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In Gabon, few data exist on extended-spectrum beta-lactamases-producing Enterobacteriaceae (ESBL-PE). This study investigated ESBL-PE prevalence and the associated resistance genes in clinical samples (n = 5,956) and anal swabs (n = 78) analyzed in eight hospitals and a medical analysis laboratory in Gabon from January 2016 to March 2018. Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) mass spectrometry analysis identified 790 Enterobacteriaceae isolates (n = 712 clinical samples and n = 78 fecal samples). ESBL-PE prevalence (Müller-H