, domestic animals, and into the environment, important knowledge given the One-Health approach.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics10081013/s1, Figure S1: Geographic location of the different areas where the faecal samples from sick dogs were collected in Portugal. Table S1: Primers sequences and PCR conditions used for genes encoding antibiotic resistance in E. coli. conceptualization, methodology, validation, resources, data curation, writing-review and editing, visualization, supervision, project administration, funding acquisition; C.T.: conceptualization, methodology, validation, resources, data curation, writing-review and editing, visualization, supervision, project administration, funding acquisition; P.P. (Patrícia Poeta): conceptualization, methodology, validation, resources, data curation, writing-review and editing, visualization, supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.
Funding: I.C. gratefully acknowledges the financial support of "Fundação para a Ciência e Tecnologia" (FCT-Portugal) related to PhD grant, through the reference SFRH/BD/133266/2017 (Medicina Clínica e Ciências da Saúde), as well as MCTES (Ministério da Ciência, Tecnologia e Ensino Superior) and European Union (EU), with reference to Fundo Social Europeu (FSE). The experimental work carried out in the University of La Rioja (Spain) was financed by the project SAF2016-76571-R from the Agencia Estatal de Investigation (AEI) of Spain and FEDER of EU. N.S.C. was awarded a grant for the year 2018, from the Algerian Ministry of Higher Education and Scientific Research (The PNE Program), under the direction of Carmen Torres. This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Spain; grant number RTI2018-098267-R-C33), the Junta de Castilla y León (Consejería de Educación, Spain; grant number LE018P20) and the Associate Laboratory for Green Chemistry-LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in Supplementary Material.