In-vitro analysis of biofilm formation and synergistic antibiotic-phage therapy for amoxicillin-resistant Escherichia coli

Abstract

Biofilm formation by Escherichia coli (E. coli) significantly enhances bacterial resistance to antibiotics, complicating treatment, particularly in amoxicillin-resistant strains. Bacteriophage therapy demonstrates potential in treating biofilm-related infections, and the combination of phages and antibiotics (phage-antibiotic synergy, PAS) further enhances efficacy. This is the first study to evaluate PAS using penicillin-class antibiotics against multidrug-resistant (MDR) Gram-negative bacteria, E. coli. E. coli-specific lytic bacteriophages were isolated and characterized. PAS was evaluated in both planktonic and biofilm forms using sub-inhibitory concentrations of AMC. The viability of biofilm and planktonic forms was assessed not only by colony counts but also by flow cytometry. Moreover, morphological alterations were evaluated by scanning electron microscopy (SEM), and genomic alterations by PAS were analyzed through whole genome fingerprinting using ERIC PCR. In biofilm and planktonic form, phage first achieved effective bacterial killing after 24 h, when 106 PFU/mL was supplemented with amoxicillin clavulanic acid (AMC) combination after 7 h for optimal PAS killing. PAS treatment significantly reduced biofilm viability compared to phage therapy only, while AMC was not effective at all. SEM revealed disrupted cell walls, detachment of flagella, and rupture of bacterial cells, as well as changes in morphology and biofilm matrix in combination therapy. Phage-first treatment with ɸA3 followed by AMC after 7 h effectively eradicates multidrug-resistant E. coli, causing genomic changes that restore antibiotic sensitivity at subinhibitory doses, potentially addressing antimicrobial resistance. In PAS, a cocktail of phages may be advised to avoid the emergence of phage mutant strains. © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2025.