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Background: Extended-spectrum β lactamases (ESBLs), have the ability to hydrolyze and cause resistance to various types of the β-lactam antibiotics, including the extended-spectrum (or third-generation) cephalosporins (e.g., cefotaxime, ceftriaxone, ceftazidime) and monobactams (e.g., aztreonam). ESBL-producing Gram negative bacteria is still posing significant therapeutic challenges. Objectives: To assess the prevalence and molecular characteristics of ESBL producing Gram negative bacilli, isolated from a cohort of pediatric patients in Gaza hospitals. Methods: A total of 322 isolates of Gram-negative bacilli were collected from four referral pediatric hospitals in Gaza, namely: Al-Nasr, Al-Rantisi, Al-Durra and Beit Hanoun hospitals. These isolates were tested for ESBL production using the double disk synergy and CHROMagar phenotypic methods. Molecular characterization of the ESBL producing strains was performed using PCR targeting the CTX-M, TEM and SHV genes. Antibiotic profile was done using Kirby Bauer method according to Clinical and Laboratory Standard Institute. Results: Out of 322 isolates tested by phenotypic methods, 166 (51.6%) were ESBL positive. The prevalence of ESBL production in Al-Nasr, Al-Rantisi, Al-Durra and Beit Hanoun hospitals was 54%, 52.5%, 45.5% and 52.8% respectively. The prevalence of ESBL production among Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter spp., Proteus mirabilis, Enterobacter spp., Citrobacter spp., and Serratia marcescens is 55.3%, 63.4%, 17.8%, 57.1%, 33.3%, 28.5%, 38.4%, and 4% respectively. ESBL production among urine, pus, blood, CSF and sputum was 53.3%, 55.2%, 47.4%, 33.3%, and 25% respectively. Out of the 322 isolates, 144 were screened for CTX-M, TEM and SHV production. Using PCR, 85 (59%) had at least one gene. The prevalence rate of CTX-M, TEM and SHV genes was 60%, 57.6%, and 38.3% respectively. Meropenem and amikacin were highest rates of susceptibility antibiotics against ESBLs producers (83.1% and 82.5% respectively), while the least effective antibiotics were amoxicillin (3.1%) and cephalexin (13.9%). Moreover, ESBLs producers showed high resistance rate to cefotaxime, ceftriaxone and ceftazidime (79.5%, 78.9% and 79.5% respectively). Conclusion: Our results show high prevalence of ESBL production among Gram negative bacilli isolated from children in different pediatric hospitals in Gaza strip. A substantial level of resistance to first and second generation cephalosporins was also observed. This ascertains the need for a rational antibiotic prescription and consumption policy. [ABSTRACT FROM AUTHOR]