Next Article in Journal
Antibiotics Use for Dental or Oral Cavity Infections in Pediatric Dentistry: Knowledge and Prescribing Practices Between Italian Dentists
Previous Article in Journal
Microbiome and Resistome in Poultry Litter-Fertilized and Unfertilized Agricultural Soils
Previous Article in Special Issue
Antimicrobial Resistance Genes in Clinical Escherichia coli Strains from Livestock and Poultry in Shandong Province, China During 2015–2020
 
 
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 4
10.3390/antibiotics14040356
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

Serogrouping and Molecular Characterization of ESBL-Producing Avian Pathogenic Escherichia coli from Broilers and Turkeys with Colibacillosis in Algeria

by
Nadia Safia Chenouf
1,2,3,
Chafik Redha Messaï
1,4,
Isabel Carvalho
5,
Tamara Álvarez-Gómez
6,
Vanessa Silva
5,7,
Abdelghani Zitouni
3,
Ahcene Hakem
8,
Patricia Poeta
5,8,9,10 and
Carmen Torres
6,*
1
Faculty of Natural and Life Sciences, Earth and Universe Sciences, University Mohamed El Bachir El Ibrahimi of Bordj Bou Arreridj, El Anasser, Bordj Bou Arreridj 34000, Algeria
2
Laboratory for Exploration and Valorization of Steppe Ecosystems (EVES), Department of Biology, Faculty of Natural Sciences and Life, University of Djelfa, Moudjbara Road BP 3117, Djelfa 17000, Algeria
3
Laboratoire de Biologie des Systèmes Microbiens (LBSM), Ecole Normale Supérieure Cheikh Mohamed El Bachir El Ibrahimi, BP 92, Kouba, Algiers 16000, Algeria
4
Laboratory of Research Health and Animal Production, High National Veterinary School, Issad Abbes Street, Oued Smar, Algiers 16000, Algeria
5
Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal
6
Area Biochemistry and Molecular Biology, OneHealth-UR Research Group, University of La Rioja, 26006 Logroño, Spain
7
LAQV-REQUIMTE, Department of Chemistry, NOVA School of Science and Technology, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal
8
Agropastoralism Research Center of Djelfa, Djelfa 17000, Algeria
9
CECAV—Veterinary and Animal Research Centre, University of Traìs-os-Montes and Alto Douro (UTAD), 5000-801 Vila Real, Portugal
10
Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS), 5000-801 Vila Real, Portugal
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(4), 356; https://doi.org/10.3390/antibiotics14040356
Submission received: 1 February 2025 / Revised: 10 March 2025 / Accepted: 17 March 2025 / Published: 31 March 2025
(This article belongs to the Special Issue Molecular Characterization and Antimicrobial Resistance of Pathogens Isolated from Animals)
Browse Figure
Versions Notes

Abstract

:
Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) strains is a bacterial disease responsible for enormous economic losses in the poultry industry, due to high mortality rates in farms, antibiotic therapy costs, and seizures at slaughterhouses. The aim of this study was to characterize the serogroups and molecular features of extended spectrum β-lactamase (ESBL)-producing APEC isolates recovered from 248 liver samples of 215 broilers and 33 turkeys with colibacillosis lesions in northeast Algeria. For this, microbiological tests were carried out, according to the recommended standards: E. coli isolates were recovered using standard microbiological protocols, and identification was carried out by MALDI-TOF MS. Serogrouping was performed using a rapid agglutination slide and the antisera of three O somatic groups (O1, O2, O78). Antimicrobial susceptibility was determined by the disk diffusion method. PCR assays and sequencing were used to detect antimicrobial resistance genes, integrons, phylogrouping, and MLST. Conjugation experiments were also conducted to determine the transferability of the retrieved ESBL-encoding genes. Overall, 211 (85.1%) APEC isolates were collected (one per positive sample), and 164 (77.7%) of them were typable. The O2 and O1 serogroups were the most detected (46.1% in broiler typable isolates and 61.5% in turkey typable isolates). Seventeen APEC isolates were ESBL-producers and harbored the following genes (number of isolates): blaCTX-M-1 (14), blaCTX-M-15 (2), and blaSHV-12 (1). They belonged to phylogroups D (10 isolates), B1 (6 isolates), and B2 (1 isolate). The MLST of 13 ESBL producers revealed seven STs: ST23, ST38, ST48, ST117, ST131, ST1146, and ST5087. The ESBL-encoding genes were transferred by conjugation among 15 ESBL-producing isolates, and transconjugants acquired either the IncK or IncI1 plasmids. Concerted efforts from all poultry actors are needed to establish surveillance monitoring strategies to mitigate the spread of ESBL-producing isolates implicated in avian colibacillosis.

1. Introduction

Avian infections caused by avian pathogenic Escherichia coli (APEC), so-called avian colibacillosis (ACL), have been noticed to be among the most reported devastating infectious diseases in poultry husbandry, resulting in weighty economic losses worldwide. These diseases can present as localized or systemic infections, manifesting in various forms across avian species., including colisepticemia, chronic respiratory disease (CRD), yolk sac infection/omphalitis, and osteomyelitis in turkey [1,2].
APEC is known to be either a primary pathogen or occasionally secondary to other.predisposing factors, such as initial respiratory viral (Newcastle disease, infectious bronchitis, avian influenza) and Mycoplasma infections [3,4]. APEC strains are classified as a subset of extra-intestinal pathogenic E. coli (ExPEC), a large group of pathogens with diverse virulent properties. They are serologically categorized into a variety of serogroups according to the serotyping method established by Kauffmann, in which the O antigen structure confers the main basis [5]. Earlier on, most APEC strains associated with ACL were ascribed to three major serogroups, O1, O2, and O78 [6]; nevertheless, although a greater diversity was shown thereafter via WGS [7,8,9,10], serotyping remains an established tool for APEC typing [11,12].
For decades, antibiotics have been extensively used in poultry farming for treatment, prevention, and growth promotion (the latter was banned in European countries in 2006 and later in some other countries). This promoted the dissemination of resistant and/or multidrug-resistant (MDR) bacteria, including APEC strains. Antibiotic resistance (AR) is recognized as a global, escalating health crisis, and poultry is regarded as a worrying source of resistant bacteria that may be transmitted to humans via the food supply chain [13,14]. The production of extended-spectrum β-lactamases (ESBLs) is considered a major player in the AR phenomenon and one of the most significant AR mechanisms used by E. coli among other Enterobacteriaceae species [15,16]. ESBLs are a group of diverse and rapidly evolving enzymes that inactivate third- and fourth-generation cephalosporins and monobactams but not cephamycin or carbapenems. Multiple ESBL types have been discovered and can be categorized into different families. While TEM- and SHV-type ESBLs are genetically related, CTX-M-type ESBLs have been found to be more diverse and remain the most dominant worldwide [17]. The successful spread of blaCTX-M-encoding genes is mainly due to their location on conjugative plasmids that may co-harbor other AR genes, resulting in resistance to other multiple antibiotic classes, such as aminoglycosides and fluoroquinolones [15].
In Algeria, poultry constitutes the most dominant livestock, notably in the northeast, and chickens and turkeys were the predominant sources of poultry meat in 2021 (91% and 6.1% of total poultry production, respectively). Although some studies characterized the virulence factors of APEC strains causing ACL in this country [18,19], only a small number investigated the genetic characteristics of antimicrobial-resistant and/or ESBL-producing APEC from diseased animals [20]. Therefore, the objective of this study was to determine the serogrouping and provide an in-depth molecular characterization of ESBL-producing strains collected from broilers and turkeys with colibacillosis lesions in northeast Algeria.

2. Results

2.1. APEC Isolation, Serogrouping, and Antimicrobial Susceptibility Testing

Out of the 248 liver samples, 211 contained APEC isolates (85.1%) and one isolate per positive sample was selected. These isolates originated from 185 of 215 broilers (86%) and 26 of 33 turkeys (78.8%), as shown in Table 1.
Regarding serogrouping, among the 185 broiler isolates, 143 (77.3%) of them were typable: the O2 serogroup was the predominant one, with 66 isolates (35.7%), followed by O1 with 50 isolates (27%), and then O78 with 27 isolates (14.6%). Moreover, 42 (22.7%) of the broiler APEC isolates were non-typable with these sera reagents. Concerning turkey isolates, 21 (80.8%) were typable: the dominant serogroup was O1 (61.5%), followed by O2 (15.4%), and then O78 (1) (3.8%). Conversely, five turkey isolates (19.2%) were non-typable.
As for antimicrobial susceptibility, high resistance rates were observed in APEC isolates from broilers and turkeys, respectively, towards tetracycline (87.9% and 100%), nalidixic acid (94.6% and 96.1%), ampicillin (81.1% and 100%), trimethoprim/sulfamethoxazole (75.7% and 88.5%), amoxicillin/clavulanic acid (75.7% and 69.2%), and ciprofloxacin (65.4% and 73%). On the contrary, all isolates exhibited susceptibility to imipenem (Table 2). MDR (resistance to at least three antimicrobial classes) was detected in 94% of broiler APEC isolates and in all turkey APEC isolates (Table 3).

2.2. Prevalence and Antimicrobial Resistance Genotype of ESBL-Producing APEC Isolates

The ESBL phenotype was detected in 17 out of 211 total APEC isolates (rates of 7% and 15.4% in broiler and turkey samples analyzed, respectively). All ESBL-producing isolates were MDR but showed susceptibility to colistin (MIC ≤ 1 μg/mL). PCR and sequencing assays revealed three ESBL-encoding genes (number of isolates): blaCTX-M-1 (14), blaCTX-M-15 (2), and blaSHV-12 (1) (Table 4). The blaTEM-1 gene, encoding a widespread β-lactamase, was also found in five ESBL-producing isolates. Regarding resistance to non-β-lactam agents, all ESBL-positive isolates carried the tet(A) gene, and three of them co-harbored the tet(B) gene. Sulfonamide resistance, detected in 12 isolates, was encoded by sul1 (5 isolates), sul2 (6 isolates), and sul3 genes (1 isolate). The two chloramphenicol-resistant isolates contained the catA gene, and the gentamicin-resistant isolate carried the aac(3)-II gene. Class 1 integrons were detected in 8 of the 12 trimethoprim/sulfamethoxazole-resistant ESBL producers (Table 4).

2.3. Molecular Typing and Conjugation Transference

Out of the 17 ESBL-producing isolates, 10 (58.8%) were found to belong to the D phylogenetic group. Six isolates (35.3%) were assigned to the B1 phylogroup, while only one isolate belonged to the B2 phylogroup (5.9%). MLST revealed seven different STs: ST23, ST38, ST48, ST117, ST131, ST1146, and ST5087 (Table 4).
Fifteen isolates (all blaCTX-M-1-harboring isolates and the blaSHV-12-harboring isolate) were found to be involved in the transferability of the plasmid-borne blaCTX-M and blaSHV-12, respectively. Transconjugants that carried the blaCTX-M-1 gene acquired the IncK plasmid in five isolates and the IncI1 plasmid in association with tetracycline resistance encoded by tet(A) in one isolate. However, the conjugative plasmids in the remaining transconjugants were non-typable (Table 5).

3. Discussion

Avian colibacillosis is a bacterial infection affecting poultry farming, and antimicrobial-resistant APEC strains have been continuously reported globally. In Algeria, several studies have reported the occurrence of ACL in broilers nationwide [21,22,23,24]. Other authors have investigated the virulence determinants of APEC strains [18,19]. However, data on the AR genetic traits of APEC strains, notably ESBL-APEC isolates, are still lacking, and the available figures have been sourced from a sparse number of surveys [20]. To fill this gap, this study was conducted to provide a better molecular characterization of ESBL-producing APEC isolates collected in northeast Algeria throughout a seven-year period (2016–2021).
Out of the 248 liver samples collected from broilers and turkeys with ACL lesions at four slaughterhouses, 211 (85.1%) contained APEC isolates (isolation rates of 86% in broilers and 78.8% in turkeys). These findings corroborate previous studies conducted nationally [21,22,23,24] and internationally [25], in which the APEC isolation rates exceeded 80%. Our study provides more evidence that ACL remains one of the most prevailing avian diseases encountered in broilers and turkeys and a primary cause of the seizure and destruction of carcasses in slaughterhouses, as well as that E. coli is the major etiological agent in Algeria and across the world [26]. It is important to mention that the small sample size of turkeys is attributed to the limited number of turkey farms in the study region, which may generate several limitations.
In our study, 77.8% of APEC isolates belonged to three serogroups: O1 (31.3%), O2 (33.2%), and O78 (12.3%). These serogroups are the most often recovered amongst APEC isolates from cases of colibacillosis, with O78 being the most pathogenic [27,28,29]. In fact, the prediction of the pathogenicity of microorganisms isolated from sick animals is crucial. For E. coli, serotyping remains the basis of any pathogenicity diagnosis, even if it allows for the identification of a limited number of APEC isolates [30,31].
Our results are in line with those reported by numerous previous studies, but with variable frequencies. This variability could be attributed, on the one hand, to the use of different serotyping methods and, on the other hand, to the variable number of isolates studied from one study to another [9,27]. However, the distribution and frequencies of these dominant serogroups vary considerably geographically and temporally [32].
Also, it was reported that E. coli strains most commonly belonging to those serogroups possess typical APEC virulence factors such as lipopolysaccharide (LPS) (colibacillary endotoxin), the hemagglutinin protein Tsh, resistance to serum, and phagocytosis and have the ability to colonize the internal organs of chickens [27,33]. Likewise, possession of certain chromosomal or plasmid genes encoding virulence factors contributes significantly to APEC isolates’ biology and gives them a specific pathogenicity. Therefore, APEC strains may be a source of genes and plasmids that encode virulence factors [9,34].
Overall, 17 APEC isolates were resistant to CTX and/or CAZ. Basically, resistance to third-generation cephalosporins is a feature of Enterobacteriaceae that produce either ESBLs or AmpC β-lactamases, notably the CMY type. The latter are less prevalent than ESBLs but are still of great interest. In this study, all third-generation cephalosporin-resistant isolates were ESBL producers. Therefore, we only focused on ESBL genes, and no typical AmpC phenotype was detected.
The prevalence of ESBL-producing APEC in this study (8% from both avian species) seems much higher than the ones revealed in other studies, with rates of 5% and 1.9%, respectively [20,23]. Other previous surveys of poultry in Algeria investigating ESBL-producing E. coli carriage in fecal samples of healthy animals and broiler livers commercialized in the center of Algeria reported rates of 26.2% and 5.9%, respectively [13,35]. In Tunisia, a carriage rate of 25.5% of ESBL-producing E. coli was recorded in healthy chickens at laying hen farms [36]. Likewise, Misumi et al. [37] reported a percentage of 26.3% in chickens with ACL in Japan. Similar frequencies were observed in APEC causing ACL in broilers in Spain [38]. In Brazil, among 15 APEC isolates recovered from spiced chicken meat, 13 were ESBL producers [39]. Aside from the number of samples, the discrepancies in ESBL prevalence recorded in the different studies cited above could be traced back to the variability of the isolation protocols (with or without selective media supplemented with antibiotics).
The dramatically increasing rates of ESBL in avian E. coli strains could be primarily associated with the misuse of third-generation cephalosporins, among other β-lactams such as amoxicillin and ampicillin in poultry farms to control the early mortality caused by E. coli infections [40]. Ceftiofur, a third-generation cephalosporin that is indicated to treat cattle and swine respiratory infections, is not approved for use in poultry in many countries, including Algeria and the European Union. Nonetheless, numerous studies have propounded a possible link between the administration of ceftiofur and the expansion of third-generation cephalosporin-resistant and/or ESBL-producing E. coli strains in poultry [41,42,43]. Moreover, several surveys found that in certain poultry farms and hatcheries, Marek’s disease and avian influenza vaccines are administered jointly with ceftiofur to promote growth and prevent secondary bacterial infections [41,44].
Unsurprisingly, the blaCTX-M group 1 subtypes—blaCTX-M-1 (n = 14) and blaCTX-M-15 (n = 2)—were the most predominant ESBL-encoding genes detected in our study. Indeed, it is well known that most outbreaks implicating ESBLs have been spawned by CTX-M subtypes rather than TEM or SHV. The two variants detected in this study are the most widely spread ESBL genes among animals and humans, respectively [15,17], and the CTX-M-1 enzyme is known to be the most frequent ESBL subtype in avian E. coli from healthy and sick chickens in Algeria and universally [20,45,46,47]. Throughout the globe, blaCTX-M-carrying isolates have been found in many ecological niches, including hospital and community settings, food-producing animals, wild animals, pets, food, and the environment [17,48]. The drastic explosion of CTX-M-type β-lactamases, also known as “the CTX-M pandemic”, might be the consequence of the dissemination of blaCTX-M genes through mobile genetic platforms such as self-transmissible plasmids and transposons [49]. The blaSHV-12 gene was also retrieved in one specimen. SHV-producing Enterobacteriaceae have been previously reported in poultry and poultry products in different countries, including Algeria [13,50,51]. This low frequency of the SHV type among our APEC isolates confirmed that, together with the TEM ESBL types, SHV types are thought to have almost been displaced by CTX-M subtypes [49,52].
In this study, ESBL genes were carried by plasmids belonging to incompatibility groups IncK and IncI1. These two plasmids, referred to as epidemic plasmids, were successfully transferred through conjugation experiments, which demonstrate the significant role of horizontal gene transfer in the spread of ESBL genes and the genomic plasticity of our ESBL-producing APEC isolates.
Our ESBL-containing APEC isolates were distributed in three phylogroups: D (10 isolates), B1 (6 isolates), and B2 (1 isolate). These phylogroups differ considerably in terms of AR phenotype, genetic content, ecological niches, and pathogenicity [53]. While the B1 phylogroup is common in animals and encompasses commensal and intestinal pathogenic E.coli strains (InPEC), the D and B2 phylogroups comprise ExPEC strains, including greatly virulent ones [54,55]. Among the B1 phylogenetic group, four different STs—ST23, ST48, ST5087, and ST1146—were detected, underlining the diversity of our isolates. Previous reports indicated the circulation of the first three clones among avian E. coli isolates, highlighting the dissemination of these international lineages and their contribution to the wide spread of ESBL-encoding genes [13,56,57]. As for ST1146, it has been described to occur among E. coli isolates from poultry [58] and in grain culture soil samples [59]. Two genetic lineages assigned to the D phylogroup (ST38 and ST117) and one in the B2 phylogroup (ST131) were also observed in our isolates. ESBL-producing E. coli isolates of these lineages have been widely described across different ecologies and hosts [60,61], designating their role as ESBL drivers and high-risk clonal lineages.
Importantly, several studies have revealed the relationship between serotyping and STs in APEC isolates, with special attention paid to APEC O78 serotypes [12,62]. In our study, two ESBL-producing APEC isolates assigned to the O78 serogroup were sequenced as ST23. This finding is in line with previous reports that indicated that this serogroup is mainly represented by two distinct sequence types, ST23 and ST88 [62]. Also, the O78 ST117 APEC sequence type has largely spread and was implicated in large colibacillosis outbreak cases in Nordic broilers [63]. This lineage is known to be associated with high virulence and MDR determinants [64]. However, in our study, ESBL isolates with ST117 belonged to serotype O1. This could be explained by the fact that APEC O78 ST23 strains encode the H4 flagella antigens, while ST117 strains can encode a variety of O-antigens in place of O78, as mentioned by [64,65]. Genomic epidemiological studies, typing of H-antigens, and determination of virulence genes methods need to be further designed to comprehend the phylogenetic relationships of APEC types.

4. Materials and Methods

4.1. Research Approval

As this study was performed on dead animals, it was deemed to be exempt from needing protocol approval.

4.2. Sample Collection

From July 2016 to May 2021, 248 liver samples of condemned carcasses of 215 broilers and 33 turkeys diagnosed with ACL on post mortem examination carried out according to [66] were obtained at four licensed poultry slaughterhouses and one veterinary clinic in northeast Algeria, located in Setif, Algiers, Boumerdes, and Bordj Bou Arréridj (BBA) cities. As illustrated in Figure 1, live animals originated from 55 supply poultry houses located in 25 communes belonging to 11 wilayas in northeast Algeria (Sétif, Algiers, Batna, Béjaia, Tizi Ouzou, Boumerdes, Annaba, Constantine, Mila, Skikda, and Bordj Bou Arréridj). Liver samples were accurately labeled and then transported to the laboratory in cooled sterile containers to be processed.

4.3. Sample Processing and APEC Strain Isolation

A Bunsen burner was used to sterilize the surface of each organ sample before cutting it down sterilely into small cubes using sterile forceps and scissors. Enrichment was performed by placing some pieces of each organ sample in a 10 mL nutrient broth tube (Pasteur Institute of Algeria, PIA) and incubating overnight at 37 °C. Next, a drop of each enriched culture was streaked onto MacConkey selective and differential media (BiotechLab, Algiers, Algeria) and incubated at 37 °C for 24 h. Growth of E. coli gave rise to pink colonies (lactose-positive) often surrounded by precipitated bile salts. For each of the positive samples, one pure presumptive E. coli colony was picked up and identified using Gram staining, an oxidase test, a catalase test, and a triple sugar iron agar (TSI) test (PIA). Ultimately, API 20E system kits (BioMérieux, Marcy-l’Étoile, France) and the MALDI-TOF Biotyper system were used to identify E. coli isolates.

4.4. Serogrouping

According to the manufacturer’s instructions, APEC isolates were first streaked onto trypto-casein soy agar (TSA) media (Biolab, Algeria) and incubated at 37 °C for 24 h. Serogrouping was then carried out using a rapid agglutination slide (Citoglas, London, UK) and the antisera of the 3 O somatic groups (O1, O2, O78) (Biovac, Anger, France) as described by [67].

4.5. Antimicrobial Susceptibility and ESBL Testing

According to the guidelines of the Clinical Laboratory Standards Institute [68], the antimicrobial susceptibility of APEC isolates was determined using the Kirby–Bauer disk diffusion method. The following antimicrobial agents were used (μg/disk): ampicillin (AMP, 10), amoxicillin/clavulanic acid (AMC, 20 + 10), cefotaxime (CTX, 30), ceftazidime (CAZ, 30), imipenem (IMP, 10), nalidixic acid (NA, 30), ciprofloxacin (CIP, 5), neomycin (NEO, 30), gentamicin (GEN, 10), trimethoprim/sulfamethoxazole (SXT, 1.25 + 23.75), chloramphenicol (CLR, 30), tetracycline (TET, 30), and nitrofurantoin (FUR, 300). The quality control strain E. coli ATCC 25,922 was used for antimicrobial susceptibility testing.
Phenotypic ESBL screening was conducted for all APEC isolates through the double-disk synergy test (DDST), which uses cefotaxime and ceftazidime disks (third-generation cephalosporins) along with an amoxicillin/clavulanic acid disk (β-lactamase inhibitor) placed 30 mm apart, center to center [69]. ESBL-producing K. pneumoniae ATCC 700,603 were adopted as a reference strain. Additionally, the colistin minimum inhibitory concentration (MIC) which delineates in vitro levels of susceptibility or resistance was evaluated in ESBL-producing APEC isolates using the agar dilution method, as prescribed by the European Committee on Antimicrobial Susceptibility Testing guidelines [70].

4.6. Antimicrobial Resistance Genes in ESBL-Producing APEC Isolates

In ESBL-producing APEC isolates, the rapid boiling method was used to extract genomic DNA [71]. Afterwards, PCR and consecutive sequencing assays were used to investigate the presence of ESBL-encoding genes (blaCTX-M groups, blaTEM, and blaSHV), colistin resistance genes (mcr-1 to mcr-3), tetracycline resistance genes (tet(A) and tet(B)), sulfonamides resistance genes (sul1, sul2, and sul3), gentamicin resistance genes (aac(3)-I and aac(3)-II), and chloramphenicol resistance genes (cmlA, and florR and catA). Moreover, the detection of class 1 and 2 integrons was screened through the presence of integrase genes (intI and intII) [13,72]. The beta-lactamase genes obtained by PCR (blaCTX-M groups, blaTEM, and blaSHV) were sequenced to determine the variants of the genes [13,72].

4.7. Phylogrouping, MLST, and Conjugation Experiments

ESBL-producing APEC isolates were categorized into one of the four phylogenetic groups (A, B1, B2, or D) using a rapid protocol previously described [8]. Multilocus sequence typing (MLST) was used to characterize selected ESBL-producing APEC isolates using the sequences of internal fragments of the seven house-keeping genes specific to E. coli (adk, fumC, gyrB, icd, mdh, purA and recA). The sequence type (ST) of each isolate was then determined using the E. coli MSLT database (https://enterobase.warwick.ac.uk/species/ecoli/allele_st_search, accessed on 20 April 2019).
Conjugation experiments were performed to determine the transferability of the previously retrieved ESBL-encoding genes. Selected ESBL-harboring APEC isolates were selected as donors, and E. coli CSH26 (plasmid-free, lactose-negative, and RifR) was chosen as recipient. Rifampicin (50 µg/mL) and cefotaxime (4 µg/mL) were added to MacConkey agar to select transconjugants, which were subsequently subjected to antimicrobial susceptibility testing and ESBL gene amplification using PCR assays. Ultimately, PCR-based replicon typing (PBRT) of the donors and the ESBL-containing transconjugants was carried out to look into the acquired genes [13,73].

5. Conclusions

In this study, we report the high prevalence of O2 and O1 serogroups among APEC isolates from Algerian poultry and the dominance of the blaCTX-M-1-encoding gene in ESBL-producing APEC isolates. MLST showed a great diversity of ESBL-producing APEC isolates, with detection of high-risk clonal lineages. We also report the detection of successful transferred epidemic plasmids (IncK and IncI1), highlighting the genomic plasticity of our ESBL-producing APEC isolates. Concerted efforts from all poultry actors are needed to establish vigilant infection monitoring strategies and mitigate the spread of ESBL-producing APEC strains in Algeria.

Author Contributions

Conceptualization, N.S.C., C.R.M., I.C., P.P. and C.T.; methodology, N.S.C., C.R.M., I.C., T.Á.-G., V.S. and C.T.; software, N.S.C., C.R.M., I.C., T.Á.-G., V.S. and C.T.; validation, N.S.C., C.R.M., I.C., T.Á.-G., V.S., A.H., A.Z., P.P. and C.T.; formal analysis, N.S.C., C.R.M., I.C., T.Á.-G., V.S. and C.T.; investigation, N.S.C., C.R.M. and C.T.; resources, C.T; data curation, N.S.C., C.R.M., I.C., T.Á.-G., V.S. and C.T.; writing—original draft preparation, N.S.C., C.R.M., I.C. and C.T.; writing—review and editing, N.S.C., I.C. and C.T.; visualization, N.S.C., C.R.M., I.C., T.Á.-G., V.S. and C.T.; supervision, C.T., C.R.M., P.P., A.H. and A.Z.; project administration, C.T.; funding acquisition, C.T. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported in part by projects UI/00772 and LA/P/0059/2020 funded by the Portuguese Foundation for Science and Technology (FCT).

Institutional Review Board Statement

Ethical review and approval were waived as sampling activities were performed for diagnostic purposes.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Acknowledgments

The authors gratefully acknowledge Chorfa Abdelhafid, veterinary practitioner, and Guechtouli Sihem, teacher and researcher at the University of Boumerdes, Algeria, as well as the veterinarians at the sampled slaughterhouses, for their valuable help with sampling.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lutful Kabir, S.M. Avian Colibacillosis and Salmonellosis: A Closer Look at Epidemiology, Pathogenesis, Diagnosis, Control and Public Health Concerns. Int. J. Environ. Res. Public. Health 2010, 7, 89–114. [Google Scholar] [CrossRef] [PubMed]
  2. Nolan, L.K.; Vaillancourt, J.P.; Barbieri, N.L.; Logue, C.M. Colibacillosis. In Diseases of Poultry, 14th ed.; Swayne, D.E., Ed.; Wiley-Blackwell: Hoboken, NJ, USA, 2020; Volume 1, pp. 770–830. [Google Scholar]
  3. Ewers, C.; Janssen, T.; Wieler, L.H. Avian pathogenic Escherichia coli (APEC). Berl. Munch. Tierarztl. Wochenschr. 2003, 116, 381–395. [Google Scholar]
  4. Kathayat, D.; Lokesh, D.; Ranjit, S.; Rajashekara, G. Avian Pathogenic Escherichia coli (APEC): An Overview of Virulence and Pathogenesis Factors, Zoonotic Potential, and Control Strategies. Pathogens 2021, 10, 467. [Google Scholar] [CrossRef] [PubMed]
  5. Kauffmann, F. The Serology of the Coli Group. J. Immunol. Baltim. Md 1950 1947, 57, 71–100. [Google Scholar]
  6. Dziva, F.; Stevens, M.P. Colibacillosis in Poultry: Unravelling the Molecular Basis of Virulence of Avian Pathogenic Escherichia coli in Their Natural Hosts. Avian Pathol. 2008, 37, 355–366. [Google Scholar] [CrossRef]
  7. Newman, D.M.; Barbieri, N.L.; de Oliveira, A.L.; Willis, D.; Nolan, L.K.; Logue, C.M. Characterizing Avian Pathogenic Escherichia coli (APEC) from Colibacillosis Cases, 2018. PeerJ 2021, 9, e11025. [Google Scholar] [CrossRef]
  8. Clermont, O.; Bonacorsi, S.; Bingen, E. Rapid and Simple Determination of the Escherichia coli Phylogenetic Group. Appl. Environ. Microbiol. 2000, 66, 4555–4558. [Google Scholar] [CrossRef] [PubMed]
  9. Mehat, J.W.; van Vliet, A.H.M.; La Ragione, R.M. The Avian Pathogenic Escherichia coli (APEC) Pathotype Is Comprised of Multiple Distinct, Independent Genotypes. Avian Pathol. J. WVPA 2021, 50, 402–416. [Google Scholar] [CrossRef]
  10. Wirth, T.; Falush, D.; Lan, R.; Colles, F.; Mensa, P.; Wieler, L.H.; Karch, H.; Reeves, P.R.; Maiden, M.C.J.; Ochman, H.; et al. Sex and Virulence in Escherichia coli: An Evolutionary Perspective. Mol. Microbiol. 2006, 60, 1136–1151. [Google Scholar] [CrossRef]
  11. Kravik, I.H.; Kaspersen, H.; Sjurseth, S.K.; Dean, K.R.; David, B.; Aspholm, M.; Sekse, C. A Molecular Epidemiological Study on Escherichia coli in Young Chicks with Colibacillosis Identified Two Possible Outbreaks across Farms. Vet. Res. 2023, 54, 10. [Google Scholar] [CrossRef]
  12. Cordoni, G.; Woodward, M.J.; Wu, H.; Alanazi, M.; Wallis, T.; La Ragione, R.M. Comparative Genomics of European Avian Pathogenic E. Coli (APEC). BMC Genomics 2016, 17, 960. [Google Scholar] [CrossRef] [PubMed]
  13. Chenouf, N.S.; Carvalho, I.; Messaï, C.R.; Ruiz-Ripa, L.; Mama, O.M.; Titouche, Y.; Zitouni, A.; Hakem, A.; Torres, C. Extended Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae from Broiler Liver in the Center of Algeria, with Detection of CTX-M-55 and B2/ST131-CTX-M-15 in Escherichia coli. Microb. Drug Resist. Larchmt. N 2021, 27, 268–276. [Google Scholar] [CrossRef]
  14. Helmy, Y.A.; Kathayat, D.; Closs, G.J.; Galgozy, K.; Fuchs, J.R.; Rajashekara, G. Efficacy of Quorum Sensing and Growth Inhibitors Alone and in Combination against Avian Pathogenic Escherichia coli Infection in Chickens. Poult. Sci. 2023, 102, 102543. [Google Scholar] [CrossRef] [PubMed]
  15. Husna, A.; Rahman, M.M.; Badruzzaman, A.T.M.; Sikder, M.H.; Islam, M.R.; Rahman, M.T.; Alam, J.; Ashour, H.M. Extended-Spectrum β-Lactamases (ESBL): Challenges and Opportunities. Biomedicines 2023, 11, 2937. [Google Scholar] [CrossRef]
  16. Ur Rahman, S.; Ali, T.; Ali, I.; Khan, N.A.; Han, B.; Gao, J. The Growing Genetic and Functional Diversity of Extended Spectrum Beta-Lactamases. BioMed Res. Int. 2018, 2018, 9519718. [Google Scholar] [CrossRef] [PubMed]
  17. Castanheira, M.; Simner, P.J.; Bradford, P.A. Extended-Spectrum β-Lactamases: An Update on Their Characteristics, Epidemiology and Detection. JAC-Antimicrob. Resist. 2021, 3, dlab092. [Google Scholar] [CrossRef]
  18. Meguenni, N.; Chanteloup, N.; Tourtereau, A.; Ahmed, C.A.; Bounar-Kechih, S.; Schouler, C. Virulence and Antibiotic Resistance Profile of Avian Escherichia coli Strains Isolated from Colibacillosis Lesions in Central of Algeria. Vet. World 2019, 12, 1840–1848. [Google Scholar] [CrossRef]
  19. Mohamed, L.; Ge, Z.; Yuehua, L.; Yubin, G.; Rachid, K.; Mustapha, O.; Junwei, W.; Karine, O. Virulence Traits of Avian Pathogenic (APEC) and Fecal (AFEC) E. coli Isolated from Broiler Chickens in Algeria. Trop. Anim. Health Prod. 2018, 50, 547–553. [Google Scholar] [CrossRef]
  20. Meguenni, N.; Le Devendec, L.; Jouy, E.; Le Corvec, M.; Bounar-Kechih, S.; Rabah Bakour, D.; Kempf, I. First Description of an Extended-Spectrum Cephalosporin- and Fluoroquinolone- Resistant Avian Pathogenic Escherichia coli Clone in Algeria. Avian Dis. 2015, 59, 20–23. [Google Scholar] [CrossRef]
  21. Messaï, C.; Khelef, D.; Boukhors, K.; Radji, N.; Goucem, R.; Hamdi, T. Antimicrobial Susceptibility of Escherichia coli Strains Isolated from Broiler Chickens Affected by Colibacillosis in Setif. Afr. J. Microbiol. Res. 2013, 7, 2668–2672. [Google Scholar] [CrossRef]
  22. Messaï, C.R.; Aït-Oudhia, K.; Khelef, D.; Hamdi, T.M.; Chenouf, N.S.; Messaï, M.R. Serogroups and Antibiotic Susceptibility Pattern of Avian Pathogenic Escherichia coli Strains Responsible for Colibacillosis in Broiler Breeding Farms in the East of Algeria. Afr. J. Microbiol. Res. 2015, 9, 2358–2363. [Google Scholar] [CrossRef]
  23. Halfaoui, Z.; Menoueri, N.M.; Bendali, L.M. Serogrouping and Antibiotic Resistance of Escherichia coli Isolated from Broiler Chicken with Colibacillosis in Center of Algeria. Vet. World 2017, 10, 830–835. [Google Scholar] [CrossRef]
  24. Aberkane, C.; Messaï, A.; Messaï, C.R.; Boussaada, T. Antimicrobial Resistance Pattern of Avian Pathogenic Escherichia coli with Detection of Extended-Spectrum β-Lactamase-Producing Isolates in Broilers in East Algeria. Vet. World 2023, 16, 449–454. [Google Scholar] [CrossRef] [PubMed]
  25. Goudarztalejerdi, A.; Mohammadzadeh, A.; Najafi, S.V.; Nargesi, F.; Joudari, S. Serogrouping, Phylotyping, and Virulence Genotyping of Commensal and Avian Pathogenic Escherichia coli Isolated from Broilers in Hamedan, Iran. Comp. Immunol. Microbiol. Infect. Dis. 2020, 73, 101558. [Google Scholar] [CrossRef]
  26. Delago, J.; Miller, E.A.; Flores-Figueroa, C.; Munoz-Aguayo, J.; Cardona, C.; Smith, A.H.; Johnson, T.J. Survey of Clinical and Commensal Escherichia coli from Commercial Broilers and Turkeys, with Emphasis on High-Risk Clones Using APECTyper. Poult. Sci. 2023, 102, 102712. [Google Scholar] [CrossRef]
  27. Mellata, M.; Dho-Moulin, M.; Dozois, C.M.; Curtiss, R., 3rd; Lehoux, B.; Fairbrother, J.M. Role of Avian Pathogenic Escherichia coli Virulence Factors in Bacterial Interaction with Chicken Heterophils and Macrophages. Infect. Immun. 2003, 71, 494–503. [Google Scholar] [CrossRef] [PubMed]
  28. Kim, Y.B.; Yoon, M.Y.; Ha, J.S.; Seo, K.W.; Noh, E.B.; Son, S.H.; Lee, Y.J. Molecular Characterization of Avian Pathogenic Escherichia coli from Broiler Chickens with Colibacillosis. Poult. Sci. 2020, 99, 1088–1095. [Google Scholar] [CrossRef]
  29. Hu, R.; Li, J.; Zhao, Y.; Lin, H.; Liang, L.; Wang, M.; Liu, H.; Min, Y.; Gao, Y.; Yang, M. Exploiting Bacterial Outer Membrane Vesicles as a Cross-Protective Vaccine Candidate against Avian Pathogenic Escherichia coli (APEC). Microb. Cell Factories 2020, 19, 119. [Google Scholar] [CrossRef]
  30. Durso, L.M.; Bono, J.L.; Keen, J.E. Molecular Serotyping of Escherichia coli O26:H11. Appl. Environ. Microbiol. 2005, 71, 4941–4944. [Google Scholar] [CrossRef]
  31. Schouler, C.; Schaeffer, B.; Brée, A.; Mora, A.; Dahbi, G.; Biet, F.; Oswald, E.; Mainil, J.; Blanco, J.; Moulin-Schouleur, M. Diagnostic Strategy for Identifying Avian Pathogenic Escherichia coli Based on Four Patterns of Virulence Genes. J. Clin. Microbiol. 2012, 50, 1673–1678. [Google Scholar] [CrossRef]
  32. Frydendahl, K. Prevalence of Serogroups and Virulence Genes in Escherichia coli Associated with Postweaning Diarrhoea and Edema Disease in Pigs and a Comparison of Diagnostic Approaches. Vet. Microbiol. 2002, 85, 169–182. [Google Scholar] [CrossRef]
  33. La Ragione, R.M.; Woodward, M.J. Virulence Factors of Escherichia coli Serotypes Associated with Avian Colisepticaemia. Res. Vet. Sci. 2002, 73, 27–35. [Google Scholar] [CrossRef]
  34. Dziva, F.; Hauser, H.; Connor, T.R.; van Diemen, P.M.; Prescott, G.; Langridge, G.C.; Eckert, S.; Chaudhuri, R.R.; Ewers, C.; Mellata, M.; et al. Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms. Infect. Immun. 2013, 81, 838–849. [Google Scholar] [CrossRef] [PubMed]
  35. Belmahdi, M.; Bakour, S.; Al Bayssari, C.; Touati, A.; Rolain, J.-M. Molecular Characterisation of Extended-Spectrum β-Lactamase- and Plasmid AmpC-Producing Escherichia coli Strains Isolated from Broilers in Béjaïa, Algeria. J. Glob. Antimicrob. Resist. 2016, 6, 108–112. [Google Scholar] [CrossRef]
  36. 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]
  37. Misumi, W.; Magome, A.; Okuhama, E.; Uchimura, E.; Tamamura-Andoh, Y.; Watanabe, Y.; Kusumoto, M. CTX-M-55-Type ESBL-Producing Fluoroquinolone-Resistant Escherichia Coli Sequence Type 23 Repeatedly Caused Avian Colibacillosis in Kagoshima Prefecture, Japan. J. Glob. Antimicrob. Resist. 2023, 35, 325–331. [Google Scholar] [CrossRef] [PubMed]
  38. Solà-Ginés, M.; Cameron-Veas, K.; Badiola, I.; Dolz, R.; Majó, N.; Dahbi, G.; Viso, S.; Mora, A.; Blanco, J.; Piedra-Carrasco, N.; et al. Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain. PLoS ONE 2015, 10, e0143191. [Google Scholar] [CrossRef]
  39. da Silva, F.B.; Ferreira, M.R.A.; Sobrinho, I.d.S.J.; Dias, M.; Rodrigues, R.R.; Moreira, C.N. Occurrence of ESBL-Producing Avian Pathogenic Escherichia coli (APEC) Isolates in Spiced Chicken Meat in Goias, Brazil. Lett. Appl. Microbiol. 2023, 76, ovac070. [Google Scholar] [CrossRef]
  40. Seo, K.W.; Shim, J.B.; Kim, Y.B.; Son, S.H.; Bi Noh, E.; Yoon, S.; Lim, S.-K.; Ju Lee, Y. Impacts and Characteristics of Antimicrobial Resistance of Escherichia coli Isolates by Administration of Third-Generation Cephalosporins in Layer Hatcheries. Vet. Microbiol. 2020, 243, 108643. [Google Scholar] [CrossRef]
  41. Baron, S.; Jouy, E.; Larvor, E.; Eono, F.; Bougeard, S.; Kempf, I. Impact of Third-Generation-Cephalosporin Administration in Hatcheries on Fecal Escherichia coli Antimicrobial Resistance in Broilers and Layers. Antimicrob. Agents Chemother. 2014, 58, 5428–5434. [Google Scholar] [CrossRef]
  42. Saraiva, M.M.S.; Moreira Filho, A.L.B.; Freitas Neto, O.C.; Silva, N.M.V.; Givisiez, P.E.N.; Gebreyes, W.A.; Oliveira, C.J.B. Off-Label Use of Ceftiofur in One-Day Chicks Triggers a Short-Term Increase of ESBL-Producing E. coli in the Gut. PLoS ONE 2018, 13, e0203158. [Google Scholar] [CrossRef]
  43. Heinrich, K.; Chan, D.; Fussell, R.J.; Kay, J.F.; Sharman, M. Can the Unauthorised Use of Ceftiofur Be Detected in Poultry? Food Addit. Contam. Part Chem. Anal. Control Expo. Risk Assess. 2013, 30, 1733–1738. [Google Scholar] [CrossRef]
  44. Shen, X.; Zhang, A.; Zhao, R.; Yin, L.; Yin, D.; Dai, Y.; Hou, H.; Wang, J.; Hu, X.; Pan, X.; et al. Effects of Adding Antibiotics to an Inactivated Oil-Adjuvant Avian Influenza Vaccine on Vaccine Characteristics and Chick Health. Poult. Sci. 2024, 103, 104135. [Google Scholar] [CrossRef] [PubMed]
  45. Hassen, B.; Abbassi, M.S.; Ruiz-Ripa, L.; Mama, O.M.; Hassen, A.; Torres, C.; Hammami, S. High Prevalence of Mcr-1 Encoding Colistin Resistance and First Identification of Bla(CTX-M-55) in ESBL/CMY-2-Producing Escherichia coli Isolated from Chicken Faeces and Retail Meat in Tunisia. Int. J. Food Microbiol. 2020, 318, 108478. [Google Scholar] [CrossRef]
  46. Irrgang, A.; Hammerl, J.A.; Falgenhauer, L.; Guiral, E.; Schmoger, S.; Imirzalioglu, C.; Fischer, J.; Guerra, B.; Chakraborty, T.; Käsbohrer, A. Diversity of CTX-M-1-Producing E. coli from German Food Samples and Genetic Diversity of the Bla(CTX-M-1) Region on IncI1 ST3 Plasmids. Vet. Microbiol. 2018, 221, 98–104. [Google Scholar] [CrossRef] [PubMed]
  47. Girlich, D.; Poirel, L.; Carattoli, A.; Kempf, I.; Lartigue, M.-F.; Bertini, A.; Nordmann, P. Extended-Spectrum Beta-Lactamase CTX-M-1 in Escherichia coli Isolates from Healthy Poultry in France. Appl. Environ. Microbiol. 2007, 73, 4681–4685. [Google Scholar] [CrossRef] [PubMed]
  48. Liu, C.M.; Stegger, M.; Aziz, M.; Johnson, T.J.; Waits, K.; Nordstrom, L.; Gauld, L.; Weaver, B.; Rolland, D.; Statham, S.; et al. Escherichia coli ST131-H22 as a Foodborne Uropathogen. mBio 2018, 9, e00470-18. [Google Scholar] [CrossRef]
  49. Cantón, R.; González-Alba, J.M.; Galán, J.C. CTX-M Enzymes: Origin and Diffusion. Front. Microbiol. 2012, 3, 110. [Google Scholar] [CrossRef]
  50. Chiaretto, G.; Zavagnin, P.; Bettini, F.; Mancin, M.; Minorello, C.; Saccardin, C.; Ricci, A. Extended Spectrum Beta-Lactamase SHV-12-Producing Salmonella from Poultry. Vet. Microbiol. 2008, 128, 406–413. [Google Scholar] [CrossRef]
  51. Briñas, L.; Moreno, M.A.; Zarazaga, M.; Porrero, C.; Sáenz, Y.; García, M.; Dominguez, L.; Torres, C. Detection of CMY-2, CTX-M-14, and SHV-12 Beta-Lactamases in Escherichia coli Fecal-Sample Isolates from Healthy Chickens. Antimicrob. Agents Chemother. 2003, 47, 2056–2058. [Google Scholar] [CrossRef]
  52. Liakopoulos, A.; Mevius, D.; Ceccarelli, D. A Review of SHV Extended-Spectrum β-Lactamases: Neglected Yet Ubiquitous. Front. Microbiol. 2016, 7, 1374. [Google Scholar] [CrossRef]
  53. Touchon, M.; Hoede, C.; Tenaillon, O.; Barbe, V.; Baeriswyl, S.; Bidet, P.; Bingen, E.; Bonacorsi, S.; Bouchier, C.; Bouvet, O.; et al. Organised Genome Dynamics in the Escherichia coli Species Results in Highly Diverse Adaptive Paths. PLoS Genet. 2009, 5, e1000344. [Google Scholar] [CrossRef] [PubMed]
  54. Singh, A.; Upadhyay, V.; Upadhyay, A.K.; Singh, S.M.; Panda, A.K. Protein Recovery from Inclusion Bodies of Escherichia coli Using Mild Solubilization Process. Microb. Cell Factories 2015, 14, 41. [Google Scholar] [CrossRef]
  55. Lagerstrom, K.M.; Hadly, E.A. Under-Appreciated Phylogroup Diversity of Escherichia coli within and between Animals at the Urban-Wildland Interface. Appl. Environ. Microbiol. 2023, 89, e0014223. [Google Scholar] [CrossRef]
  56. Blaak, H.; Hamidjaja, R.A.; van Hoek, A.H.A.M.; de Heer, L.; de Roda Husman, A.M.; Schets, F.M. Detection of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli on Flies at Poultry Farms. Appl. Environ. Microbiol. 2014, 80, 239–246. [Google Scholar] [CrossRef]
  57. Zahoor, M.A.; Nawaz, Z.; Jamil, A.; Yasmin, A.; Alagawany, M.; Othman, S.I.; Allam, A.A.; El-Shall, N.A. Determining the Prevalence and Genetic Diversity of Plasmid-Mediated Sulfonamide Resistance in Escherichia coli from Commercial Broiler Samples. Poult. Sci. 2024, 103, 103258. [Google Scholar] [CrossRef]
  58. Zurfluh, K.; Wang, J.; Klumpp, J.; Nüesch-Inderbinen, M.; Fanning, S.; Stephan, R. Vertical Transmission of Highly Similar Bla CTX-M-1-Harboring IncI1 Plasmids in Escherichia coli with Different MLST Types in the Poultry Production Pyramid. Front. Microbiol. 2014, 5, 519. [Google Scholar] [CrossRef]
  59. Furlan, J.P.R.; Gallo, I.F.L.; Stehling, E.G. Genomic Characterization of Multidrug-Resistant Extraintestinal Pathogenic Escherichia coli Isolated from Grain Culture Soils. Pedosphere 2022, 32, 495–502. [Google Scholar] [CrossRef]
  60. Lübcke, P.; Heiden, S.E.; Homeier-Bachmann, T.; Bohnert, J.A.; Schulze, C.; Eger, E.; Schwabe, M.; Guenther, S.; Schaufler, K. Multidrug-Resistant High-Risk Clonal Escherichia coli Lineages Occur along an Antibiotic Residue Gradient in the Baltic Sea. Npj Clean Water 2024, 7, 94. [Google Scholar]
  61. Soncini, J.G.M.; Cerdeira, L.; Sano, E.; Koga, V.L.; Tizura, A.T.; Tano, Z.N.; Nakazato, G.; Kobayashi, R.K.T.; Aires, C.A.M.; Lincopan, N.; et al. Genomic Insights of High-Risk Clones of ESBL-Producing Escherichia coli Isolated from Community Infections and Commercial Meat in Southern Brazil. Sci. Rep. 2022, 12, 9354. [Google Scholar] [CrossRef]
  62. Denamur, E.; Clermont, O.; Bonacorsi, S.; Gordon, D. The Population Genetics of Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2021, 19, 37–54. [Google Scholar] [CrossRef] [PubMed]
  63. Ronco, T.; Stegger, M.; Olsen, R.H.; Sekse, C.; Nordstoga, A.B.; Pohjanvirta, T.; Lilje, B.; Lyhs, U.; Andersen, P.S.; Pedersen, K. Spread of Avian Pathogenic Escherichia coli ST117 O78:H4 in Nordic Broiler Production. BMC Genom. 2017, 18, 13. [Google Scholar] [CrossRef]
  64. Clermont, O.; Dixit, O.V.A.; Vangchhia, B.; Condamine, B.; Dion, S.; Bridier-Nahmias, A.; Denamur, E.; Gordon, D. Characterization and Rapid Identification of Phylogroup G in Escherichia coli, a Lineage with High Virulence and Antibiotic Resistance Potential. Environ. Microbiol. 2019, 21, 3107–3117. [Google Scholar] [CrossRef] [PubMed]
  65. Kim, S.-W.; Karns, J.S.; Van Kessel, J.A.S.; Haley, B.J. Genome Sequences of Five Multidrug-Resistant Escherichia coli Sequence Type 117 Isolates Recovered from Dairy Calves. Genome Announc. 2017, 5, e00732-17. [Google Scholar] [CrossRef]
  66. Majó, N.; Dolz, R. Autopsie Des Volailles: Diagnostic Macroscopique et Méthodes de Prélèvements; Les éditions du Point Vétérinaire: Evreux à l’Eure, France, 2012; 82p. [Google Scholar]
  67. Ørskov, F.; Ørskov, I. The Serology of Capsular Antigens. Curr. Top. Microbiol. Immunol. 1990, 150, 43–63. [Google Scholar] [PubMed]
  68. Wayne, P. Clinical and Laboratory Standards Institute. M100: Performance Standards for Antimicrobial Susceptibility Testing, 32nd ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2022; ISBN 9781684400331. [Google Scholar]
  69. Jarlier, V.; Nicolas, M.H.; Fournier, G.; Philippon, A. Extended Broad-Spectrum Beta-Lactamases Conferring Transferable Resistance to Newer Beta-Lactam Agents in Enterobacteriaceae: Hospital Prevalence and Susceptibility Patterns. Rev. Infect. Dis. 1988, 10, 867–878. [Google Scholar] [CrossRef]
  70. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 12.0. Available online: http://www.eucast.org/clinical_breakpoints/ (accessed on 25 June 2024).
  71. Holmes, D.S.; Quigley, M. A Rapid Boiling Method for the Preparation of Bacterial Plasmids. Anal. Biochem. 1981, 114, 193–197. [Google Scholar] [CrossRef]
  72. Carvalho, I.; Safia Chenouf, N.; Cunha, R.; Martins, C.; Pimenta, P.; Pereira, A.R.; Martínez-Álvarez, S.; Ramos, S.; Silva, V.; Igrejas, G.; et al. Antimicrobial Resistance Genes and Diversity of Clones among ESBL- and Acquired AmpC-Producing Escherichia coli Isolated from Fecal Samples of Healthy and Sick Cats in Portugal. Antibiotics 2021, 10, 262. [Google Scholar] [CrossRef]
  73. Carattoli, A.; Bertini, A.; Villa, L.; Falbo, V.; Hopkins, K.L.; Threlfall, E.J. Identification of Plasmids by PCR-Based Replicon Typing. J. Microbiol. Methods 2005, 63, 219–228. [Google Scholar] [CrossRef]
Antibiotics 14 00356 g001
Figure 1. Distribution of poultry houses at cities level (Wilayas). (a): Map of Africa; (b): Locations of sampled poultry houses. Maps were established using https://www.mapsland.com and https://www.mapchart.net/, accessed on 12 September 2024.
Figure 1. Distribution of poultry houses at cities level (Wilayas). (a): Map of Africa; (b): Locations of sampled poultry houses. Maps were established using https://www.mapsland.com and https://www.mapchart.net/, accessed on 12 September 2024.
Antibiotics 14 00356 g001
Table 1. Serogroups of broiler and turkey APEC isolates (n = 211).
Table 1. Serogroups of broiler and turkey APEC isolates (n = 211).
SerogroupBroiler Isolates
(n = 185)		Turkey Isolates
(n = 26) 		Total Isolates
(n = 211)
O150 (27%)143 (77.3%)16 (61.5%)21 (80.8%)66 (31.3%)164 (77.8%)
O266 (35.7%)4 (15.4%)70 (33.2%)
O7827 (14.6%)1 (3.8%)28 (13.3%)
Non-Typable isolates42 (22.7%)5 (19.2%)47 (22.2%)
Table 2. Antimicrobial resistance rates of broiler and turkey APEC isolates (n = 211).
Table 2. Antimicrobial resistance rates of broiler and turkey APEC isolates (n = 211).
Antibiotic Disks UsedBroiler APEC Isolates
(n = 185) 		Turkey APEC Isolates
(n = 26)		Total APEC Isolates
n = 211
Ampicillin150 (81.1%)26 (100%)176 (83.4%)
Amoxicillin/clavulanic acid140 (75.7%)18 (69.2%)158 (74.9%)
Cefotaxime12 (6.5%)5 (19.2%)17 (8%)
Ceftazidime2 (1.1%)1 (3.8%)3 (1.4%)
Imipenem0 (0%)0 (0%)0 (0%)
Nalidixic acid175 (94.6%)25 (96.1%)200 (94.8%)
Ciprofloxacin121 (65.4%)19 (73%)140 (66.3%)
Neomycin63 (34.1%)8 (30.8%)71 (33.6%)
Gentamicin11 (5.9%)3 (11.5%)14 (6.6%)
Trimethoprim/sulfamethoxazole140 (75.7%)23 (88.5%)163 (77.2%)
Chloramphenicol36 (19.5%)11 (42.3%)47 (22.3%)
Tetracycline181 (97.8%)26 (100%)207 (98.1%)
Nitrofurantoin65 (35.1%)10 (38.5%)75 (35.5%)
Table 3. Multidrug resistance in broiler and turkey APEC isolates (n = 211).
Table 3. Multidrug resistance in broiler and turkey APEC isolates (n = 211).
Antimicrobial Classes
with Resistance		Broiler APEC Isolates (n = 185)Turkey APEC Isolates (n = 26)
Number of IsolatesPrevalence (%)Number of IsolatesPrevalence (%)
021.100
10000
294.900
3 *3720311.5
4 *5127.6830.8
5 *4222.7934.6
6 *3016.2519.2
7 *147.613.8
Total18510026100%
*: MDR (multidrug resistance).
Table 4. Genetic characterization of ESBL-producing APEC isolates (n = 17).
Table 4. Genetic characterization of ESBL-producing APEC isolates (n = 17).
ESBL Isolate CodePoultry House LocationSampling Point (Year)Breed Type (Age)SerogroupPhylogroup/STAntimicrobial Resistance PhenotypeMIC of COL (µg/µL)Antimicrobial Resistance Genotype
C8195 Djemila (Setif)Slaughterhouse (2016)Broiler (7–8 weeks)O2B1/ST5087AMP-AMC-CTX-SXT-FUR-CLR-NAL-CIP-TET≤1blaCTX-M-1, tet(A), Intl1, Cat, sul2
C8196 Hamma Bouziane (Constantine)Slaughterhouse (2016)Broiler (7–8 weeks)O2B1/ST48AMP-CTX-SXT-TET≤1blaCTX-M-1, tet(A), Intl1, sul2
X800 Ain Arnat (Setif)Slaughterhouse (2016)Broiler (7–8 weeks)O1D/ST38AMP-AMC-CTX-SXT-NAL-CIP-TET≤1blaCTX-M-1, tet(A), Intl1, sul2
X801
X802
X803 		BejaiaSlaughterhouse (2016)Broilers (7–8 weeks)O78
O78
O1		B1/ST23
B1/ST23
B1/ST23		AMP-AMC-CTX-SXT-NAL-CIP-TET
AMP-AMC-CTX-NAL-TET
AMP-AMC-CTX-NAL-TET		≤1blaCTX-M-1, tet(A), sul1
blaCTX-M-1, tet(A)
blaCTX-M-1, tet(A)
X804 Remada (Setif)Veterinary clinic (2018)Turkey (12 weeks)O2B2/ST131AMP-CTX-GEN-SXT-CLR-NAL-CIP-TET≤1blaCTX-M-1, tet(A), Intl1, cat, sul1, aac(3)-II
X805
X806 		Bir Hadada (Setif)Veterinary clinic (2018)Broilers (3 weeks)O1
O1		D/ST117
D/ND		AMP-CTX-SXT-NAL-CIP-TET
AMP-CTX-SXT-NAL-CIP-TET		≤1blaCTX-M-1, blaTEM-1, tet(A), tet(B), Intl1, sul1
blaCTX-M-1, blaTEM-1, tet(A), tet(B), Intl1, sul2
X807
X808		Bellaa (Setif)Veterinary clinic (2018)Turkeys (4 weeks)O1
O1		D/ST117
D/ST117		AMP-CTX-SXT-NAL-TET
AMP-CTX-SXT-NAL-CIP-TET		≤1blaCTX-M-1, blaTEM-1, tet(A), tet(B), Intl1, sul2
blaCTX-M-1, blaTEM-1, tet(A), Intl1, sul2
X809
X810		Bir Hadada (Setif)Veterinary clinic (2018)Broilers (5 weeks)O1
O1		D/ST117
D/ND		AMP-CTX-NAL-TET
AMP-CTX-NAL-TET		≤1blaCTX-M-1, tet(A),
blaCTX-M-1, tet(A)
X811Bir Hadada (Setif)Veterinary clinic (2018)Turkey (7 weeks)O1D/ST117AMP-CTX-NAL-TET≤1blaCTX-M-1, tet(A)
X1241Beida Bordj (Setif)Slaughterhouse (2018)Turkey (7–8 weeks)NTB1/ST1146AMP-CTX-CAZ-NEO-SXT-TET≤1blaSHV-12, blaTEM-1, tet(A), sul3
BBA001(Bordj Bou Arréridj)Slaughterhouse (2021)Broiler (7–8 weeks)NTD/NDAMP-CTX-CAZ-TET-SXT≤1blaCTX-M-15, tet(A), sul1
BBA002(Bordj Bou Arréridj)Slaughterhouse (2021)Broiler (7–8 weeks)NTD/NDAMP-CTX-CAZ-TET-SXT≤1blaCTX-M-15, tet(A), sul1
AMP: ampicillin; AMC: amoxicillin/clavulanic acid; CTX: cefotaxime; CAZ: ceftazidime; COL: colistin; NAL: nalidixic acid; CIP: ciprofloxacin; GEN: gentamicin; NEO: neomycin; SXT: trimethoprim/sulfamethoxazole; FUR: nitrofurantoin; CLR: chloramphenicol; TET: tetracycline; MIC: minimum inhibitory concentration; ESBL: extended-spectrum β-lactamase; ST: sequence type; NT: non-typable; ND: not determined.
Table 5. Conjugation in ESBL-producing APEC isolates (n = 17).
Table 5. Conjugation in ESBL-producing APEC isolates (n = 17).
ESBL Isolate (Donor)Conjugation/Plasmids in DonorsTransconjugantsAntimicrobial Resistance Phenotype in TCPlasmids/Antimicrobial Resistance Genotype in TC
C8195+/IncI1, FIB, FIC, IncKX1223ESBL, RIF RIncK/blaCTX-M-1
C8196+/IncI1, IncKX1224ESBL, RIF RIncK/blaCTX-M-1
X800+/IncI1X1225ESBL, RIF R, TET RIncI1/blaCTX-M-1, tet(A)
X801+/IncI1, FIB, IncKX1468ESBL, RIF RIncK/blaCTX-M-1
X802+/IncI1, FIB, IncKX1226ESBL, RIF RIncK/blaCTX-M-1
X803+/IncI1, FIBX1469ESBL, RIF RIncK/blaCTX-M-1
X804+/IncI1, FIBX1227ESBL, RIF R, GEN RNon-typable/blaCTX-M-1, aac(3)-II
X805+/IncI1, FIB, FICX1228ESBL, RIF RNon-typable/blaCTX-M-1
X806+/IncI1, FIBX1470ESBL, RIF RNon-typable/blaCTX-M-1
X807+/IncI1, FIB, FICX1229ESBL, RIF RNon-typable/blaCTX-M-1
X808+/IncI1, FIBX1471ESBL, RIF RNon-typable/blaCTX-M-1
X809+/IncI1, FIBX1472ESBL, RIF RNon-typable/blaCTX-M-1
X810+/IncI1, FIBX1473ESBL, RIF RNon-typable/blaCTX-M-1
X811+/IncI1, FIBX1474ESBL, RIF RNon-typable/blaCTX-M-1
X1241+/K, IncI1X1475ESBL, RIF RNon-typable/blaSHV-12
BBA001−///
BBA002−///
+: positive conjugation; −: negative conjugation; TC: transconjugants; RIF: rifampicin; TET: tetracycline; GEN: gentamicin; R: resistance.
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

Chenouf, N.S.; Messaï, C.R.; Carvalho, I.; Álvarez-Gómez, T.; Silva, V.; Zitouni, A.; Hakem, A.; Poeta, P.; Torres, C. Serogrouping and Molecular Characterization of ESBL-Producing Avian Pathogenic Escherichia coli from Broilers and Turkeys with Colibacillosis in Algeria. Antibiotics 2025, 14, 356. https://doi.org/10.3390/antibiotics14040356

AMA Style

Chenouf NS, Messaï CR, Carvalho I, Álvarez-Gómez T, Silva V, Zitouni A, Hakem A, Poeta P, Torres C. Serogrouping and Molecular Characterization of ESBL-Producing Avian Pathogenic Escherichia coli from Broilers and Turkeys with Colibacillosis in Algeria. Antibiotics. 2025; 14(4):356. https://doi.org/10.3390/antibiotics14040356

Chicago/Turabian Style

Chenouf, Nadia Safia, Chafik Redha Messaï, Isabel Carvalho, Tamara Álvarez-Gómez, Vanessa Silva, Abdelghani Zitouni, Ahcene Hakem, Patricia Poeta, and Carmen Torres. 2025. "Serogrouping and Molecular Characterization of ESBL-Producing Avian Pathogenic Escherichia coli from Broilers and Turkeys with Colibacillosis in Algeria" Antibiotics 14, no. 4: 356. https://doi.org/10.3390/antibiotics14040356

APA Style

Chenouf, N. S., Messaï, C. R., Carvalho, I., Álvarez-Gómez, T., Silva, V., Zitouni, A., Hakem, A., Poeta, P., & Torres, C. (2025). Serogrouping and Molecular Characterization of ESBL-Producing Avian Pathogenic Escherichia coli from Broilers and Turkeys with Colibacillosis in Algeria. Antibiotics, 14(4), 356. https://doi.org/10.3390/antibiotics14040356

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