Objective:To investigate the in vitro interference of cefotaxime at subinhibitory concentrations [sub-minimal inhibitory concentrations(MIC)] on biofilm formation by nontypeable Haemophilus influenzae(NTHi).Methods:The interference of subinhibitory concentrations of cefotaxime on biofilm formation of the clinical strong-biofilm forming isolates of NTHi was evaluated by a microtiter plate biofilm formation assay.The effect of sub-MIC cefotaxime on bacterial cell-surface hydrophobicity was determined using a standard microbial adhesion to n-hexadecane test.Additionally,the effects on bacterial adherence to human fibronectin and expression of bacterial adhesins were also investigated.Results:Subinhibitory concentrations of cefotaxime,both at 0.1× and 0.5× MIC levels,efficiently reduced the NTHi biofilm formation,and this effect was independent of decreasing bacterial viability.Sub-MIC cefotaxime also decreased bacterial cell-surface hydrophobicity and reduced adherence to human fibronectin.Inhibition in the P2 and P6 gene expressions upon exposure to sub-MIC cefotaxime was also noted.Conclusions:Taken together,our results indicate that sub-MIC cefotaxime interferes with the formation of NTHi biofilm,and this effect is feasibly related to the interference with cell-surface hydrophobicity,fibronectin-binding activity as well as alteration of the P2 and P6 gene expression.The findings of the present study therefore provide a rationale for the use of subinhibitory concentrations of cefotaxime for treatment of NTHi-related diseases. Objective: To investigate the in vitro interference of cefotaxime at subinhibitory concentrations [sub-minimal inhibitory concentrations (MIC)] on biofilm formation by nontypeable Haemophilus influenzae (NTHi). Methods: The interference of subinhibitory concentrations of cefotaxime on biofilm formation of the clinical strong -biofilm forming isolates of NTHi was evaluated by a microtiter plate biofilm formation assay. The effect of sub-MIC cefotaxime on bacterial cell-surface hydrophobicity was determined using a standard microbial adhesion to n-hexadecane test. Additionally, the effects on bacterial adherence to Results: Subinhibitory concentrations of cefotaxime, both at 0.1 × and 0.5 × MIC levels, efficiently reduced the NTHi biofilm formation, and this effect was independent of late-bacterial viability. Sub-MIC cefotaxime also decreased bacterial cell-surface hydrophobicity and reduced adherence to human fibronec tin.Inhibition in the P2 and P6 gene expressions upon exposure to sub-MIC cefotaxime was also noted. Conclusions: Taken together, our results indicate that sub-MIC cefotaxime interferes with the formation of NTHi biofilm, and this effect is feasible respect to the interference with cell-surface hydrophobicity, fibronectin-binding activity as well as alteration of the P2 and P6 gene expression. findings of the present study therefore provide a rationale for the use of subinhibitory concentrations of cefotaxime for treatment of NTHi-related diseases.