Next Article in Journal
Time Trends in Prevalence and Antimicrobial Resistance of Respiratory Pathogens in a Tertiary Hospital in Rome, Italy: A Retrospective Analysis (2018–2023)
Previous Article in Journal
Cold Plasma as a Revolutionary Antimicrobial Modality: A Multifaceted Weapon Against Antibiotic Resistance
Previous Article in Special Issue
Prevalence and Characterization of the Antimicrobial Resistance and Virulence Profiles of Staphylococcus aureus in Ready-to-Eat (Meat, Chicken, and Tuna) Pizzas in Mansoura City, Egypt
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antibiotics
Volume 14
Issue 9
10.3390/antibiotics14090931
antibiotics-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phenotypic and Molecular Study of Multidrug-Resistant Escherichia coli Isolates Expressing Diverse Resistance and Virulence Genes from Broilers in Tunisia

by
Ghassan Tayh
1,*,
Fatma Nsibi
1,
Khaled Abdallah
1,
Omar Abbes
2,
Ismail Fliss
3 and
Lilia Messadi
1,*
1
Department of Microbiology and Immunology, National School of Veterinary Medicine, University of Manouba, LR16AGR01, Sidi Thabet, Ariana 2020, Tunisia
2
DICK Company, Poulina Group Holding, Ben Arous 2034, Tunisia
3
Department of Food Sciences and Nutrition, University of Laval, Québec City, QC G1K 7P4, Canada
*
Authors to whom correspondence should be addressed.
Antibiotics 2025, 14(9), 931; https://doi.org/10.3390/antibiotics14090931
Submission received: 20 July 2025 / Revised: 25 August 2025 / Accepted: 26 August 2025 / Published: 15 September 2025
(This article belongs to the Special Issue The Antimicrobial Resistance in the Food Chain)
Browse Figures
Review Reports Versions Notes

Abstract

This study investigated the molecular and phenotypic characteristics of antimicrobial resistance in Escherichia coli isolates recovered from the ceca of healthy broilers in Tunisia. A total of 111 E. coli isolates were obtained from chicken samples collected at slaughterhouses and cultured on cefotaxime-supplemented MacConkey agar. All isolates exhibited a multidrug-resistant (MDR) phenotype, and 72.1% were confirmed as extended-spectrum β-lactamase (ESBL) producers. The most frequent β-lactamase gene was blaCTX-M-G1, followed by blaTEM and blaSHV. Carbapenem resistance genes (blaOXA-48 and blaIMP) were detected in 12.6% and 6.3% of isolates, respectively, while six isolates harbored the colistin resistance gene mcr-1. Among the tested virulence genes, fimH, traT, and iutA were the most prevalent, detected in over 70% of isolates. Class 1 integrons were present in 83% of isolates, and class 2 integrons in 39.6%, with gene cassettes encoding resistance to trimethoprim (dfrA) and streptomycin (aadA). These findings highlight the widespread presence of MDR and ESBL-producing E. coli strains with virulence traits and integrons in poultry, underscoring the risk of transmission to humans. This study provides essential data supporting the implementation of integrated surveillance strategies in line with the One Health approach.

1. Introduction

Escherichia coli belongs to the commensal microbiota that exists as harmless bacteria in the intestine of humans and warm-blooded animals [1]. However, many pathogenic strains can cause intestinal or extraintestinal infections in humans and animals [2]. The pathogenic E. coli strains are categorized into intestinal pathogenic isolates (InPEC) for diarrheagenic E. coli (DEC) that cause gastroenteritis and extraintestinal pathogenic E. coli (ExPEC) that cause urinary tract infection (UTI), meningitis, and pneumonia [2].
The capacity of these strains to cause infections is associated with several virulence determinants such as adhesins, hemolysin, fimbriae, and others, enhancing the bacterial survival in several conditions and potentiating the bacterial pathogenesis [3]. In bacterial pathogens, these factors are encoded by specific genes found on the chromosome or within mobile genetic elements, such as plasmids or transposons [4]. ExPEC strains possess various virulence factors that enhance their ability to infect the host by facilitating adhesion, iron acquisition, invasion, and toxin production. Adhesion molecules such as fimH, BfpA, papGIII, sfa/focDE, and eae promote bacterial attachment to host tissues, while the iron acquisition gene iutA aids in survival by securing essential nutrients. The invasion-associated gene traT contributes to immune evasion, and toxins, including stx-1, stx-2, ehxA, and cdt3, play a crucial role in pathogenicity. These factors collectively enable ExPEC to establish and sustain infections [5,6].
E. coli strains can be classified into seven phylogenetic groups (A, B1, B2, C, D, E, and F, and the eighth is the Escherichia cryptic clade I) by employing a multiplex PCR assay that was developed by Clermont et al. [7]. Different studies revealed that ExPEC strains belong to phylogroups B2 and D, while commensal and diarrhoeagenic strains belong to other phylogroups [8].
Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in poultry, including chickens, turkeys, ducks, and other avian species, leading to systemic colibacillosis characterized by common postmortem lesions. Mortality rates in young chickens can reach up to 53.5% [9]. Avian colibacillosis is considered one of the major causes of economic losses in the poultry sector worldwide. In the United States, more than USD 40 million is expected to be lost in the broiler sector each year as a result of carcass condemnation [10]. APEC shares significant genetic similarity with human extraintestinal pathogenic E. coli (ExPEC), particularly uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC), raising concerns about potential zoonotic transmission from poultry to humans [11]. ExPEC, including APEC, can cause a range of extraintestinal infections in humans, such as urinary tract infections, neonatal meningitis, and sepsis [12], making them important to both public health and the poultry industry.
The global rise of infections caused by microorganisms resistant to antimicrobials, including first-line drugs such as cephalosporins, colistin, carbapenems, and fluoroquinolones, is a critical global problem, leading to serious morbidity, mortality, and health costs and a significant decrease in livestock production [13]. It is predicted that by 2050, antimicrobial resistance (AMR) will become the leading cause of death, according to global estimation, indicating that AMR caused over 1.2 million deaths in 2019, and it is projected to reach about 10 million annually by 2050 [14]. Furthermore, the resistant bacteria from poultry and other food animal sources have been on the increase around the world [15].
Antibiotic resistance is considered a key factor in the development of infection, as a virulence-like factor in specific ecological niches, where antibiotic-resistant bacteria are able to proliferate. This is particularly true in the hospital settings (intensive care units, burn units, etc.), where drug-resistant opportunistic pathogens can spread and cause disease more easily [16]. In Enterobacterales, especially in E. coli and Klebsiella pneumoniae, resistance to β-lactams due to production of extended-spectrum β-lactamases (ESBL) is a significant resistance mechanism that prevents the success of antimicrobial treatment of infections caused by these bacteria [17].
ESBL-producing bacteria have become a major worry in clinical settings worldwide, causing outbreaks related to enzymes of the CTX-M class being the most common, particularly CTX-M-15 [18]. Recently, among different Enterobacteriaceae members globally, ESBLs of the CTX-M type have supplanted SHV- and TEM-ESBLS types [19]. ESBL-producing isolates exhibited multidrug-resistant (MDR) effects to antimicrobials like aminoglycosides, tetracyclines, chloramphenicol, trimethoprim, sulfonamides, and quinolones [20].
Although carbapenems are considered the drug of choice for bacterial infections, resistance to carbapenems has now become a major clinical worry worldwide. This type of resistance is caused by enzymes hydrolyzing carbapenems, and the most influential carbapenemases on the hydrolysis of carbapenems and their geographic distribution are KPC, VIM, IMP, NDM, and OXA-48 types [21]. The presence of transferable genetic elements, such as plasmids, transposons, and integrons, plays a crucial role in the spread of multiple resistance genes [2].
Poultry is considered a highly significant source for the spread of antibiotic-resistant bacteria in the population and environment. Pathogenic E. coli in poultry is a direct menace to both poultry production and human health due to difficulty in treating infections [12].
The spread of antimicrobial-resistant bacteria has emerged as a One Health concern, as these bacteria can be transferred among humans, animals, and the environment. The use of antimicrobials in any of these sectors contributes to the increasing AMR burden [22]. Research indicates that AMR bacteria and their resistance determinants can be transmitted from food-producing animals to humans through direct contact or consumption of animal-derived products [23].
The objective of this study was to assess the prevalence of multidrug-resistant E. coli isolates from broilers intended for human consumption. It also aimed to characterize the genotype of antimicrobial resistance, phylogenetic groups, virulence factors, and integrons in these E. coli isolates.

2. Results

In our study, 111 E. coli isolates were obtained from MacConkey agar supplemented with cefotaxime (CTX 1 µg/mL) from the ceca of chicken broilers from three suppliers, distributed as 51 samples from supplier A, 30 from supplier B, and 30 from supplier C. The isolates were investigated for the determination and characterization of antibiotic resistance.

2.1. Antimicrobial Resistance Pattern

Antimicrobial susceptibility testing showed high resistance to amoxicillin (100%), cephalothin (96.4%), piperacillin (93.7%), cefotaxime (83.8%), cefepime (82%), tetracycline (79.3%), streptomycin (79.3%), aztreonam (77.5%), ceftazidime (75.7%), cefuroxime (73.9%), chloramphenicol (71.2%), and florfenicol (64.9%). Low resistance in E. coli isolates against cefotixin and colistin was observed, as the resistance average was lower than 20% (Figure 1). All isolates were multidrug-resistant (MDR). ESBL phenotype was confirmed in 72.07% (80/111) of isolates.

2.2. Detection of β-Lactamases Genes and Colistin Resistance Genes

The identification of resistance genes by PCR revealed that the most common broad-spectrum β-lactamase genes in the isolates were blaCTX-M-G1 (n = 85), followed by blaTEM (n = 47) and blaSHV (n = 36). The carbapenem resistance genes blaOXA-48 and blaIMP were detected in fourteen (12.6%) and seven (6.3%) isolates, respectively. Eleven isolates were positive for the cephalosporinase CMY gene, and six isolates carried the plasmid-mediated colistin resistance mcr-1 gene (Table 1).
Four β-lactamase genes (blaCTX-M-G1, blaSHV, blaTEM, blaCMY) and (blaCTX-M-G1, blaSHV, blaTEM, blaCMY) were detected in two strains. Three β-lactamase genes 3 (blaCTX-M-G1, blaTEM, blaOXA-48), 3 (blaCTX-M-G1, blaTEM, blaIMP), 2 (blaCTX-M-G1, blaSHV, blaOXA-48), 2 (blaCTX-M-G1, blaSHV, blaTEM), 1(blaSHV, blaTEM, blaCMY), 1 (blaCTX-M-G1, blaTEM, blaCMY), 1 (blaSHV, blaCMY, blaOXA-48), and 1 (blaTEM, blaCMY, blaOXA-48) were present in 14 E. coli isolates (Table S3).

2.3. Identification of Antimicrobial Resistance Genes

A variety of non-β-lactam antimicrobial resistance genes were found in the isolates. The plasmid-mediated quinolone resistance genes qnrS, qnrB, and qnrA were found in twenty-four (21.6%), twenty-two (19.8%), and two (1.8%), respectively. The aminoglycosides (gentamycin and streptomycin) resistance genes aac(3)-II, aac (6)-Ib-cr, aadA-1, and aadA-5 were found in 30 (27%), 32 (28.8%), 80 (72%), and 33 (29.7%), respectively. Twenty-two (19.8%) of the E. coli isolates harbored sul genes [sul1 and sul2] (Table S3, Table 1).

2.4. Virulence Factors Genes

A total of 17 virulence factors were identified in 111 E. coli isolates, with traT, fimH, and iutA having the highest detection rates as they appeared in two-thirds of the isolates. Among the adhesion factors, fimH was the most dominant (83.8%), followed by bfpA (43.2%), eae (11.7%), papGIII (7.2%), and sfa/focDE (3.6%). Toxin genes stx-1, stx-2, cdt3, and ehxA were detected in 38.7%, 7.2%, 2.7%, and 2.7%, respectively. Among the invasion factors, traT was the most predominant (83.8%), followed by iutA (73.9%) and ibeA (0.9%).

2.5. Phylogenetic Groups

The phylogenetic analysis classified the E. coli isolates into A 43 (38.7%), B1 18 (16.2%), F 16 (14.4%), D 15 (13.5%), E 8 (7.2%), B2 6 (5.4%), C 3 (2.7%), and clade I 2 (1.8%).

2.6. Association of Virulence Genes and Resistance Genes Distribution Among Phylogenetic Groups

The analysis of phylogroups distribution among the resistance genes revealed that the resistance genes blaCTX-M-1, blaTEM, blaSHV, blaOXA-48, qnrB, and aac(6)Ib-cr were distributed among phylogenetic groups, whereas mcr-1, blaIMP, qnrS, and qnrA were present only in groups A and B1. β-lactamase genes CTX-M-ESBL, TEM, and SHV were the most dominant in the A group, followed by the F and B1 groups, with a statistically significant difference (p value = 0.003). In addition, quinolone resistance genes qnrS and aac(6)Ib-cr were classified in the A1 and D groups (p value = 0.003 and 0.054, respectively) (Table 2 and Figure 2).
Figure 3 shows the association between virulence genes and phylogenetic groups. The genes traT, fimH, BfpA, iutA, fyuA, papGIII, stx-1, hly, and cnf1 were widely disseminated in the groups. Genes stx-2, eae, and cdt3 were found in groups A, B1, and B2. Group A was the dominant group that carried the virulence genes, whereas ehxA and sfa/focDE were less present in group A isolates.

2.7. Characterization of Integrons

Class 1 integrons were found in 92 (82.9%) of the isolates, and the 3′CS that contains qacEΔ1 and sul1 was detected in 43 (38.7%) of positive class 1 integrons. Class 2 integrons were demonstrated in 44 (39.6%) isolates. Thirty-four isolates harbored both integrons. The sequencing of variable regions (VR) of class 1 and 2 integrons revealed different gene cassette arrangements. In VR of class 1 integrons, gene cassettes containing genes encoding resistance to trimethoprim (dfrA) and streptomycin (aadA) were detected in three different arrangements: dfrA1-aadA1 (eight isolates), dfrA12-aadA2 (five isolates), and aadA1 (three isolates). For the VR of class 2 integrons, the gene cassettes sat2-dfrA1 and sat2-aadA1 were detected in nine and three isolates, respectively (Table S3).

3. Discussion

Poultry and poultry products are mostly popular and consumed foods by humans worldwide. However, few data are available regarding the molecular characterization of E. coli isolated from poultry in Tunisia. In this study, we assessed the molecular characterization of E. coli isolates collected from the ceca of chicken broilers in poultry slaughterhouses from three main geographical regions, extending from Great Tunis in the north, Mahdia in the central–east, and Sfax in the southeast of Tunis.
Our findings revealed that the broilers intended for human consumption could be contaminated with broad-spectrum antibiotic-resistant E. coli, and the possible spread of these strains to humans and the environment is alarming. This is a big threat for Tunisia as a developing country that has limited sufficiently qualified staff and challenges of the economic situation that impact health situations [24].
The ESBL rate among E. coli was confirmed in 80 isolates (72.07%). This rate is consistent with the frequency of ESBL production (76.5%) reported in E. coli isolates from chicken meat in Tunisia [25]. This finding was similar to that of Liu et al. [26] and higher than the rates of ESBL-producing E. coli detected in chicken from Ghana, Algeria, and Tunisia, which were 56.2%, 55.5%, and 20.1%, respectively [27,28,29], as well as in Egypt, where the frequency related to chicken meat was 61.6% [17]. ESBL production has also been reported in cloacal swabs of chickens in recent studies from Malaysia (37.2%) [30] and Taiwan (36.3%) [31]. The comparatively higher rate observed in our isolates may be attributed to the fact that they were collected from the cecum, which generally yields a high frequency of bacterial isolates and resistance traits within the intestinal environment. A Korean study found an occurrence rate of ESBL in chicken of about 94.1% [32]. Many studies confirmed that the high prevalence of ESBL-producing E. coli in broilers is becoming common and more significant than in other food animals. They also revealed that the chicken origin of ESBL–E. coli is related to more resistance to antibiotics compared with ESBL from other food-producing animals [26,33]. From the previous findings, the ESBL–E. coli rates may differ by the sample sources and region. The high ESBL rate in E. coli from poultry is driven by excessive antibiotic use, facilitating resistance selection. Horizontal gene transfer via plasmids accelerates spread, while poor biosecurity, contaminated feed, and environmental persistence further sustain transmission [30,34].
This study reported a high percentage of resistance compared with another Tunisian study conducted in E. coli isolated from 150 healthy poultry, with resistance rates to tetracycline, streptomycin, sulfamethoxazole/trimethoprim, nalidixic acid, ciprofloxacin, amoxicillin/clavulanic acid, cefotaxime, and ceftazidime of about 74.7%, 57%, 57%, 54.4%, 34.2%, 15.2%, 5.1%, and 3.8%, respectively [35]. The higher resistance observed in our study may indicate an increased use of antimicrobials in veterinary medicine in recent years, potentially driving the selection and spread of resistant E. coli strains. In Tunisia, a broad range of antimicrobials is used in food-producing animals, including poultry, for both prophylactic and therapeutic purposes. Commonly administered antibiotics include penicillins (e.g., amoxicillin), tetracyclines, macrolides, colistin, sulfonamides, aminoglycosides, and quinolones, raising concerns about antimicrobial resistance (AMR) and its potential transmission to humans via the food chain and environment.
A study conducted in Qatar recorded high resistance in E. coli isolated from broiler chickens; the percentage of resistance to antibiotic agents (ampicillin, cephalothin, ciprofloxacin, tetracycline, and fosfomycin) in strains was high (76.5–100%), and MDR isolates were 99.3% [11]. In another study from South Korea, they observed highly resistant E. coli isolates to cefotaxime, cefazolin, ampicillin, and piperacillin from fecal samples of chickens collected from the intestinal tracts at slaughterhouses, and all isolates were MDR [32].
All of the isolates were MDR, which indicates that the E. coli isolates may be a reservoir of antibiotic resistance. The use of antimicrobials for disease treatment and prophylaxis in poultry farming may lead to an increase in bacterial resistance. Hundreds of tons of antibiotics are used annually in the agricultural industry [36]. The high frequency of MDR strains is considered a significant health risk because these isolates may disseminate to food products and the environment and, thus, be transferred to humans. Colistin is a last-resort antibiotic in the treatment of MDR Gram-negative bacteria, and fortunately, colistin was susceptible against 92.8% of our isolates. Consequently, this antibiotic may be suggested as a drug of choice for MDR E. coli in our country.
The CTX-M family encoding extended-spectrum β-lactamases is dominant and has become a major worry in health settings worldwide [37]. In this study, the blaCTX-M gene was found in 86 (77.5%) strains. Similar findings were reported in Ghana, where the blaCTX-M gene was the most dominant and confirmed in 95.1% of the strains [27]. The blaTEM and blaSHV genes encoding β-lactamase have been reported in 47 (42.3%) and 36 (32.4%) strains, respectively. The predominance of the CTX-M gene over other β-lactamases genes was recorded in a previous Tunisian study from healthy poultry [38]. However, many studies reported the dominance of the blaTEM and blaSHV genes over blaCTX-M among E. coli from chicken in Algeria, Egypt, and Poland [39,40,41].
The plasmid-mediated colistin resistance determinant (mcr-1) is associated with colistin resistance and is widely detected in different bacteria isolated from humans, food, and livestock [42]. In our study, mcr-1 was detected in six colistin-resistant strains with a prevalence rate of 5.4% (six of all tested isolates). This finding is lower than in previous reports from Malaysia and Egypt [40,43], which found mcr-1 determinant in 27.6%, 41.83%, and 64.3% of the tested E. coli strains, respectively, and they suggested that chicken farms may be a source of colistin-resistant E. coli. A Tunisian study reported mcr-1 from chicken gut microbiota in Tunisia [42]. It has been demonstrated that most mcr-1-positive bacteria include various lactamase genes, such as blaCTX-M, blaOXA-48, blaTEM, blaSHV, and blaCMY. This combination further restricts the therapeutic alternatives because the last two last-resort medications, colistin and carbapenems, are challenged by this occurrence.
The current study reported the carbapenem resistance genes blaOXA-48 and blaIMP in fourteen (12.6%) and seven (6.3%) isolates, respectively. These genes were confirmed with other β-lactamases, particularly CTX-M in the same isolate, and, in some cases, with TEM, SHV, and CMY. There are a few reports about the prevalence of carbapenem resistance in food-producing animals in Tunisia. A recent study reported blaOXA-48 and blaIMP genes among ESBL-producing E. coli isolates from Tunisian diarrheic calves in Tunisia [44]. Another Tunisian study documented the presence of blaVIM and blaIMP genes in E. coli isolates from healthy rabbits [45]. In an Egyptian study, Hamza et al. [46] investigated blaNDM, blaKPC, and blaOXA48 genes among broiler chickens and reported 15% of Klebsiella pneumoniae resistant to carbapenems, with all the isolates harboring the blaNDM gene. The presence of carbapenem resistance genes blaNDM, blaOXA, and blaIMP was reported in E. coli from chickens in poultry farms in Malaysia [47]. While carbapenems are not administered in poultry or other food animal production, resistance to them in E. coli isolates could be linked to resistance against other antibiotics commonly used there. These co-resistant bacteria might be disseminated through direct contact, insect vectors, or other animals [48,49].
The simultaneous production of four or three βlactamase types in the same strain, consisting of ESBLs (CTX-M, SHV, TEM), cephalosporinase (CMY), and carbapenemases (OXA-48 and IMP), is remarkable in 16 (14.4%) E. coli isolates. The existence of more than two βlactamase genes, ESBLs, and carbapenemases in the same isolate has been reported in Enterobacteriaceae from several sources in Tunisia [50,51,52]. The coexistence of multiple β-lactamase genes within the same bacterium is concerning, as it indicates the presence of important resistance determinants and may predict the emergence and dissemination of multidrug-resistant (MDR) strains. This could ultimately lead to treatment failures, resulting in significant morbidity and mortality.
We screened genes encoding antimicrobial resistance in the isolates that exhibited phenotype resistance in the disc diffusion test. Increased use of quinolones is associated with the development of quinolone resistance issues in humans and veterinary medicine [53]. Plasmid-mediated quinolone resistance (PMQR) genes, which cause quinolone resistance, were detected in 44% (49/111), with aac (6)-Ib-cr being the most frequent gene, followed by qnrS and qnrB. The presence of PMQR genes is linked with MDR plasmids, mostly associated with β-lactamases genes [54]. Furthermore, these genes increase resistance to other antimicrobials like aminoglycosides, β-lactams, chloramphenicol, sulfonamides, tetracyclines, and trimethoprim [41]. The sulfonamide resistance genes sul1 and sul2 were detected in 20 (18%) and 21 (18.9%), respectively. In agreement with our study, these elements were widely distributed in E. coli from poultry chicken [41,55]. These genes were more prevalent in isolates containing integrons than integron-negative isolates, with a statistically significant difference. This finding is consistent with reports from Tunisia and Algeria [39,55].
One of the significant findings of this study is the virulence factors (VFs). Several VFs associated with extraintestinal pathogenic E. coli (ExPEC) were identified in the isolates. All isolates had one or more genes. These factors are common to ExPEC strains in animals and humans, allowing these bacteria to colonize, invade, and lyse the host cell with toxins and cause infections outside the gut [56]. The most frequent genes were traT, fimH, and iutA, followed by ibeA, papGIII, cdt3, and sfa/focDE. Other studies reported similar VFs in E. coli isolated from poultry. The fimH and iutA were detected in Nigeria and Zimbabwe [57,58], iutA, papC, and sfa/focDE in Brazil [56], and iutA and ibeA in France [59]. The VFs associated with diarrheagenic E. coli (stx-1, stx-2, eae, and ehxA) were confirmed among the isolates, similar to findings in a previously reported study [8]. These factors enhance the capacity of E. coli to cause severe diseases in humans due to their ability to excrete toxins [60]. Our isolates contained the genetic content of the ExPEC, and this increases the emerging challenges to public health because they can be transmitted to humans by contaminated meat, knowing that bacteria were isolated from healthy chicken intended for slaughter. These strains belong to gut microbiota, whereas ExPEC has the ability to invade organs and cause infections outside the intestine in humans and animals by possessing these virulence factors [56]. The gene iutA encoding a protein associated with increased serum survival in humans and animals and colonizing capacity was found in 73.9% of our isolates. The TraT gene expresses a lipoprotein located in the outer membrane that inhibits the classical pathway of complement activation and leads to serum survival [3], which was found with a high frequency in 85.6% of the strains. This gene was also the most detected in E. coli isolated from hens at three industrial laying hen farms in the north of Tunisia [61]. Studies indicated that poultry may be a source of ExPEC for humans, in the case of consumption of highly contaminated meat. The similarity of genetic relatedness between isolates from healthy poultry and processed chicken products suggests the spread of these bacteria via the food chain [62].
Class 1 integrons were identified in 92 (82.9%) isolates, and class 2 integrons in 44 (39.6%), and our finding is consistent with other studies that indicated domination of class 1 among E. coli strains [61,63]. Integrons are a genetic system that contains a site-specific recombination, allowing integration, expression, and exchange of several gene cassettes that play a major role in the global dissemination of antibiotic resistance, particularly in Enterobacteriaceae [64]. Integrons can spread resistance genes from one organism to another, which poses a significant problem for the livestock and poultry sectors, given how poorly infection control systems operate [65]. We sequenced the VR in classes 1 and 2 integrons. In class 1, the VR revealed the presence of resistance genes to trimethoprim (dfrA1&12) and streptomycin (aadA1&2), and the gene cassette array dfrA1-aadA1 was the most dominantly found. These observations are in agreement with the findings of a poultry study in Tunisia [55]. Gene cassettes in class 2 included resistance genes dfrA1 and aadA1 with the sat2 gene. These determinants were detected in class 2 integrons worldwide [55,66]. Class 1 integrons are distinguished by a conserved segment at the 5’ end (known as 5CS), which contains the integrase gene (intl1), and a conserved segment at the 3’ end (known as 3CS), which includes the qacEΔ gene responsible for conferring resistance to quaternary ammonium compounds. Additionally, the 3CS also contains the sul1 gene, which encodes resistance to sulfonamides [67]. This structure was found in 43 (38.7%) among class 1 positive isolates, and the results of the presence and lack of this structure were interesting; in fact, the deficient integrons missing some of these genes in the 3′CS region have been described [63,68].
One of the significant findings of this study is the detection of four β-lactamase genes (blaCTX-M-G1, blaSHV, blaTEM, blaCMY) and (blaCTX-M-G1, blaSHV, blaTEM, blaCMY) in two strains, along with the presence of class 1 and class 2 integrons carrying gene cassettes. This is a public health concern, as these bacteria also harbor other important resistance genes, such as mcr-1, in addition to multiple virulence genes, highlighting the alarming potential for their dissemination.
Clermont et al. improved the PCR method for phylogenetic typing, which has become widely used, allowing the differentiation of virulent strains (B2 or D) and commensal lineages (A and B1) [7]. The 111 CTX-resistant E. coli isolates were classified mainly in group A (38.7%) and in group B1 (16.2%), which were considered as commensal lineages. Similar results were found in Brazil [69], Portugal [70], and India [12]. In our results, 13.5% of the CTX-resistant isolates were classified in group D, 7.2% in group E, and 5.9% of isolates were assigned to the B2 category, which has the highest pathogenicity. These results agree with other studies [12,39]. The group B2 and D isolates are deemed ExPEC [8], while phylogroup E is linked to both human and animal intestinal strains, as well as human E. coli O157:H7 strains [71]. These findings propose that our strains belonging to the B2 and D groups are ExPEC, as well as the prospect of phylogroup E, which may be linked with ExPEC.
The limitations of this study include the absence of plasmid profiling to assess horizontal gene transfer and the lack of Multilocus Sequence Typing (MLST) to characterize bacterial isolates by determining their sequence types (STs). Accordingly, future investigations should incorporate these methods, particularly MLST, and analyze selected isolates using whole-genome sequencing for comprehensive characterization.

4. Material and Methods

4.1. Samples

Samples were collected from healthy broiler chickens intended for slaughter for human consumption. A total of 111 cecal samples were obtained during the first four months of 2022 from the three main poultry meat suppliers in Tunisia, distributed as follows: 51 samples from the first supplier, 30 from the second, and 30 from the third. These suppliers are the major providers of poultry meat, located in Greater Tunis, Sousse (central–east Tunisia), and Sfax (southeast Tunisia). The samples were collected randomly from healthy broiler chickens at approximately 35 days of age. They were kept at 4 °C and rapidly transported to the laboratory for bacterial analysis.

4.2. Bacterial Isolation and Identification

A gram of cecal contents from each sample was suspended in a 1.5 mL Eppendorf tube containing buffered peptone water and was incubated overnight at 37 °C. A 10 µL loop of bacterial suspension was plated onto MacConkey agar (MAC) (Oxoid, Basingstoke, UK). MAC was supplemented with cefotaxime (CTX 1 µg/mL) and incubated at 37 °C overnight, as previously described [72]. One bacterial colony was picked up from MAC supplemented with CTX culture and plated on brain heart infusion agar (Merck, Darmstadt, Germany) for further investigation, whereas the E. coli isolates from non-supplemented MAC plates were included for prevalence rate purposes. The bacterial isolates were identified by API 20E gallery (bioMérieux, Marcy-l’Étoile, France) and were confirmed by a PCR targeting the uidA gene (486 bp) using specific primers (Forward: 5′-ATCACCGTGGTGACGCATGTCGC-3′ and Reverse: 5′-CACCACGATGCCATGTTCATCTGC-3′) (Bio Basic, Markham, ON, Canada) based on the method previously described by [73]. Bacterial isolates were stored at −20 °C in brain heart infusion broth supplemented with 20% glycerol.

4.3. Antimicrobial Sensitivity Test and ESBL Phenotype Detection

Antimicrobial sensitivity was carried out by the disk diffusion method on Mueller-Hinton agar (BioRad, Marne la Coquette, France) plates according to the guidelines of the Antibiogram Committee of the French Society (CA-SFM) [74] using twenty antibiotic discs (Bio-Rad France) comprising μg/disk: twelve β-lactams represented by amoxicillin (25), amoxicillin/clavulanic acid (20/10), ticarcillin/clavulanic acid (75/10), cefotaxime (30), ceftazidime (30), cefepime (30), cefoxitin (30), aztreonam (30), ertapenem (10), piperacillin (30), cephalothin (30), cefuroxime (30), and nine non-β-lactams antibiotics represented by (chloramphenicol (30), gentamicin (15), nalidixic acid (30), enrofloxacin (5), tetracycline (30), sulfamethoxazole/trimethoprim (1.25/23.75), streptomycin (10), and florfenicol (30). Furthermore, colistin susceptibility was examined by Colispot [75].
The ESBL phenotype was detected by a synergy test using a disk of cefotaxime, ceftazidime, and cefepime around a disk containing clavulanic acid, which is placed on Mueller-Hinton agar plates with a distance of 30 mm between the two discs. The enhanced inhibition zone of any discs combined with clavulanic acid by 5 mm or more was considered an ESBL producer [76]. All isolates exhibited resistance to third-generation cephalosporins (cefotaxime and/or ceftazidime), and positive ESBL confirmation disk tests were selected for molecular analyses. Furthermore, the multidrug-resistant (MDR) percentage was searched. The MDR is known as an isolate exhibiting resistance to at least one agent in three or more antimicrobial classes [55]. The E. coli ATCC 25922 reference strain was used as a control.

4.4. Detection of β-Lactamase Genes

PCR amplification was performed to search for the presence of the β-lactamases encoding genes: blaCTX-M, blaTEM, blaOXA, blaSHV, and blaCMY. Carbapenem resistance genes blaVIM, blaIMP, blaNDM, blaKPC, and blaOXA-48 were searched by PCR [21]. The primers used are listed in Table S1.

4.5. Detection of Antimicrobial Resistance Genes to Non-β-Lactam Agents

The presence of genes that confer resistance to sulfamethoxazole (sul1, sul2, and sul3), aminoglycosides (aac(3)-I, aac(3)-II, aac(3)-IV, AadA-1 and AadA-5), quinolones (qnrA, qnrB, qnrS, and aac(6’)-1b, and colistin (mcr-1)) were tested by PCR amplification [77].

4.6. Detection and Characterization of Integrons

The presence of intI1 and intI2 genes was screened in all isolates by PCR. The presence of qacED1-sul1 genes within 3’-conserved regions of class 1 integrons was identified by PCR. The gene cassettes in the variable region of class 1 integrons were screened by PCR and sequencing. The primers used are listed in Table S1 [77].

4.7. Determination of Phylogenetic Groups and Virulence Factors

Using the revised technique previously described by Clermont, Christenson, Denamur, and Gordon [7] and the primers given in Table S2, the phylogenetic groups (A, B1, B2, D, C, E, and Clade 1) to which the strains belong were identified.
The presence of virulence genes stx1, stx2, ehxA, eae, cdt3, cnf1, hly, aer, papA, bfpA, papG allele III, fimH, traT, ibeA, sfa/foc, iutA, and fyuA was searched by PCR (Table S2).
Bacterial strains carrying the target genes, previously isolated from animals in the Laboratory of Microbiology and Immunology at the National School of Veterinary Medicine, Tunisia, were used in this study as positive controls for antimicrobial resistance genes or virulence genes.

4.8. Data Analysis

Statistical Package for the Social Sciences (SPSS) was used to analyze the data and determine the frequency of bacterial isolates, resistance genes, virulence factors, and phylogenetic groups. Pearson’s Chi-square test was used to investigate the significant differences in phylogroups distribution among the resistance genes and virulence genes, using SPSS version 26 software (IBM Corporation, Somers, NY, USA). The level of statistical significance was set at p < 0.05.

5. Conclusions

The high rate of antimicrobial resistance and the development of MDR E. coli in poultry intended for human consumption, as well as the spreading risk of these bacteria to humans and the environment, are concerning. This study proved a high prevalence of ESBL-producing E. coli in chicken. It also demonstrated a high level of blaCTX-M-group1-producing isolates. Chicken farms may be a significant source of the ESBL-producing bacteria, causing illnesses in people that are difficult to cure. The presence of multiple antimicrobial resistance genes and several virulence factors associated with integrons makes E. coli a potential food safety issue and poses a significant threat to public health. This is due to the possibility of horizontal transfer of these genes to bacteria in the environment and humans, complicating the concern over antibiotic resistance. Limiting the use of antimicrobials in chicken production may help minimize the emergence of antimicrobial resistance genes and virulence factors, hence lowering the risk of human infection. Regulatory control of the administration of antimicrobials is required to prevent the development of antibiotic-resistant strains and to safeguard public health.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antibiotics14090931/s1, Table S1: Primers used in the present study for the detection of antimicrobial resistance and integron genes of E. coli isolated from ceca of healthy poultry; Table S2: Primers used in this study for the detection of virulence genes and phylogenetic grouping of E. coli isolated from ceca of healthy poultry; Table S3: Characteristics of the 111 E. coli isolates were obtained from ceca of chicken broiler samples [78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93].

Author Contributions

G.T. designed the study, performed the experimental work (the microbiological and molecular tests), collected the data, analyzed and interpreted the data, and drafted the manuscript. F.N. collected samples and helped in performing the experimental part of the manuscript. K.A. helped in performing the experimental part. O.A. participated in the project design. I.F. participated in the project design and contributed to the final writing and editing of the manuscript. L.M. designed and supervised the study and contributed to the final writing and editing of the manuscript. They agree to be accountable for all aspects of the work, ensuring that any questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the International Development Research Center (IDRC)—Innovet-Initiative under the research program Project Innovet-AMR n°110298-001 Avibiocin 2 project (2024–2026).

Institutional Review Board Statement

Not applicable. The study did not require ethical approval.

Data Availability Statement

The datasets generated and analyzed during the current study are included within this manuscript.

Conflicts of Interest

Author Omar Abbes was employed by the company DICK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Escobar-Páramo, P.; Grenet, K.; Le Menac’h, A.; Rode, L.; Salgado, E.; Amorin, C.; Gouriou, S.; Picard, B.; Rahimy, M.C.; Andremont, A. Large-scale population structure of human commensal Escherichia coli isolates. Appl. Environ. Microbiol. 2004, 70, 5698–5700. [Google Scholar] [CrossRef]
  2. Ramos, S.; Silva, V.; Dapkevicius, M.L.E.; Caniça, M.; Tejedor-Junco, M.T.; Igrejas, G.; Poeta, P. Escherichia coli as Commensal and Pathogenic Bacteria Among Food-Producing Animals: Health Implications of Extended Spectrum β-lactamase (ESBL) Production. Animals 2020, 10, 2239. [Google Scholar] [CrossRef]
  3. Rezatofighi, S.E.; Mirzarazi, M.; Salehi, M. Virulence genes and phylogenetic groups of uropathogenic Escherichia coli isolates from patients with urinary tract infection and uninfected control subjects: A case-control study. BMC Infect. Dis. 2021, 21, 361. [Google Scholar] [CrossRef]
  4. Wu, H.-J.; Wang, A.H.; Jennings, M.P. Discovery of virulence factors of pathogenic bacteria. Curr. Opin. Chem. Biol. 2008, 12, 93–101. [Google Scholar] [CrossRef] [PubMed]
  5. Pakbin, B.; Brück, W.M.; Rossen, J.W.A. Virulence Factors of Enteric Pathogenic Escherichia coli: A Review. Int. J. Mol. Sci. 2021, 22, 9922. [Google Scholar] [CrossRef] [PubMed]
  6. Kim, B.; Kim, J.H.; Lee, Y. Virulence Factors Associated with Escherichia coli Bacteremia and Urinary Tract Infection. Ann. Lab. Med. 2022, 42, 203–212. [Google Scholar] [CrossRef] [PubMed]
  7. Clermont, O.; Christenson, J.K.; Denamur, E.; Gordon, D.M. The Clermont Escherichia coli phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups. Environ. Microbiol. Rep. 2013, 5, 58–65. [Google Scholar] [CrossRef]
  8. Laarem, M.; Barguigua, A.; Nayme, K.; Akila, A.; Zerouali, K.; El Mdaghri, N.; Timinouni, M. Occurrence of plasmid-mediated quinolone resistance and virulence genes in avian Escherichia coli isolates from Algeria. J. Infect. Dev. Ctries. 2017, 11, 143–151. [Google Scholar] [CrossRef]
  9. Mellata, M. Human and avian extraintestinal pathogenic Escherichia coli: Infections, zoonotic risks, and antibiotic resistance trends. Foodborne Pathog. Dis. 2013, 10, 916–932. [Google Scholar] [CrossRef]
  10. de Brito, B.G.; Gaziri, L.C.J.; Vidotto, M.C. Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis. Infect. Immun. 2003, 71, 4175–4177. [Google Scholar] [CrossRef]
  11. Johar, A.; Al-Thani, N.; Al-Hadidi, S.H.; Dlissi, E.; Mahmoud, M.H.; Eltai, N.O. Antibiotic resistance and virulence gene patterns associated with avian pathogenic Escherichia coli (APEC) from broiler chickens in qatar. Antibiotics 2021, 10, 564. [Google Scholar] [CrossRef]
  12. Hussain, A.; Shaik, S.; Ranjan, A.; Nandanwar, N.; Tiwari, S.K.; Majid, M.; Baddam, R.; Qureshi, I.A.; Semmler, T.; Wieler, L.H. Risk of transmission of antimicrobial resistant Escherichia coli from commercial broiler and free-range retail chicken in India. Front. Microbiol. 2017, 8, 2120. [Google Scholar] [CrossRef] [PubMed]
  13. de Been, M.; Lanza, V.F.; de Toro, M.; Scharringa, J.; Dohmen, W.; Du, Y.; Hu, J.; Lei, Y.; Li, N.; Tooming-Klunderud, A. Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages. PLoS Genet. 2014, 10, e1004776. [Google Scholar] [CrossRef]
  14. O’Neill, J. Review on Antimicrobial Resistance: Tackling Drug-Resistant Infections Globally: Final Report and Recommendations; Wellcome Trust: London, UK, 2016. [Google Scholar]
  15. Mohamed, M.-Y.I.; Abu, J.; Zakaria, Z.; Khan, A.R.; Abdul Aziz, S.; Bitrus, A.A.; Habib, I. Multi-Drug Resistant Pathogenic Escherichia coli Isolated from Wild Birds, Chicken, and the Environment in Malaysia. Antibiotics 2022, 11, 1275. [Google Scholar] [CrossRef]
  16. Lye, D.; Earnest, A.; Ling, M.; Lee, T.-E.; Yong, H.-C.; Fisher, D.; Krishnan, P.; Hsu, L.-Y. The impact of multidrug resistance in healthcare-associated and nosocomial Gram-negative bacteraemia on mortality and length of stay: Cohort study. Clin. Microbiol. Infect. 2012, 18, 502–508. [Google Scholar] [CrossRef] [PubMed]
  17. Abdallah, H.; Reuland, E.; Wintermans, B.; Al Naiemi, N.; Koek, A.; Abdelwahab, A.; Ammar, A.; Mohamed, A.; Vandenbroucke-Grauls, C. Extended-spectrum β-lactamases and/or carbapenemases-producing Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt. PLoS ONE 2015, 10, e0136052. [Google Scholar] [CrossRef]
  18. Canton, R.; Novais, A.; Valverde, A.; Machado, E.; Peixe, L.; Baquero, F.; Coque, T. Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 2008, 14, 144–153. [Google Scholar] [CrossRef]
  19. Cantón, R.; Coque, T.M. The CTX-M β-lactamase pandemic. Curr. Opin. Microbiol. 2006, 9, 466–475. [Google Scholar] [CrossRef]
  20. Changkaew, K.; Intarapuk, A.; Utrarachkij, F.; Nakajima, C.; Suthienkul, O.; Suzuki, Y. Antimicrobial Resistance, Extended-Spectrum β-Lactamase Productivity, and Class 1 Integrons in Escherichia coli from Healthy Swine. J. Food Prot. 2015, 78, 1442–1450. [Google Scholar] [CrossRef]
  21. Poirel, L.; Walsh, T.R.; Cuvillier, V.; Nordmann, P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn. Microbiol. Infect. Dis. 2011, 70, 119–123. [Google Scholar] [CrossRef]
  22. Jin, M.; Osman, M.; Green, B.A.; Yang, Y.; Ahuja, A.; Lu, Z.; Cazer, C.L. Evidence for the transmission of antimicrobial resistant bacteria between humans and companion animals: A scoping review. One Health 2023, 17, 100593. [Google Scholar] [CrossRef]
  23. Muloi, D.; Ward, M.J.; Pedersen, A.B.; Fevre, E.M.; Woolhouse, M.E.; van Bunnik, B.A. Are food animals responsible for transfer of antimicrobial-resistant Escherichia coli or their resistance determinants to human populations? A systematic review. Foodborne Pathog. Dis. 2018, 15, 467–474. [Google Scholar]
  24. WHO. Available online: https://www.who.int/about/accountability/results/who-results-report-2020-2021/country-profile/2021/tunisia (accessed on 1 March 2023).
  25. Abdallah, K.; Tayh, G.; Maamar, E.; Mosbah, A.; Abbes, O.; Fliss, I.; Messadi, L.J.M.R. Genotypic Characterisation and Risk Assessment of Virulent ESBL-Producing E. coli in Chicken Meat in Tunisia: Insights from Multi-Omics Machine Learning Perspective. Microbiol. Res. 2025, 16, 131. [Google Scholar]
  26. Liu, Z.; Wang, K.; Zhang, Y.; Xia, L.; Zhao, L.; Guo, C.; Liu, X.; Qin, L.; Hao, Z. High prevalence and diversity characteristics of blaNDM, mcr, and blaESBLs harboring multidrug-resistant Escherichia coli from chicken, pig, and cattle in China. Front. Cell. Infect. Microbiol. 2022, 1364, 755545. [Google Scholar] [CrossRef]
  27. Akenten, C.W.; Ofori, L.A.; Khan, N.A.; Mbwana, J.; Sarpong, N.; May, J.; Thye, T.; Obiri-Danso, K.; Paintsil, E.K.; Fosu, D.; et al. Prevalence, Characterization, and Antimicrobial Resistance of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli from Domestic Free-Range Poultry in Agogo, Ghana. Foodborne Pathog. Dis. 2023, 20, 59–66. [Google Scholar] [CrossRef]
  28. Mezhoud, H.; Boyen, F.; Touazi, L.-h.; Garmyn, A.; Moula, N.; Smet, A.; Haesbrouck, F.; Martel, A.; Iguer-Ouada, M.; Touati, A. Extended spectrum β-lactamase producing Escherichia coli in broiler breeding roosters: Presence in the reproductive tract and effect on sperm motility. Anim. Reprod. Sci. 2015, 159, 205–211. [Google Scholar] [CrossRef]
  29. Mnif, B.; Ktari, S.; Rhimi, F.; Hammami, A. Extensive dissemination of CTX-M-1-and CMY-2-producing Escherichia coli in poultry farms in Tunisia. Lett. Appl. Microbiol. 2012, 55, 407–413. [Google Scholar] [CrossRef] [PubMed]
  30. Aliyu, A.B.; Jalila, A.; Saleha, A.A.; Zunita, Z. ESBL Producing E. coli in Chickens and Poultry Farms Environment in Selangor, Malaysia: A Cross-Sectional Study on Their Occurrence and Associated Risk Factors with Environment and Public Health Importance. Zoonoses Public Health 2024, 71, 962–971. [Google Scholar] [CrossRef] [PubMed]
  31. Kuan, N.-L.; Chen, Y.-P.; Shien, J.-H.; Yeh, K.-S. Characteristics of the extended-spectrum-β-lactamase-producing Escherichia coli isolated from diseased livestock and poultry in Taiwan. Sci. Rep. 2024, 14, 29459. [Google Scholar] [CrossRef]
  32. Song, J.; Oh, S.-S.; Kim, J.; Park, S.; Shin, J. Clinically relevant extended-spectrum β-lactamase–producing Escherichia coli isolates from food animals in South Korea. Front. Microbiol. 2020, 11, 604. [Google Scholar] [CrossRef]
  33. Ho, P.-L.; Chow, K.; Lai, E.L.; Lo, W.-U.; Yeung, M.; Chan, J.; Chan, P.; Yuen, K. Extensive dissemination of CTX-M-producing Escherichia coli with multidrug resistance to ‘critically important’antibiotics among food animals in Hong Kong, 2008–2010. J. Antimicrob. Chemother. 2011, 66, 765–768. [Google Scholar] [CrossRef]
  34. Kasparaviciene, B.; Novoslavskij, A.; Aksomaitiene, J.; Stankeviciene, J.; Kasetiene, N.; Sinkevicius, R.; Malakauskas, M. Prevalence and Antimicrobial Resistance of ESBL E. coli in Early Broiler Production Stage and Farm Environment in Lithuania. Microorganisms 2025, 13, 425. [Google Scholar] [CrossRef]
  35. Abbassi, M.; Kilani, H.; Zouari, M.; Mansouri, R.; Oussama, E.; Hammami, S.; Chehida, N.B. Antimicrobial resistance in Escherichia coli isolates from healthy poultry, bovine and ovine in Tunisia: A real animal and human health threat. J. Clin. Microbiol. Biochem. Technol. 2017, 3, 019–123. [Google Scholar]
  36. Ardakani, Z.; Aragrande, M.; Canali, M. Global antimicrobial use in livestock farming: An estimate for cattle, chickens, and pigs. Animal 2024, 18, 101060. [Google Scholar] [CrossRef]
  37. Tayh, G.; Al Laham, N.; Ben Yahia, H.; Ben Sallem, R.; Elottol, A.E.; Ben Slama, K. Extended-spectrum β-lactamases among Enterobacteriaceae isolated from urinary tract infections in Gaza strip, Palestine. BioMed Res. Int. 2019, 2019, 4041801. [Google Scholar] [CrossRef]
  38. Ben Sallem, R.; Ben Slama, K.; Sáenz, Y.; Rojo-Bezares, B.; Estepa, V.; Jouini, A.; Gharsa, H.; Klibi, N.; Boudabous, A.; Torres, C. Prevalence and characterization of extended-spectrum beta-lactamase (ESBL)–and CMY-2–producing Escherichia coli isolates from healthy food-producing animals in Tunisia. Foodborne Pathog. Dis. 2012, 9, 1137–1142. [Google Scholar] [CrossRef] [PubMed]
  39. Messaili, C.; Messai, Y.; Bakour, R. Virulence gene profiles, antimicrobial resistance and phylogenetic groups of fecal Escherichia coli strains isolated from broiler chickens in Algeria. Vet. Ital. 2019, 55, 35–46. [Google Scholar] [CrossRef]
  40. Abdel-Rahman, M.A.; Hamed, E.A.; Abdelaty, M.F.; Sorour, H.K.; Badr, H.; Hassan, W.M.; Shalaby, A.G.; Mohamed, A.A.-E.; Soliman, M.A.; Roshdy, H. Distribution pattern of antibiotic resistance genes in Escherichia coli isolated from colibacillosis cases in broiler farms of Egypt. World 2023, 16, 1. [Google Scholar] [CrossRef]
  41. Racewicz, P.; Majewski, M.; Biesiada, H.; Nowaczewski, S.; Wilczyński, J.; Wystalska, D.; Kubiak, M.; Pszczoła, M.; Madeja, Z.E. Prevalence and characterisation of antimicrobial resistance genes and class 1 and 2 integrons in multiresistant Escherichia coli isolated from poultry production. Sci. Rep. 2022, 12, 6062. [Google Scholar] [CrossRef] [PubMed]
  42. Maamar, E.; Alonso, C.A.; Hamzaoui, Z.; Dakhli, N.; Abbassi, M.S.; Ferjani, S.; Saidani, M.; Boubaker, I.B.-B.; Torres, C. Emergence of plasmid-mediated colistin-resistance in CMY-2-producing Escherichia coli of lineage ST2197 in a Tunisian poultry farm. Int. J. Food Microbiol. 2018, 269, 60–63. [Google Scholar] [CrossRef]
  43. Aklilu, E.; Raman, K. MCR-1 gene encoded colistin-resistant Escherichia coli in raw chicken meat and bean sprouts in Malaysia. Int. J. Microbiol. 2020, 2020, 8853582. [Google Scholar] [CrossRef]
  44. Ben Haj Yahia, A.; Tayh, G.; Landolsi, S.; Maamar, E.; Galai, N.; Landoulsi, Z.; Messadi, L. First Report of OXA-48 and IMP Genes Among Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolates from Diarrheic Calves in Tunisia. Microb. Drug Resist. 2023, 29, 150–162. [Google Scholar] [CrossRef]
  45. Lengliz, S.; Benlabidi, S.; Raddaoui, A.; Cheriet, S.; Ben Chehida, N.; Najar, T.; Abbassi, M.S. High occurrence of carbapenem-resistant Escherichia coli isolates from healthy rabbits (Oryctolagus cuniculus): First report of blaIMI and blaVIM type genes from livestock in Tunisia. Lett. Appl. Microbiol. 2021, 73, 708–717. [Google Scholar] [CrossRef]
  46. Hamza, E.; Dorgham, S.M.; Hamza, D.A. Carbapenemase-producing Klebsiella pneumoniae in broiler poultry farming in Egypt. J. Glob. Antimicrob. Resist. 2016, 7, 8–10. [Google Scholar] [CrossRef] [PubMed]
  47. Aklilu, E.; Harun, A.; Singh, K.K.B.; Ibrahim, S.; Kamaruzzaman, N.F. Phylogenetically Diverse Escherichia coli Strains from Chicken Coharbor Multiple Carbapenemase-Encoding Genes (blaNDM-blaOXA-blaIMP). BioMed Res. Int. 2021, 2021, 5596502. [Google Scholar] [CrossRef]
  48. Bonardi, S.; Pitino, R. Carbapenemase-producing bacteria in food-producing animals, wildlife and environment: A challenge for human health. Ital. J. Food Saf. 2019, 8, 7956. [Google Scholar] [CrossRef]
  49. Ahmad, K.; Khattak, F.; Ali, A.; Rahat, S.; Noor, S.; Mahsood, N.; Somayya, R. Carbapenemases and extended-spectrum β-lactamase–producing multidrug-resistant Escherichia coli isolated from retail chicken in peshawar: First report from Pakistan. J. Food Prot. 2018, 81, 1339–1345. [Google Scholar] [PubMed]
  50. Ktari, S.; Arlet, G.; Mnif, B.; Gautier, V.; Mahjoubi, F.; Ben Jmeaa, M.; Bouaziz, M.; Hammami, A. Emergence of multidrug-resistant Klebsiella pneumoniae isolates producing VIM-4 metallo-β-lactamase, CTX-M-15 extended-spectrum β-lactamase, and CMY-4 AmpC β-lactamase in a Tunisian university hospital. Antimicrob. Agents Chemother. 2006, 50, 4198–4201. [Google Scholar] [CrossRef]
  51. Yahia, A.B.H.; Tayh, G.; Landolsi, S.; Maazaoui, A.; Chehida, F.B.; Mamlouk, A.; Dâaloul-Jedidi, M.; Messadi, L. Characterization of Extended-Spectrum β-Lactamase Producing-and Carbapenem–Resistant Escherichia coli Isolated from Diarrheic Dogs in Tunisia: First Report of bla IMP Gene in Companion Animals. Microbiol. Res. 2024, 15, 1119–1133. [Google Scholar] [CrossRef]
  52. Tayh, G.; Jebali, G.; Selmi, R.; Kaboudi, K.; Dâaloul-Jedidi, M.; Messadi, L. Prevalence, characterization, and emergence of extended-spectrum Β-lactamase producing-and carbapenem-resistant gram-negative bacteria isolated from houseflies (Musca domestica) in tunisia. Slov. Vet. Res. 2025. [Google Scholar] [CrossRef]
  53. Seo, K.W.; Lee, Y.J. Prevalence and characterization of plasmid mediated quinolone resistance genes and class 1 integrons among multidrug-resistant Escherichia coli isolates from chicken meat. J. Appl. Poult. Res. 2019, 28, 761–770. [Google Scholar] [CrossRef]
  54. Piekarska, K.; Rzeczkowska, M.; Zacharczuk, K.; Chróst, A.; Januszkiewicz, A.; Bareja, E.; Olak, M.; Gierczyński, R. Prevalence of qnr genes in clinical Enterobacteriaceae non-susceptible to fluoroquinolone in Poland. Med. Dosw. I Mikrobiol. 2012, 64, 211–219. [Google Scholar]
  55. Abbassi, M.S.; Kilani, H.; Abid, I.; Sáenz, Y.; Hynds, P.; Lengliz, S.; Ben Chehida, N.; Boutiba-Ben Boubaker, I. Genetic background of antimicrobial resistance in multiantimicrobial-resistant Escherichia coli isolates from feces of healthy broiler chickens in Tunisia. BioMed Res. Int. 2021, 2021, 1269849. [Google Scholar] [CrossRef]
  56. Ferreira, J.C.; Penha Filho, R.A.C.; Kuaye, A.P.Y.; Andrade, L.N.; Chang, Y.-F.; Darini, A.L.C. Virulence potential of commensal multidrug resistant Escherichia coli isolated from poultry in Brazil. Infect. Genet. Evol. 2018, 65, 251–256. [Google Scholar] [CrossRef]
  57. Ugwu, I.; Lee-Ching, L.; Ugwu, C.; Okoye, J.; Chah, K. In vitro assessment of pathogenicity and virulence encoding gene profiles of avian pathogenic Escherichia coli strains associated with colibacillosis in chickens. Iran. J. Vet. Res. 2020, 21, 180. [Google Scholar]
  58. Mbanga, J.; Nyararai, Y.O. Virulence gene profiles of avian pathogenic Escherichia coli isolated from chickens with colibacillosis in Bulawayo, Zimbabwe. Onderstepoort J. Vet. Res. 2015, 82, 1–8. [Google Scholar] [CrossRef]
  59. Chouikha, I.; Bree, A.; Moulin-Schouleur, M.; Gilot, P.; Germon, P. Differential expression of iutA and ibeA in the early stages of infection by extra-intestinal pathogenic E. coli. Microbes Infect. 2008, 10, 432–438. [Google Scholar] [CrossRef] [PubMed]
  60. Kargar, M.; Homayoon, M. Prevalence of shiga toxins (stx1, stx2), eaeA and hly genes of Escherichia coli O157: H7 strains among children with acute gastroenteritis in southern of Iran. Asian Pac. J. Trop. Med. 2015, 8, 24–28. [Google Scholar] [CrossRef]
  61. Maamar, E.; Hammami, S.; Alonso, C.A.; Dakhli, N.; Abbassi, M.S.; Ferjani, S.; Hamzaoui, Z.; Saidani, M.; Torres, C.; Boubaker, I.B.-B. High prevalence of extended-spectrum and plasmidic AmpC beta-lactamase-producing Escherichia coli from poultry in Tunisia. Int. J. Food Microbiol. 2016, 231, 69–75. [Google Scholar] [CrossRef] [PubMed]
  62. Manges, A.R.; Johnson, J.R. Food-borne origins of Escherichia coli causing extraintestinal infections. Clin. Infect. Dis. 2012, 55, 712–719. [Google Scholar] [CrossRef]
  63. Soufi, L.; Sáenz, Y.; Vinué, L.; Abbassi, M.S.; Ruiz, E.; Zarazaga, M.; Hassen, A.B.; Hammami, S.; Torres, C. Escherichia coli of poultry food origin as reservoir of sulphonamide resistance genes and integrons. Int. J. Food Microbiol. 2011, 144, 497–502. [Google Scholar] [CrossRef]
  64. Rowe-Magnus, D.A.; Mazel, D. The role of integrons in antibiotic resistance gene capture. Int. J. Med. Microbiol. 2002, 292, 115–125. [Google Scholar] [CrossRef]
  65. Fletcher, S. Understanding the contribution of environmental factors in the spread of antimicrobial resistance. Environ. Health Prev. Med. 2015, 20, 243–252. [Google Scholar] [CrossRef]
  66. Dessie, H.K.; Bae, D.H.; Lee, Y.J. Characterization of integrons and their cassettes in Escherichia coli and Salmonella isolates from poultry in Korea. Poult. Sci. 2013, 92, 3036–3043. [Google Scholar] [CrossRef]
  67. Recchia, G.D.; Hall, R.M. Gene cassettes: A new class of mobile element. Microbiology 1995, 141, 3015–3027. [Google Scholar] [CrossRef]
  68. Guerra, B.; Junker, E.; Schroeter, A.; Malorny, B.; Lehmann, S.; Helmuth, R. Phenotypic and genotypic characterization of antimicrobial resistance in German Escherichia coli isolates from cattle, swine and poultry. J. Antimicrob. Chemother. 2003, 52, 489–492. [Google Scholar] [CrossRef]
  69. Coura, F.M.; Diniz, S.A.; Silva, M.X.; Arcebismo, T.L.; Minharro, S.; Feitosa, A.C.; Lage, A.P.; Knöbl, T.; Mussi, J.M.S.; Heinemann, M.B. Phylogenetic group of Escherichia coli isolates from broilers in Brazilian poultry slaughterhouse. Sci. World J. 2017, 2017, 5898701. [Google Scholar] [CrossRef] [PubMed]
  70. Pais, S.; Costa, M.; Barata, A.R.; Rodrigues, L.; Afonso, I.M.; Almeida, G. Evaluation of antimicrobial resistance of different phylogroups of Escherichia coli isolates from feces of breeding and laying hens. Antibiotics 2023, 12, 20. [Google Scholar] [CrossRef]
  71. Clermont, O.; Olier, M.; Hoede, C.; Diancourt, L.; Brisse, S.; Keroudean, M.; Glodt, J.; Picard, B.; Oswald, E.; Denamur, E. Animal and human pathogenic Escherichia coli strains share common genetic backgrounds. Infect. Genet. Evol. 2011, 11, 654–662. [Google Scholar] [CrossRef] [PubMed]
  72. Hendriksen, R.S.; Cavaco, L.M.; Guerra, B.; Bortolaia, V.; Agersø, Y.; Svendsen, C.A.; Nielsen, H.N.; Kjeldgaard, J.S.; Pedersen, S.K.; Fertner, M.; et al. Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples. Front. Microbiol. 2023, 14, 1229542. [Google Scholar] [CrossRef] [PubMed]
  73. Heininger, A.; Binder, M.; Schmidt, S.; Unertl, K.; Botzenhart, K.; Döring, G. PCR and blood culture for detection of Escherichia coli bacteremia in rats. J. Clin. Microbiol. 1999, 37, 2479–2482. [Google Scholar] [CrossRef] [PubMed]
  74. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 7.1. 2017. Available online: www.eucast.org (accessed on 24 August 2025).
  75. Jouy, E.; Haenni, M.; Le Devendec, L.; Le Roux, A.; Châtre, P.; Madec, J.Y.; Kempf, I. Improvement in routine detection of colistin resistance in E. coli isolated in veterinary diagnostic laboratories. J. Microbiol. Methods 2017, 132, 125–127. [Google Scholar] [CrossRef] [PubMed]
  76. Jacoby, G.A.; Han, P. Detection of extended-spectrum beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli. J. Clin. Microbiol. 1996, 34, 908–911. [Google Scholar] [CrossRef] [PubMed]
  77. Sáenz, Y.; Brinas, L.; Domínguez, E.; Ruiz, J.; Zarazaga, M.; Vila, J.; Torres, C. Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins. Antimicrob. Agents Chemother. 2004, 48, 3996–4001. [Google Scholar] [CrossRef]
  78. Batchelor, M.; Hopkins, K.; Threlfall, E.; Clifton-Hadley, F.; Stallwood, A.; Davies, R.; Liebana, E. bla CTX-M genes in clinical Salmonella isolates recovered from humans in England and Wales from 1992 to 2003. Antimicrob. Agents Chemother. 2005, 49, 1319–1322. [Google Scholar] [CrossRef]
  79. Jouini, A.; Vinué, L.; Slama, K.B.; Saenz, Y.; Klibi, N.; Hammami, S.; Boudabous, A.; Torres, C. Characterization of CTX-M and SHV extended-spectrum β-lactamases and associated resistance genes in Escherichia coli strains of food samples in Tunisia. J. Antimicrob. Chemother. 2007, 60, 1137–1141. [Google Scholar] [CrossRef]
  80. Literacka, E.; Empel, J.; Baraniak, A.; Sadowy, E.; Hryniewicz, W.; Gniadkowski, M. Four variants of the Citrobacter freundii AmpC-Type cephalosporinases, including novel enzymes CMY-14 and CMY-15, in a Proteus mirabilis clone widespread in Poland. Antimicrob Agents Chemother 2004, 48, 4136–4143. [Google Scholar] [CrossRef]
  81. Candan, E.D.; Aksöz, N. Escherichia coli: Characteristics of carbapenem resistance and virulence factors. Braz. Arch. Biol. Technol. 2017, 60, e17160416. [Google Scholar] [CrossRef]
  82. Liu, Y.Y.; Wang, Y.; Walsh, T.R.; Yi, L.X.; Zhang, R.; Spencer, J.; Doi, Y.; Tian, G.; Dong, B.; Huang, X.; et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: A microbiological and molecular biological study. Lancet. Infect. Dis. 2016, 16, 161–168. [Google Scholar] [CrossRef]
  83. Cattoir, V.; Poirel, L.; Rotimi, V.; Soussy, C.J.; Nordmann, P. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J. Antimicrob. Chemother. 2007, 60, 394–397. [Google Scholar] [CrossRef]
  84. Park, C.H.; Robicsek, A.; Jacoby, G.A.; Sahm, D.; Hooper, D.C. Prevalence in the United States of aac(6’)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother 2006, 50, 3953–3955. [Google Scholar] [CrossRef]
  85. Van De Klundert, J.; Vliegenthart, J. Nomenclature of aminoglycoside resistance genes: A comment. Antimicrob. Agents Chemother. 1993, 37, 927–928. [Google Scholar] [CrossRef][Green Version]
  86. Feng, P.; Monday, S. Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes. Mol. Cell Probes 2000, 14, 333–337. [Google Scholar] [CrossRef] [PubMed]
  87. Johnson, J.R.; Stell, A.L. Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise. J. Infect. Dis. 2000, 181, 261–272. [Google Scholar] [CrossRef] [PubMed]
  88. Tóth, I.; Hérault, F.; Beutin, L.; Oswald, E. Production of Cytolethal Distending Toxins by Pathogenic Escherichia coli Strains Isolated from Human and Animal Sources: Establishment of the Existence of a New cdt Variant (Type IV). J. Clin. Microbiol. 2003, 41, 4285–4291. [Google Scholar] [CrossRef] [PubMed]
  89. Vidal, M.; Kruger, E.; Durán, C.; Lagos, R.; Levine, M.; Prado, V.; Toro, C.; Vidal, R. Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections. J. Clin. Microbiol. 2005, 43, 5362–5365. [Google Scholar] [CrossRef]
  90. Besser, T.E.; Shaikh, N.; Holt, N.J.; Tarr, P.I.; Konkel, M.E.; Malik-Kale, P.; Walsh, C.W.; Whittam, T.S.; Bono, J.L. Greater diversity of Shiga toxin-encoding bacteriophage insertion sites among Escherichia coli O157:H7 isolates from cattle than in those from humans. Appl. Environ. Microbiol. 2007, 73, 671–679. [Google Scholar] [CrossRef]
  91. Ruiz, J.; Simon, K.; Horcajada, J.P.; Velasco, M.; Barranco, M.; Roig, G.; Moreno-Martínez, A.; Martínez, J.A.; Anta, T.J.d.; Mensa, J.; et al. Differences in Virulence Factors among Clinical Isolates of Escherichia coli Causing Cystitis and Pyelonephritis in Women and Prostatitis in Men. J. Clin. Microbiol. 2002, 40, 4445–4449. [Google Scholar] [CrossRef]
  92. Yamamoto, S.; Terai, A.; Yuri, K.; Kurazono, H.; Takeda, Y.; Yoshida, O. Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction. FEMS Immunol. Med. Microbiol. 1995, 12, 85–90. [Google Scholar] [CrossRef]
  93. Hinenoya, A.; Naigita, A.; Ninomiya, K.; Asakura, M.; Shima, K.; Seto, K.; Tsukamoto, T.; Ramamurthy, T.; Faruque, S.M.; Yamasaki, S. Prevalence and characteristics of cytolethal distending toxin-producing Escherichia coli from children with diarrhea in Japan. Microbiol. Immunol. 2009, 53, 206–215. [Google Scholar] [CrossRef]
Antibiotics 14 00931 g001
Figure 1. Antimicrobial resistance of 111 E. coli isolates.
Figure 1. Antimicrobial resistance of 111 E. coli isolates.
Antibiotics 14 00931 g001
Antibiotics 14 00931 g002
Figure 2. Prevalence of resistance genes and their distribution according to phylogenetic groups.
Figure 2. Prevalence of resistance genes and their distribution according to phylogenetic groups.
Antibiotics 14 00931 g002
Antibiotics 14 00931 g003
Figure 3. Prevalence of the virulence genes and distribution of phylogenetic groups.
Figure 3. Prevalence of the virulence genes and distribution of phylogenetic groups.
Antibiotics 14 00931 g003
Table 1. Frequency of the detected genes in 111 E. coli isolates obtained from the ceca of chicken broiler samples.
Table 1. Frequency of the detected genes in 111 E. coli isolates obtained from the ceca of chicken broiler samples.
CategoryDeterminantn (%)
Virulence genestraT93 (83.8%)
fimH88 (79.3%)
iutA82 (73.9%)
bfpA48 (43.2%)
fyuA35 (31.5%)
stx-1/shiga toxin34 (30.6%)
stx-28 (7.2%)
Resistance genesblaCTX-M-G185 (76.6%)
blaTEM47 (42.3%)
blaSHV36 (32.4%)
blaCMY12 (10.8%)
blaIMP7 (6.3%)
blaOXA-4814 (12.6%)
mcr-16 (5.4%)
aadA-180 (72%)
aadA-533 (29.7%)
aac (6)-Ib-cr32 (28.8%)
qnrS24 (21.6%)
qnrB22 (19.8%)
IntegronsInt1+92 (82.9%)
Int2+44 (39.6%)
Table 2. Prevalence of resistance genes and their distribution according to phylogenetic groups.
Table 2. Prevalence of resistance genes and their distribution according to phylogenetic groups.
GenesA (n = 43) Percent (%)B1 (n = 18)Percent (%)B2 (n = 6)Percent (%)C (n = 3)Percent (%)Clade I (n = 2)Percent (%)D (n = 15)Percent (%)E (n = 8)Percent (%)F (n = 16)Percent (%)TotalPercent (%)p-Value
blaCTX-M-14093950583.3266.72100960787.51168.88576.60.001
blaSHV1125.6633.3116.7266.700640225743.83632.40.001
blaTEM1637.2633.3233.331002100426.76758504742.30.001
blaCMY24.7316.7000000213.300531.31210.80.156
mcr-149.3211.100000000000065.40.741
blaIMP511.615.600000016.7000076.30.819
blaOXA-4849.3211.100133.300426.7112.5212.51412.60.661
qnrA12.315.600000000000021.80.975
qnrB1023.3426.7233.3133.30032000212.52219.80.705
qnrS1841.915.600133.300426.7000002421.60.003
aac(6)Ib-cr1739.5316.7350266.700533.300212.53228.80.054
acc(3)-II1432.6426.7233.30000746.722516.330270.011
aadA-13479.11477.83503100150960562.51062.580720.001
aadA-51637.2426.7233.30000533.300637.53329.70.001
sul1818.6633.300133.300426.70016.320180.27
sul21023.3527.8233.30000426.700002219.80.231
IntI137861688.96100310021001173.34501381.39282.90.001
IntI21432.6950466.7002100213.3337.51062.54439.60.001
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Tayh, G.; Nsibi, F.; Abdallah, K.; Abbes, O.; Fliss, I.; Messadi, L. Phenotypic and Molecular Study of Multidrug-Resistant Escherichia coli Isolates Expressing Diverse Resistance and Virulence Genes from Broilers in Tunisia. Antibiotics 2025, 14, 931. https://doi.org/10.3390/antibiotics14090931

AMA Style

Tayh G, Nsibi F, Abdallah K, Abbes O, Fliss I, Messadi L. Phenotypic and Molecular Study of Multidrug-Resistant Escherichia coli Isolates Expressing Diverse Resistance and Virulence Genes from Broilers in Tunisia. Antibiotics. 2025; 14(9):931. https://doi.org/10.3390/antibiotics14090931

Chicago/Turabian Style

Tayh, Ghassan, Fatma Nsibi, Khaled Abdallah, Omar Abbes, Ismail Fliss, and Lilia Messadi. 2025. "Phenotypic and Molecular Study of Multidrug-Resistant Escherichia coli Isolates Expressing Diverse Resistance and Virulence Genes from Broilers in Tunisia" Antibiotics 14, no. 9: 931. https://doi.org/10.3390/antibiotics14090931

APA Style

Tayh, G., Nsibi, F., Abdallah, K., Abbes, O., Fliss, I., & Messadi, L. (2025). Phenotypic and Molecular Study of Multidrug-Resistant Escherichia coli Isolates Expressing Diverse Resistance and Virulence Genes from Broilers in Tunisia. Antibiotics, 14(9), 931. https://doi.org/10.3390/antibiotics14090931

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop