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

Foodborne illnesses cause hundreds of thousands of deaths annually, with children under five accounting for nearly a third of these fatalities. Bacteriophages (phages), natural bacterial predators, offer a targeted approach to biocontrol, minimizing disruption to beneficial bacterial populations. Phage-based products have received FDA approval for controlling bacterial contaminants in food, offering advantages over traditional antimicrobials by not affecting food quality. However, challenges in delivery and stability limit their widespread use. Filamentous phages, essentially protein nanoparticles, are attractive building blocks for antimicrobial materials due to their biocompatibility, self-propagation, and customizable surface chemistry. Previous research demonstrated the creation of bulk phage hydrogels; however, the current study focuses on miniaturizing these materials to the microscale in the form of microgels. Microgels offer increased surface area for bacterial contact, improved flow properties for spray or injection delivery, and enhanced protection against harsh environments compared to bulk materials. This research aims to develop a high-throughput, biomolecule-friendly method to produce phage microgels, preserving their antimicrobial activity and enabling versatile applications in food safety and other fields.

The use of bacteriophages for biocontrol is gaining traction, particularly in food safety and agriculture. Research highlights the targeted nature of phage infection, their ability to eliminate specific bacterial strains without harming beneficial commensals, and their potential for food decontamination without compromising taste or nutritional value. However, limitations exist regarding delivery methods and the stability of phage products, hindering widespread adoption. Studies have explored the use of phages as building blocks for various materials, capitalizing on their inherent properties like self-assembly and customizable surface chemistry. The creation of phage-based hydrogels and other macroscopic materials has shown promise; however, the potential of phage microgels for enhanced applications remains largely unexplored.

The researchers developed a novel high-throughput method for producing phage microgels using a biomolecule-friendly approach. This involved utilizing a microporous polystyrene honeycomb film template created via the breath figure method. The template consisted of uniform, open-ended spherical micropores, enabling the generation of a large array of microgels. Two crosslinkers, glutaraldehyde (GA) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), were employed to crosslink the filamentous M13 phages. GA crosslinking resulted in Schiff base formation, leading to significant autofluorescence, while EDC crosslinking produced amide bonds with minimal fluorescence. The addition of bovine serum albumin (BSA) further enhanced gelation efficiency and modulated fluorescence properties. The microgels, formed within the template pores, were easily detached by peeling and sonication, yielding a suspension of individual microgels. The size, porosity, and preparation efficiency of the microgels were characterized using microscopy and image analysis. Scanning electron microscopy (SEM) was used to examine the nanostructure of the microgels, revealing a self-organized, highly aligned nanofibrous texture in phage-exclusive microgels. The fluorescence properties of the microgels were investigated using fluorescence microscopy and Fourier transform infrared (FTIR) spectroscopy. The antimicrobial activity of the pure and hybrid phage microgels (incorporating virulent phages T7 and HER262) was evaluated in three scenarios: an undetached microgel array patch, a microgel spray, and direct addition to bacterial suspensions. Antimicrobial activity was assessed against multidrug-resistant *E. coli* O157:H7 in both nutrient-rich (TSB) and nutrient-deficient (PBS) environments, measuring bacterial titers and optical density. Finally, the effectiveness of the microgels was tested in decontaminating lettuce and beef contaminated with *E. coli* O157:H7.

The study successfully demonstrated the fabrication of phage microgels with several key features: 1. **High-Throughput Production:** The novel template-based method yielded over 35,000 microgels per square centimeter, each containing over 700,000 M13 phages, representing a highly efficient manufacturing process. 2. **Tunable Properties:** The use of different crosslinkers (GA and EDC) allowed for the control of microgel autofluorescence, expanding their applicability for various purposes. The addition of BSA modulated the fluorescence intensity and improved gelation speed. 3. **Self-Organized Nanostructure:** SEM analysis revealed a highly aligned nanofibrous texture in the phage-exclusive microgels, directly resulting from the self-assembly of phage nanofilaments. 4. **Effective Antimicrobial Activity:** The microgels, particularly when loaded with virulent phages (HER262 and T7), exhibited significant antimicrobial activity against multidrug-resistant *E. coli* O157:H7. In both PBS and TSB, the microgels reduced bacterial loads substantially, resulting in up to a 6-log reduction within 9 hours. 5. **Versatile Applications:** The microgels were successfully tested as antimicrobial patches, sprays, and direct additions to bacterial solutions, showcasing their versatility for various biocontrol applications. 6. **Food Safety Application:** In tests using contaminated lettuce and beef, the phage microgel spray effectively reduced *E. coli* O157:H7 contamination to undetectable levels (<100 CFU/g for lettuce and a >99.94% reduction for beef).

The findings address the research question by successfully demonstrating the development and application of phage microgels as effective antimicrobials. The high-throughput, biocompatible fabrication method significantly advances the feasibility of utilizing phages for large-scale biocontrol applications. The tunable properties of the microgels, particularly their autofluorescence and gelation kinetics, offer design flexibility for diverse applications. The superior antimicrobial performance, especially the significant reduction of multidrug-resistant *E. coli* O157:H7 in food products, highlights the potential of this technology for enhancing food safety. The successful demonstration in multiple delivery formats (patch, spray, suspension) broadens the applicability of this technology. The results contribute significantly to the field by providing a scalable and effective strategy for combating multidrug-resistant bacteria, offering a promising alternative to traditional antibiotics.

This research successfully established a novel high-throughput method for producing phage microgels with tunable properties and high antimicrobial efficacy against multidrug-resistant bacteria. The resulting microgels offer a promising solution for food safety and biocontrol applications. Future research could focus on expanding the range of target bacteria, optimizing phage selection and microgel formulation, and exploring additional applications in diverse environments (e.g., medical, environmental). Investigating the long-term stability and efficacy of the microgels under various storage and application conditions is also warranted.

While the study demonstrates significant antimicrobial efficacy, limitations exist. The study focused primarily on *E. coli* O157:H7; further research is needed to assess efficacy against a broader range of bacterial species. The long-term stability and shelf-life of the microgels need more investigation, and cost-effectiveness compared to other antimicrobial strategies should also be assessed. The impact of potential immune responses in vivo, should the technology be used for medical applications, needs careful consideration.