1. IntroductionAnimal food products, such as eggs, meat, and milk, are abundant in proteins essential for the body’s maintenance, repair, and growth [1]. Poultry is among the most reported carriers of foodborne pathogens [2]. Hughes et al. [3] also asserted that poultry meat, red meat, and eggs are recognized as major vectors for the transmission of pathogens.E. coli is a typical inhabitant of the gut of warm-blooded animals and is used frequently as an indicator bacterium of fecal contamination in the food industry. E. coli is a non-spore-forming, Gram-negative rod, usually motile by peritrichous flagella that is a member of the Enterobacteriaceae [4]. Many monitoring programs include E. coli because they are established markers of fecal contamination, ubiquitous in food-producing animals, easy to cultivate, and readily acquire resistance mechanisms to combat agents with activity against Gram-negative organisms [5]. They are also known reservoirs of ARGs that can be transferred horizontally to and from other closely related bacteria [6]. E. coli is considered a good indicator of the selective pressure imposed by antimicrobial use in food animals and has been hypothesized to be a potential predictor of emerging resistance in pathogenic bacteria that cannot be recovered from meat or animal samples Furthermore, annual trends indicate a possible correlation between Salmonella spp. and E. coli resistance [7]. The reason for using E. coli as an indicator is that it appears only at low background levels in the environment but possesses high survival rates [8].Microorganisms from animal, environmental, and human sources normally contaminate raw foods [9]. The initial number of living microorganisms, including pathogens, will be substantially reduced when properly processed. However, the prevalence of pathogenic microorganisms and deterioration in ready-to-eat (RTE) foods can substantially increase through post-processing handling activities, the duration of exposure at points of sale and storage conditions [10].At slaughter, resistant strains from the gut readily soil poultry carcasses, and as a result, poultry meat is often contaminated with resistant E. coli, likewise eggs become contaminated during laying [11,12,13,14,15,16,17,18]. Hence, resistant fecal E. coli from poultry can infect humans both directly and via food. These resistant bacteria may colonize the human intestinal tract and may also transfer resistance genes to human endogenous flora [19]. In the case of eggs, microbial contamination has a major outcome for the poultry industry and contaminated eggs are a serious public health problem worldwide. The importance of these diseases in humans can range from mild symptoms to life-threatening situations [20]. Egg and its products are an important component source of necessary nutrients. Eggs can act as a vector in the transmission of food poisoning microorganisms. Many investigations have already reported contamination of eggs with Salmonella spp., Listeria monocytogenes [21], Campylobacter spp. [22], and E. coli [23], and if the appropriate treatment does not occur, these pathogens can reach consumers’ homes and become a food safety problem.Over the past 50 years, the use of antibiotics combined with strict biosecurity and hygiene measures has helped the poultry industry grow, preventing the negative impacts of many avian diseases caused by previously referred microorganisms [24]. The use of antibiotics to control gastrointestinal infections can lead to a change in the intestinal microbiota of hens, which can influence their immunity and health [25].Scientific evidence suggests that the use of antimicrobials in animal production may promote bacterial resistance in treated animals [26]. Bacterial resistance of E. coli to antibiotics has been the subject of several studies in recent years [4,27]. Bacterial resistance to animal antibiotics is a public health problem. Antibiotic abuse and associated selection pressure led to decreased therapeutic efficacy and created populations of antibiotic-resistant microorganisms. Antibiotic resistance can spread over time, despite the suspension of antibiotic use. Several studies have suggested that antimicrobial resistance (AMR) bacteria and their AMR determinants can be transmitted from food animals to humans by direct contact and/or through animal products [28,29].The use of antibiotics for growth promotion purposes is prohibited in the European Union. In intensive production systems, animals are exposed to a high risk of infection, as they live under stressful conditions and are driven to increase productivity. In these systems, the frequent application of antibiotics are perfect circumstances for bacterial strains to develop and resist antibiotics [30,31,32]. In Portugal, antibiotics used for application in animals, authorized for the treatment of infections, are oxytetracycline (OCT), amoxicillin (AMX), tylosin (TYL), colistin (CL), doxycycline (DOX), ampicillin (AMP), tiamulin (TIA), sulfadiazine (SFD), and enrofloxacin (ENR) [33].E. coli strains have been classified based on genetic substructures associated with different phylogenies in different phylogroups that present different phenotypic and genotypic characteristics [34]. The PCR-based assay developed by Clermont et al. [35] is intended for the classification of E. coli strains into the major phylogroups A, B1, B2, and D; however, this method could only validate 80–85% of all E. coli phylogroups and it is sometimes necessary to use more alternatives [36,37]. A modification was made to the triplex method by adding one gene, resulting in a quadruple PCR [38]. Five strains or clades (I–V) were also found in E. coli strains, of which clade I is currently included in the phylogenetic grouping, making eight groups: A, B1, B2, C, D, E, F, and clade I [39]. Studies have shown that strains associated with virulent extraintestinal infection generally belong to phylogroups B2, D, or E and that commensal isolates of E. coli are generally affiliated with groups A and B1 [40,41].Although this microorganism is considered a commensal, there are strains that can cause diarrheal diseases [42]. The virulence attributes have been used to differentiate pathogenic strains of E. coli and divided into diarrheal pathogens causing diarrhea (DEC) and extraintestinal E. coli (ExPEC) [43] based on the site of infection. There are six classic pathotypes of DEC: enteropathogenic (EPEC), shiga toxin–producing (STEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), and diffusely adherent E. coli (DAEC) [44]. Two additional E. coli pathotypes, belonging to ExPEC, are responsible for extraintestinal infections: uropathogenic E. coli (UPEC) causing urinary tract infections and neonatal meningitis associated E. coli (NMEC) [45,46]. Avian pathogenic E. coli (APEC) are a member of DEC, closely related to EPEC, which are frequently assigned to specific phylogenetic groups along with human UPEC and NMEC that cause disease outside the intestine [47,48]. During the last decades, the emergence of AMR bacteria has been enormously announced worldwide. In relation to an extensive use of β-lactam antibiotics in both clinical and nonclinical settings, a great diversity of β-lactamase types has consequently emerged [49]. In this context, ESBL constitute a mechanism of resistance of great clinical relevance that is spreading not only in humans but also among domestic animals [50]. ESBL-producing Enterobacteriaceae have been recognized as highly prevalent in food-producing animals and derived food, in the Mediterranean countries [51,52,53,54,55].

This study was conducted to investigate the prevalence and characterization of β-glucoronidase positive E. coli in fecal samples collected from breeding and laying hens in Portugal. The isolates were characterized by their phylogroups and a search for virulence genes was performed (pathotypes). Antimicrobial resistance assays and ESBL-associated genes detection were also performed. The main objectives were to increase knowledge regarding E. coli carriage in hens and to raise aware of the risks that consumers may be exposed to with the spread of MDR strains, especially if good hygiene practices are not completely fulfilled, causing a public health and/or food safety problem.

3. DiscussionE. coli isolates from breeding hens were more resistant to AMP (83.3%) than to ENRO (69.2%). Likewise, results from laying hens also demonstrate that the isolates were more resistant to AMP (75.1%) than to ENRO (47%). Of the total samples, isolates were more resistant to AMP (79.3%) than ENRO (58.4%), obtaining a higher number of isolates resistant to AMP than resistant to ENRO. The use of both antimicrobials is allowed for disease treatments in animals and their spread in farms may lead to an increase in microbial resistance in the population. Seventy-nine E. coli strains were isolated from the fecal samples of laying hens. E. coli strains are now classified into eight phylogroups: A, B1, B2, C, D, E, F, and clade I [41]. Studies have shown that isolates belonging to the A and B1 phylogroups are commensals, while those that belong to the B2, D, and E groups are the extraintestinal pathogenic strains [58,59,60]. Obeng et al. [61] determined the phylogenetic groups of E. coli isolates from the feces of intensively farmed and free-range poultry from South Australia. They found that the predominant phylogenetic groups were phylogroup B1 with 39.4% and phylogroup A with 32.3%. In a more recent study, Hayashi et al. [62], revealed that the 70 E. coli isolates from hens’ samples in Japan majorly belonged to group B1 (25.7%) and group A (14.3%). Similar to our results, these studies also demonstrate the predominance of B1 and A in samples from hens. In the study by Projahn et al. [63], broiler breeder lots and the corresponding eggs were analyzed. Of the eggs tested, 0.9% (n = 560) were contaminated on the outer surface of the shell. Additional analysis showed a relationship between the species found in the eggs and those isolated from the corresponding lots of origin, which demonstrates a pseudo-vertical transfer of Enterobacteriaceae to the hatchery. Isolates of the four positive eggs of flock were all found to be E. coli of the phylogroup B1. This study demonstrates the contamination of eggshells through E. coli contaminated feces from egg laying hens, presenting a risk to the health of consumers. It is also possible that they constitute a possible source of contamination for the chicks, given the detection of their presence in the feces of breeding hens, thus representing a risk for chicken and egg consumers. Furthermore, isolates belonging to phylogroups D and E that have been associated with virulent extraintestinal infection were found. Adefioye et al. [64] concluded in their study that most of the human isolates from fecal samples of apparently healthy individuals belonged to phylogroup B1. They only found a few isolates belonging to B2 and D phylogroups and concluded that these isolates were mostly commensals, which as a result of antibiotic exposure and other environmental and genetic factors, may revert to being pathogenic [65].Tenaillon et al. [34] referred that genetic diversity of E. coli exhibits host taxonomic and environmental components. This can be illustrated by the prevalence of the four main phylogenetic groups in various human and animal populations. In humans, group A strains are predominant (40.5%), followed by B2 strains (25.5%), while B1 and D strains (17% each) are less common. In animals, there is a predominance of B1 strains (41%), followed by A strains (22%), B2 (21%) and, to a lesser extent, D strains (16%). Our results corroborate this statement since the most predominant phylogroups found in hens were B1 and A.The presence of antibiotic-resistant foodborne pathogens in food can lead to gastrointestinal disturbances in humans [66]. On the other hand, antibiotic-resistant pathogens can transfer the gene to other microorganisms, resulting in the spread of AMR pathogens [67,68]. There are not many studies available concerning AMR in hens’ fecal samples. Two studies used a similar number of isolates from the same source. In the study by Langata et al. [69], AMR patterns among 85 resistant hen fecal isolates in Kenya were characterized and Abassi et al. [70] characterized 83 E. coli fecal isolates recovered from hens, in Tunisia. NAL was used in the three studies; Portugal has the higher number of resistant isolates (65.8%) while Kenya only has 18.8%. In the case of TET, the three studies presented a higher number of isolates being resistant between 90% in Tunisia and 42% in Kenya. Resistance to CTX was found in Tunisia and the number of isolates resistant to CAZ were similar in Tunisia and Portugal, and were not tested in Kenya. CIP was tested in Portugal and Kenya with 60% of Portuguese isolates being resistant and only 1.2% in Kenya. The differences found can be related to different use of antibiotics in agriculture and chicken or egg production.In Portugal, the Directorate General for Food and Veterinary (DGAV) [33] controls the use of antibiotics and reports those authorized for the treatment of infections. Our results demonstrate antimicrobial-resistant isolates in hens’ feces that are not present in these reports, that is, that are not permitted for animal use.

When analyzing the results of antimicrobial susceptibility differentiating the types of hens between breeding and egg laying hens, we found that isolates from the feces of breeding hens showed a higher percentage of resistance to a greater number of antibiotics. Excluding that for Azithromycin, a greater percentage of resistant isolates was observed in breeding hens. This can be related to prophylactic use of antibiotics in poultry production.

Based on these results, it is not possible to make any correlation between the resistances and phylogroups.

Knowledge about MDR load and resistance patterns in isolates extracted from food-producing animals is imperative to design targeted interventions to limit antibiotic use. The use of commensal intestinal E. coli as a marker for the presence of resistance in bacterial flora is a critical component of MDR surveillance programs in food producing and wild animals [71]. In Liu et al. [72], fecal samples were obtained from six broiler fattening farms in China. They describe that the MDR of E. coli isolates was 91%. According to Koju et al. [73], hen caecum samples were collected from slaughterhouses/stores in Nepal and it was found that 71% showed resistance to at least three categories of antimicrobials. Comparing the MDR value obtained in our study (57%) with the values from those reports, we found that the number of the MDR of E. coli isolates from hen samples in Portugal is not as high as in other countries, despite this, it is still a worrying reality. The monitoring and treatment of drug-resistant bacteria in the poultry industry will be a long and difficult task, and one which will require a collaborative effort and should include aspects of chick breeding, the breeding environment, and feed additives.

The most common pattern of antibiotic resistance is the one that conjugates penicillins, fluoroquinolones, tetracyclines, macrolides, and sulfonamides. When we compare these data with those published by DGAV, we find that three of the categories (PEN, TET, and MA) present in the most common pattern presented by our isolates, correspond to the three classes of antibiotics most commercialized in Portugal.

Isolates from breeding hens showed higher AMR and more MDR isolates when compared to isolates from laying hens. Once again, these results demonstrate the relationship between the prophylactic use of antibiotics and poultry production.

According to the European Food Safety Authority (EFSA), the prevalence of presumptive ESBL E. coli producers in the different animal species and their products varies within the EU countries [74]. In Denmark, isolates of CTX-M producing E. coli from healthy egg laying hens (not exposed to antimicrobial agents) were found in stool samples collected from the ground and cloacal swabs, so there is a possibility to find ESBL–E. coli between eggshells, which can be contaminated by contact with feces [75]. In the present study, the blaTEM and blaCTX-M genes were detected in three E. coli isolates (5%) and could be considered as potential ESBL producers. Both the blaTEM and blaCTX-M genes have been detected among food producing meat at retail and broiler products in recent studies in Portugal [76,77]. Machado et al. [76] identified that 60% of uncooked hen carcasses (n = 20) and 10% of feces from healthy hens (n = 20) were positive for blaTEM and blaCTX-M genes. Clemente et al. [77] examined the existence of ESBL in meat collected at retail stores in Portugal and found a prevalence of 30.3% in poultry meat, while this was 11.8% and 10.5% for beef and pork, respectively. The results of these studies demonstrate the importance of monitoring the ESBL genes in E. coli isolates and how their study is essential for food chain safety and human health. In fact, and interesting to point out, is that the three isolates harboring ESBL genes were from three different farms, from distinct types of hens (breeding and laying) and from two different phylogroups (B1 and E) and all showing MDR. Mahmud et al. [78] have already described a high prevalence of MDR in ESBL-producing E. coli and 71% of ESBL-producing E. coli isolates were MDR. The fact that isolates came from different places indicates the potential spread of microorganisms highly resistant to antimicrobials that can reach consumers’ homes, thus changing their environmental microbiota, and if food becomes contaminated, it could accumulate and proliferate in the intestine, where genetic transfer can occur.