Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that bla TEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of bla TEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4g/0 . 5g intravenously. The P/T MIC was tested using broth microdilution, and β-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of bl a TEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and bla TEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant) We found a higher copy number of bla TEM gene in P/T-R isolates, generated by three different genetic events: (1) IS 26 -mediated duplication of the bla TEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying bla TEM , and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.