Phenotypic and multiplex PCR-based genotypic characterization of ESBL-producing E. coli (ESBL-Ec) isolates from commercial layer poultry

Introduction

Enterobacteriaceae is a family of Gram-negative, facultative anaerobic, non-spore-forming rods. Although they are part of the normal gut microflora, many members, particularly E. coli, are responsible for clinical conditions involving the gastrointestinal and urinary tract, and various systemic infections (Paterson et al., 2006) [1] . Antibiotics, primarily beta-lactam groups, aminoglycosides, and fluoroquinolones are commonly used to treat the infections caused by these organisms. In recent times, there is an increasing threat of the emergence of antimicrobial resistance among animals due to the imprudent use of antibiotics in them. The indiscriminate and continuous use of beta-lactam antibiotics has led to the development of resistance to penicillin and lower-generation cephalosporins. Consequently, in the 1980s third-generation cephalosporins emerged as a solution to the increased resistance towards lower-generation drugs. But, the bacteria are smart enough to evolve and rapidly develop resistance even against the third generation cepholosporins. Hence, the effectiveness of this group is further enhanced by combining them with betalactam inhibitors such as clavulanic acid, tazobactam and sulbactam. However, the bacteria developed mechanisms to survive in the presence of beta-lactam inhibitors and render these drugs ineffective by inactivating the drugs with enzymes, modifying the antibiotic's target sites, or preventing the drug from reaching its target. The resistance to beta-lactam antibiotics in bacteria are conferred majorly by beta-lactamase enzymes including the Extended Spectrum Beta-Lactamases (ESBLs) encoded in transferable plasmids or chromosomes, which hydrolyse the betalactams, making them ineffective against penicillin, cephalosporins, and monobactams except for cephamycins and carbapenems (Smet et al., 2008; Teklu et al., 2019) [6,4] . Additionally, the plasmid-mediated spread of ESBL resistance makes it easier to propagate resistance to other members of Enterobacteriaceae present in the intestinal microflora. E. coli is a primary driver in the dissemination of ESBL mediated resistance via horizontal gene transfer and high-risk bacterial clones as it is ubiquitous and resilient gut microbe in humans and animals and acts as a massive environmental reservoir. These resistant strains and their plasmids are continuously exchanged between humans, livestock/food products, and the environment (via wastewater), making the dissemination cycle widespread as a part of the "One Health" cycle (Marcantonio et al., 2025) [14] . [12,13] . CTX-M enzymes represent a growing family of class A ESBLs that preferentially hydrolyze cefotaxime over ceftazidime, both oxyimino cephalosporins, including more than 30 members organized in six groups (Bonnet et al., 2004) [10] .

The spread of ESBL producing E. coli from poultry to humans is unneglectable as poultry meat and eggs contribute a major part of the human diet. [2,3,6] and layers (Wibisono et al., 2020; Olopade et al., 2022) [5,7] outside India, but minimal studies are available in India. Hence, it is important to assess the presence of ESBL-producing bacteria in poultry birds to formulate an effective mitigation and control strategies and prevent the spread of ESBL resistance to humans. The study area, Namakkal is popularly known as the poultry hub of south India as it houses a high density of layer birds. The present work aims to study the prevalence of ESBLproducing Enterobacteriaceae group of bacteria by phenotypic characterization as well as to determine the genes responsible for conferring resistance in layer birds around Namakkal.

Materials and Methods

Collection of sample

A total of 25 cloacal swabs from the live layer chickens at varying stages of laying were collected in a sterile container from Namakkal region using random sampling during August to December 2022 and shipped to laboratory on ice.

Isolation of E. coli from cloacal swabs

The collected swabs were inoculated in nutrient broth and incubated overnight at 37 °C. The inoculum from the nutrient broth was streaked onto MacConkey agar and incubated at 37 °C overnight for isolation of Gram-negative bacteria from the samples. The pink colonies (lactose fermenters) from the MacConkey agar were randomly selected and inoculated into eosin methylene blue agar to confirm the isolates as E. coli. Simultaneously, the genus of the isolates was determined by the by colony morphology, gram staining and biochemical tests using Hi24 Enterobacteriaceae Identification Kit (Cat. No. KB016). A single colony of each isolate was inoculated in nutrient broth and incubated at 37 °C overnight (Lupindu et al., 2017) [8] .

Phenotypic detection of ESBL production

The antibiotic sensitivity pattern of the isolates were determined in Mueller Hinton Agar (MHA) by Kirby Bauer disc diffusion method as per the CLSI standards using antibiotic discs such as amoxicillin/clavulanate (30 μg[20/10]), cefotaxime (30 μg), ceftazidime (30 μg), Cotrimazole (25 μg) and Amikacin (30 μg) (HiMedia India Pvt. Ltd) to determine the sensitivity patterns. The E. coli isolates which were not susceptible (either resistant or intermediate) to 3 or more antibiotics classes were identified as multidrug resistant (MDR) (Magiorakos et al., 2012) [17] . The E. coli isolates found to be resistant to third generation cephalosporins such as cefotaxime (30 μg), ceftazidime (30 μg) were subjected to combination disc test to determine their ability to produce ESBL by inactivating clavulanic acid and to grow in the presence of clavulanic acid inhibitors. A disc of ceftazidime (30 μg), ceftazidime and clavulanic acid (30 μg/10 μg), cefotaxime (30 μg), cefotaxime and clavulanic acid (30 μg/10 μg) was placed at an appropriate distance on a MHA plate inoculated with a suspension of each resistant isolate equating 0.5 McFarland turbidity standards and incubated overnight (18-24 h) at 37 °C. The isolates showing a difference of ≥ 5 mm in the inhibition zone diameter around the respective combination disc versus cefotaxime (30 μg) or ceftazidime (30 μg) disc alone were identified phenotypically as ESBL producers (Fröding et al., 2017) [18] . The data obtained was analysed in MS Excel and depicted in percentages.

Genotypic characterization of ESBL-producing isolates

The E. coli isolates phenotypically identified as ESBL producers were subjected to genotypic characterization targeting four common genes encoding the ESBL production (bla OXA , bla SHV , and bla TEM ) by multiplex PCR (Dallenne et al., (2010) [19] and bla CTX-M by uniplex PCR (Hasman et al., 2005) [9] . The whole-cell DNA from the resistant bacterial isolates was prepared by suspending a colony of ESBL isolate in 100 µL of distilled water and boiling at 100 °C for 10 min, cooled on ice followed by brief centrifugation at 4,000 rpm for 10 s (Queipo-Ortuño et al., 2008) [11] and the concentration of the DNA in the supernatant was determined in a Nano drop equipment. The primers and the amplifying conditions mentioned by Dallenne et al., 2010 [19] & Hasman et al., 2005 [9] were followed in this study (Table 1).

The PCR was subsequently performed in a 50 µL reaction mixture containing 25 µL of Taq DNA polymerase Master mix RED (Amplicon), 2 µL of the supernatant containing the DNA as template, 1 µL of forward and reverse primer each of bla OXA , bla SHV , and bla TEM gene-specific primers and 17 µL nuclease free water. PCR amplification was performed at an initial temperature of 94 °C for 10 min, followed by 30 cycles of 94 °C for 40 sec, 60 °C for 40 sec, and 72 °C for 1 min, and then a final extension of 72 °C for 7 min. The PCR amplification conditions for the bla CTX-M gene were 94 °C for 5 min, 35 cycles of 94 °C for 30 sec, 60 °C for 15 sec, 72 °C for 30 sec, and 72 °C for 5 min. The amplified PCR products were analysed by 1.5% agarose gel electrophoresis (w/v) at 100 V for one hour, and viewed in a gel documentation apparatus. The amplicon size of products was compared alongside a 100 bp DNA ladder.

Results

E. coli could be successfully isolated from all the cloacal swabs collected from the layer chickens. The colonies were confirmed as E. coli by the characteristic pink colour colonies produced by the lactose fermenters in the MacConkey agar (Fig. 1A), and green metallic sheen colonies in the EMB agar (Fig. 1B), as well as by Gram staining revealing the presence of uniform Gram-negative rods (Fig. 1C), and biochemical characteristics like IMViC reaction (+ + --) (Fig. 1D) The antibiotic sensitivity test to determine the susceptibility of the E. coli isolates in MHA agar using five antibiotics revealed the highest percentage of resistance against amikacin (72%) followed by cotrimoxazole (36%), cefotaxime (24%), ceftazidime (20%) and amoxicillinclavulanic acid combination (8%) and about 24% and 16% of isolates were found to be partially resistant to amikacin and ceftazidime respectively (Fig . 2). With regard to the resistance to third-generation cephalosporins, comparatively, a higher percentage [24% (6/25)] of isolates displayed resistance to cefotaxime against 20% (5/25) for ceftazidime. With respect to the ESBL production, among the 25 E. coli isolates, 28% (7/25) isolates were found to be resistant to third generation cephalosporins of either cefotaxime or ceftazidime by combination disk method (Fig. 3,4). Further, 57.14% (4/7) of the resistant isolates were exhibiting resistance to both cefotaxime and ceftazidime, and the remaining 42.85% (3/7) isolates were found to be resistant to either cefotaxime or ceftazidime.

Interestingly, only 50% (3/6) of the cefotaxime-resistant isolates were found to be ESBL producers, whereas all the ceftazidime-resistant isolates were identified as ESBL producer (Fig. 4) in the combination disc method. Notably, 12% (3/25) of isolates were discovered as multidrugresistant which were non-susceptible to at least 3 antibiotics belonging to different categories. Namakkal region, which is one of the most densely populated poultry belts in India that supplies a major portion of the eggs to the state of Tamil Nadu and other neighbouring states. The results of the phenotypic observations correlated well with the molecular characterization underscoring the role of either of the gene encoding ESBL in conferring resistance to the isolates. The presence of ESBL-E among the layer birds could significantly impact the food products obtained from them as there is a high probability for the dissemination of the genes conferring resistance through either via plasmids, transposons, and integrons (Carattoli, 2013; Zheng et al., 2025) [20,21] to other pathogens in humans as well as into the environment. This transfer of resistance to human beings happens through the animal-sourced food products, resulting in a major public health risk (Seiffert et al., 2013) [22] . The mode of transfer of ESBL resistance in low-and middleincome countries most often occurs between humans, animals and the environment rather than human-to-human transmission, which is quite common in high-income countries (Beaumont et al., 2026) [15] implying the role of poultry and the dissemination of resistance to manpower associated with poultry operations and eventually to the environment. Furthermore, the present study is in accordance with the observation made by Beaumont et al., (2026) [15] revealing the potential for resistance transfer from animals to humans. The occurrence of ESBL producing bacterial isolates could be majorly attributed to the usage of antibiotics as growth promoters which is quite a common practice among commercial poultry farm owners. The main contributors to the development of antimicrobial resistance in livestock rearing are the use of antibiotics to promote growth and for prophylactic measures and therapeutic purposes. The reckless usage of massive amounts of antibiotics in livestock rearing, often outweighing the human usage could potentially render antibiotics useless in fighting against the pathogens in the near future (Van Boeckel et al., 2017) [16] . Overall, a cautious usage of antibiotics and routine monitoring of the prevalence of ESBL producing E. coli among food animals is a crucial step to prevent the emergence and dissemination of antimicrobial resistance and to provide a safe and healthy environment where the all these biotic coexist. Additionally, the availability of fitting and comprehensive data plays an essential role in policymaking regarding the reduction in the usage of antibiotics in livestock rearing. The presence of resistance in the livestock microbiome poses a significant threat to public health given the close contact between humans and animals and the dependency of human beings on livestock for various food commodities. Given these scenario, strategic studies on antimicrobial resistance at regular intervals covering the most possible interfaces could contribute towards the antimicrobial resistance stewardship in near future.

References

1. Paterson DL. Resistance in gram-negative bacteria: Enterobacteriaceae. American Journal of Infection Control. 2006;34(5 Suppl 1):S20-S28.
2. Saliu EM, Vahjen W, Zentek J. Types and prevalence of extended-spectrum beta-lactamase producing Enterobacteriaceae in poultry. Animal Health Research Reviews. 2017;18(1):46-57.
3. Kola A, Kohler C, Pfeifer Y, Schwab F, Kühn K, Schulz K, et al. High prevalence of extended-spectrumβ-lactamase-producing Enterobacteriaceae in organic and conventional retail chicken meat, Germany. Journal of Antimicrobial Chemotherapy. 2012;67(11):26312634.
4. Teklu DS, Negeri AA, Legese MH, Bedada TL, Woldemariam HK, Tullu KD. Extended-spectrum betalactamase production and multi-drug resistance among Enterobacteriaceae isolated in Addis Ababa, Ethiopia. Antimicrobial Resistance & Infection Control. 2019;8:39.
5. Wibisono FJ, Sumiarto B, Untari TRI, Effendi MH, Permatasari DA, Witaningrum AM. Pattern of antibiotic resistance on extended-spectrum betalactamase genes producing Escherichia coli on laying hens in Blitar, Indonesia. Biodiversitas Journal of Biological Diversity. 2020;21(10):4631-4635.
6. Smet A, Martel A, Persoons D, Dewulf J, Heyndrickx M, Catry B, et al. Diversity of extended-spectrum βlactamases and class C β-lactamases among cloacal Escherichia coli isolates in Belgian broiler farms. Antimicrobial Agents and Chemotherapy. 2008;52(4):1238-1243.
7. Olopade A, Bitrus AA, Momoh-Zekeri AHH, Bamaiyi PH. Multi-drug resistant phenotypes of extendedspectrum β-lactamase (ESBL)-producing E. coli from layer chickens. Iraqi Journal of Veterinary Sciences. 2022;36(4):945-951.
8. Lupindu AM. Isolation and characterization of Escherichia coli from animals, humans, and environment. In: Escherichia coli-Recent Advances on Genotype and Phenotype. London: IntechOpen; 2017.
9. Hasman H, Mevius D, Veldman K, Olesen I, Aarestrup FM. β-Lactamases among extended-spectrum betalactamase (ESBL)-resistant Salmonella from poultry, poultry products and human patients in The Netherlands. Journal of Antimicrobial Chemotherapy. 2005;56(1):115-121.
10. Bonnet R. Growing group of extended-spectrum βlactamases: the CTX-M enzymes. Antimicrobial Agents and Chemotherapy. 2004;48(1):1-14.
11. Queipo-Ortuño MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P. Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clinical and Vaccine Immunology. 2008;15(2):293-296.
12. Carvalho I, Silva N, Carrola J, Silva V, Currie C, Igrejas G, et al. Antibiotic resistance. Hoboken (NJ): John Wiley & Sons; 2019.
13. Caneiras C, Lito L, Melo-Cristino J, Duarte A. Community-and hospital-acquired Klebsiella pneumoniae urinary tract infections in Portugal: virulence and antibiotic resistance. Microorganisms. 2019;7(5):138.
14. Di Marcantonio L, Chiatamone Ranieri S, Toro M, Marchegiano A, Cito F, Sulli N, et al. Comprehensive regional study of ESBL Escherichia coli: genomic insights into antimicrobial resistance and inter-source dissemination of ESBL genes. Frontiers in Microbiology. 2025;16:1595652.
15. Escherichia coli and carriage in the community worldwide: epidemiology, transmission pathways, molecular insights, and practical aspects. Clinical Microbiology Reviews. 2026;39:e00274-25.
16. Van Boeckel TP, Glennon EE, Chen D, Gilbert M, Robinson TP, Grenfell BT, et al. Reducing antimicrobial use in food animals. Science. 2017;357(6358):1350-1352.
17. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clinical Microbiology and Infection. 2012;18(3):268-281.
18. Fröding I, Vondracek M, Giske CG. Rapid EUCAST disc diffusion testing of MDR Escherichia coli and Klebsiella pneumoniae: inhibition zones for extendedspectrum cephalosporins can be reliably read after 6 h of incubation. Journal of Antimicrobial Chemotherapy. 2017;72(4):1094-1102.
19. Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy. 2010;65(3):490-495.
20. Carattoli A. Plasmids and the spread of resistance. International Journal of Medical Microbiology. 2013;303(6-7):298-304.
21. Zheng XE, Xu D, Yan J, Qian M, Wang P, Zaeim D, et al. Mobile genetic elements facilitate the transmission of antibiotic resistance genes in multidrug-resistant Enterobacteriaceae from duck farms. Food Science and Human Wellness. 2024;13(2):729-735.
22. Seiffert SN, Hilty M, Perreten V, Endimiani A. Extended-spectrum cephalosporin-resistant Gramnegative organisms in livestock: an emerging problem for human health. Drug Resistance Updates. 2013;16(12):22-45.
23. Kürekci C, Yüksel M, Celil Ozaslan BG, Tan S, Jäckel C, Grobbel M, et al. Diversity and resistance profiles of ESBL-producing gram-negative bacteria from dairy farms in Southern Türkiye. Antibiotics. 2024;13(12):1134.