(A) Transmission electron micrograph of phage YC#06 virions negatively stained with 2% phosphotungstic acid. (B to E) Biological properties of phage YC#06. Determination of the optimal MOI (B), temperature tolerance (C), pH sensitivity (D), and one-step growth curve (E). (F) Dynamic curves of bacterial escape from phage control based on viable bacteria counting observation. Phage titers were measured by the double-layer agar method. Data are presented as the mean ± standard deviation (SD).

Effect of phage dose on PAS between YC#06 and antibiotic combination. The y axis represents the active bacteria amount, which is calculated by log (CFU/mL). The x axis represents the effective antibiotic concentration. “×MIC” shows that the effective antibiotic concentrations used are at n times the MIC. MICs were chosen according to the Clinical and Laboratory Standards Institute guidelines. Lower, phage dose at a low MOI (0.01); middle, phage dose at a middle MOI (1); high, phage dose at a high MOI (100).

Time-kill analyses of antibiotic mixtures alone and in combination with bacteriophage YC#06 against B.m#4015 at different MICs at the end of 8 h exposure (A), at the end of 16 h exposure (B), and at the end of 24 h exposure (C). Antibiotic mixtures include chloramphenicol, imipenem, and cefotaxime. “×MIC” shows that the effective antibiotic concentrations used are at n times the MIC. MICs were chosen according to the Clinical and Laboratory Standards Institute guidelines. Synergy was defined as a 2-log₁₀ CFU/mL kill compared to the most effective agent (or double-combination regimen) alone at 24 h. Bactericidal activity was defined as a 3-log₁₀ CFU/mL reduction from baseline. Data are presented as the mean ± standard deviation (SD).

Antibiofilm effect of antibiotic mixtures alone and in combination with bacteriophage YC#06 on biofilm formation inhibition (A, B) and mature biofilm reduction (C, D) in vitro. Antibiotic mixtures include chloramphenicol, imipenem, and cefotaxime. (A and C) Optical density at 600 nm (OD₆₀₀) was measured using the microplate reader. (B and D) Viable bacteria counting was detected using dilution plate counting method. I, bacteria added alone as control group; II, phage added as treatment group; III and IV, antibiotic mixtures added as treatment group; V and VI, antibiotic mixtures plus phage added as treatment group. For the biofilm formation inhibition experiment in panels A and B, the phage and B.m#4015, antibiotic mixtures and B.m#4015, and antibiotic mixtures plus phage and B.m#4015 were cocultured at the initial time. For the mature biofilm-reducing experiment in panels C and D, phage, antibiotic mixtures, and antibiotic mixtures plus phage were added after mature biofilm was observed. “×MIC” shows that the effective antibiotic concentrations used are at n times the MIC. MICs were chosen according to the Clinical and Laboratory Standards Institute guidelines. Data are presented as the mean ± standard deviation (SD) (*, P < 0.05).

In vivo efficacy of YC#06 and antibiotic mixtures alone or combination against B.m#4015 in a zebrafish model. Monitoring lasted for a period of 7 days. Statistical analyses were performed using a t test for two comparisons and analysis of variance (ANOVA) test (*, P < 0.05).