Two-pronged attack strategy for biofilm removal with enzymatically active DspB-expressing T7_DspB phage. Initial infection of E. coli biofilm results in rapid multiplication of phage and expression of DspB. Both phage and DspB are released upon lysis, leading to subsequent infection as well as degradation of the crucial biofilm EPS component, β-1,6-N-acetyl-d-glucosamine (22).

Assays for E. coli TG1 biofilm levels and phage counts after 24 h with no treatment or with treatment with phage T7_wt, phage T3_wt, non-DspB-expressing phage T7_control, or DspB-expressing phage T7_DspB. Error bars indicate SEM. (A) Mean absorbance (600 nm) for n = 16 biofilm pegs stained with 1% CV, solubilized in 33% acetic acid, and diluted 1:3 in 1× PBS (50). (B) Mean cell densities [log₁₀(CFU per peg)] for n = 12 biofilm pegs. Pegs treated with T7_DspB resulted in a 3.65 log₁₀(CFU per peg) reduction in viable cells recovered from E. coli biofilm compared with untreated biofilm. (C) Mean phage counts [log₁₀(PFU per peg)] recovered from media in n = 3 microtiter plate wells (wells) or sonication of n = 3 biofilm pegs (biofilm), as indicated, after 24 h of treatment with initial inoculations of 10³ PFU per well. Both T7_control and T7_DspB showed evidence of replication with phage counts obtained from the microtiter plate wells or with phage counts recovered from the biofilms after sonication.

Genomes of engineered phage used for biofilm treatment. (A) Genome of T7select415-1 shows a unique BclI site and capsid gene 10B. (B) DspB-expressing phage T7_DspB was created by cloning T3 gene 1.2 into the unique BclI site and cloning the ϕ10-dspB construct after capsid gene 10B. (C) Non-DspB-expressing control phage T7_control was created by cloning T3 gene 1.2 into the unique BclI site and cloning the control S·Tag insert (included in the T7select415-1 kit) as a fusion with the capsid gene 10B.

Time-course curves, dosage–response curves, and SEM images for engineered phage treatment targeting E. coli TG1 biofilm. (A and E) Each data point represents the mean log₁₀-transformed cell density of n = 12 biofilm pegs. (D and F) Each data point represents the mean log₁₀-transformed phage counts obtained from n = 3 microtiter plate wells. Error bars indicate SEM. (A) Time course (up to 48 h) of viable cell counts for no treatment (red squares), treatment with T7_control (black circles), or treatment with T7_DspB (blue crosses) demonstrates that T7_DspB significantly reduced biofilm levels compared with T7_control. (B) SEM image of T7_DspB-treated biofilm after 20 h shows significant disruption of the bacterial biofilm. (C) SEM image of untreated biofilm after 20 h shows a dense biofilm. (D) Time course of phage counts obtained after initial inoculation of E. coli TG1 biofilm with 10³ PFU per well of T7_control (black circles) or T7_DspB (blue crosses). Both T7_control and T7_DspB began to replicate rapidly after initial inoculation. (E) Dose–response curves of mean cell densities (measured after 24 h of treatment) for T7_control (black circles) and T7_DspB (blue crosses). For all initial phage inoculations, T7_DspB-treated biofilm had significantly lower mean cell densities compared with T7_control-treated biofilm. (F) Dose–response curves of mean phage counts (measured after 24 h of treatment) for T7_control (black circles) and T7_DspB (blue crosses). For all initial phage inoculations, both T7_control and T7_DspB multiplied significantly. (Scale bars, 10 μm.)