# Exploring the effect of sequential antibiotic exposure in resistant Escherichia coli causing urinary tract infections: a proof of principle study

## TL;DR

This study explores if using two antibiotics in sequence can reduce resistance in E. coli causing urinary tract infections.

## Contribution

The study demonstrates that sequential antibiotic exposure may improve treatment efficacy against resistant E. coli.

## Key findings

- Sequential exposure to mecillinam followed by ciprofloxacin significantly inhibited E. coli growth.
- No synergistic effect was observed between ciprofloxacin and mecillinam in checkerboard assays.
- Phenotypic resistance to antibiotics was reduced through sequential treatment.

## Abstract

Antimicrobial resistance development, particularly in infections such as urinary tract infections (UTIs), is a global clinical concern. The objective of this study was to determine if sequential antibiotic exposure with ciprofloxacin and mecillinam can reduce the growth of resistant clinical Escherichia coli strains, thus improving the effectiveness of antibiotic therapy. Six E. coli isolates with heterogeneous resistance to ciprofloxacin and/or mecillinam obtained from patients with UTIs were exposed to one of the antibiotics (0.75 × minimum inhibitory concentration, MIC) for 1 h. This was followed by treatment with the second antibiotic at different concentrations (0.0375/0.075/0.375/0.75 × MIC). Continuous growth measurements were conducted in order to assess the impact of sequential exposure. One representative strain was selected for intact cell counting. In addition, a checkerboard assay was conducted to investigate the synergistic impact of ciprofloxacin and mecillinam, and genetic analyses were performed to identify the mechanisms of resistance for all isolates. The six E. coli strains were phylogenetically different, and none exhibited a synergistic effect for ciprofloxacin and mecillinam. Our data suggest that sequential exposure to mecillinam and ciprofloxacin appears to reduce the growth capacity of clinical E. coli isolates with phenotypic resistance to either or both agents. Sequential antibiotic exposure may be an interesting strategy to improve the antibiotic efficacy of current agents to overcome phenotypic resistance in E. coli.

As global rates of antibiotic resistance increase and the development of new antibiotics become more difficult and costly, it is important to explore alternative strategies to improve the effectiveness of existing antibiotics. Previous studies have shown that sequential exposure of Pseudomonas aeruginosa to two antibiotics can effectively kill the bacteria and reduce the likelihood of resistance developing. However, the potential of this sequential approach for the treatment of Escherichia coli infections has not been thoroughly investigated. In our study, we conducted a proof-of-principle study to determine whether sequential exposure to mecillinam and ciprofloxacin can overcome phenotypic resistance to one or both drugs. We found that when E. coli were treated with subinhibitory doses of mecillinam followed by ciprofloxacin, their growth was significantly inhibited compared to treatment with ciprofloxacin alone, suggesting that sequential antibiotic exposure may be a viable strategy for treating infections caused by resistant E. coli.