Prevalence of multidrug-resistant and extended-spectrum β–lactamase-producing Escherichia coli from chicken farms in Egypt

Background and Aim: Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains exhibit antibiotic resistance and are known to infect humans worldwide. This study assessed the phenotypic and genotypic prevalence of ESBL-resistant E. coli isolates recovered from the respiratory tracts of chickens in El-Sharkia Governorate, Egypt. Materials and Methods: We obtained 250 lung samples (one lung/bird) from 50 chicken farms (5 chickens/farm) to isolate, identify, and serotype E. coli. Antimicrobial resistance susceptibility was determined using the disk diffusion method, while the ESBL phenotype was identified using double disk synergy. We detected the β-lactamase genes, blaTEM, and blaSHV, using a polymerase chain reaction. Results: The results showed that 140/250 (56%) were infected with E. coli. All the serogroups of isolated E. coli exhibited high multi-antimicrobial resistance index values (>0.2), and 65.7% were confirmed to have ESBL. Among the isolates with the ESBL phenotypes, 55 (60%) and 32 (35%) contained the blaTEM and blaSHV genes, respectively. Conclusion: The widespread distribution of multidrug-resistant and ESBL-producing E. coli among poultry farms is a significant human health hazard. These results will help the Egyptian authorities to implement a national one-health approach to combat the antimicrobial resistance problem.


Introduction
Meat production from chicken, one of the most frequently farmed animals, accounts for over 90 billion tons annually worldwide [1]. The antibiotics used to treat bacterial infections in humans and food-producing animals can also be used as growth promoters to improve the productivity of poultry. However, the treatment efficacy of antibiotics is hindered by the rapid spread of antibiotic-resistant (ABR) pathogens. In the US, over 2.8 million ABR bacterial infections have been reported, with a mortality rate of approximately 35,000 [2]. Reports have shown that using antibiotics at sub-therapeutic concentrations in poultry feed increases their production [3]. As growth promoters, antibiotics exert several beneficial effects, such as combating subclinical diseases, reducing illness and mortality rates, enhancing growth rates, minimizing feed costs, and increasing the feed conversion ratio [4,5]. Certain antibiotic families are used as growth promoters in poultry and livestock, including penicillins, aminoglycosides, and tetracyclines. These are also used to treat bacterial infections in humans [6]. Excessive and inconsistent use of antibiotics in animals and humans has led to the emergence of muti-drug-resistant bacterial strains that can spread antibiotic resistance globally [7].
Avian pathogenic Escherichia coli (APEC) causing systemic or local infections outside the gut are known as extraintestinal pathogenic E. coli (ExPEC). Colibacillosis due to ExPEC affects broiler chicken aged 4-6 weeks and is characterized by subacute fibrinous aracialities, peritonitis, pericarditis, salpingitis, or septicemia [8,9]. Colibacillosis negatively impacts poultry production by increasing costs due to treatment and prophylaxis, mortality rates, and rejection of diseased carcasses at slaughterhouses [9]. Avian pathogenic E. coli exhibits various resistance patterns against antibiotics approved for poultry, including penicillin, sulfonamides, chloramphenicol, tetracyclines, fluoroquinolones, and aminoglycosides [10]. Extended-spectrum beta-lactamase (ESBL) producers are resistant to penicillin and oxyimino-cephalosporins, such as cefotaxime, ceftazidime, and monobactams. However, they cannot degrade cephamycins and are inactivated by clavulanic acid [11]. Several drugs permitted for veterinary medicine have been implicated in the development of ESBL-producing Gram-negative Available at www.veterinaryworld.org/Vol.16/May-2023/12.pdf Enterobacteriaceae [12,13], contributing to the emergence of ESBL genes among APEC strains [14,15]. Antibiotic-resistant commensal E. coli strains can distribute the ABR genes to other pathogenic bacteria, spreading resistant genes from poultry to humans [16]. In addition to chicken farms, ESBL-producing E. coli strains have also been found in other farm animals and meat products [17,18]. The most ubiquitous ESBL genes, TEM and SHV [19], have been found in food-producing animals, indicating that the food chain might be a possible transmission route from animals to humans [15,20].
Hence, monitoring the ABR patterns of human pathogens and pathogenic commensal bacteria in animals is crucial. In this study, we aimed to examine the prevalence of multidrug-resistant and ESBL-producing E. coli and perform genotypic characterization of the ESBL-related beta-lactamase (bla) genes, including blaTEM and blaSHV in poultry farms in the El-Sharkia Governorate, Egypt.

Ethical approval
This study was conducted according to the guidelines of the Institutional Animal Care and Use Committee of Zagazig University, Egypt (Approval no. ZU-IACUC/12/F/5/2019).

Study period and location
The study was conducted from December 2019 to April 2021 at the Faculty of Veterinary Medicine, Zagazig University, and the Reference Laboratory for Quality Control on Poultry Production (RLQP), Dokki, Egypt.

Sample collection, bacterial isolation, and identification
We collected 250 samples (lungs and trachea) from birds exhibiting clinical signs and post-mortem lesions of colibacillosis from 50 poultry farms, including commercial, intensive, low-scale (5000-10,000 birds), and rural farms with poor biosecurity levels and no official veterinary supervision from different regions in El-Sharkia Governorate, Egypt, between December 2019 and April 2021. The antibiotics used in these poultry farms either as growth promoters and prophylactics or for treatment included bacitracin, colistin, tetracycline, amoxicillin, cefotaxime, ampicillin, doxycycline, lincomycin, spectinomycin, fluorophenol, and ceftiofur Na.
After aseptically collecting the samples in sterile bags, they were immediately transported to the Reference Laboratory for Quality Control on Poultry Production (RLQP), Dokki, for bacteriological examination. To identify the E. coli strains, 25 g of the samples were homogenized in 250 mL of buffered peptone water and incubated at 37°C for 24 h for pre-enrichment [21]. Then, the strains were isolated and identified using a technique recommended by Swayne [22].

Serological identification of E. coli
According to Edwards and Ewing [23], the isolated strains were serotyped at the Animal Health Research Institute, Dokki, Giza, using diagnostic polyvalent and monovalent E. coli antisera (Denka Seiken Co. Ltd, Japan).

Antimicrobial susceptibility
The antimicrobial susceptibility of all E. coli isolates was tested using agar diffusion against 13 antibiotics (Oxoid, Hampshire, UK) according to Clinical and Laboratory Standards Institute guidelines and was clinically categorized with breakpoints [24]. Multiresistant isolates, that is, resistant to three or more antimicrobial categories, were selected for further examination [25].

Phenotypic screening of ESBL
We cultured suspensions containing 0.5 McFarland units of the confirmed multi-resistant strains on Mueller-Hinton agar plates (Oxoid) with cefotaxime and ceftazidime disks. The inhibition zone surrounding the disk/tablet with cephalosporin alone was compared to the zone around the disk/tablet containing cephalosporin and clavulanic acid. A size difference of ≥5 mm between the zones formed the disks with clavulanic acid and those without represents positive results [26].

Bacterial DNA extraction
The isolates were streaked on nutrient agar and incubated for 14-16 h at 37°C. Then, 5 mL liquid culture media was inoculated with a single colony and incubated overnight at 37°C. Genomic DNA was extracted at the RLQP using the G-spin™ Total DNA Extraction Kit (INTRON Biotechnology, Korea) according to the manufacturer's recommendations.

Detection of ABR genes
The polymerase chain reaction (PCR) oligonucleotide primers were obtained from Metabion (Germany) and Biobasic (Canada) ( Table-1) [27]. The 25 μL-PCR reaction mix consisted of 12.5 μL of Emerald Amp Max PCR Master Mix (Takara, Japan), 6 μL of template DNA, 1 μL of 20 pmol of each primer, and 4.5 μL of water. The PCR reactions were conducted using the Applied Biosystems 2720 thermal cycler under the following conditions: Initial denaturation for 3 min at 95°C; 30 cycles of 95°C for 30 s; annealing at a specific temperature for 1 min; extension at 72°C for 30 s; and final extension at 72°C for 5 min. To analyze the products, 15 μl of each product was loaded into each lane of a 1.5% agarose gel (Applichem, Darmstadt, Germany). Electrophoresis was performed in 1× Tris Boric acid EDTA buffer at a 5 V/cm gradient. The fragment sizes were determined using a 100 bp DNA ladder (Fermentas, Waltham, Massachusetts, USA). The gel photographs were obtained and analyzed using a gel documentation system (Alpha Innotech, Biometra, Germany).

Prevalence of E. coli among the examined poultry samples
Of the 250 chickens examined for E. coli infections from 50 broiler poultry farms, 140 specimens (56%) were positive for E. coli, while 110 were negative (44%) ( Table-2).

Serotyping of E. coli isolates
The E. coli isolates were serotyped using eight specific polyvalent and 43 monovalent group O somatic antisera. The results are shown in Table-3.

Sensitivity of E. coli serotypes to different antibiotics
The antibiotic resistance of the 140 E. coli strains was tested against 14 antibiotics using the disk diffusion method. As shown in Table-4, E. coli O groups were 100% resistant to streptomycin, cephalexin, oxytetracycline, and doxycycline, followed by ampicillin (94.6%) and sulphamethoxazole-trimethoprim, cefotaxime, and ceftazidime (92.9%). Furthermore, 24 out of 140 E. coli strains were resistant to apramycin, and 115 strains (82.1%) were resistant to norfloxacin, ciprofloxacin, and colistin sulfate.

Multidrug resistance and phenotypic ESBL production in the isolated E. coli strains
Tables-5 and 6 show the multiple antimicrobial resistance (MAR) index and ESBL production in the E. coli strains isolated from the chickens.

Detection of the blaSHV gene
The blaSHV gene imparts resistance against β-lactam antibiotics. Of the 92 ESBL-producing strains, 32 isolates (−35%) exhibited positive amplification of a 516 bp fragment using the primer specific to the blaSHV gene. The positive control also showed this 516 bp fragment, whereas the negative control showed no amplification (Figure-1).

Detection of the blaTEM gene
Escherichia coli strains containing the blaTEM gene are resistant to β-lactam antibiotics. We found that 60% of the E. coli isolates (55/92) with the ESBL phenotype contained this gene, as shown by the amplification of the 516 bp fragment using the primer specific for the blaTEM gene. The positive control also showed this 516 bp fragment, whereas the negative control did not (Figure-2).

Discussion
Our study investigated the prevalence of multidrug-resistant pathogenic or commensal E. coli isolated from the respiratory tracts of infected chickens with chronic respiratory disease signs. We also detected the genes causing ABR against β-lactam antibiotics, which can be transferred to zoonotic or commensal bacteria and, subsequently, to humans, causing potential infections. Colibacillosis in chickens might be a primary or secondary infection, which induces diverse localized or systemic infections caused by E. coli [28,29].
In this study, we found that E. coli was found in 56% of the chickens, possibly due to intensive rearing of birds in poorly aerated houses or because of Mycoplasma galliseptecum infection of the flock, which causes or aggravates colisepticaemia. Environmental stresses and respiratory viral infections may also predispose the chickens to this disease [30]. Similar results were shown by El-Tawab et al. [31], who reported a higher incidence of E. coli in chickens during winter (60.9%) than in summer (41%). These results agreed with that of Ruzauskas et al. [32], who reported a 41.7% prevalence of E. coli in raw chicken livers. Furthermore, similar results were obtained by Sarba et al. [33], who isolated E. coli from 40.4% of samples from colisepticaemia chickens.
Here, 140 of the 250 E. coli isolates obtained from chickens were categorized into seven O serogroups. The most dominant serotype was E. coli O 125, occurring in 35.7% of isolates (50/140), which is consistent with the previous studies of Ozaki et al. [34], who reported that O125 was the most prevalent (61.3%) serogroup associated with colibacillosis in poultry [33].
Due to the significant losses caused by colibacillosis in poultry production globally, different antibiotic groups are used to combat this infection, including β-lactams, aminoglycosides, fluoroquinolones,     ESBL=Extended-spectrum beta-lactamase sulfonamides, and tetracyclines, which has led to the emergence of multi-resistant E. coli strains [35]. The antibiogram performed for different E. coli serotypes against 13 antibiotics revealed that approximately 100% of the E coli isolates were multi-resistant as they were resistant to at least three antibiotics, displaying a so-called MAR pattern (against ≥3 antimicrobials). E. coli isolates showed high resistance against 13 antibiotics. A previous study examining broiler chickens in Egypt detected a high phenotypic resistance rate of E. coli to penicillin, streptomycin, trimethoprim/sulphamethoxazole, and tetracycline [31,36]. Other studies also showed the prevalence of multi-resistant E. coli among poultry farms in Tunisia [16] and Jordan [37]. Our results showed that 65.7% of the E. coli isolates were phenotypically ESBL-positive, consistent with Abdallah et al. [18], who found that 65.09% Available at www.veterinaryworld.org/Vol.16/May-2023/12.pdf of E. coli isolates (69/106) were ESBL producers. However, a recent survey showed that 46.7% of the chicken in poultry farms (56/120) in Egypt were phenotypically positive for ESBL [15]. In Sweden, ESBL-producing E. coli was found in the guts of approximately 34% of broilers [38]. In Malaysia, 48.8% of the E. coli isolates obtained from retail chicken meat shops were ESBL-positive [39].
Studies have found the blaTEM gene in 94.73% of isolated E. coli strains (18/19) [31], which agrees with the results of Colom et al. [27], who detected this gene in 88.2% of amoxicillin-clavulanate resistant E. coli isolates (45/51). However, these results contradicted with the study by Overdevest et al. [41], who obtained a lower percentage (−14%). The blaSHV and blaTEM genes were found in 1.8% and 72.9% of APEC isolates in Jordan, respectively [37]. This varies from the results of Huijbers et al. [42], who found that the incidence of blaSHV (17%) was higher than blaTEM (9.1%) among the ESBL-producing E. coli strains isolated from infected broilers and people employed in these farms [31].
ESBLs produced from Gram-negative bacteria, especially enterobacteriaceae, are encoded by plasmids and can cause resistance against all three generations of cephalosporins [43]. As cephalosporins are used to treat several Gram-negative bacterial infections in humans, the spread of ESBL-producing bacteria poses a significant public health threat to humans [44].
As antibiotic resistance is a global health issue [45], countries need to urgently adopt a national one approach to combat antimicrobial resistance, such as strengthening and implementing regulations for antibiotics use in veterinary and public health fields, focused surveillance to curb the spread of antimicrobial resistance in animals and humans, updating the guidelines of antimicrobial treatments, continuing education for prescribers on antimicrobial use, and monitoring drug abuse by animal owners and unskilled people for treating animals [46][47][48].

Conclusion
This study examined the different E. coli serotypes isolated from chicken farms in the El-Sharkia Governorate, Egypt. We found that 100% of the strains were multidrug-resistant, of which 65.7% were ESBL producers. We also detected high rates of blaTEM and blaSHV genes among these isolates. These results suggest that these resistance genes can be potentially transferred to humans, causing a significant public health threat. We recommend that the Egyptian authorities plan and implement a national one-health approach to combat ABR, including widening the surveillance of antimicrobial resistance, enforcing the available regulations, and monitoring the production, storage, sale, and usage of veterinary drugs.

Authors' Contributions
GAS: Conceptualized the study, wrote the manuscript, and curated and analyzed the data. EAA and MAK: Supervised the study, validated the results, conceptualized the work, and reviewed and edited the manuscript. RIA: Conceived the work, performed all experiments, and contributed to writing of the manuscript. NRR: Analyzed the data and drafted and revised the manuscript. All authors have read, reviewed, and approved the final manuscript.