To establish a stable fluorescent neutrophil-Escherichia coli (E.coli)-metals binary fluorescence dynamic model, and to evaluate the practicability and application value of this model.

pUC57-mCherry plasmid was isolated and transferred to clinical E. coli isolates (ST131) through electroporation, ST131-mCherry was then constructed. ST131-mCherry was screened using in-vivo imaging (IVIS) system. Dilution plate method was utilized to compare the growth curve of ST131-mCherry and ST131, crystal violet staining was used to compare the biofilms in 96-well plates. After the co-culture of ST131-mCherry with titanium, the biofilms at different time points were visualized by fluorescence microscope and quantified by ultrasonic concussion and dilution plate method. All-trans-retinoid acid (ARTA) was used to induce HL60-enhanced green fhcorescent protein (eGFP) to differentiate into neutrophil like cells. Induced differentiated HL60-eGFP, ST131-mCherry and metals were co-cultured to establish the binary fluorescence triple components dynamic model. The antibacterial efficiencies of neutrophil like cells on different metal-surfaces were compared through fluorescent observation and bacterial count. Independent t test was used to evaluate the statistical significance.

Red fluorescence clinical E. coli (ST131-mCherry) was constructed through clinical strains screening and plasmid electroporation. The fluorescence of ST131-mCherry was proved to be stable in the 48 h consecutive culture. There was no significant difference of growth curve between ST131-mCherry and ST131 wild strain. OD value of biofilms stained with crystal violet in tryptic soy broth (TSB) at 490 nm was (1.04±0.06) and (1.04±0.05) (t=0.890, P>0.05) for ST131 and ST131-mCherry respectively, it was (1.55±0.06) and (1.49±0.17) (t=0.822, P>0.05) in 0.5% glucose tryptic soy broth (TSBG), (2.13±0.16) and (2.124±0.13)(t=0.833, P>0.05) in 10% synovial fluid (SF). ST131-mCherry can form biofilms on the surface of titanium, the fluorescent intensity of bacteria was enhanced with the growth of biofilm. The co-culture of differentiated HL60-eGFP, ST131-mCherry and titanium/tantalum revealed that neutrophils could devour more E. coli in the tantalum system after 60 min, bacterial survival rate was (33±2.5)% and (15.7±1.2)% for Ti and Ta system respectively (t=10.47, P<0.01).

E. coli-fluorescent neutrophil-metals binary fluorescence dynamic model is constructed in current study, which is a practical tool to investigate antimicrobial biomaterials in vitro.