Protecting bacteriophages under UV irradiation with brilliant blue FCF for targeted bacterial control

Abstract

Compared to the standard methods for treating bacterial diseases, bacteriophages are eco-friendly and chemical-free. Exposure to ultraviolet (UV) light or sunlight hampers the efficacy of phage-based approaches. This is crucial when phages are i) exposed to sunlight (e.g., in agriculture) or ii) are to be used simultaneously with UV for sterilization. Here, we develop a method utilizing a food dye, brilliant blue FCF (BB), that selectively stabilizes bacteriophages against exposure to UV irradiation. In the absence of BB, all tested phages and bacteria are completely inactivated by UV exposure. However, with the addition of BB, all tested non-enveloped phages are effectively protected, while gram-negative bacteria remain vulnerable to UV inactivation. The mechanism of protection requires selective binding of BB to the virion. The simultaneous action of BB-stabilized bacteriophages and UV allows for the removal of up to 99.99 % of bacteria within only 30-60 min. We demonstrate the method's applicability in combating biofouling of membranes and food sterilization. We envision using the developed approach against biofouling in industrial processes, agriculture, and the food industry.

Keywords: Antimicrobial combinations; Bacteriophages; Biofilms; Food dyes; Food preservation; Membranes; Stabilization.

Fig. 1

a) The evaluation of UV-protective properties of the food dyes to T4 phages. The overnight incubation in 0.5 % solutions of tartrazine (TR), quinoline yellow (QY), sunset yellow FCF (SY), Ponceau 4R (PC), Allura red (AR), azorubine (AZ), brilliant blue FCF (BB) and indigo carmine (IC) prevented the titer drop caused by 1 min UV exposure. Additional control compounds were tested, i.e., Congo red, rhodamine B, eosin Y, SYBR Green, crystal violet, and quinine. b) TR, QY, SY, PC, AR, AZ, and IC protected both gram-negative (E. coli, A. baumannii) and gram-positive (B. subtilis, S. aureus) bacteria. Only BB was selective, i.e., inefficient in protecting gram-negative bacteria. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

a) BB was effective in protecting a plethora of bacteriophages, including T4, T7, MS2, M13 (host: E. coli), P22 (host: S. enterica), vB_SauS_CS1 (host: S. aureus) and LR1_PAO1 (phage isolated from the environment, host: P. aeruginosa). BB negatively impacted enveloped phage Phi6 (host: P. syringae) (data not shown). b) BB prevented the titer drop upon exposure of T4 phages to artificial sunlight. c) EC₅₀ concentration of BB was established (T4 - 0.32 %, MS2 - 0.42 %, M13–0.41 %, P22–0.32 %). Here, exemplary data for T4 was shown. d) Molecular structure of BB.

Fig. 3

a) The evaluation of the molecular basis of dye-mediated UV protection of bacteriophages. The schematic illustration of experiments in which phages were either mixed with BB or placed in separate cuvettes. Phages were protected only when BB was added directly to the suspension. b) The evaluation of phage (T4)/bacteria (E. coli) -protective properties of sodium dodecyl sulfate (SDS), 2-(N-morpholino)ethanesulfonic acid (MES), sodium toluene sulfonate (TSA), sodium 4-phenol sulfonate (HBSA), sodium 2-naphthol-6-sulfonate (NSA), sodium isatin-5-sulfonate (ISA), and isatin along with the molecular structures of the compounds. The analysis allowed us to identify three domains required for the molecule to have phage-protective properties: i) sulfonic group, ii) UV-absorbing domain (here – aromatic rings), and iii) side group able to form hydrogen bonds.

Fig. 4

a) The application of BB-stabilized bacteriophages for the dual-mode (phages + UV) sterilization of membranes. The surfaces of syringe filter membranes were spiked with bacteria (E. coli or B. subtilis), and then BB-stabilized bacteriophages (T4 or Phi29, respectively) were introduced to the filters. UV irradiation was used as in previous experiments. b) The simultaneous application of UV and phages stabilized with BB for food sterilization against bacterial contamination. The sterilization of lettuce leaves surface spiked with E. coli or S. enterica (both gram-negative) contamination showed significantly better results when BB was added to T4 or P22 phages, respectively. More detailed statistical analysis is presented in Tables S1–S4 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).