Developing cost-effective, consumer-accessible platforms for point-of-use environmental and clinical pathogen testing is a priority, to reduce reliance on laborious, time-consuming culturing approaches. Unfortunately, a system offering ultrasensitive detection capabilities in a form that requires little auxiliary equipment or training has remained elusive. Here, a colorimetric DNAzyme-crosslinked hydrogel sensor is presented. In the presence of a target pathogen, DNAzyme cleavage results in hydrogel dissolution, yielding the release of entrapped gold nanoparticles in a manner visible to the naked eye. Recognizing that Escherichia coli holds high relevance within both environmental and clinical environments, an E. coli-responsive DNAzyme is incorporated into this platform. Through the optimization of the hydrogel polymerization process and the discovery of bacteriophage-induced DNAzyme signal amplification, 101 CFU mL-1 E. coli is detected within real-world lake water samples. Subsequent pairing with an artificial intelligence model removed ambiguity in sensor readout, offering 96% true positive and 100% true negative accuracy. Finally, high sensor specificity and stability results supported clinical use, where 100% of urine samples collected from patients with E. coli urinary tract infections are accurately identified. No false positives are observed when testing healthy samples. Ultimately, this platform stands to significantly improve population health by substantially increasing pathogen testing accessibility.

Engineered by nature, biological entities are exceptional building blocks for biomaterials. These entities can impart enhanced functionalities on the final material that are otherwise unattainable. However, preserving the bioactive functionalities of these building blocks during the material fabrication process remains a challenge. We describe a high-throughput protocol for the bottom-up self-assembly of highly concentrated phages into microgels while preserving and amplifying their inherent antimicrobial activity and biofunctionality. Each microgel is comprised of half a million cross-linked phages as the sole structural component, self-organized in aligned bundles. We discuss common pitfalls in the preparation procedure and describe optimization processes to ensure the preservation of the biofunctionality of the phage building blocks. This protocol enables the production of an antimicrobial spray containing the manufactured phage microgels, loaded with potent virulent phages that effectively reduced high loads of multidrug-resistant Escherichia coli O157:H7 on red meat and fresh produce. Compared with other microgel preparation methods, our protocol is particularly well suited to biological materials because it is free of organic solvents and heat. Bench-scale preparation of base materials, namely microporous films (the template for casting microgels) and pure concentrated phage suspension, requires 3.5 h and 5 d, respectively. A single production run, that yields over 1,750,000 microgels, ranges from 2 h to 2 d depending on the rate of cross-linking chemistry. We expect that this platform will address bottlenecks associated with shelf-stability, preservation and delivery of phage for antimicrobial applications, expanding the use of phage for prevention and control of bacterial infections and contaminants.

Abstract Background Patients with IBD are increasingly experiencing treatment failures on frontline therapies. While corticosteroids are an effective frontline intervention, 16% of patients fail to respond, and 20%–30% show only partial responses. Bacteriophages have recently garnered attention as a potential adjunctive therapy for IBD to target bacterial strains associated with IBD, including adherent-invasive Escherichia coli (AIEC). Aims Our aim was to determine whether prophylactic bacteriophage therapy could enhance the therapeutic efficacy of a sub-therapeutic dose of budesonide in a gnotobiotic mouse model of E. coli-driven colitis. Methods Adult germ-free C57BL/6 mice were co-colonized with altered Schaedler-like flora (ASF) plus E. coli NRG857c, a Crohn’s disease-associated bacterial isolate. Three weeks later, mice were treated with phage HER259 (1x109 PFU/dose; 0.1% bicarbonate) 3 times/ week or vehicle (PBS with 0.1% bicarbonate) 3 times/ week (n=5/group). Mice were then exposed to low-dose dextran sulfate sodium (2%; DSS) in drinking water for 5 days, followed by 2 days of water. All mice were administered a subtherapeutic dosage of 2ug/day of Budesonide halfway through DSS exposure to endpoint. Mice were monitored daily for weight loss, stool consistency, and fecal occult blood. At sacrifice, colon tissue was collected for histological analysis. Results Compared with budesonide treatment alone, prophylactic phage treatment, followed by a sub-therapeutic dose of budesonide, led to reduced stool consistency scores (p ampersand:003C0.001) and reduced presence of occult blood (p ampersand:003C 0.001). No difference in fecal E. coli load was observed between groups. At endpoint, the combined phage-budesonide treatment was associated with lower histological scores as compared with mice treated with budesonide alone (p ampersand:003C 0.01). Conclusions While underlying mechanisms remain elusive, our results suggest a beneficial effect of prophylactic phage intervention to the subsequent administration of a frontline immunosuppressive compound used to treat IBD. Future work will investigate the mechanisms by which adjunctive phage therapy can enhance existing therapies used to treats colitis, to better inform clinical guidelines. Funding Agencies CIHR

Three-dimensional (3D) hierarchical wrinkled materials built with biological entities have so far remained exclusive to nature. Herein, multiscale functional ultraporous 3D bio-networks of bioprinted phage-built wrinkled microarrays are created through establishing a universal heat- and solvent-independent substrate-shrinkage method induced by high-pressure carbon dioxide (HPCD). This method results in diverse wrinkled patterns on soft materials and is particularly powerful for solvent- and heat-sensitive biomaterials, for which other methods have failed. The phage nanofilaments (7 nm width) self-assemble into orderly-aligned submicron bundles (100 nm width), which crimp into tunable microscale wrinkles (0.7-5.0 mu m width) on size-controllable micro-arrays (200-600 mu m width) exhibiting a four-level hierarchical nano-reticular structure. The HPCD method also protects the bioactivity of biorecognition molecules loaded into the microarrays, leading to the design of bacteria-sensing chips, made with in-house deoxyribozyme-loaded 3D phage microarrays. The developed bacteria-sensing chips achieve a limit of detection that is 100 x more sensitive with greater reproducibility compared to two-dimensional (2D) microdot arrays and correctly identify Legionella pneumophila in contaminated water samples collected from industrial cooling towers, highlighting phage-built wrinkled networks as a platform for bottom-up assembly of biological building blocks into biofunctional material. Virus-assembled microdots with a wrinkled pattern induced by the high-pressure carbon dioxide method show a morphology similar to sunflowers. image

Abstract Background Recent evidence has linked ultra-processed foods, which are high in salt, to increased risk of inflammatory bowel disease (IBD). A high salt diet (HSD) was also shown to be colitogenic in specific pathogen-free mice, in part, through modulation of the microbiota. We recently described high microbial proteolytic activity (PA) in feces from IBD patients, that when transferred to germ-free mice, increased acute colitis severity. However, the drivers of high microbial PA are unknown. Aims To determine whether HSD increases proteolytic activity, thereby exacerbating colitis. Methods Adult germ-free (GF) C57BL/6 mice were colonized with feces from a patient with UC in flare (n=13). Mice were fed either a control diet (CD; 7004, Teklad) or a HSD (7004 supplemented with 4% NaCl) plus 1% NaCl in drinking water. Three weeks following colonization, chronic colitis was induced in half of the mice by three cycles (five days each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a five-day wash-out period between cycles. Fecal PA and predicted microbial protease profiles were measured before colitis induction. Weight loss and disease activity (e.g., occult blood in feces and stool consistency) was evaluated for the duration of colitis and microscopic colitis was evaluated at endpoint. Results In mice colonized with UC microbiota, a HSD increased elastolytic activity (p=.03 versus CD) and induced a unique predicted protease and peptidase profile. After colitis induction, mice consuming a HSD lost more weight than mice consuming the CD (p=.0001) and had higher disease activity (p=.0004). Additionally, HSD induced more severe microscopic colitis scores compared with the CD (p=.0001). Conclusions These results raise the hypothesis that a HSD drives microbial proteolytic activity of IBD microbiota. However, whether this shift is causally associated with the worsening of colitis remains unclear. Mechanistic insight underlying this HSD-microbial proteolytic function could lead to specific dietary modifications or anti-proteolytic therapies for IBD in patients with proteolytic-driven inflammation. Funding Agencies CCC, CIHR

Abstract Background An imbalance in host proteases has been implicated in inflammatory bowel disease (IBD). Recent evidence implicates microbial proteolytic activity (PA) in ulcerative colitis but whether it also plays a role in Crohn’s disease (CD) remains unclear. Purpose We therefore investigated the colitogenic potential and underlying pathways of proteolytic CD microbiota. Method Adult germ-free (GF) C57BL/6 mice were colonized with CD microbiota selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), and from healthy controls with LPA (HC-LPA), after which total fecal proteolytic, elastolytic and mucolytic activity were analyzed in the mice. Microbial community was assessed by 16S rRNA gene sequencing. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colitis severity and underlying pathways were investigated in C57BL/6, Nucleotide-binding Oligomerization Domain-2 knock-out (Nod2-/-), and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (R38E-PAR2) subjected to 2% dextran sodium sulfate in drinking water for 5 days followed by 2 days on water. Result(s) Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity compared with GF mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with GF mice. Fecal supernatants from CD-LPA or HC-LPA colonized mice had lower in vitro PAR2 cleavage compared to supernatants from GF and CD-HPA colonized mice. Several genes, such as Map kinases, Rhoa, Myd88, and Tollip, were increased in GF mice compared to colonized mice. 18 genes related to inflammation and barrier function (e.g., Mapk2k6, Tnf, Claudin1) were differentially expressed between CD-LPA and CD-HPA. CD-HPA mice had lower alpha diversity, distinct microbial profiles, and higher fecal proteolytic activity compared with CD-LPA. Abundance of several beneficial species (e.g., Akkermansia muciniphilia) was decreased while other taxa were increased (e.g., Hungattella hathewayi) in CD-HPA compared to CD-LPA. H. hathewayi as well as the serine protease K04772 were transcriptionally increased in fecal samples from CD-HPA colonized mice. C57BL/6 and Nod2-/- mice, but not R38E-PAR2 mice, colonized with CD-HPA developed earlier and more severe colitis compared with mice colonized with CD-LPA. Conclusion(s) CD proteolytic microbiota is proinflammatory through a PAR2 pathway. H. hathewayi correlates with the proinflammatory phenotype through the serine protease K04772 in this model. The results support a role of microbial PA in CD, which could constitute a biomarker for identifying patients who would benefit from anti-proteolytic therapies. Disclosure of Interest None Declared

As cases of multidrug resistant bacterial infections increase, scientists and clinicians around the world are increasingly turning to bacteriophages as alternatives to antibiotics. Even though our understanding of phage has increased significantly since the early days of its discovery, over a century ago, the currently used tools and technologies for phage purification for therapeutic applications are severely limited. Bacteriophages are produced by bacterial cultures, and impurities such as endotoxins must therefore be removed before clinical use. We present an anion exchange bind-and-elute membrane chromatographic method for purifying T7 bacteriophage from Escherichia coli culture supernatant that removes undesirable impurities, while ensuring a high viable phage count in the purified product. Our method does not involve the use of chemicals such as organic solvents and caesium chloride that could typically leave residual toxicity in the final product. It also does not require expensive equipment, such as an ultracentrifuge. Using our method, that is based on an in-house designed membrane module, 65% of viable T7 phage was recovered, and up to 94% endotoxins could be removed. The method, which took approximately 15 min, is rapid and scalable, and produces quite pure bacteriophage samples in a single step. It therefore potentially represents a major improvement over the status quo, and shows the way ahead for streamlining phage manufacturing for therapeutic use.

Emerging evidence implicates microbial proteolytic activity in ulcerative colitis (UC), but whether it also plays a role in Crohn's disease (CD) remains unclear. We investigated the effects of colonizing adult and neonatal germ-free C57BL/6 mice with CD microbiota, selected based on high (CD-HPA) or low fecal proteolytic activity (CD-LPA), or microbiota from healthy controls with LPA (HC-LPA) or HPA (HC-HPA). We then investigated colitogenic mechanisms in gnotobiotic C57BL/6, and in mice with impaired Nucleotide-binding Oligomerization Domain-2 (NOD2) and Protease-Activated Receptor 2 (PAR2) cleavage resistant mice (Nod2 (-/-); R38E-PAR2 respectively). At sacrifice, total fecal proteolytic, elastolytic, and mucolytic activity were analyzed. Microbial community and predicted function were assessed by 16S rRNA gene sequencing and PICRUSt2. Immune function and colonic injury were investigated by inflammatory gene expression (NanoString) and histology. Colonization with HC-LPA or CD-LPA lowered baseline fecal proteolytic activity in germ-free mice, which was paralleled by lower acute inflammatory cell infiltrate. CD-HPA further increased proteolytic activity compared with germ-free mice. CD-HPA mice had lower alpha diversity, distinct microbial profiles and higher fecal proteolytic activity compared with CD-LPA. C57BL/6 and Nod2 (-/-) mice, but not R38E-PAR2, colonized with CD-HPA had higher colitis severity than those colonized with CD-LPA. Our results indicate that CD proteolytic microbiota is proinflammatory, increasing colitis severity through a PAR2 pathway.

Abstract Background Opportunistic pathogens have been postulated to drive dysregulated inflammation in inflammatory bowel disease (IBD). Indeed, adherent-invasive Escherichia coli (AIEC) isolated from IBD patients have pathobiont and pro-inflammatory characteristics. Current treatments for IBD suppress the immune response and do not target key microbial drivers, therefore novel strategies are required. Purpose Our aim was to determine whether bacteriophage therapy targeted against AIEC could reduce the severity of E. coli-driven colitis in gnotobiotic mice. Method Adult germ-free C57BL/6 mice were colonized with altered Schaedler-like flora (ASF) and E. coli NRG857c, a Crohn’s disease-associated bacterial isolate. Three weeks later, mice were treated with daily phage (selected by killing curves bioassays against E. coli NRG857c) or PBS for 2 weeks (n=6/group). Mice were then exposed to low-dose dextran sulfate sodium (2%; DSS) in drinking water for 5 days, followed by 2 days of water. PBS-treated mice (n=6) that received no DSS were used as additional negative controls. Mice were monitored daily for weight, stool consistency, and occult blood. At sacrifice, colon tissue was collected for histological analysis and fecal contents were cultured to determine bacterial load. In separate experiments, C57BL/6NTac-Il10em8Tac (IL-10-/-) mice were colonized with ASF-like microbiota and E. coli NRG857c. Three weeks later, mice (n=5) were treated with weekly phage or PBS (n=5) for 7 weeks. Mice were monitored weekly as described above. Result(s) Daily phage treatment reduced the severity of clinical symptoms induced by acute DSS administration (p < 0.001 vs. DSS-PBS treated mice). At endpoint, phage treatment was associated with lower histological scores as compared with DSS-PBS controls (p < 0.0001). A 1-log reduction in AIEC bacterial load was observed in phage treated mice as compared with DSS-PBS controls (p < 0.001). In IL-10-/- mice, weekly phage treatment delayed the spontaneous onset of colitis (p < 0.0001 vs. PBS-treated mice). At endpoint, mice treated with phage had lower colitis scores. Reduced weekly AIEC bacterial load was observed in phage-treated mice. Conclusion(s) Lytic phages, targeting a known AIEC pathobiont isolated from Crohn’s disease patients, ameliorate acute intestinal injury and delay onset of spontaneous colitis. Future work will investigate the mechanisms by which phage therapy prevents and treats colitis, to better inform clinical trial design. Disclosure of Interest None Declared

Nanofilamentous bacteriophages (bacterial viruses) are biofunctional, self-propagating, and monodisperse natural building blocks for virus-built materials. Minifying phage-built materials to microscale offers the promise of expanding the range function for these biomaterials to sprays and colloidal bioassays/biosensors. Here, we crosslink half a million self-organized phages as the sole structural component to construct each soft microgel. Through an in-house developed, biologics-friendly, high-throughput template method, over 35,000 phage-built microgels are produced from every square centimetre of a peelable microporous film template, constituting a 13-billion phage community. The phage-exclusive microgels exhibit a self-organized, highly-aligned nanofibrous texture and tunable auto-fluorescence. Further preservation of antimicrobial activity was achieved by making hybrid protein-phage microgels. When loaded with potent virulent phages, these microgels effectively reduce heavy loads of multidrug-resistant Escherichia coli O157:H7 on food products, leading to up to 6 logs reduction in 9 hours and rendering food contaminant free.

Abstract Background Ulcerative colitis (UC) is one of two forms of inflammatory bowel disease (IBD). The exact cause of IBD is unknown but altered host-microbe interactions and genetic susceptibility are involved in its pathogenesis. Many patients with IBD do not respond to biological therapies targeting single cytokines, therefore new therapies that target common immune pathways are being developed and need to be tested in relevant preclinical models. Previously we have shown that mice colonized with UC microbiota upregulated genes related to inflammation without induction of colitis compared to healthy volunteer-colonized mice. We thus investigated whether IRAK4 and TPL2-induced pathways, new therapeutic targets in development upstream of inflammatory cytokine gene activation, are upregulated in mice colonized with UC microbiota and chronic colitis. Aims Our aim was to characterize TPL2 and IRAK4 signalling pathways and T cell phenotypes in UC-colonized mice following chronic low-dose dextran sodium sulfate (DSS) colitis. Methods 10-16-week-old germ-free C57BL/6 mice were colonized with fecal microbiota from a patient with UC experiencing a flare (n=16). Mice were housed in a gnotobiotic facility during the experiment. Three weeks following colonization, colitis was induced in half of the mice by three cycles (5 d each; 2.0%, 1.5% and 1.5%, respectively) of DSS in drinking water with a 5-d wash-out period between cycles. All mice were fed a control diet (7004, Teklad). Fecal samples were collected weekly. At sacrifice, disease activity (colon length, occult blood in feces, stool consistency, and spleen weight) was measured and colon tissue was collected for histological analysis and RNA sequencing. Mesenteric lymph nodes (MLNs) were acquired for flow cytometry to analyze T cell phenotypes. Results In UC-colonized mice, chronic low-dose DSS induced softer feces (p<0.0001), shorter colon length (p<0.0001), and increased spleen weight (p<0.0001), compared with water treated mice. This was paralleled by detectable blood in stool, development of moderate colitis (DSS: 1.9+/-1.4 vs H2O: 0.5+/-0.2; p<0.02) and higher proportion of IL-17 (p=0.003) and IFN-γ-producing T cells (p=0.06) in MLNs compared with water treated mice. RNA sequencing revealed that inflammatory genes, mainly related to the IRAK4/TPL2 pathway (e.g., Gadd45b, Socs3, Il1b), were significantly increased (p≤0.05) in the colon of mice treated with DSS compared with water. Conclusions When challenged with a chronic low-dose chemical injury agent, mice colonized with UC microbiota develop clinical and histological signs of colitis and upregulation of genes involved in inflammation like Gadd45b, Socs3, and Il1b. Thus, this model represents a new valuable tool for preclinical testing of new drug candidates, such as those related to the modulation of IRAK4/TPL2 pathways. Funding Agencies CCC

The World Health Organization has designated Staphylococcus aureus as a global health concern. This designation stems from the emergence of multiple drug-resistant strains that already account for hundreds of thousands of deaths globally. The development of novel treatment strategies to eradicate S. aureus or mitigate its pathogenic potential is desperately needed. In the effort to develop emerging strategies to combat S. aureus, phage display is uniquely positioned to assist in this endeavor. Leveraging bacteriophages, phage display enables researchers to better understand interactions between proteins and their antagonists. In doing so, researchers have the capacity to design novel inhibitors, biosensors, disinfectants, and immune modulators that can target specific S. aureus strains. In this review, we highlight how phage display can be leveraged to design novel solutions to combat S. aureus. We further discuss existing uses of phage display as a detection, intervention, and prevention platform against S. aureus and provide outlooks on how this technology can be optimized for future applications.

Phage display is broadly used for detection, mapping, disinfection and prevention of Staphylococcus aureus.

Bacteriophages, or phages (bacterial viruses), have seen a resurgence in their applications following the emergence of antimicrobial-resistant superbugs that pose enormous risks to human health and food supplies. Phages present numerous advantages over conventional small-molecule antibiotics, including that they are highly selective bacteria-killers and demonstrate low inherent cytotoxicity to human health. Notwithstanding the direct therapeutic applications of these innate bacteria-killing viruses, they have also garnered attention as biological nanoparticles. Due to the diversity in sizes and shapes of phages, self-replicating capacity, geometrical batch-to-batch consistency, and ease of synthetic modifications, phages are excellent building blocks for creating bioactive biomaterial platforms. In this review, we provide a brief history of the development of phage-based materials and identify key stakeholders who are driving innovation in this space. In addition, we explore various phage-based gel structures and provide a critical analysis of how their structures produce distinct advantageous properties that can be exploited for applications in solving challenges in biomedical engineering.

Abstract Background Bacterial infections have been postulated to drive the dysregulated inflammation found in inflammatory bowel disease (IBD). In particular, adherent-invasive Escherichia coli (AIEC) isolated from patients with IBD have pathobiont characteristics and have been implicated in IBD pathogenesis. Aims Our aim was to characterize and compare the level of intestinal inflammation and potential microbiota shifts induced by E. coli clinical isolates using a gnotobiotic mouse model of colitis. Methods Adult germ-free C57BL/6 mice were transferred to ISO positive cages in a gnotobiotic facility and colonized with altered Schaedler flora-like (ASF) microbiota and one of three clinical E. coli isolates: E. coli C0004 (n = 5), E. coli LF82 (n = 9), E. coli NRG857c (n = 6), or ASF alone (n = 6). Three weeks later, mice were treated for 5 days with low dose dextran sodium sulphate in drinking water (2%; DSS), followed by 2 days of water. Mice were monitored daily for clinical symptoms (weight, stool consistency, and occult blood). At sacrifice, colon tissue was collected for histological analysis. Cecum contents were cultured to determine bacterial load. Fecal samples were collected for 16S rRNA gene sequencing analysis before and after DSS treatment. Results All mice colonized with an E. coli isolate displayed significantly greater clinical and microscopic scores of colitis compared to ASF alone, but the severity was dependent on the colonized E. coli strain. E. coli NRG857c-colonized mice exhibited more severe symptoms (p < 0.001) two days earlier than mice colonized with other E. coli isolates. Mice colonized with E. coli LF82 or E. coli NRG857c had higher histological scores of colitis compared to mice colonized with E. coli C0004, which were also significantly greater than ASF alone (p < 0.0001). 16S rRNA gene sequencing revealed that ASF-alone-colonized mice lacked Proteobacteria. All E. coli-colonized mice had comparable bacterial loads, which were verified by 16S rRNA analysis. Following DSS, E. coli LF82 and E. coli NRG857c relative abundance remained stable, whereas the relative abundance of Roseburia/ASF 492 declined significantly in all E. coli colonized mice. Conclusions The presence of E. coli pathobionts in mice drive the severity of chemically induced colitis, with AIEC NRG857c inducing the greatest severity. This gnotobiotic mouse model enables us to control the severity of colitis in a well-defined microbiota that is dependent on the colonized E. coli isolate. Using this model, we will be able to assess therapeutic candidates that aim to treat colitis at varying stages of its disease progression. Funding Agencies CCC, CIHRNSERC

We report the development of ordered shape-controllable microbump structures on protein hydrogels using polystyrene honeycomb templates. Addition of protein nanogels results in the formation of hierarchical nano-on-micro structures and increases surface hydrophilicity by over 55%, exhibiting bacteria repellency 100 times stronger than a flat hydrogel surface composed of the same protein.

Biological hydrogels play important physiological roles in the body. These hydrogels often contain ordered subdomains that provide mechanical toughness and other tissue-specific functionality. Filamentous bacteriophages are nanofilaments with a high aspect ratio that can self-assemble into liquid crystalline domains that could be designed to mimic ordered biological hydrogels and can thus find application in biomedical engineering. We have previously reported hydrogels of pure crosslinked liquid crystalline filamentous phage formed at very high concentrations exhibiting a tightly packed microstructure and high stiffness. In this work, we report a method for inducing self-assembly of filamentous phage into liquid crystalline hydrogels at concentrations that are several orders of magnitude below that of lyotropic liquid crystal formation, thus creating structural order, but a less densely packed hydrogel. Hybrid hydrogels of M13 phage and bovine serum albumin (0.25 w/v%) were formed and shown to adsorb up to 16 its weight in water. Neither component gelled on its own at the low concentrations used, suggesting synergistic action between the two components in forming the hydrogel. The hybrid hydrogels exhibited repetitive self-healing under physiological conditions and at room temperature, autofluorescence in three channels, and antibacterial activity towards Escherichia coli host cells. Furthermore, the hybrid hydrogels exhibited more than 2 higher ability to pack water compared to BSA-only hydrogels and 2 higher flexibility (lower compression modulus) compared to tightly packed M13-only hydrogels, suggesting that our method could be used to create hydrogels with tunable mechanical properties through the addition of globular proteins, while maintaining structural order at the microscale.

Filamentous bacteriophages (bacterial viruses) are semiflexible proteinous nanofilaments with high aspect ratios for which the surface chemistry can be controlled with atomic precision via genetic engineering. That, in addition to their ability to self-propagate and replicate a nearly monodisperse batch of biologically and chemically identical nanofilaments, makes these bionanofilaments superior to most synthetic nanoparticles and thus a powerful tool in the bioengineers' toolbox. Furthermore, filamentous phages form liquid crystalline structures at high concentrations; these ordered assemblies create hierarchically ordered macro-, micro-, and nanostructures that, once cross-linked, can form hierarchically ordered hydrogels, hydrated soft material with a variety of physical and chemical properties suitable for biomedical applications (e.g., wound dressings and tissue engineering scaffolds) as well as biosensing, diagnostic assays. We provide a critical review of these hydrogels of filamentous phage, and their physical, mechanical, chemical, and biological properties and current applications, as well as an overview of limitations and challenges and outlook for future applications. In addition, we present a list of design parameters for filamentous phage hydrogels to serve as a guide for the (bio)engineer and (bio)chemist interested in utilizing these powerful bionanofilaments for designing smart, bioactive materials and devices.