Self-assembling nanofibrous bacteriophage microgels as sprayable antimicrobials targeting multidrug-resistant bacteria

Bacteriophages are natural, highly specific bacterial predators that can selectively target and kill pathogenic strains without broadly disrupting commensal microbiota, making them attractive for applications in food safety, agriculture, and therapeutics. Phage-based products for biocontrol in foods (e.g., targeting Escherichia coli, Salmonella, Listeria) have received regulatory acceptance, offering advantages such as minimal impact on taste, texture, or nutrition. However, widespread adoption is hindered by challenges in delivery and stability that limit efficacy. Phage virions are proteinaceous, monodisperse nanoparticles whose surfaces can be precisely engineered, positioning them as powerful building blocks for multifunctional antimicrobial materials. Prior work has shown that filamentous phage can be crosslinked into bulk hydrogels, but translating these materials to microscale formats is necessary to enable sprayable systems and colloidal applications while preserving bioactivity. This study addresses these gaps by developing a biomolecule-friendly, high-throughput template method to fabricate phage-exclusive and phage–protein hybrid microgels that maintain phage functionality, exhibit tunable fluorescence, and demonstrate targeted antimicrobial efficacy against multidrug-resistant bacteria in environmental, liquid, and food matrices.

The authors situate their work within several domains: (1) Phage biocontrol for food safety has a regulatory track record (FDA GRAS notices) and is recognized by global health organizations for its potential impact on reducing foodborne illness. (2) Phages as nano-engineering tools have been used in biosensing, materials assembly, and as building blocks for functional biomaterials; filamentous phage hydrogels have been reported previously as bulk soft materials. (3) Conventional microgel fabrication methods (e.g., microfluidics, emulsions) often involve heat or organic solvents, which compromise bioactivity of sensitive biomolecules like proteins and viruses, limiting their suitability. (4) Breath figure methods offer facile fabrication of large-area honeycomb films with ordered, uniform micropores at tunable sizes/shapes and have been used as templates for micro-architectures. The present work leverages these advances to create microgels from phages under gentle, solvent-free, heat-free conditions, overcoming manufacturing and stability challenges and enabling new delivery formats (patches, sprays).

Materials and phages: Filamentous phage M13 served as the primary structural building block. Virulent phages HER262 (targeting E. coli O157:H7) and T7 (targeting E. coli BL21) were used to impart strong bactericidal activity. Phages were propagated in their respective E. coli hosts, purified via PEG/NaCl precipitation followed by ultrafiltration, and titered by plaque assay. Honeycomb template fabrication (breath figure method): A 5 wt% polystyrene solution in chloroform (600 µL) was cast on glass in a ~55% RH chamber. After 20 min, a white honeycomb film formed and was peeled after ~1 h. Resulting films exhibited a single-layer of closely packed, open-ended spherical micropores with inner diameter 35.73 ± 2.86 µm. Phage microgel formation: Honeycomb films were O2 plasma-treated (5 min) to increase hydrophilicity. A mixture containing M13 (typically 5 × 10^8 PFU mL−1 for methods; higher titers used in characterization) and a crosslinker was cast on the film; vacuum (∼5–10 min) aided pore filling. Crosslinkers: (i) Glutaraldehyde (GA, 0.1 M unless noted) forms Schiff bases and related linkages between lysine amines on phage coat proteins; (ii) EDC (0.1 M) promotes zero-length amide bond formation between carboxyl and amine groups, yielding water-soluble isourea by-products that wash out, producing phage-exclusive gels. Films were incubated in sealed humid containers at 4 °C for 1–2 days to gel. Excess surface gel was removed by a glass slide. Template peeling and microgel isolation: Adhesive tape was applied to peel off the top half of the pores, exposing the microgels lodged in the lower film surface. The peeled film was immersed in sterile water or PBS and sonicated (~10 min) to release microgels into suspension. Microgels were stored at 4 °C. SEM confirmed intact pore networks and successful detachment. Hybrid microgels and bioactivity preservation: Bovine serum albumin (BSA) was incorporated with M13 and GA to form M13+BSA+GA hybrid microgels, accelerating gelation (from >12 h to ~30 min) and consuming excess crosslinker to preserve phage bioactivity. For embedding virulent phages (e.g., HER262 at 1 × 10^10 PFU mL−1), GA concentration was reduced to 0.02 M and BSA was required for gelation, minimizing intramolecular crosslinking that could impair tail fibers. Characterization: Size distributions of template pores and hydrated/dried microgels were measured by inverted microscopy. Porosity was inferred from volume shrinkage upon dehydration. Preparation efficiency was determined by counting microgels produced per cm^2 of template. Surface nanostructure was imaged by SEM/FE-SEM after critical point drying and metal coating. Autofluorescence was assessed via inverted fluorescence microscopy across blue (ex/em 340/435 nm), green (465/515), orange (528/590), and red (625/670) channels. FTIR spectra (4000–500 cm−1) probed chemical functionalities and crosslinking signatures. Antimicrobial assays: - Patch mode: Undetached microgel arrays (in-template patches) were washed to remove free phage and placed on bacterial lawns (E. coli ER2738, O157:H7, or BL21) on LB agar overlays; lysis zones were imaged after overnight incubation at 37 °C. - Spray mode: Microgel suspensions (>3 × 10^9 microgels mL−1) were sprayed onto bacterial lawns; plaques/lysis zones imaged after overnight incubation. - Suspension assays: In PBS (nutrient-deficient) and TSB (nutrient-rich), bacterial cultures at defined initial titers (10^4–10^9 CFU mL−1 in PBS; 10^4–10^8 in TSB) were treated with microgels (~1500 microgels mL−1 added) or PBS control. OD600 (TSB) was monitored over 9 h and CFU enumerated at endpoints. Food decontamination tests: Romaine lettuce squares (~0.4 g) were contaminated to 10^6 CFU g−1 with E. coli O157:H7, sprayed with microgels (200 µL) or water, wrapped, and held at room temperature for 9 h. Bacteria were recovered by vortexing in PBS and enumerated on MacConkey-Sorbitol agar (detection limit 100 CFU g−1). Beef cubes (~3 g) were contaminated to 10^8 CFU g−1, treated similarly (detection limit 34 CFU g−1).

- High-throughput, gentle fabrication: Peelable honeycomb templates yielded ordered arrays of phage microgels that could be used as patches or released into suspensions. Production reached >35,000 microgels per cm^2 of template (consistent with template pore density and isolation yields), with each microgel comprising on the order of 7 × 10^5 M13 phages based on input concentration and size estimates. - Size and porosity: Hydrated microgel diameters (mean ± SD): GA 25.34 ± 5.72 µm; EDC 24.39 ± 4.92 µm; M13+BSA+GA 30.77 ± 3.83 µm, all smaller and broader than the ~35.73 ± 2.86 µm template pores. Upon drying, diameters reduced to 11.13 ± 2.32 µm (GA) and 13.16 ± 1.99 µm (EDC), indicating 91.5% and 84.3% volume reduction, respectively; BSA-containing microgels shrank less (to 21.97 ± 3.04 µm; 63.6% volume reduction), consistent with higher density/lower porosity. - Preparation efficiency: Template pore density was 83,862 ± 5,241 pores cm−2. Isolated microgels per cm^2: GA 35,295 ± 5,490; EDC 41,226 ± 6,878; M13+BSA+GA 31,431 ± 6,185, corresponding to 42–49% of pores yielding microgels (losses attributed to partial filling and isolation losses). - Nanofibrous architecture: FE-SEM revealed highly aligned nanofibrous textures (7–20 nm widths) consistent with bundled M13 filaments; g3p tips appeared as bright protrusions. Hybrid M13+BSA microgels lacked long-range order, indicating BSA interfered with phage alignment but preserved functional exposure of g3p. - Tunable autofluorescence: GA-crosslinked microgels exhibited strong autofluorescence (green/orange/red; weak blue) due to Schiff base (C=N) formation; EDC-crosslinked microgels showed minimal fluorescence, particularly absent in red. Quantitatively, GA and M13+BSA+GA microgels were 294.7% and 320.9% brighter than EDC microgels in green and orange channels; BSA addition increased GA microgel fluorescence by ~23.5% (green) and ~26.1% (orange). FTIR confirmed amide bands common to all microgels and GA-specific features (sp2 C–H, aldehyde C–H, imine C=N). - Antimicrobial efficacy in vitro: Hybrid microgels embedding virulent HER262 formed lysis zones on E. coli ER2738 and O157:H7 lawns (patch and spray formats). In PBS, HER262-embedded microgels reduced E. coli O157:H7 by 6 logs within 9 h at initial 10^8 CFU mL−1 and fully eradicated bacteria within 9 h when initial titers were ≤10^4 CFU mL−1. In TSB, microgels suppressed growth, maintaining titers at 10^4–10^7 CFU mL−1 versus 10^9 CFU mL−1 in controls after 9 h. Specificity was demonstrated by lack of killing against non-host strains (ER2738, BL21) when inappropriate host-phage pairs were tested. T7-embedded microgels produced lysis on BL21 lawns and yielded ≥6-log reductions in PBS and ~5-log differences in TSB. - Food decontamination: On lettuce inoculated at 10^6 CFU g−1, spraying HER262-embedded microgels reduced E. coli O157:H7 to below detection (<100 CFU g−1) after 9 h (up to 6-log reduction). On beef inoculated at 10^8 CFU g−1, microgels reduced counts from 2.5 × 10^8 to 1.4 × 10^6 CFU g−1 (reported as 99.94% reduction).

The study demonstrates that filamentous phage can be crosslinked into microscale, highly porous, nanofibrous gels using a biologics-friendly template approach, enabling new delivery formats (patches, sprays, suspensions) while preserving bioactivity. Self-assembled alignment of M13 nanofilaments yields mechanically coherent microgels with large surface areas that facilitate host contact and can encapsulate additional virulent phages and proteins. Choice of crosslinker allows tuning of autofluorescence: GA provides strong intrinsic fluorescence useful for imaging, whereas EDC yields low-background microgels suitable for fluorescence-based sensing. Incorporating BSA accelerates gelation and mitigates crosslinker-induced bioactivity loss, especially for delicate virulent phages with tail fibers, enabling robust antibacterial performance. The microgels performed across environmental conditions (nutrient-rich and -deficient) and matrices (agar lawns, liquid suspensions, real food surfaces), achieving up to 6-log reductions of multidrug-resistant E. coli O157:H7 and substantial reductions on meat surfaces. The hydrated microgel environment also offers protection against desiccation compared with free phage, addressing a key stability challenge. Collectively, the findings validate microgels as effective, targeted, and scalable phage delivery vehicles for biocontrol applications.

This work establishes virus-built microgels as a versatile, high-throughput, and biologics-friendly platform for targeted antimicrobial applications. Using breath-figure honeycomb templates and small-molecule crosslinkers (GA or EDC), the authors produced peelable arrays and suspensions of phage-exclusive and phage–protein hybrid microgels with highly aligned nanofibrous architecture and tunable autofluorescence. When loaded with virulent phages, these microgels delivered potent, host-specific antibacterial action, achieving up to 6-log reductions of E. coli O157:H7 in vitro and on food products (lettuce, beef). The approach is heat- and solvent-free, scalable, and compatible with desiccation-sensitive biomolecules, suggesting broad applicability to antimicrobial packaging, produce sprays, and household decontamination. Future work could explore broader pathogen targets, alternative bioactive payloads, optimization of microgel mechanical properties and storage stability, and integration into industrial food processing and packaging workflows.

- Yield relative to template capacity: Only 37–49% of template pores yielded isolated microgels, attributed to partial filling and isolation losses, which may affect large-scale process efficiency. - Crosslinker trade-offs: GA imparts strong autofluorescence that can interfere with fluorescence-based sensing; EDC minimizes fluorescence but may yield different gelation kinetics and properties. GA concentration had to be reduced (to 0.02 M) and BSA added to preserve virulent phage bioactivity. - Structural order vs. bioactivity: Adding BSA disrupted nanofiber alignment but was important for maintaining phage activity, indicating a balance between structural order and functional preservation. - Desiccation sensitivity of free phages: Free M13, HER262, and T7 lost ~4 logs of titer after 1 h desiccation and rehydration, underscoring dependence on the hydrated microgel environment for stability. - Scope of testing: Antimicrobial validation focused on E. coli strains (O157:H7, ER2738, BL21) and two food matrices; broader pathogen panels, complex food environments, and long-term shelf-life or storage conditions were not reported.