Capsid and Tail
A weekly phage periodical
April 25, 2019
Issue 25: Phages as diagnostic tools

Phages as diagnostic tools

Beyond therapeutics, phage-based diagnostics show the versatility of phages in biotech. This week, guest writer Cathy Nguyen takes us through three examples of phage-based diagnostics.

What’s New

Andrei Bolocan and colleagues from APC Microbiome, the University College Cork and the Teagasc Food Research Centre in Ireland have published a new review evaluating the prospects of E. faecalis phage therapy. They’ve categorized phages with respect to suitability for therapy by looking at in vitro and in vivo potency, characterizing their genomes (including checks for antibiotic resistance genes) and looking at endolysin architecture.

ReviewPhage Therapy

Phigaro is a scalable command-line tool by Elizaveta Starikova and colleagues for phage and prophage prediction from nucleid acid sequences (including metagenomes). It can also produce annotated prophage genome maps and distinguish tailed from non-tailed phages!
Preprint | Github.

Bioinformatics ToolProphages

The same group that brought us the fascinating study on filamentous phage Pf’s influence on P. aeruginosa virulence has published another study on the same phage. This time, they describe Pf prevalence (high) and implications (increased antibiotic resistance, reduced lung function) in CF lungs.

Cystic FibrosisResearchPhage Biology

CBS 60 Minutes covered superbugs this week. Buying colistin off the shelf, anyone?

Antibiotic ResistanceNews

A new paper (published today!) by Ann Gregory and colleagues shows identification of almost 200,000 new viral populations in the ocean (this work comes in part from the Tara Oceans expedition). The work identifies interesting new biodiversity hotspots in the ocean (including in the Arctic!) and virome diversity patterns that don’t always follow diversity patterns of larger organisms.

Marine ViromeResearchTara Oceans

Latest Jobs

Research Aide: Bacteriophage Discovery
Texas Tech University, Lubbock, TX

The successful candidate will be involved in laboratory activities mainly on the detection, isolation, and characterization of bacteriophages from environmental samples, as well as identification of Shiga Toxin-producing E. coli (STEC).

Research Assistant Phage Discovery
Postdoctoral position: Staphylococcal phage biology
University of Copenhagen, Frederiksberg, Denmark

We are looking for a highly motivated and dynamic researcher for a 2 year position, to commence August 1, 2019. The position is related to role of bacteriophages in the transfer of antibiotic resistance genes in Staphylococcus and the extent to which such transfer takes place in biofilms. We have a multinational research environment with strong national and international collaborators.

Post Doc Phage BiologyAntimicrobial resistanceBiofilms
Postdoctoral positon: Biocontrol of Clostridium perfringens in poultry
University of Lincoln, Lincoln, GB

A Research Fellow is required within the School of Life Sciences to support a new project in collaboration with partners in China entitled ‘Novel biocontrol to combat Clostridium perfringens in poultry flocks’. Ideally you will have a strong background in microbiology/molecular biology and some knowledge of bacteriophage technology.

Post Doc AgricultureBiocontrol

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Phages as diagnostic tools

Cathy Nguyen, MS
LinkedIn
ResearchGate

Phage-based diagnostics have been implemented in various areas from biodefense to clinical settings. In this article, I highlight three FDA-approved or available products for phage-mediated detection of bacterial pathogens in the agricultural, clinical and biodefense environments.

Current methods used for microbial diagnostics

Currently, bacterial identification is still heavily culture-dependent. Though for many decades, phage typing was used to identify and distinguish different strains in a variety of sample types, the robustness of this assay is still lacking in that it requires meticulous maintenance of phage stocks and propagating strains. As they can be rapid and highly specific, more modern phage-based detection systems have the ability to bridge this gap.

What are examples of phage diagnostics products currently available?

How do they work?

Gamma Phage Assay: a traditional phage lysis assay

Initially developed in the 50s, the Gamma Phage Assay was modified in 2005 to become the first phage-based diagnostic test to gain FDA approval for human use within the CDC Laboratory Response Network for the detection of the biowarfare pathogen, Bacillus anthracis.

The traditional phage lysis assay is used alongside capsule detection to determine the presence and virulence of B. anthracis.

Bluephage®: a colorimetric assay

Coliphages have been researched extensively as possible indicators of fecal contamination. Bluephage® implements the use of somatic coliphages for fecal and viral detection and quantification in contaminated water. The technique provides detection of 1 somatic coliphage within 3.5 h, and is applicable to water, food and sludge samples.

The method utilizes a modified E. coli strain (knocked out uidB and uidC genes) incapable of intracellular glucuronic acid transportation. This subsequently overexpresses the uidA gene which encodes for the enzyme β-glucuronidase. Once the phages lyses the cell, the amassed enzyme within the bacterial cells is released into medium containing a substrate (chromogen) analogous to glucuronic acid, producing a color transition from yellow to blue to indicate a positive result.

Lumiphage: a bioluminescence assay

Guild BioSciences has engineered a reporter phage system that produces a signal upon infection of target bacteria. Bacterial (Vibrio harveyii) luciferase genes (luxA and luxB) were integrated into nonessential regions of a temperate phage genome to create a reporter phage capable of conferring bioluminescence.

The purified reporter phage is typically added directly to bacterial cultures, and as the phage is lysogenic and not lytic, bioluminescence increases exponentially with microbial growth.

Concluding remarks

Though there has been renewed interest in using phage as therapeutic tools and as an a potential answer to antibiotic resistance, the technologies highlighted here serve to remind us of the diversity in phage applications.

Beyond bacterial biocontrol and detection, phages have been and are used in areas including phage-derived enzymes, drug discovery and nanotechnology.

Further reading

Phage detection methods can be improved by combining them with techniques like real-time PCR and mass spectrometry to detect phage amplification. To learn more, have a look at these two examples:

  • Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., & Filippov, A. A. (2010). Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR. PLoS One, 5(6), e11337. DOI: 10.1371/journal.pone.0011337
  • Rees, J. C., & Barr, J. R. (2017). Detection of methicillin-resistant Staphylococcus aureus using phage amplification combined with matrix-assisted laser desorption/ionization mass spectrometry. Analytical and bioanalytical chemistry, 409(5), 1379-1386. DOI:
    10.1007/s00216-016-0070-3

Cathy Nguyen

LinkedIn
ResearchGate

Charles River Laboratories, Charleston, South Carolina

Cathy Nguyen has been interested in bacteriophages since her undergraduate years at Coastal Carolina University in Conway, SC where she obtained a BSci in Biology. She continued her education with a MS in Biomedical Sciences from Medical University of South Carolina (MUSC) in Charleston, SC where she studied and worked collaboratively with Guild BioSciences (GBS), a local biotech company focused on phage diagnostics. After graduation, she continued working with GBS as a research specialist, focusing on genetically modified reporter phages for environmental and clinical samples. Currently, she works as a product development lab technologist at Charles River Laboratories in Charleston, SC.

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