High Prevalence and Diversity of Cephalosporin-Resistant Enterobacteriaceae Including Extraintestinal Pathogenic E. coli CC648 Lineage in Rural and Urban Dogs in Northwest Spain

The aim of this work was to assess the prevalence of extended spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in fecal samples recovered from rural and urban healthy dogs in Northwest Spain (Galicia) to identify potential high-risk clones and to molecularly characterize positive isolates regarding the genes coding for ESBL/pAmpC resistance and virulence. Thirty-five (19.6%) out of 179 dogs were positive for cephalosporin-resistant Enterobacteriaceae, including Escherichia coli and Klebsiella pneumoniae (39 and three isolates, respectively). All the isolates were multidrug resistant, with high rates of resistance to different drugs, including ciprofloxacin (71.4%). A wide diversity of ESBL/pAmpC enzymes, as well as E. coli phylogroups (A, B1, C, D, E, F and clade I) were found. The eight isolates (20.5%) found to conform to the ExPEC status, belonged to clones O1:H45-clade I-ST770 (CH11-552), O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58), with the latter also complying the uropathogenic (UPEC) status. The three K. pneumoniae recovered produced CTX-M-15 and belonged to the ST307, a clone previously reported in human clinical isolates. Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistance.


Introduction
The increase of antibiotic resistance represents a global threat to human and animal health, being that therapeutic options to combat infections have drastically reduced in recent years [1,2]. According to

Results
Thirty-five (19.6%) of the 179 dogs tested carried cephalosporin-resistant Enterobacteriaceae (10 from urban and 25 from rural environments, 20.8% and 19.1%, respectively). From the 35 positive dogs, 39 ESBL-and/or pAmpC-producing E. coli and three K. pneumoniae ESBL-producing isolates were recovered. Six dogs (14.3%) carried more than one ESBL-and/or AmpC-producing Enterobacteriaceae (Table S1): E. coli and K. pneumoniae (three dogs), or two and three different E. coli isolates (two and a single dog, respectively).
Three ESBL-producing K. pneumoniae were also recovered from three individual dogs (two from urban and one from rural areas). All were positive for the bla CTX-M-15 and belonged to the ST307. Interestingly, these K. pneumoniae isolates were found together with pAmpC-producing E. coli (in two dogs) and with one SHV-12 producing E. coli (in one dog).
In summary, we have detected a high rate of fecal colonization by ESBL-and pAmpC-producing Enterobacteriaceae in urban and rural dogs in Galicia, with no statistically significant differences (20.8% and 19.1%, respectively).

Discussion
The rate of colonization by MDR bacteria in companion animals has been assessed in several studies, where the prevalence of colonization by ESBL-producing Enterobacteriaceae among dogs and cats (including healthy and sick animals) ranged widely-between 3.1% and 55% [11,12]-while the figures reported only for healthy dogs were around 20% [12]. These data match the results obtained here, with a prevalence of ca. 20%, regardless of the origin of the dogs (rural or urban). This is also consistent with data from previous studies, which did not find significant differences with respect to the urbanization level of the dogs analyzed [11]. Colonization by ESBL/pAmpC-producing Enterobacteriaceae in companion animals could be related to several factors, including a selective pressure due to previous antibiotic exposure [10] but also to indirect acquisition through raw feeding with meat or carcasses from food-producing animals, which is not uncommon in rural areas. We know that the dogs included in our study had not received any antimicrobial treatment during the previous four weeks before sampling, but, unfortunately, earlier data on antibiotic consumption were not available. A recent study carried out in three European countries (Belgium, Italy, and the Netherlands), found that antimicrobial consumption in companion animals was lower than consumption in food-producing animals. However, the authors reported a high use of WHO critically important antimicrobials, including cefovecin (a third-generation cephalosporin) and quinolones, being that this consumption is higher for dogs than cats [21]. Third and fourth generation cephalosporins, as well as quinolones, have been classified as restricted by the recent categorization of antimicrobials of the European Medicines Agency [22], but they were not prohibited. In a recent survey carried out in Spain, β-lactams and quinolones were the most prescribed antimicrobials in dogs [23]. It is well known that the use of quinolones typically selects ESBL-producing Enterobacteriaceae [24]. Considering that 71.4% of the analyzed isolates from our study were resistant to ciprofloxacin, it is tempting to speculate that quinolone exposure in these dogs could have been involved in the selection of ESBL-producing Enterobacteriaceae.
The high genetic diversity and ESBL/pAmpC types found in the present study has been previously described within E. coli recovered from dogs [17,25], as well as the STs 93, 453, and 770, found here in isolates conforming the ExPEC status [17,25,26]. Five bla CTX-M-14 -carrying isolates recovered in this study belonged to ST770 Escherichia clade I. The clade I was considered as a phylogroup of E. coli based on the extent of recombination detected with strains belonging to E. coli sensu stricto [27]. ST770 is an infrequently reported clone, which has been associated with bla CTX-M-1 carriage in broilers and poultry in the Netherlands and Switzerland [28,29] and with bla CTX-M-14 in a patient diagnosed with a urinary tract infection (UTI) in Spain [30]. Also, this clone, harboring mcr-1 and bla CTX-M-2 , has been recently recovered from a dog with a UTI in Argentina [31] and associated with pAmpC production, specifically CMY-2, from rooks wintering in Czechia and from broilers in Sweden [32,33].
It is of note that in a recent study performed in our region (Galicia) on chicken and turkey meat, we recovered five (5%) ESBL-producing Escherichia clade I ST770 (CH116-552) from different samples, all of them positive for the ExPEC status [34]. Furthermore, three of these isolates were O1:H45. In the same study, we recovered ExPEC-positive isolates belonging to the clones O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58). All those isolates from poultry meat were MDR and most of them fluoroquinolone-resistant. As stated above, dogs may acquire antimicrobial resistant Enterobacteriaceae via various routes, including raw feeding with chicken meat or carcasses, which is quite common in rural areas. Poultry products can act as a reservoir for human extraintestinal Enterobacteriaceae pathogens [34][35][36], so we also hypothesize that such products could be playing a role in their transmission between animals, particularly in rural environments. Importantly, MDR E. coli of human clinical origin and characterized as A-ST93 (CH11-neg), B1-ST453 (CH6-31), and F-ST1485 (CH231-58) were also recently reported in the same health area (Galicia) [37,38]. This reinforces the importance of "One-Health" actions against dissemination of antimicrobial resistance.
Other STs detected in E. coli from dogs in the present study were ST93 and ST453. E. coli ST93 were reported in wild birds in Pakistan, associated with the carriage of bla CTX-M-15 [28]; in beef, veal, pork and poultry, associated with bla CTX-M-1 in Switzerland [28]; and in broiler chickens carrying bla CTX-M-2 in Brazil [39]. In addition, ST93 was also associated to the spread of the mcr-1 gene in companion animals and retail food in China [40,41]. Regarding infections in humans, mcr-1-carrying ST93 E. coli was recovered from a patient with bacteremia in Uruguay [42]. E. coli ST453 harboring bla CTX-M-1 was isolated from pigs and their breeders [43], associated with extraintestinal disease in humans and metritis in cattle in Australia and, carrying mcr-1, with wastewater in Japan [44].
Despite the fact that the pandemic E. coli ST131-B2 was not detected here, eight out of the 39 (20.5%) isolates conformed to the ExPEC status, including one isolate of the global ExPEC lineage F-CC648 belonging to the ST1485 [6]. The phylogroup F together with phylogroup B2 comprise most human clinical ExPEC isolates. Among phylogroup F, the clonal complex 648 (CC648) is a resistance-associated lineage recovered from different sources (human, animal, or environmental) and increasingly associated with extraintestinal pathologies [45]. Importantly, the dog isolate also fulfilled the UPEC status, conjugating in the same isolate a high number of resistance and virulence genes.
In addition to E. coli isolates, we have found three dogs carrying CTX-M-15-producing K. pneumoniae, which is a major nosocomial pathogen able to persist in many different reservoirs, including not only health care settings but also retail meat, livestock, and wastewater [46,47]. This species belongs to the ESKAPE list and is considered as a pathogen that represents a global threat to human health, especially in hospital environments [1]. Information about ESBL carriage in this species recovered from companion animals, such as dogs, is limited [10]. The ST307 clone of K. pneumoniae found in our study is considered a potential high-risk clone for humans and has been associated with different ESBL-and carbapenemase-encoding genes [48,49]. Recently, it has been obtained from sick and healthy dogs in Vila Real, a city in northern Portugal very close to Galicia, carrying bla CTX-M-15 and bla SHV-28 [50]. This clone (including CTX-M-15-SHV-28-producing isolates) was also detected in 27% of the poultry meat samples analyzed by Díaz-Jiménez et al. in Galicia [34].
Fortunately, no carbapenemase-producing Enterobacteriaceae were recovered among the dogs studied. In a previous work, a 0.6% prevalence of carbapenemase-producing Enterobacteriaceae in the fecal microbiota of companion dogs attending a veterinary hospital in the Community of Madrid (Spain) was reported [13]. Probably because carbapenems are not used in veterinary medicine, bacteria resistant to these drugs are less common than ESBL-producing bacteria in companion animals [14].
The present study has limitations, such as its cross-sectional design, which did not allow subsequent follow up of the dogs studied. Moreover, information about risk factors for MDR bacteria colonization/infection was not available. However, we provide data on the prevalence of ESBL-and pAmpC-producing Enterobacteriaceae among healthy rural and urban dogs in northwest Spain, including extraintestinal pathogenic E. coli lineages, such as CC648, highlighting the potential Antibiotics 2020, 9, 468 7 of 12 role of these animals in the transmission to humans of high-risk pathogens and resistance genes. Therefore, within a "One-Health" approach, their surveillance should be a priority line in the fight against antimicrobial resistance.

Sample Collection, Culture, and Bacterial Identification
A total of 179 fresh fecal specimens were collected during May and June 2019 from individual healthy dogs living in rural and urban environments in Galicia, a ca. 29,500 km 2 region in Northwest Spain. The healthy status of the dogs was established by the veterinary team in charge of the sampling. Sampling was designed to be representative of the entire territory studied. Thus, a total of 43 different geographical areas were screened, selecting dogs from different rural environments of the four provinces of Galicia (A Coruña, Lugo, Ourense, and Pontevedra), as well as from the main cities in the same region. Urban refers to dogs that live in flats with their owners in large or medium-sized towns in Galicia. Their function is as a companion animal (pets) and they do not contact with livestock. In contrast, rural refers to dogs which usually live in rural areas, in smaller towns or villages. But the most important is that the latter are used as guard dogs in farms, or for hunting. Most of these animals are in contact with livestock (poultry, ruminants -bovine and ovine-and porcine) and with wildlife. The sampling size was calculated based on the dog population in Galicia, which according to official data for 2019 (Galician Registry of Identification of Companion Animals, Department of the Environment of the Xunta de Galicia, Spain) is 609,804 dogs. Of these, 147,284 (24.2%) are urban dogs and 462,520 (75.8%) are rural dogs. In the present study, we have sampled 179 dogs, of which 48 are urban (26.8%) and 131 rural (73.2%) [ Table S1]. The rural vs. urban proportions of this work were adjusted to the values of the geographic area. In order to avoid biases, in those cases in which the same owner had several dogs or several dogs lived together in the same area, a single sample of a representative individual was collected. Dogs included in the study had not received any antimicrobial treatment during the previous four weeks. Samples were kept refrigerated (4 • C) in sterile swabs until processing in the laboratory within 24 h after sampling. For this, they were plated on Chromagar ESBL (bioMérieux, Marcy l´Étoile, France), Chromid Carba Smart (bioMérieux), Chromagar OXA-48 (bioMérieux), and also on Columbia agar with 5% sheep blood (bioMérieux) used as a growth control. Bacterial isolates growing in selective media were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS, Bruker Daltronics GmbH, Bremen, Germany).
All isolates were tested for ESBL-encoding genes (bla TEM , bla SHV , and bla CTX-M ) and for pAmpCs by PCR amplification followed by sequencing of the positive amplicons using specific primers [ Table S2]. Genes encoding plasmid-mediated colistin resistance (mcr-1 to mcr-5) were also screened as previously described [52].
The phylogroup of E. coli isolates was determined following the scheme of Clermont et al. [27] [ Table S4]. Isolates with ExPEC and/or UPEC status were further characterized for their serotypes, clonotypes and STs. Serotyping was established using the method previously described by Guinee et al. [55] with antisera against O (O1 to O185) and H (H1 to H56) antigens. Clonotyping was accomplished by sequencing 469 nucleotides (nt) internal to the fumC gene and 489 nt internal to fimH, which allowed us to define the CH type [56] [ Table S5]. ST assignment for E. coli and for K. pneumoniae isolates was performed according to the Achtman and the Diancourt MLST schemes, respectively [57,58] [Table S6 and Table S7].
Pulsed-field gel electrophoresis (PFGE) was performed to E. coli isolates as previously described using XbaI [34], and the profiles obtained were compared and analyzed by InfoQuest™FP v.4.5 software (Bio-Rad Laboratories). A dendrogram was constructed by the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method, based on Dice's similarity coefficient (1.5% band tolerance; 1.5% optimization).

Statistical Analysis
Differences in colonization between urban and rural dogs were analyzed by a two-tailed Fisher's exact test, with p values of less than 0.05 being considered as statistically significant.

Conclusions
Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistance.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/8/468/s1, Table S1: dog sampling data; Table S2: primers used for the detection and/or sequencing of bla CTX-M , bla SHV , bla TEM , bla CMY , and mcr genes; Table S3: targets and primers associated with extraintestinal pathogenic E. coli; Table S4: targets and primers to determine phylogroups of E. coli (Clermont et al., 2013); Table S5: targets and primers used to determine clonotypes; Table S6: targets and primers to determine sequence types by MLST (E. coli), Table S7: targets and primers to determine sequence types by MLST (K. pneumoniae).