Bacterial resistance to temperate phage is influenced by the frequency of lysogenic establishment.

Temperate phages can shape bacterial community dynamics and evolution through lytic and lysogenic life cycles. In response, bacteria that resist phage infection can emerge. This study explores phage-based factors that influence bacterial resistance using a model system of temperate P22 phage and Salmonella both inside and outside the mammalian host. Phages that remained functional despite gene deletions had minimal impact on lysogeny and phage resistance except for deletions in the immI region that substantially reduced lysogeny and increased phage resistance to levels comparable to that observed with an obligately lytic P22. This immI deletion does not make the lysogen less competitive but instead increases the frequency of bacterial lysis. Thus, subtle changes in the balance between lysis and lysogeny during the initial stages of infection can significantly influence the extent of phage resistance in the bacterial population. Our work highlights the complex nature of the phage-bacteria-mammalian host triad.

Characterization of prophages in bacterial genomes from the honey bee (Apis mellifera) gut microbiome.

The gut of the European honey bee (Apis mellifera) possesses a relatively simple bacterial community, but little is known about its community of prophages (temperate bacteriophages integrated into the bacterial genome). Although prophages may eventually begin replicating and kill their bacterial hosts, they can also sometimes be beneficial for their hosts by conferring protection from other phage infections or encoding genes in metabolic pathways and for toxins. In this study, we explored prophages in 17 species of core bacteria in the honey bee gut and two honey bee pathogens. Out of the 181 genomes examined, 431 putative prophage regions were predicted. Among core gut bacteria, the number of prophages per genome ranged from zero to seven and prophage composition (the compositional percentage of each bacterial genome attributable to prophages) ranged from 0 to 7%. Snodgrassella alvi and Gilliamella apicola had the highest median prophages per genome (3.0 ± 1.46; 3.0 ± 1.59), as well as the highest prophage composition (2.58% ± 1.4; 3.0% ± 1.59). The pathogen Paenibacillus larvae had a higher median number of prophages (8.0 ± 5.33) and prophage composition (6.40% ± 3.08) than the pathogen Melissococcus plutonius or any of the core bacteria. Prophage populations were highly specific to their bacterial host species, suggesting most prophages were acquired recently relative to the divergence of these bacterial groups. Furthermore, functional annotation of the predicted genes encoded within the prophage regions indicates that some prophages in the honey bee gut encode additional benefits to their bacterial hosts, such as genes in carbohydrate metabolism. Collectively, this survey suggests that prophages within the honey bee gut may contribute to the maintenance and stability of the honey bee gut microbiome and potentially modulate specific members of the bacterial community, particularly S. alvi and G. apicola.

Towards the Characterization and Engineering of Bacteriophages in the Gut Microbiome.

The gut microbiome and its importance to human health are a rapidly evolving area of study. Bacteria often take center stage. However, the composition is much more complex with other microbial members of the gut also playing key roles. Bacteriophages (phages), the viruses that infect bacteria, are an integral component of gut microbiomes and can often be found cocolonizing with their commensal bacterial hosts. Recent studies have shown associations between the composition of resident phage communities and human health and disease, but the mechanisms of these associations remain elusive. My research laboratory is focused on understanding the role of phages in the gut microbiome and exploring their possible therapeutic applications.

In situ reprogramming of gut bacteria by oral delivery.

Abundant links between the gut microbiota and human health indicate that modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration. We demonstrate that an engineered temperate phage λ expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, we develop an aqueous-based encapsulation formulation with a microbiota-based release mechanism and show that it facilitates oral delivery of phage in vivo. Finally we combine these technologies and show that bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.

Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut.

Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria. Here, we deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered λ phage to transcriptionally repress Shiga toxin by using genetic hybrids between λ and other lambdoid phages to overcome resistance encoded by the virulence-expressing prophage. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces Shiga toxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. Our work reveals a new framework for transferring functions to bacteria within their native environment.IMPORTANCE With the increasing frequency of antibiotic resistance, it is critical to explore new therapeutic strategies for treating bacterial infections. Here, we use a temperate phage, i.e., one that integrates itself into the bacterial genome, to neutralize the expression of a virulence factor by modifying bacterial function at the genetic level. We show that Shiga toxin production can be significantly reduced in vitro and in the mammalian gut. Alternative to traditional applications of phage therapy that rely on killing bacteria, our genetics-based antivirulence approach introduces a new framework for treating bacterial infections.

Engineered Interspecies Amino Acid Cross-Feeding Increases Population Evenness in a Synthetic Bacterial Consortium.

In nature, microbes interact antagonistically, neutrally, or beneficially. To shed light on the effects of positive interactions in microbial consortia, we introduced metabolic dependencies and metabolite overproduction into four bacterial species. While antagonistic interactions govern the wild-type consortium behavior, the genetic modifications alleviated antagonistic interactions and resulted in beneficial interactions. Engineered cross-feeding increased population evenness, a component of ecological diversity, in different environments, including in a more complex gnotobiotic mouse gut environment. Our findings suggest that metabolite cross-feeding could be used as a tool for intentionally shaping microbial consortia in complex environments.IMPORTANCE Microbial communities are ubiquitous in nature. Bacterial consortia live in and on our body and in our environment, and more recently, biotechnology is applying microbial consortia for bioproduction. As part of our body, bacterial consortia influence us in health and disease. Microbial consortium function is determined by its composition, which in turn is driven by the interactions between species. Further understanding of microbial interactions will help us in deciphering how consortia function in complex environments and may enable us to modify microbial consortia for health and environmental benefits.

Dynamic Modulation of the Gut Microbiota and Metabolome by Bacteriophages in a Mouse Model.

The human gut microbiome is comprised of densely colonizing microorganisms including bacteriophages, which are in dynamic interaction with each other and the mammalian host. To address how bacteriophages impact bacterial communities in the gut, we investigated the dynamic effects of phages on a model microbiome. Gnotobiotic mice were colonized with defined human gut commensal bacteria and subjected to predation by cognate lytic phages. We found that phage predation not only directly impacts susceptible bacteria but also leads to cascading effects on other bacterial species via interbacterial interactions. Metabolomic profiling revealed that shifts in the microbiome caused by phage predation have a direct consequence on the gut metabolome. Our work provides insight into the ecological importance of phages as modulators of bacterial colonization, and it additionally suggests the potential impact of gut phages on the mammalian host with implications for their therapeutic use to precisely modulate the microbiome.

Clotting Mimicry from Robust Hemostatic Bandages Based on Self-Assembling Peptides.

Uncontrolled bleeding from traumatic wounds is a major factor in deaths resulting from military conflict, accidents, disasters and crime. Self-assembling peptide nanofibers have shown superior hemostatic activity, and herein, we elucidate their mechanism by visualizing the formation of nanofiber-based clots that aggregate blood components with a similar morphology to fibrin-based clots. Furthermore, to enhance its direct application to a wound, we developed layer-by-layer assembled thin film coatings onto common materials used for wound dressings-gauze and gelatin sponges. We find these nanofibers elute upon hydration under physiological conditions and generate nanofiber-based clots with blood. After exposure to a range of harsh temperature conditions (-80 to 60 °C) for a week and even 5 months at 60 °C, these hemostatic bandages remain capable of releasing active nanofibers. In addition, the application of these nanofiber-based films from gauze bandages was found to accelerate hemostasis in porcine skin wounds as compared to plain gauze. The thermal robustness, in combination with the self-assembling peptide's potent hemostatic activity, biocompatibility, biodegradability, and low cost of production, makes this a promising approach for a cheap yet effective hemostatic bandage.

Multifunctional Self-Assembled Films for Rapid Hemostat and Sustained Anti-infective Delivery.

Uncontrolled bleeding and infection are the major causes of death and morbidity from traumatic wounds during military conflicts, disasters, and accidents. Because immediate treatment is critical to survival, it is desirable to have a lightweight and rapidly applicable bandage-one capable of delivering a hemostat that can quickly resolve bleeding while addressing infection over short and longer time frames. It is challenging to design thin film coatings capable of multidrug release, particularly when the drugs are quite different in nature (biologic versus small molecule, charged versus neutral) and the desired release profiles are different for each drug. Herein we have adopted a layer-by-layer film assembly technique to create a linear combination of two independently functional films capable of rapidly releasing thrombin within minutes while sustaining vancomycin elution for more than 24 h. By conjugating vancomycin to a hydrolytically degradable polyacid, poly(ß-L-malic acid), we were able to create a robust thin film with loading and release kinetics that remain unaffected by the additional deposition of a thrombin-based film, demonstrating the possibility for future multitherapeutic films with independently tunable release kinetics.

Multimonth controlled small molecule release from biodegradable thin films.

Long-term, localized delivery of small molecules from a biodegradable thin film is challenging owing to their low molecular weight and poor charge density. Accomplishing highly extended controlled release can facilitate high therapeutic levels in specific regions of the body while significantly reducing the toxicity to vital organs typically caused by systemic administration and decreasing the need for medical intervention because of its long-lasting release. Also important is the ability to achieve high drug loadings in thin film coatings to allow incorporation of significant drug amounts on implant surfaces. Here we report a sustained release formulation for small molecules based on a soluble charged polymer-drug conjugate that is immobilized into nanoscale, conformal, layer-by-layer assembled films applicable to a variety of substrate surfaces. We measured a highly predictable sustained drug release from a polymer thin film coating of 0.5-2.7 µm that continued for more than 14 mo with physiologically relevant drug concentrations, providing an important drug delivery advance. We demonstrated this effect with a potent small molecule nonsteroidal anti-inflammatory drug, diclofenac, because this drug can be used to address chronic pain, osteoarthritis, and a range of other critical medical issues.

Ordered and kinetically discrete sequential protein release from biodegradable thin films.

Multidrug regimens can sometimes treat recalcitrant diseases when single-drug therapies fail. Recapitulating complex multidrug administration from controlled release films for localized delivery remains challenging because their release kinetics are frequently intertwined, and an initial burst release of each drug is usually uncontrollable. Kinetic control over protein release is demonstrated by cross-linking layer-by-layer films during the assembly process. We used biodegradable and naturally derived components and relied on copper-free click chemistry for bioorthogonal covalent cross-links throughout the film that entrap but do not modify the embedded protein. We found that this strategy restricted the interdiffusion of protein while maintaining its activity. By depositing a barrier layer and a second protein-containing layer atop this construct, we generated well-defined sequential protein release with minimal overlap that follows their spatial distribution within the film.

Multilayer films assembled from naturally-derived materials for controlled protein release.

Herein we designed and characterized films composed of naturally derived materials for controlled release of proteins. Traditional drug delivery strategies rely on synthetic or semisynthetic materials or utilize potentially denaturing assembly conditions that are not optimal for sensitive biologics. Layer-by-layer (LbL) assembly of films uses benign conditions and can generate films with various release mechanisms including hydrolysis-facilitated degradation. These use components such as synthetic polycations that degrade into non-natural products. Herein we report the use of a naturally derived, biocompatible and degradable polyanion, poly(ß-l-malic acid), alone and in combination with chitosan in an LbL film, whose degradation products of malic acid and chitosan are both generally recognized as safe (GRAS) by the FDA. We have found that films based on this polyanion have shown sustained release of a model protein, lysozyme that can be timed from tens of minutes to multiple days through different film architectures. We also report the incorporation and release of a clinically used biologic, basic fibroblast growth factor (bFGF), which demonstrates the use of this strategy as a platform for controlled release of various biologics.

Hydrophobic polycationic coatings that inhibit biofilms and support bone healing during infection.

Adhesion of microorganisms to biomaterials with subsequent formation of biofilms on such foreign bodies as orthopedic trauma hardware is a critical factor in implant-associated infections; once a biofilm has been established, its microorganisms become recalcitrant to the host's immune surveillance and markedly resistant to drugs. We have previously reported that painting with the hydrophobic polycation N,N-dodecyl,methyl-PEI (PEI = polyethylenimine) renders solid surfaces bactericidal in vitro. Herein we observe that N,N-dodecyl,methyl-PEI-derivatized titanium and stainless steel surfaces resist biofilm formation by Staphylococcus aureus compared to the untreated ones. Using imaging, microbiology-, histopathology-, and scanning electron microscopy (SEM) experiments in a clinically relevant large-animal (sheep) trauma model, we subsequently demonstrate in vivo that orthopedic fracture hardware painted with N,N-dodecyl,methyl-PEI not only prevents implant colonization with biofilm but also promotes bone healing. Functionalizing orthopedic hardware with hydrophobic polycations thus holds promise in supporting bone healing in the presence of infection in veterinary and human orthopedic patients.

An antimicrobial polycationic sand filter for water disinfection.

A new sand filtration water disinfection technology is developed which relies on the antimicrobial properties of hydrophobic polycations (N-hexylated polyethylenimine) covalently attached to the sand's surface. The efficacy of the filter disinfection process was evaluated both with water spiked with E. coli and with real aqueous effluent from a wastewater treatment plant. For the former, over 7-log reduction in bacterial count was achieved. With real environmental wastewater secondary effluent samples, the E. coli concentration reduction declined to under 2 logs. This reduced inactivation efficiency compared to the model aqueous sample is likely due to the particulate or colloidal matter present that diminishes the contact between the immobilized polycation and the suspended bacteria. Preliminary sand washing methods were tested to assess potential 'regeneration' approaches. Potential advantages of the proposed approach over conventional disinfection in terms of eliminating harmful by-products and reducing energy consumption are discussed.

On structural damage incurred by bacteria upon exposure to hydrophobic polycationic coatings.

Hydrophobic polycations previously developed by us efficiently kill E. coli and Staphylococcus aureus on contact. As visualized by electron microscopy herein, these pathogenic bacteria incur marked morphological damage from the exposure to these N-alkylated-polyethylenimine "paints" which results in the leakage of an appreciable fraction of the total cellular protein. The quantity and composition of that leaked protein is similar to that released upon traditional lysozyme/EDTA treatment, thus providing insights into the mechanism of action of our microbicidal coatings.

Hydrophobic polycationic coatings disinfect poliovirus and rotavirus solutions.

Coating surfaces with N-alkylated polyethylenimines (PEIs), namely branched N,N-hexyl,methyl-PEI via covalent attachment to glass or linear N,N-dodecyl,methyl-PEI by physical deposition ("painting") onto polyethylene, enables the resultant materials to quickly and efficiently disinfect aqueous solutions of (non-enveloped) poliovirus and rotavirus.

Mechanism of inactivation of influenza viruses by immobilized hydrophobic polycations.

N,N-dodecyl,methyl-polyethylenimine coatings applied to solid surfaces have been shown by us to disinfect aqueous solutions of influenza viruses. Herein we elucidate the mechanism of this phenomenon. Infectivity-, protein-, RNA-, and scanning electron microscopy-based experiments reveal that, upon contact with the hydrophobic polycationic coating, influenza viruses (including pathogenic human and avian, both wild-type and drug-resistant, strains) irreversibly adhere to it, followed by structural damage and inactivation; subsequently, viral RNA is released into solution, while proteins remain adsorbed.

Light-activated covalent coating of cotton with bactericidal hydrophobic polycations.

A methodology is developed and validated whereby a cotton fabric is impregnated with a photosensitive hydrophobic N-alkyl-polyethylenimine, followed by its covalent immobilization triggered by ultraviolet light. The resultant fabric efficiently kills on contact waterborne pathogenic bacteria E. coli and S. aureus.