Bacteriophage-Based Bioanalysis.

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.

Monomeric streptavidin phage display allows efficient immobilization of bacteriophages on magnetic particles for the capture, separation, and detection of bacteria.

Immobilization of bacteriophages onto solid supports such as magnetic particles has demonstrated ultralow detection limits as biosensors for the separation and detection of their host bacteria. While the potential impact of magnetized phages is high, the current methods of immobilization are either weak, costly, inefficient, or laborious making them less viable for commercialization. In order to bridge this gap, we have developed a highly efficient, site-specific, and low-cost method to immobilize bacteriophages onto solid supports. While streptavidin-biotin represents an ideal conjugation method, the functionalization of magnetic particles with streptavidin requires square meters of coverage and therefore is not amenable to a low-cost assay. Here, we genetically engineered bacteriophages to allow synthesis of a monomeric streptavidin during infection of the bacterial host. The monomeric streptavidin was fused to a capsid protein (Hoc) to allow site-specific self-assembly of up to 155 fusion proteins per capsid. Biotin coated magnetic nanoparticles were functionalized with mSA-Hoc T4 phage demonstrated in an E. coli detection assay with a limit of detection of < 10 CFU in 100 mLs of water. This work highlights the creation of genetically modified bacteriophages with a novel capsid modification, expanding the potential for bacteriophage functionalized biotechnologies.

A phage-based magnetic relaxation switching biosensor using bioorthogonal reaction signal amplification for Salmonella detection in foods.

Phages are uniquely suited for bacterial detection due to their low cost and ability to recognize live bacteria. Herein, our work establishes the proof-of-concept detection of Salmonella in orange juice based on a phage-mediated portable magnetic relaxation switching (MRS) biosensor. The limit of quantification (LOQ) could reach 5 CFU/mL (95 % confidence interval [CI]: 4-7, N = 4) with a linear range of 102-108 CFU/mL, which has improved 10-fold than that without bioorthogonal signal amplification. The recovery rate of the phage-based MRS biosensor was 95.0 % (95 % confidence interval [CI]: 89.0 %-100.9 %, N = 6). The specificity of the phage-based MRS biosensor was 100 % without false-positive results. In addition, this sensor was able to detect <10 CFU per 25 mL of Salmonella in orange juice with 4-h pre-enrichment. The result from the phage-based MRS biosensor is consistent with that from the standard plate count method. This sensor provides a reliable and ultrasensitive detection platform for pathogens.

Enterobacteria Phage Ac3's Genome Annotation and Host Range Analysis Against the ECOR Reference Library.

Host range analyses and genome sequencing/annotation of bacteriophage isolates allow more effective development of tools for applications in medicine, agriculture, and the environment and expand our understanding of phage biology. Here we present the complete sequence of phage Ac3's assembled and annotated genome (accession OK040907). Originally referred to simply as "3," Ac3 has previously been described as a T4-like bacteriophage belonging to the Myoviridae family in the Caudovirales order of tailed bacteriophages. Using a combination of spot tests and full plate plaque assays, Ac3's permissive and adsorptive host range were evaluated against the ECOR Reference Library; a panel of 72 Escherichia coli isolates meant to represent the diversity of E. coli. Spot assays revealed that Ac3 could adsorb to 43 of the 72 strains (59.7%), whereas plaque assays demonstrated Ac3's ability to complete replication within 27 of the 72 strains (37.5%). By overlaying spot test and plaque assay results, 16 of the 45 nonpermissive ECOR strains (35.5%) were highlighted as being able to support Ac3's adsorption and tail contraction, but not its replication. Further characterization of Ac3 is still needed, however, the study presented here provides a solid starting point for future research.

In Vivo Capsid Engineering of Bacteriophages for Oriented Surface Conjugation.

The current state-of-the-art in bacteriophage (phage) immobilization onto magnetic particles is limited to techniques that are less expensive and/or facile but nonspecific or those that are more expensive and/or complicated but ensure capsid-down orientation of the phages, as necessary to preserve infectivity and performance in subsequent applications (e.g., therapeutics, detection). These cost, complexity, and effectiveness limitations constitute the major hurdles that limit the scale-up of phage-based strategies and thus their accessibility in low-resource settings. Here, we report a plasmid-based technique that incorporates a silica-binding protein, L2, into the T7 phage capsid, during viral assembly, with and without inclusion of a flexible linker peptide, allowing for targeted binding of the phage capsid to silica without requiring the direct modification of the phage genome. L2-tagged phages were then immobilized onto silica-coated magnetic nanoparticles. Inclusion of the flexible linker between the phage capsid protein and the L2 protein improved immobilization density compared to both wild type T7 phages and L2-tagged phages without the flexible linker. Taken together, this work demonstrates phage capsid modification without engineering the phage genome, which provides an important step toward reducing the cost and increasing the specificity/directionality of phage immobilization methods and could be more broadly applied in the future for other phages for a range of other capsid tags and nanomaterials.

A microfluidic device and instrument prototypes for the detection of Escherichia coli in water samples using a phage-based bioluminescence assay.

Current quantification methods of Escherichia coli (E. coli) contamination in water samples involve long incubation, laboratory equipment and facilities, or complex processes that require specialized training for accurate operation and interpretation. To address these limitations, we have developed a microfluidic device and portable instrument prototypes capable of performing a rapid and highly sensitive bacteriophage-based assay to detect E. coli cells with detection limit comparable to traditional methods in a fraction of the time. The microfluidic device combines membrane filtration and selective enrichment using T7-NanoLuc-CBM, a genetically engineered bacteriophage, to identify 4.1 E. coli CFU in 100 mL of drinking water within 5.5 hours. The microfluidic device was designed and tested to process up to 100 mL of real-world drinking water samples with turbidities below 10 NTU. Prototypes of custom instrumentation, compatible with our valveless microfluidic device and capable of performing all of the assay's units of operation with minimal user intervention, demonstrated similar assay performance to that obtained on the benchtop assay. This research is the first step towards a faster, portable, and semi-automated, phage-based microfluidic platform for improved in-field water quality monitoring in low-resource settings.

Enterobacteria Phage SV76 Host Range and Genomic Characterization.

Background: Increasing the quantity and detail of bacteriophage genomic data is critical to broadening our understanding of how bacteriophages operate to allow us to harness their unique properties for biotechnology advancements. Here we present the complete sequence of phage SV76's assembled and annotated genome (Accession OM339528). SV76 has previously been classified as a T4-like bacteriophage belonging to the Tequatrovirus genus within the Myoviridae family of contractile tailed bacteriophages. Materials and Methods: Whole genome sequencing, assembly, and annotation was performed on SV76. Double-agar spot assays were utilized to determine SV76's host range against a panel of 72 Escherichia coli isolates meant to represent the diversity of E. coli, as well as a series of knockouts designed to identify required receptor binding proteins. The genome and host range were compared to the closely related phage, T2. Results: Spot assays revealed that SV76 could plaque on 10 of the 72 strains (13.9 %) and nine of the nine E. coli K12 single gene knockout of known phage receptors (100%). SV76 did not plate on a ΔfadL E. coli indicating suggesting a requirement as a receptor binding protein. Conclusions: SV76 is closely related to T2 with similar host ranges within ECOR. This study presents novel host range and genomic data on SV76 phage, providing a foundation for future studies to further characterize SV76 to understand more about SV76 and other T4-like phages that can be applied to create novel biotechnologies.

An Engineered Reporter Phage for the Fluorometric Detection of Escherichia coli in Ground Beef.

Despite enhanced sanitation implementations, foodborne bacterial pathogens still remain a major threat to public health and generate high costs for the food industry. Reporter bacteriophage (phage) systems have been regarded as a powerful technology for diagnostic assays for their extraordinary specificity to target cells and cost-effectiveness. Our study introduced an enzyme-based fluorescent assay for detecting the presence of E. coli using the T7 phage engineered with the lacZ operon which encodes beta-galactosidase (ß-gal). Both endogenous and overexpressed ß-gal expression was monitored using a fluorescent-based method with 4-methylumbelliferyl ß-d-galactopyranoside (MUG) as the substrate. The infection of E. coli with engineered phages resulted in a detection limit of 10 CFU/mL in ground beef juice after 7 h of incubation. In this study, we demonstrated that the overexpression of ß-gal coupled with a fluorogenic substrate can provide a straightforward and sensitive approach to detect the potential biological contamination in food samples. The results also suggested that this system can be applied to detect E. coli strains isolated from environmental samples, indicating a broader range of bacterial detection.

Bacteriophage Capsid Modification by Genetic and Chemical Methods.

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g., fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation, but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

FEAST of biosensors: Food, environmental and agricultural sensing technologies (FEAST) in North America.

We review the challenges and opportunities for biosensor research in North America aimed to accelerate translational research. We call for platform approaches based on: i) tools that can support interoperability between food, environment and agriculture, ii) open-source tools for analytics, iii) algorithms used for data and information arbitrage, and iv) use-inspired sensor design. We summarize select mobile devices and phone-based biosensors that couple analytical systems with biosensors for improving decision support. Over 100 biosensors developed by labs in North America were analyzed, including lab-based and portable devices. The results of this literature review show that nearly one quarter of the manuscripts focused on fundamental platform development or material characterization. Among the biosensors analyzed for food (post-harvest) or environmental applications, most devices were based on optical transduction (whether a lab assay or portable device). Most biosensors for agricultural applications were based on electrochemical transduction and few utilized a mobile platform. Presently, the FEAST of biosensors has produced a wealth of opportunity but faces a famine of actionable information without a platform for analytics.

Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay.

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage's ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage's "adsorptive host range." The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage's adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein's targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage's adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4's long tail fiber binding tip (gp37) to evaluate and quantify gp37's relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay's ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

Optimization of T4 phage engineering via CRISPR/Cas9.

A major limitation hindering the widespread use of synthetic phages in medical and industrial settings is the lack of an efficient phage-engineering platform. Classical T4 phage engineering and several newly proposed methods are often inefficient and time consuming and consequently, only able to produce an inconsistent range of genomic editing rates between 0.03-3%. Here, we review and present new understandings of the CRISPR/Cas9 assisted genome engineering technique that significantly improves the genomic editing rate of T4 phages. Our results indicate that crRNAs selection is a major rate limiting factor in T4 phage engineering via CRISPR/Cas9. We were able to achieve an editing rate of > 99% for multiple genes that functionalizes the phages for further applications. We envision that this improved phage-engineering platform will accelerate the fields of individualized phage therapy, biocontrol, and rapid diagnostics.

Phage-based biosensors: in vivo analysis of native T4 phage promoters to enhance reporter enzyme expression.

Phage-based biosensors have shown significant promise in meeting the present needs of the food and agricultural industries due to a combination of sufficient portability, speed, ease of use, sensitivity, and low production cost. Although current phage-based methods do not meet the bacteria detection limit imposed by the EPA, FDA, and USDA, a better understanding of phage genetics can significantly increase their sensitivity as biosensors. In the current study, the signal sensitivity of a T4 phage-based detection system was improved via transcriptional upregulation of the reporter enzyme Nanoluc luciferase (Nluc). An efficient platform to evaluate the promoter activity of reporter T4 phages was developed. The ability to upregulate Nluc within T4 phages was evaluated using 15 native T4 promoters. Data indicates a six-fold increase in reporter enzyme signal from integration of the selected promoters. Collectively, this work demonstrates that fine tuning the expression of reporter enzymes such as Nluc through optimization of transcription can significantly reduce the limits of detection.

A Syringe-Based Biosensor to Rapidly Detect Low Levels of Escherichia Coli (ECOR13) in Drinking Water Using Engineered Bacteriophages.

A sanitized drinking water supply is an unconditional requirement for public health and the overall prosperity of humanity. Potential microbial and chemical contaminants of drinking water have been identified by a joint effort between the World Health Organization (WHO) and the United Nations Children's Fund (UNICEF), who together establish guidelines that define, in part, that the presence of Escherichia coli (E. coli) in drinking water is an indication of inadequate sanitation and a significant health risk. As E. coli is a nearly ubiquitous resident of mammalian gastrointestinal tracts, no detectable counts in 100 mL of drinking water is the standard used worldwide as an indicator of sanitation. The currently accepted EPA method relies on filtration, followed by growth on selective media, and requires 24-48 h from sample to results. In response, we developed a rapid bacteriophage-based detection assay with detection limit capabilities comparable to traditional methods in less than a quarter of the time. We coupled membrane filtration with selective enrichment using genetically engineered bacteriophages to identify less than 20 colony forming units (CFU) E. coli in 100 mL drinking water within 5 h. The combination of membrane filtration with phage infection produced a novel assay that demonstrated a rapid, selective, and sensitive detection of an indicator organism in large volumes of drinking water as recommended by the leading world regulatory authorities.

Engineering Biorthogonal Phage-Based Nanobots for Ultrasensitive, In Situ Bacteria Detection.

Advances in synthetic biology, nanotechnology, and genetic engineering are allowing parallel advances in areas such as drug delivery and rapid diagnostics. Although our current visions of nanobots may be far off, a generation of nanobots synthesized by engineering viruses is approaching. Such tools can be used to solve complex problems where current methods do not meet current demands. Assuring safe drinking water is crucial for minimizing the spread of waterborne illnesses. Although extremely low levels of fecal contamination in drinking water are sufficient to cause a public health risk, it remains challenging to rapidly detect Escherichia coli, the standard fecal indicator organism. Current methods sensitive enough to meet regulatory standards suffer from either prohibitively long incubation times or requirement of expensive, impractical equipment. Bacteriophages, tuned by billions of years of evolution to bind viable bacteria and readily engineered to produce custom proteins, are uniquely suited to bacterial detection. We have developed a biosensor platform based on magnetized phages encoding luminescent reporter enzymes. This system utilizes bio-orthogonally functionalized phages to enable site-specific conjugation to magnetic nanoparticles. The resulting phage-based nanobots, when combined with standard, portable field equipment, allow for detection of <10 cfu/100 mL of viable E. coli within 7 h, faster than any methods published to date.

Colorimetric detection of Escherichia coli using engineered bacteriophage and an affinity reporter system.

Reporter phage systems have emerged as a promising technology for the detection of bacteria in foods and water. However, the sensitivity of these assays is often limited by the concentration of the expressed reporter as well as matrix interferences associated with the sample. In this study, bacteriophage T7 was engineered to overexpress mutated alkaline phosphatase fused to a carbohydrate-binding module (ALP*-CBM) following infection of E. coli to enable colorimetric detection in a model system. Magnetic cellulose particles were employed to separate and concentrate the overexpressed ALP*-CBM in bacterial lysate. Infection of E. coli with the engineered phage resulted in a limit of quantitation of 1.2 × 105 CFU, equating to 1.2 × 103 CFU/mL in 3.5 h when using a colorimetric assay and 100 mL sample volume. When employing an enrichment step, < 101 CFU/mL could be visually detected from a 100 mL sample volume within 8 h. These results suggest that affinity tag modified enzymes coupled with a material support can provide a simple and effective means to improve signal sensitivity of phage-based assays. Graphical abstract.

Detection of protease and engineered phage-infected bacteria using peptide-graphene oxide nanosensors.

A peptide-graphene oxide nanosensor has been developed to detect tobacco etch virus (TEV) protease and bacteria infected with an engineered bacteriophage. In the detection strategy, a peptide (sequence: RKRFRENLYFQSCP) is tagged with fluorophores and graphene oxide (GO) is used to adsorb the peptides while quenching their fluorescence. In the presence of TEV protease, fluoropeptides are cleaved between glutamine (Q) and serine (S), resulting in the recovery of fluorescence signal. Based on the fluorescent intensity, the detection limit of TEV protease is 51 ng/µL. Additionally, we have utilized the sensing system to detect bacteria cells. Bacteriophages, which were engineered to carry TEV protease genes, were used to infect target bacteria (Escherichia coli) resulting in the translation and release of the protease. This allowed the estimation of bacteria at the concentration of 104 CFU/mL. This strategy has the potential to be developed as a multiplex detection platform of multiple bacterial species. Graphical abstract.

Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection.

Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages. We present here a synthetic biology strategy to engineer phages using a simple in vitro method. We used this method to insert a NanoLuc luciferase expression cassette into the T7 phage, in order to construct the NRGp6 reporter phage. The synthetic NRGp6 phage was used to efficiently detect low concentrations of Escherichia coli from liquid culture. We envision that our approach will benefit synthetic biologists for constructing different kinds of engineered phages, and enable new approaches for phage-based therapeutics and diagnostics.

Phage based electrochemical detection of Escherichia coli in drinking water using affinity reporter probes.

The monitoring of drinking water for indicators of fecal contamination is crucial for ensuring a safe supply. In this study, a novel electrochemical method was developed for the rapid and sensitive detection of Escherichia coli (E. coli) in drinking water. This strategy is based on the use of engineered bacteriophages (phages) to separate and concentrate target E. coli when conjugated with magnetic beads, and to facilitate the detection by expressing gold binding peptides fused alkaline phosphatase (GBPs-ALP). The fusion protein GBPs-ALP has both the enzymatic activity and the ability to directly bind onto a gold surface. This binding-peptide mediated immobilization method provided a novel and simple approach to immobilize proteins on a solid surface, requiring no post-translational modifications. The concentration of E. coli was determined by measuring the activity of the ALP on gold electrodes electrochemically using linear sweep voltammetry (LSV). This approach was successfully applied in the detection of E. coli in drinking water. We were able to detect 105 CFU mL-1 of E. coli within 4 hours. After 9 hours of preincubation, 1 CFU of E. coli in 100 mL of drinking water was detected with a total assay time of 12 hours. This approach compares favorably to the current EPA method and has the potential to be applied to detect different bacteria in other food matrices.

A phage-based assay for the rapid, quantitative, and single CFU visualization of E. coli (ECOR #13) in drinking water.

Drinking water standards in the United States mandate a zero tolerance of generic E. coli in 100 mL of water. The presence of E. coli in drinking water indicates that favorable environmental conditions exist that could have resulted in pathogen contamination. Therefore, the rapid and specific enumeration of E. coli in contaminated drinking water is critical to mitigate significant risks to public health. To meet this challenge, we developed a bacteriophage-based membrane filtration assay that employs novel fusion reporter enzymes to fully quantify E. coli in less than half the time required for traditional enrichment assays. A luciferase and an alkaline phosphatase, both specifically engineered for increased enzymatic activity, were selected as reporter probes due to their strong signal, small size, and low background. The genes for the reporter enzymes were fused to genes for carbohydrate binding modules specific to cellulose. These constructs were then inserted into the E. coli-specific phage T7 which were used to infect E. coli trapped on a cellulose filter. During the infection, the reporters were expressed and released from the bacterial cells following the lytic infection cycle. The binding modules facilitated the immobilization of the reporter probes on the cellulose filter in proximity to the lysed cells. Following substrate addition, the location and quantification of E. coli cells could then be determined visually or using bioluminescence imaging for the alkaline phosphatase and luciferase reporters, respectively. As a result, a detection assay capable of quantitatively detecting E. coli in drinking water with similar results to established methods, but less than half the assay time was developed.