Protocol for Agar Dilution Checkerboard Analysis of Fosfomycin-Based Antibiotic Combinations Against Carbapenem-Resistant Escherichia coli

Summary

Here, we present a protocol for evaluating the synergistic activity of fosfomycin in combination with ceftriaxone, meropenem, and gentamicin against carbapenem-resistant Escherichia coli isolates using the agar dilution method. Synergy was observed with meropenem and gentamicin, but not with ceftriaxone.

Abstract

Due to the limited treatment options for carbapenem-resistant Enterobacterales, combination therapy regimens have gained increasing importance. This study aimed to investigate fosfomycin resistance rates and evaluate the in vitro synergistic interactions of fosfomycin in combination with meropenem, gentamicin, and ceftriaxone against carbapenem-resistant Escherichia coli isolates. Sixty carbapenem-resistant E. coli strains isolated from various clinical samples (urine, blood, aspirate, wound, cerebrospinal fluid, endotracheal aspirate) obtained from patients in clinical wards and intensive care units of Dicle University Medical Faculty Hospitals were included in the study. Species identification was performed using an automated microbial identification system. Antibiotic susceptibility testing and confirmation of carbapenem resistance were performed by disc diffusion with ertapenem, imipenem, and meropenem. Fosfomycin resistance and interactions among antibiotics were assessed by agar dilution. In addition, antibiotic combinations were evaluated across serial two-fold concentration gradients using the agar dilution-based protocol, and fractional inhibitory concentration indices (FICI) were calculated to determine synergy, indifference, or antagonism. Resistance rates were 76.6% for fosfomycin, 61.6% for meropenem, 85.0% for gentamicin, and 98.3% for ceftriaxone. Synergistic interactions were observed in 35% of isolates for the fosfomycin–meropenem combination, 33.4% for the fosfomycin–gentamicin combination, and 1.7% for the fosfomycin–ceftriaxone combination. Antagonism was detected in only one isolate in the fosfomycin–meropenem combination. Combinations of fosfomycin with meropenem and gentamicin showed in vitro synergistic activity against carbapenem-resistant E. coli. Further in vitro studies are required to confirm these findings.

Introduction

The Enterobacterales family comprises a highly diverse group of gram-negative bacilli, many of which are of significant clinical importance¹. Among them, Escherichia coli is the most frequently isolated species in clinical microbiology laboratories and is widely associated with infections involving nearly all organ systems, including urinary tract infections, wound infections, pneumonia in immunocompromised patients, gastroenteritis, and neonatal meningitis. It is also a major cause of Gram-negative sepsis and endotoxin-mediated septic shock. Beyond human medicine, E. coli is also an important veterinary pathogen, causing resp....