Extended-spectrum β-lactamase-producing Enterobacteriaceae from animal origin and wastewater in Tunisia: first detection of O25b-B2-CTX-M-27-ST131 Escherichia coli and CTX-M-15/OXA-204-producing Citrobacter freundii from wastewater.

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, bla, bla and bla 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 and bla genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the bla gene. The C. freundii isolated from wastewater carried bla, bla and bla. 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-CTX-M-27-ST131 E. coli isolates and of C. freundii carrying bla, bla and bla in Tunisia.