Bacterial conjugation

Bacterial conjugation was first performed between each of the 40 bla_CTX-M/mphA-carrying S. sonnei isolates associated with all the plasmid acquisitions and E. coli J53 (sodium azide resistant) by combining equal volumes (5mL) of overnight Luria-Bertani (LB) cultures. Bacteria were conjugated for 12 hours in LB broth at 37°C and E. coli transconjugants were selected on medium containing sodium azide (100 mg/l) plus ceftriaxone (6 mg/l) or sodium azide (100 mg/l) plus azithromycin (24 mg/l). To measure plasmid transfer from commensal E. coli to cipR S. sonnei and investigate the effect of ciprofloxacin on the conjugation efficiency, we first screened commensal E. coli isolates for ESBL activity and ciprofloxacin susceptibility from the pooled colony sweeps on MC agar. Subsequently, bacterial conjugation was performed between each of the 13 cipS ESBL-positive commensal E. coli isolates (donor) and the cipR ESBL-negative S. sonnei 03-0520 (recipient) in LB broth with and without supplementation of ciprofloxacin (0.25, 0.5, 0.75 x MIC of the donor organism). Successful transconjugants were selected on MC agar containing ciprofloxacin (4 mg/l) and ceftriaxone (6 mg/l). For all conjugation experiments, the conjugation frequency was calculated as the number of transconjugants per recipient cell.