Objectives: This study aimed to isolate and characterise extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) isolates from animals and wastewater in Tunisia. Methods: ESBL-E from wastewater (n = 123 samples), faeces of healthy animals (poultry, sheep, goats and calves) (n = 140) and raw milk from healthy cows (n = 42) and goats (n = 20) were investigated. Antimicrobial susceptibility was determined according to CLSI recommendations. The bla TEM , bla SHV , bla CTX-M and bla OXA-48 genes were analysed by PCR and sequencing. Phylogenetic groups were determined by PCR for Escherichia coli isolates. The clonality of E. coli and Klebsiella pneumoniae isolates was determined by XbaI-PFGE and MLST. Results: A total of 81 E. coli, 20 K. pneumoniae, 4 Enterobacter cloacae, 1 Citrobacter freundii and 1 Citrobacter braakii were isolated. The bla CTX-M-1 and bla CTX-M-15 genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the bla SHV-12 gene. The C. freundii isolated from wastewater carried bla CTX-M-15 , bla TEM-1 and bla OXA-204 . E. coli isolates belonged to phylogroups A (37), B1 (25), B2 (7) and D (12). Seventy-eight E. coli isolates were typeable by PFGE and were classified into 34 pulsotypes. The K. pneumoniae isolates belonged to 11 pulsotypes. The E. coli isolates belonged to sequence types ST131, ST224, ST162, ST845, ST5204, ST69, ST141 and ST10. The K. pneumoniae isolates belonged to ST405, ST147, ST564, ST307, ST152, ST45, ST661 and ST1564. Conclusion: This is the first report of O25b-B2 3 -CTX-M-27-ST131 E. coli isolates and of C. freundii carrying bla CTX-M-15 , bla TEM-1 and bla OXA-204 in Tunisia.
