Antimicrobial Resistance and Virulence in Escherichia coli from Broiler Production Unit: Genetic Insights for One Health ^†

Abstract

The overuse of antibiotics in livestock contributes to antimicrobial resistance and zoonotic risk. This study investigated 19 Escherichia coli isolates from broiler feces (Savinor, Portugal), characterizing resistance genes, virulence factors, integrases, and phylogenetic groups by PCR. Most isolates carried ampC, tetA, blaCTX-M, and qnrS; all harbored fimA, and six had int1. Phylogroup A predominated. Resistance was mainly found in commensal groups, highlighting adaptation to poultry environments. The findings underscore the need for regional antimicrobial resistance monitoring and One Health strategies. Stewardship, biosecurity, and alternative measures are vital to mitigate antimicrobial resistance spread and zoonotic potential.

1. Introduction

Antimicrobial resistance in livestock, particularly in poultry production, is a growing concern in Europe and globally [1]. In Portugal, the broiler industry plays a major role in the national meat supply, with intensive farming systems often relying on antimicrobial use to ensure animal health and productivity [2,3]. This practice has contributed to the selection and spread of resistant bacteria, including multidrug-resistant Escherichia coli. Recent EU surveillance reports show high resistance levels among E. coli from broilers, especially in Southern Europe. In Portugal, persistently high resistance rates to tetracyclines, sulfonamides, and quinolones have been observed, aligning with broader EU trends [4]. These resistant strains can serve as reservoirs of resistance genes that may transfer to human pathogens, posing a risk to public health and food safety [3].

E. coli is a versatile microorganism that colonizes the gastrointestinal tract of humans and animals as a commensal. However, pathogenic variants are linked to a wide range of infections, including septicemia, urinary tract infections, and gastrointestinal diseases [5,6,7]. These variants are grouped into pathotypes, each linked to specific clinical syndromes. Intestinal Pathogenic E. coli (IPEC) strains mainly cause gastrointestinal illness, ranging from mild diarrhea to severe colitis. Extraintestinal Pathogenic E. coli (ExPEC) strains, typically asymptomatic in the gut, can cause severe disease upon dissemination to other body sites [8]. Key ExPEC variants include Avian Pathogenic E. coli (APEC), Neonatal Meningitis-associated E. coli (NMEC), and Uropathogenic E. coli (UPEC) [9].

Among these, APEC strains are of relevance in poultry production, where they have been identified as a primary cause of morbidity, mortality, and economic losses [10]. Notably, some APEC strains exhibit genetic resemblance to human ExPEC, raising concerns about zoonotic potential and food safety risks [11,12]. In recent years, the increasing prevalence of antimicrobial resistance in APEC strains has emerged as a critical concern for both animal and public health, particularly within the One Health framework [13,14]. The resistance genes carried by these strains can be transferred to other bacteria, including human pathogens, through mobile genetic elements such as plasmids and integrons [15].

Fecal samples from broiler chickens are key to monitoring E. coli strains in poultry environments. These isolates reflect antimicrobial selective pressure on farms and provide insight into the spread of resistance genes within and beyond the poultry production chain [16,17]. Antimicrobial resistance studies have typically cultured indicator species, such as E. coli, to measure antimicrobial resistance [3]. While previous genomic studies have illuminated the resistance and virulence profiles of E. coli in poultry, there remains limited information on the genetic characteristics of Portuguese fecal isolates, particularly regarding their potential for resistance gene transmission and their phylogenetic background [8].

This study aims to address these gaps by performing a comprehensive molecular characterization of E. coli isolates recovered from fecal samples of broiler chickens. Resistance genes, virulence factors, integrases, and phylogenetic groups were analyzed to better understand the antimicrobial resistance burden and zoonotic risk associated with poultry farming. This research contributes to surveillance efforts and supports the development of strategies for responsible antimicrobial use and sustainable animal production. Although similar studies exist elsewhere, data on fecal E. coli from broilers in Portugal, especially with molecular detail under the One Health perspective, remain limited.

2. Methodology

2.1. Study Design and Sample Collection

This was a cross-sectional study based on 19 E. coli isolates obtained from 40 fecal samples of apparently healthy broiler chickens. The samples were collected in November 2021 at a single poultry production facility in Northern Portugal (Savinor), during routine monitoring. The birds were in the production cycle typical for broiler rearing. Feces were collected aseptically, indoors, under controlled environmental conditions typical of commercial broiler production, and transported to the laboratory under refrigeration. E. coli was isolated using Chromocult^® Coliform agar, and one isolate per positive sample was selected for molecular analysis.

2.2. Genomic DNA Extraction

Genomic DNA was extracted from 19 E. coli recovered from fecal samples of broiler chickens at a poultry production facility in Portugal (Savinor) using the boiling method. Briefly, an overnight culture of a single colony was suspended in 1 mL of Milli-Q water and heated at 100 °C for 15 min to lyse the cells. The lysate was then centrifuged at 12,000 rpm for two minutes. The resulting supernatant, which contained the genomic DNA, was collected and stored at −20 °C for future use.

2.3. Polymerase Chain Reaction-Based Genetic Analysis

PCR reactions were performed in a 50 μL final volume containing 5 μL of PCR buffer, 1.5 μL of MgCl₂, 1 μL of 2 mM dNTPs, 1 μL of each primer, 0.3 μL of Taq DNA polymerase, and 10 μL of DNA template. Nuclease-free water was added to adjust the final volume. The presence of genes encoding resistance to β-lactams (ampC, blaTEM, blaSHV, blaCTX-M, blaCTX-M-9, blaCTX-M-15, blaIMP, blaVIM, and blaOXA) and non-β-lactam antibiotics was investigated. The latter included genes conferring resistance to aminoglycosides (aac(3)-II, aac(3)-IV, aac(6′)-aph(2″), aadA1, and aadA5), streptomycin (strA and strB), tetracycline (tetA, and tetB), quinolones (qnrA, qnrS, and aac(6′)-Ib), sulfonamides (dfrA, sul1, sul2, and sul3), and chloramphenicol (cmlA and floR). Additionally, PCR screening was conducted for virulence-associated genes, including fimA (type 1 fimbriae), hlyA (hemolysin), cnf1 (cytotoxic necrotizing factor), papC (P fimbriae), and aer (aerobactin iron uptake system). The presence of int1 and int2, encoding class 1 and class 2 integrases, respectively, was also investigated. Phylogenetic grouping was performed using a triplex PCR targeting chuA, yjaA, and the DNA fragment TspE4.C2. The presence or absence of these markers allowed classification into groups A, B1, B2, or D according to Clermont’s algorithm [18].

3. Results and Discussion

3.1. Antibiotic Resistance Genes and Phylogenetic Groups

The antibiotic resistance genotypes are presented in

Table 1. Genotypic profiles of antimicrobial resistance genes and phylogenetic groups of E. coli isolates from broiler fecal samples.

Isolate	Genotype	Phylogroup
JR6	ampC, blaVIM	B1
JR7	ampC, blaCTX-M	A
JR8	ampC, blaCTX-M, tetA, qnrS	A
JR9	ampC, blaSHV, blaTEM, aadA5	B1
JR10	ampC, blaCTX-M, tetA, qnrS	A
JR11	ampC, blaSHV, blaVIM, blaIMP, blaTEM, aadA1, tetA, qnrS	A
JR12	ampC, blaCTX-M, blaVIM, tetA, qnrS	A
JR13	ampC, blaCTX-M, blaVIM, tetA, qnrS	A
JR14	ampC, blaSHV, blaTEM, aadA5, tetA, sul2, qnrS	B1
JR15	-	B1
JR16	ampC, blaSHV, blaTEM, blaCTX-M-9, tetA, sul2, strB	B1
JR17	ampC, blaCTX-M, tetA	A
JR18	ampC, blaCTX-M, tetA	A
JR19	ampC, blaCTX-M, tetA	A
JR20	ampC, blaCTX-M, tetA	A
JR21	ampC, blaCTX-M, tetA	A
JR22	ampC, aac(3)-II, aadA1	A
JR23	-	D
JR24	ampC, blaSHV, blaTEM, aadA1, tetA, sul1, sul2, strB, qnrS	D

- No resistance genes were identified.

. Overall, 17 out of the 19 isolates harbored at least one antimicrobial resistance gene. The analysis of resistance genotypes revealed a high prevalence of β-lactamase genes among the E. coli isolates, with ampC detected in 89.5% of them. This finding underscores the widespread dissemination of resistance mechanisms against β-lactam antibiotics in broiler production. Extended-spectrum β-lactamases (ESBLs), particularly blaCTX-M (52.6%), were also frequently detected, raising public health concerns due to their clinical relevance. The blaCTX-M-9 gene, which was identified in strain JR16, was first reported in Portuguese poultry in 2012 [19]. Since the use of third-generation cephalosporins is not permitted in European poultry production, the emergence of ESBL/AmpC-producing E. coli strains may be associated with co-selection pressure exerted using other antibiotics, such as sulfonamides, tetracyclines, and fluoroquinolones, as well as biocidal substances like quaternary ammonium compounds and copper sulfate [20].

A diverse range of non-β-lactam resistance genes was identified. All of these correspond to antibiotic classes that are authorized under regulated use in Portuguese poultry farming [21]. Regarding aminoglycoside resistance genes, aadA1 was the most frequently detected (15.8%), followed by strB (10.5%), aadA5 (10.5%), and aac(3)-II (5.3%). Similar values for aadA1 and aadA5 were presented in broilers in Italy [22]. The strA, aac(3)-IV, and aac(6′)-aph(2″) genes were not found. The tetA gene was detected in 68.4% of the isolates, while tetB was not detected at all. The high frequency of tetA (68.4%) suggests a historic selective pressure from tetracycline use in poultry farming. This gene is predominant in E. coli isolates from broilers [23]. In the case of quinolone resistance genes, qnrS was the only gene detected, and it was present in 36.8% of the isolates. Sulfonamide and trimethoprim resistance genes were also prevalent, with sul2 being the most common (15.8%), followed by sul1 (5.3%). Similar results were presented by Salerno [24]. Regarding chloramphenicol resistance, no genes were detected.

Several isolates (e.g., JR11, JR14, JR16) carried multiple resistance genes, indicating multidrug-resistant strains likely associated with mobile genetic elements such as plasmids or integrons. These results suggest potential horizontal gene transfer and the role of broilers as reservoirs of antimicrobial resistance, reinforcing the need for integrated surveillance and responsible antimicrobial use within a One Health framework [25].

In addition to the analysis of resistance genes, the isolates were classified into phylogroups to infer their pathogenic potential and evolutionary origin. The majority (63.2%, n = 12) of the isolates belonged to phylogroup A, followed by B1 (26.3%, n = 5) and D (10.5%, n = 2). No isolates were identified in phylogroup B2. Phylogroups A and B1 are typically associated with commensal strains or strains with limited virulence potential, commonly found in the intestinal microbiota of healthy hosts [26]. However, despite being classified as commensals, several isolates from phylogroups A and B1 carried multiple resistance genes (e.g., blaCTX-M, tetA, qnrS, aadA), indicating that even strains with low intrinsic virulence potential may serve as important reservoirs of antimicrobial resistance determinants. This raises concerns from a One Health perspective, as these strains may contribute to the spread of resistance to more virulent or zoonotic bacteria.

In contrast, phylogroup D, which has been linked to ExPEC, was identified in two isolates, and one exhibited resistance to several antibiotic classes, underscoring the potential pathogenic and public health relevance of this group. The absence of phylogroup B2, commonly associated with highly virulent ExPEC strains in human infections, may reflect host specificity or selective pressures in the poultry production environment [27].

Taken together, the combination of resistance and phylogroup data highlights the complexity of the E. coli population in broilers, with the circulation of genetically diverse, multidrug-resistant strains capable of acting as silent reservoirs for clinically relevant resistance genes.

3.2. Virulence-Associated Genes and Integrases

The virulence gene screening revealed a universal presence of fimA among all isolates (100%). The fimA gene encodes the major subunit of type 1 fimbriae, structures critical for bacterial adhesion, biofilm formation, and the colonization of mucosal surfaces [28]. Its ubiquitous presence suggests that this gene plays a fundamental role in the colonization capacity of these strains, potentially contributing to early infection processes even in non-clinical isolates. This finding may indicate a baseline virulence potential shared across the population studied. In contrast, the aer gene, encoding the siderophore aerobactin involved in iron acquisition, was detected in only five isolates (26.3%). This low prevalence suggests a limited capacity for iron uptake via this specific mechanism, which may correlate with a reduced potential for systemic pathogenicity. Alternatively, the absence of selective pressure in the isolates’ natural environments could explain the scarcity of this gene, as siderophore production is often essential in iron-limited host niches such as blood or the urinary tract [29]. No additional virulence genes were detected, which may reflect a generally low virulence profile of the isolates. However, this could also be influenced by the limited gene panel used in the PCR assays, which may not encompass the full diversity of known virulence determinants.

With respect to mobile genetic elements, class 1 integrons (int1) were identified in six isolates (31.6%). Class 1 integrons are frequently associated with the horizontal transfer of antimicrobial resistance and virulence genes, especially in clinical settings [30]. Their detection in nearly one-third of the isolates raises concerns about the potential for gene dissemination, particularly in a One Health context, where environmental or animal strains may act as reservoirs or conduits for resistance gene flow. Importantly, no isolates harbored class 2 integrons (int2), which are less prevalent and typically associated with high antibiotic selective pressure [31]. Their absence reinforces the hypothesis that the isolates may originate from sources with moderately lower exposure to antimicrobial agents. The predominance of class 1 integrons over class 2 integrons is consistent with previous studies that also showed a higher prevalence of this class in poultry isolates [32,33].

4. Conclusions

This study highlights the widespread presence of antimicrobial resistance genes among E. coli isolates from broiler chickens, with particular emphasis on β-lactamases, including a high prevalence of ampC and ESBLs such as blaCTX-M. The identification of multiple resistance genes in several isolates suggests a considerable burden of multidrug resistance, likely mediated by mobile genetic elements such as plasmids and class 1 integrons. Phylogenetic analysis revealed a predominance of phylogroups A and B1, commonly associated with commensal strains, and the absence of the highly virulent phylogroup B2. The presence of such multidrug-resistant profiles in phylogroups traditionally considered benign raises concern from a One Health perspective, as these bacteria may transfer resistance genes to more virulent or zoonotic strains under favorable ecological conditions. These findings underscore the role of poultry as a potential reservoir for resistance determinants of clinical relevance and reinforce the need for prudent antimicrobial use in animal production systems.

Despite the high resistance potential, the virulence gene profile was relatively limited, with fimA being the only universally detected gene, suggesting a conserved capacity for colonization but a lower potential for systemic pathogenicity. The low prevalence of aer and the absence of other tested virulence factors may reflect a restricted virulence repertoire or limitations of the gene panel used. The detection of class 1 integrons highlights the relevance of continued surveillance, especially in non-clinical settings that may contribute to the broader dissemination of resistance and virulence factors.

This study has some limitations that should be acknowledged. The relatively small number of isolates (n = 19) limits the generalizability of the findings, and molecular characterization was restricted to PCR screening of selected genes. Whole-genome sequencing and a broader panel of resistance and virulence markers would provide a more comprehensive understanding of genetic potential and epidemiological relevance. However, the combination of high resistance and moderate virulence in commensal E. coli from poultry underscores the need for integrated surveillance and containment strategies under a One Health approach. No epidemiological link to clinical infections was established, as the isolates came from healthy broilers and were collected during routine sampling. Future studies integrating genomic and epidemiological data are needed to assess potential cross-species transmission.