Introduction: Escherichia coli (E. coli) is a Gram-negative, rod-shaped bacterium found in the gastrointestinal tract of warm-blooded animals and humans as part of the normal gut microbiota, but is also one of the most common causes of bacterial infections in humans and animals. E. coli strains can be classified into two groups: commensal strains and pathogenic strains that cause intestinal and extraintestinal infections in healthy individuals. In contrast to commensal strains, intestinal pathogenic E. coli strains are rarely found in the normal intestinal microbiota of healthy hosts. They appear to be essentially obligate pathogens that cause gastroenteritis or colitis when ingested in sufficient amounts. Six pathotypes of intestinal pathogenic E. coli strains are recognized: enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC), and diffusely adherent (DAEC). Extraintestinal strains of E. coli (ExPEC) are usually found in the normal gut microbiota as commensals but can cause various infections. These include uropathogenic E. coli (UPEC) isolated from humans and animals with urinary tract infections, neonatal meningitis-associated E. coli (NMEC) isolated from human neonates, septicemia associated E. coli (SePEC), and also avian pathogenic E. coli (APEC) responsible for colibacillosis in poultry. E. coli is excreted with faeces and can survive for weeks or months in faecal particles, dust and water, and enter the environment via faeces and wastewater treatment plants. Pathogenic strains of E. coli can be transmitted from animals to humans through the food chain and pose a direct threat to both the poultry industry and human health as they can lead to difficult-to-treat infections. For the treatment of infections caused by E. coli, the most commonly used classes of antimicrobials include beta-lactam antibiotics, fluoroquinolones, aminoglycosides, sulfonamides, tetracyclines, and nitrofurantoin. Beta-lactam antimicrobials are among the clinically most important antimicrobials used to treat bacterial infections in both human and veterinary medicine. Because of their broad spectrum and low toxicity, they are currently the most widely used class of antimicrobial agents to treat infections caused by E. coli strains. However, the widespread and often inappropriate use of antimicrobials has led to the emergence of resistant bacterial isolates. The predominant mechanism of beta-lactam resistance in E. coli is the production of beta-lactamases, which inactivate antibiotics by hydrolyzing the beta-lactam ring. To date, many different beta-lactamases have been identified, with extended spectrum β-lactamases (ESBLs) being the most important. ESBLs are enzymes that can hydrolyze penicillins, cephalosporins, and monobactams, but not cephamycins or carbapenems, and are inhibited by beta-lactamase inhibitors such as clavulanic acid. There are three major ESBL families: TEM, SHV, and CTX-M. The predominant and clinically most important ESBLs belong to the CTXM family, followed by the TEM and SHV families, which are found worldwide. The original ESBL enzymes were variants of TEM and SHV variants that had amino acid substitutions that resulted in a change in their substrate profile to include the extended-spectrum cephalosporins. CTX-M enzymes originated from the Kluyveraa ascorbata chromosomal AmpC enzyme. Most variants can be classified into five groups based on sequence homologies: CTX-M-1, CTX-M- 2, CTX-M-8, CTX-M-9, and CTX-M-25. ESBLs are often encoded by genes located on plasmids, allowing them to spread among bacteria, and they also carry genes for resistance to other classes of antimicrobial agents, causing bacterial strains to become multidrug-resistant (MDR). Multidrug-resistant bacterial strains are resistant to at least one agent from three or more antimicrobial drug classes. Such strains of bacteria are of great public health concern, since multidrug-resistant bacterial infections are difficult to treat as there are few or no treatment options. Antimicrobial resistance has become a rapidly growing public health concern worldwide and for this reason the World Health Organization (WHO) included antimicrobial resistance as one of the top ten global health threats in 2019. The global distribution of ESBLpositive isolates is of great concern as these isolates are found in increasing numbers in livestock, including poultry, retail meat and pets. The presence of ESBL-producing E. coli in food animal production systems is of public health concern as it can be transmitted to humans trough the food chain. Also, the presence of ESBL-positive isolates in companion animals can pose a potential public health risk, as animals can serve as reservoirs of resistant bacteria for humans. Because of the limited therapeutic options and the risks associated with the acquisition of new resistance mechanisms, ESBL-producing strains pose a major challenge for microbiologists and clinical therapists. Therefore, laboratory detection of resistant strains prior to the use of an antimicrobial agent in human and veterinary medicine is of great importance. In addition, continuous education on the rational use of antibiotics is necessary to prevent the spread of resistant and multidrug-resistant bacteria. Today, the prevalence of food-producing animals carrying E. coli producing CTX-M type ESBL has reached worryingly high levels. Food safety and the fight against antibiotic resistance are particularly relevant in the One Health approach. Keeping this problem under control requires constant monitoring of the evolution of the ESBL situation and the application of an interdisciplinary One Health approach. The aim of this study is to phenotypically confirm the presence of ESBL production in E. coli isolates using combined disk diffusion method containing cefotaxime and ceftazidime with and without clavulanic acid. A further aim is to detect the presence of the blaCTX-M gene and to investigate the presence of the blaTEM and blaSHV genes in E. coli isolates. Also to identify blaCTX-M genes for CTX-M- positive isolates. Material and methods: In 2016 and 2017, a total of 498 faecal samples were collected from different poultry farms in Bosnia and Herzegovina. Samples were cultured on MacConkey agar supplemented with 1 mg/l cefotaxime after pre-enrichment with Tryptic Soy Broth containing 1 mg/l cefotaxime. In this way, the selection of cefotaxime resistant isolates was carried out. The identification of E. coli was performed according to the procedure described by Markey et al. (2013). All cefotaxime-resistant isolates were identified by standard bacteriological culture and confirmed by several biochemical tests. E. coli colonies on MacConkey agar are light pink, indicating acid production due to fermentation of lactose. Colonies were identified biochemically as indole positive and oxidase negative using indole and oxidase utilization assays. After routine bacteriological analysis, all cefotaxime-resistant E. coli isolates were screened for ESBL production using a combined disk diffusion method using cefotaxime and ceftazidime disc with and without clavulanate. Klebsiella pneumoniae ATCC 700603 was included in the study as an ESBL positive control strain and E. coli ATCC 25922 as an ESBL negative control strain. The diameters of the zones of inhibition were interpreted according to the criteria recommended in the Clinical and Laboratory Standards Institute document (CLSI, 2018). An increased zone size in the presence of clavulanate of five millimeters or more was considered indicative of ESBL production. All isolates that were phenotypically positive for ESBL production were included in further studies. Identification of the isolates was performed using the API 20 E identification system. Antimicrobial susceptibility was determined using the standard disk diffusion method for 12 antimicrobials: amoxicilin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, cefpodoxime, cefepime, meropenem, enrofloxacin, ciprofloxacin, gentamicin, trimethoprimsulfamethoxazole and tetracycline. Inhibition zone diameters were interpreted according to the criteria recommended in the documents of the Clinical and Laboratory Standards Institute (CLSI, 2015; CLSI, 2018). All isolates phenotypically confirmed to be ESBL-producing E. coli were subjected to molecular analysis using multiplex PCR screening to detect blaCTX-M, blaTEM and blaSHV genes. Additional PCR was performed for blaCTX-M positive isolates with primers amplifying a 948 bp PCR gene fragment for sequencing. PCR products were sequenced by Macrogen Europe, Amsterdam, The Netherlands and the results were analyzed using the Basic Local Alignments Tool (BLAST). For statistical analysis STATISTICA v.14.0. (Statistica, Tibco, 2020.) program was used. Fisher's exact test was used to compare statistically significant differences in the presence of individual genes, including statistically significant differences in susceptibility among isolates to antimicrobials from the same class. Results: A total of 498 faecal samples were collected from several different poultry farms in Bosnia and Herzegovina. A total of 280 cefotaxime-resistant E. coli isolates (56.2%) were isolated. The prevalence of phenotypically confirmed ESBL-producing E. coli isolates in poultry faeces was 14.9%. Based on the disk diffusion assay, all tested isolates showed resistance to cefpodoxime (100.0%) in addition to resistance to cefotaxime. The highest resistance was observed to amoxicillin-clavulanic acid (78.4%), followed by ceftazidime (54.1%). Resistance to tetracycline was 51.4%, followed by cefepime (50.0%) and cefoxitin (48.6%). Resistance to enrofloxacin was 32.4% and to ciprofloxacin 28.4%. The lowest resistance was observed to trimethoprim-sulfamethoxazole (18.9%) and there were only 6.8% of isolates resistant to gentamicin. All isolates tested were susceptible to meropenem (100.0%). Multidrug resistance was found in 85.1% of the isolates. Isolates were significantly more resistant to cefotaxime and cefpodoxime (Fisher's exact test, p < 0,05). There was no statistically significant difference between fluoroquinolones. Among 74 phenotypically confirmed ESBL-producing E. coli isolates, 54 isolates (73.0%) were positive for tested ESBL genes. Among them, 33 isolates (61.1%) carried the blaCTX-M gene, 33 (61.1%) carried the blaTEM gene and only one isolate was positive for the blaSHV gene (1.9%). The coexistence of blaCTX-M and blaTEM genes within the same isolate was detected in 13 (24.1%) isolates, while none of the isolates tested showed the coexistence of blaCTX-M, blaTEM and blaSHV genes. Sequence analysis of the blaCTX-M gene revealed that 32 (97.0%) of the isolates carried the blaCTX-M-1 gene and only one isolate (3.0%) carried the blaCTX-M-15 gene. Of 74 isolates analyzed, 20 (27.0%) isolates had no ESBL genes. Conclusions: The concentration of 1 mg/l cefotaxime as a selective antimicrobial agent is low for the selection of ESBL-producing E. coli isolates. Of 498 poultry faecal samples, 54 of the E. coli isolates (10.8%) were positive for ESBL genes tested. These isolates can be the source of resistance genes and pose a public health threat. In this study, the blaCTX-M-1 gene was the most prevalent, found in 32 out of 33 isolates (97.0%). This result suggests that isolates from poultry are not involved in human infections in Bosnia and Herzegovina, since among human isolates the most common gene variant is blaCTXM-15. Of 74 isolates analyzed, 63 (85.1%) were found to be multidrug resistant, likely as a result of the widespread and inappropriate use of antimicrobials in the poultry industry.