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(a) Schematic of in-vivo phage recombination using the CRISPR-Cas9 system. Host bacteria cells are transformed with a donor plasmid containing the reporter NanoLuc-CBM flanked with left and right homology regions to the phage (LHR and RHR) as well as selected crRNA and spacer sequences. (b) Phage plaque assay shows the presence of plaques in the bacterial lawn (top photo) and NanoLuc activity in the plaques when imaged in a dark room (bottom photo). Created with BioRender.com.
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Inadequate drinking water quality is among the major causes of preventable mortality, predominantly in young children. Identifying contaminated water sources remains a significant challenge, especially where resources are limited. The current methods for measuring Escherichia coli ( E. coli ), the WHO preferred indicator for measuring fecal contami...
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... Then, the infected bacteria release some phage particles, called plaques forming units, which are a reliable indicator for cell viability. These techniques, however, rely on virucide treatment, and false positive plaques may be acquired if this step is not entirely successful (Alonzo et al., 2022). To resolve this problem, a subsequent step is carried out to verify the DNA, such as PCR. ...
... Figures 2A, B illustrates vital biopanning steps: rinsing to remove unbound virions, selectively collecting interacting virions, and propagating phages with strong ligand affinities via E. coli infection. The CRISPR/Cas system can be applied to engineer bacteriophages, enabling targeted alterations and creating novel phage variants ( Figure 3) (47,65). Utilizing the CRISPR-Cas system for genetic manipulation of filamentous or other bacteriophages has enabled the isolation of desired phage mutants, showcasing the significant utility of CRISPR-Cas in the genetic engineering of bacteriophages (47,66). ...
... Host bacterial cells undergo transformation with a donor plasmid harboring the NanoLuc reporter gene flanked by left and right homology regions to the phage, alongside specifically chosen CRISPR RNA and genome-targeting sequences. Adapted from(65). ...
Stroke poses a critical global health challenge, leading to substantial morbidity and mortality. Existing treatments often miss vital timeframes and encounter limitations due to adverse effects, prompting the pursuit of innovative approaches to restore compromised brain function. This review explores the potential of filamentous phages in enhancing stroke recovery. Initially antimicrobial-centric, bacteriophage therapy has evolved into a regenerative solution. We explore the diverse role of filamentous phages in post-stroke neurological restoration, emphasizing their ability to integrate peptides into phage coat proteins, thereby facilitating recovery. Experimental evidence supports their efficacy in alleviating post-stroke complications, immune modulation, and tissue regeneration. However, rigorous clinical validation is essential to address challenges like dosing and administration routes. Additionally, genetic modification enhances their potential as injectable biomaterials for complex brain tissue issues. This review emphasizes innovative strategies and the capacity of filamentous phages to contribute to enhanced stroke recovery, as opposed to serving as standalone treatment, particularly in addressing stroke-induced brain tissue damage.
... 11 The most established testing methods involve cultivating samples until the E. coli grows sufficiently to produce a readily measurable signal; either through the evolution of gas in the multipletube fermentation technique, 12,13 or through a colour change brought about by the use of chromogenic nutrient media. 14,15 Novel innovations in E. coli testing have seen the development of biosensors, 16,17 E. coli specific bacteriophages, 18 DNA amplification methods 19,20 , and flow cytometry 21 amongst others (see reviews by Nurliyana 2018 22 or Tambi 2023 23 ). However, existing implementations of these testing methods are not ideal for low-resource settings 24 where the majority of institutions do not meet their water testing targets 25 . ...
Increasing access to water quality tests in low-income communities is a crucial strategy toward achieving global water equality. Recent studies in the Water Sanitation and Hygiene (WASH) sector underscore the importance of addressing practical concerns in water testing, such as robustness and results communication. In response, we present the WaterScope testing kit; an open-source, novel platform for drinking water quality assessment. It modernises the testing process with the inclusion of a unique cartridge/slider mechanism, machine-learning-enhanced classification and full digitalisation of results. WaterScope’s equivalency to conventional methods for quantifying E. coli is established through extensive validation experiments in both laboratory and field environments. This versatile platform provides potential to expand its applications to test other bacteria, perform colorimetric assays, and analyse clinical samples such as blood/urine samples. We anticipate that the system’s ease-of-use, portability, affordability, robustness, and digital nature will accelerate progress toward global water equality.
... These biosensors convert biological interactions into measurable electrical or optical signals, leading to the rapid detection of coliforms at a very low cost (Gunda et al. 2017). Phage-based assays consist of engineered phages carrying reporter genes that can infect coliforms and produce detectable bioluminescence signals (Hinkley et al. 2018;Alonzo et al. 2022). These assays offer high specificity and allow for the discrimination of viable coliform cells from non-viable ones (Hussain et al. 2021). ...
Ensuring the microbiological safety of drinking water is of paramount importance to protect public health. Coliform bacteria, including Escherichia coli, serve as key indicators of water contamination and the potential presence of harmful pathogens. Accurate and reliable detection and enumeration of coliforms in drinking water are essential for monitoring water quality and implementing appropriate interventions. This review article provides an overview of various traditional culture-based methods and rapid molecular methods employed for the analysis of coliforms in drinking water, highlighting their strengths, limitations, and advancements. Culture-based methods such as multiple-tube fermentation (most probable number) and membrane filtration techniques have long been used as standard methods for coliform detection. The emerging molecular-based approaches, including polymerase chain reaction (PCR), quantitative PCR, and nucleic acid sequencing offer improved sensitivity, specificity, and turnaround time. This comprehensive review provides a valuable resource for researchers, water quality professionals, and policymakers engaged in the detection and enumeration of coliform bacteria in drinking water. It offers an up-to-date understanding of different methods, their advancements, and the potential integration of novel technologies. By critically evaluating these approaches, this review aims to contribute to the ongoing efforts toward ensuring safe drinking water for all.
... Similarly, the phage-based assay based on 8 Nanoluc luciferase reporter phages engineered through HR was used to quantify the E. coli in the test for local sewage and drinking water. The results showed E. coli contamination in less than 10 MPN/100 mL, and the detection time was 5.5 hours (Alonzo et al., 2022). In addition, a novel phage-based MRSA diagnostic screen was developed through HR for nasal swab samples by using the Lux reporter genes capable of recognizing S. aureus. ...
Bacteriophages are the most abundant biological entity in the world and hold a tremendous amount of unexplored genetic information. Since their discovery, phages have drawn a great deal of attention from researchers despite their small size. The development of advanced strategies to modify their genomes and produce engineered phages with desired traits has opened new avenues for their applications. This review presents advanced strategies for developing engineered phages and their potential antibacterial applications in phage therapy, disruption of biofilm, delivery of antimicrobials, use of endolysin as an antibacterial agent, and altering the phage host range. Similarly, engineered phages find applications in eukaryotes as a shuttle for delivering genes and drugs to the targeted cells, and are used in the development of vaccines and facilitating tissue engineering. The use of phage display-based specific peptides for vaccine development, diagnostic tools, and targeted drug delivery is also discussed in this review. The engineered phage-mediated industrial food processing and biocontrol, advanced wastewater treatment, phage-based nano-medicines, and their use as a bio-recognition element for the detection of bacterial pathogens are also part of this review. The genetic engineering approaches hold great potential to accelerate translational phages and research. Overall, this review provides a deep understanding of the ingenious knowledge of phage engineering to move them beyond their innate ability for potential applications.
... This intensity and size, combined with commercial availability, has renewed interest in luciferase reporter phages by offering unprecedented diagnostic sensitivity. NanoLuc ® -encoding phages have now been successfully engineered to detect a variety of foodborne pathogens including E. coli, Cronobacter, Salmonella, and Listeria [6][7][8][9][10][11][12]. The continued development of this promising technology is of both scientific and commercial interest. ...
Host range is a major determinant in the industrial utility of a bacteriophage. A model host range permits broad recognition across serovars of a target bacterium while avoiding cross-reactivity with commensal microbiota. Searching for a naturally occurring bacteriophage with ideal host ranges is challenging, time-consuming, and restrictive. To address this, SPTD1.NL, a previously published luciferase reporter bacteriophage for Salmonella, was used to investigate manipulation of host range through receptor-binding protein engineering. Similar to related members of the Ackermannviridae bacteriophage family, SPTD1.NL possessed a receptor-binding protein gene cluster encoding four tailspike proteins, TSP1-4. Investigation of the native gene cluster through chimeric proteins identified TSP3 as the tailspike protein responsible for Salmonella detection. Further analysis of chimeric phages revealed that TSP2 contributed off-target Citrobacter recognition, whereas TSP1 and TSP4 were not essential for activity against any known host. To improve the host range of SPTD1.NL, TSP1 and TSP2 were sequentially replaced with chimeric receptor-binding proteins targeting Salmonella. This engineered construct, called RBP-SPTD1-3, was a superior diagnostic reporter, sensitively detecting additional Salmonella serovars while also demonstrating improved specificity. For industrial applications, bacteriophages of the Ackermannviridae family are thus uniquely versatile and may be engineered with multiple chimeric receptor-binding proteins to achieve a custom-tailored host range.
... Advantages of this system are that light production is completely dependent on phage infection of sensitive cells, and can definitely be measured with sensitivity. E. coli O157, Listeria, Salmonella, and Mycobacterium have all been identified using phage-luciferase fusions [80,81]. ...
The world is on the cusp of a post-antibiotic period. A century ago, before the advent of antibiotics, bacteriophage therapy was the treatment of choice for bacterial infections. Although bacteriophages have yet to be approved as a treatment in Western medicine, researchers and clinicians have begun to anticipate phage therapy. Bacteriophages are viruses that depend on bacterial cell metabolism to multiply. They offer a promising alternative to the use of antibiotics, and an excellent antibacterial option for combating multidrug resistance in bacteria. However, not every phage is suitable for phage therapy. In particular, prophages should not be used because they can lysogenize host cells instead of lysing them. To offer adequate therapeutic options for patients suffering from various infectious diseases, a wide selection of different phages is needed. While there is no evidence of direct toxicity induced by phage particles, it is crucial to study mammalian cell–phage interactions. This requires phage preparations to be free of bacterial cells, toxins and other compounds to avoid skewing host responses. Negative staining of purified viruses and electron microscopy remain the gold standard in the identification of bacteriophages. Interestingly, genomics has greatly changed our understanding of phage biology. Bacteriophage genome sequencing is essential to obtain a more complete understanding of their biology, and to obtain confirmation of their lifestyle. Full genetic sequencing of bacteriophage will enable a better understanding of the phage encoded proteins and biomolecules (especially phage lytic enzymes) involved in the process of bacterial cell lysis and death. Mass spectrometry can be used for the identification of phage structural proteins. The use of lytic phages as biocontrol agents requires the most appropriate and standard methods to ensure application safety. This review pursues recent research and methods in molecular biology for the isolation and characterization of phages to facilitate follow-up and implementation of work for other researchers. Patents related to this topic have been mentioned along the text.
Bacteriophages infect and replicate within bacteria and play a key role in the environment, particularly in microbial ecosystems and bacterial population dynamics. The increasing recognition of their significance stems from their wide array of environmental and biotechnological uses, which encompass the mounting issue of antimicrobial resistance (AMR). Beyond their therapeutic potential in combating antibiotic-resistant infections, bacteriophages also find vast applications such as water quality monitoring, bioremediation, and nutrient cycling within environmental sciences. Researchers are actively involved in isolating and characterizing bacteriophages from different natural sources to explore their applications. Gaining insights into key aspects such as the life cycle of bacteriophages, their host range, immune interactions, and physical stability is vital to enhance their application potential. The establishment of diverse phage libraries has become indispensable to facilitate their wide-ranging uses. Consequently, numerous protocols, ranging from traditional to cutting-edge techniques, have been developed for the isolation, detection, purification, and characterization of bacteriophages from diverse environmental sources. This review offers an exploration of tools, delves into the methods of isolation, characterization, and the extensive environmental applications of bacteriophages, particularly in areas like water quality assessment, the food sector, therapeutic interventions, and the phage therapy in various infections and diseases.
