Objectives To determine the distribution of CTX-M ESBL positive E. coli among hospitalized and non-hospitalized patients from Brod-Posavina County and Vukovarsko-Srijemska County. To determine incompatibility groups of epidemic plasmids encoding ESBL and the role of insertion sequences IS26 and ISEcp in the mobilization of the blaCTX-M gene. To determine the genetic profile of extended spectrum β-lactamases. Overview High prevalence of E. coli ESBL isolates from various samples of patients hospitalized in the regional General Hospital in Brod-Posavina County was observed at the end of 2015. At the same time, high prevalence of ESBL-producing E. coli in urine samples was observed among outpatients gravitating towards the hospital. Similar prevalence of the E. coli ESBL isolates was observed in Vukovarsko-Srijemska County. Isolates were prospectively collected over the course of 2016, and 91 isolate (without copy-isolates) from Brod-Posavina County and 11 isolate from Vukovarsko-srijemska County were collected. The patient data was collected from medical records. Materials and Methods Ninety-one isolates were collected from January 1 to December 31, 2016 from patients hospitalized in GH in Slavonski Brod and from outpatients. Eleven isolates were collected from hospital patients and outpatients from Vukovarsko-Srijemska County. The isolates showed high resistance to all antibiotics except carbapenems. Antibiotic susceptibility testing was performed by the disc-diffusion and microdilution method. Phenotypic tests were carried out to prove the production of β-lactamases. Transconjugation was performed to detect the resistance gene transfer. The isolates were genotyped using the PFGE method. Molecular characterization of ESBL β-lactamases was performed by bla gene primers using the PCR method. Plasmid incompatibility groups were determined by the PBRT method. The presence of insertion sequences was determined by the PCR mapping method. Results The blaCTX-M-1 gene was found in all of 102 isolates. 54 isolates carried only blaCTX-M. 48 isolates carried both blaCTX-M and blaTEM. Seven of 25 isolates with borderline MIK values for ertapenem were tested for the presence of the blaCARBA genes, and the presence of the blaOXA-48 genes was demonstrated. The blaCTX-M-15 gene was detected in 34 out of 35 isolates and blaCTX-M-9 was identified in one isolate. Strains obtained by transconjugation showed the same phenotypic and genotypic characteristics as the origin. Insertion sequence IS26 was located upstream of the blaCTX-M gene in 47 out of the 88 tested isolates, and ISEcp was found upstream of blaCTX-M-15 in 20 out of the 34 isolates with the blaCTX-M-15 gene. Insertion sequence IS1999 preceded the blaOXA-48 gene in 7 isolates. The presence of various plasmids groups was demonstrated, with IncB / O, IncW, Inc A / C being the most common types identified, while IncFII / IncFIC, IncX, IncL, IncK / B and IncL plasmids were less prevalent. PFGE showed the existence of four 4 different clusters with isolates from different centres and from different geographic areas. Isolates from the same centre but different clinical wards obtained in different time showed high similarity in PFGE banding patterns pointing to cross-infection. Conclusion This study suggests a significant epidemiology change in the ESBL E. coli isolates and confirms the widespread distribution of CTX-M type β-lactamases in both the inpatient and outpatient population. High incidence of ESBL E. coli in patient population has a significant influence on the choice of therapy due to limited therapeutic options. High prevalence of ESBL isolates results in an increased carbapenem use which leads to increasing resistance to carbapenems due to carbapenemase production.