History Part 15 : Phage Discovery gets its groove back with Phage DisCo

Phage discovery gets its groove back with Phage DisCo

Bacteriophage research has been all the rage, and for good reason; these bacteria killing viruses may be the solution to antibiotic resistance. Antibiotics are broad-acting drugs that kill every type of bacteria in their path. But this relief is short-lived. Bacterial pathogens can evolve to withstand antibiotics faster than the pharmaceutical industry can make them. With antibiotic resistance on the rise, it’s no wonder that bacteriophages are the newest viral sensation.  

Unlike antibiotics, phage therapy offers a more precise approach to treat bacterial infections that is harder for evolution to evade. Bacteriophages – also known as phages – act as microscopic hitmen. To kill only their specific target, a phage recognizes a unique protein receptor found on bacteria cells’ surface. Phages that use different receptors are needed for phage therapy. Multiple phages with different targets are combined into a “phage cocktail” which increases the number of methods to kill a bacterial pathogen in a single treatment. 

Resistance to phage therapy is extremely unlikely due to the diversity and size of the phage population. The number of individual bacteriophages on Earth is estimated to be 10^31, or ten thousand tredecillion! This astronomical amount of phage is both good and bad. There is a seemingly endless supply of phages waiting to be discovered, however, finding the right one for the job is like searching for a microscopic needle in a haystack. 

Part of the problem is the way phages are discovered. The current method requires scientists to wade through millions of genetically similar phages in the hopes of finding one with therapeutic potential. A more effective way of sorting samples is needed to give phage therapy a fighting chance against antibiotic resistance. Finding a rare phage is a shot in the dark, but one research group may have found a way to shed light on the issue.

Take Your Time (Do It Right)

It’s true what they say, everything old is new again. In music, fashion, and even science, trends from the past often come back, but with a twist. That’s how scientists at Harvard Medical School came up with a way to put a new spin on a classic technique. Their innovative method is called phage discovery by co-culture, or Phage DisCo for short. The abbreviation might be a stretch, but the results are as groovy as the name suggests.

Bacteriophages are currently discovered using a method designed to look at phage communities. This approach isolates phages with a single bacterial culture grown into a uniform lawn on a petri dish. Environmental samples containing a random mix of phages are added to the lawn. If a phage is able to infect the bacterial lawn, it leaves a small dead patch called a plaque (Figure 1). A single petri dish often has fifty or more plaques and the phages responsible can only be identified after a lengthy isolation process. This method is great at sampling the bacteriophage population, but terrible at finding rare phages. It’s like using the census to find the next pop music superstar! Until one research group found a way to work smarter, not harder.  

Figure 1: Diagram of original method for phage discovery. A single culture of unmodified bacteria is grown into a lawn on a petri dish before phages from an environmental sample create plaques. Phages that are rare (red) in the environment create plaques that are often indistinguishable from plaques created by common phages (green). In this example, only 3 out of the 14 plaques tested contain a rare phage.

For efficient phage discovery, two cultures are better than one! The researchers at Harvard Medical School described a way to easily identify rare phages by their plaques. Unlike the original method, Phage DisCo is designed to target phages with these desired characteristics using a co-culture of genetically modified bacteria. To do so, surface receptors were knocked out of bacteria strains one by one, then replaced with different fluorescent proteins. The modified cultures were grown into a lawn before environmental samples were added (Figure 2). 

Figure 2: Experiment design for Phage Disco. Fluorescently labeled bacteria are modified to target a phage characteristic. Modified bacteria cultures are plated and environmental samples are added. Microscopy images are taken under the different fluorescent conditions that are compiled into a final image. Phage dependencies are then determined by plaque colors. Image from original article: https://doi.org/10.1128/msystems.01644-24.

The resulting plaques were examined for fluorescence. Dark plaques, with no fluorescence, were from phages that did not rely on any of the knocked out receptors. Phages that do require a receptor will emit the color of the fluorescent protein that replaced the specific receptor (Figure 3). The method was validated using previously discovered phages and successfully characterized their receptors. When tested on actual unknown samples, Phage DisCo kept up its hot streak. For one receptor, they found a total of 11 dependent phages during their experiment. Their statistics showed that the original method would have to process 67 plaques to identify those 11 phages. Seeing is believing, and this was proof that this new method could transform phage discovery.

Figure 3: Composite image of Phage Disco experiment. A petri dish contains differentially colored plaques. Different colors denote specific characteristics of the bacteriophage making the plaque. Image from original article: https://doi.org/10.1128/msystems.01644-24.  

Ain’t No Stoppin’ Us Now

Compared to traditional methods, Phage DisCo leads the charts for phage discovery, and this is just the beginning. This study used a maximum of three modified bacteria cultures in their study, but this number could be increased. Using all the fluorescent proteins colors commercially available could scale the operation to seven strains per petri dish. Don’t put on your dancing shoes just yet! The authors note that phage isolated using Phage DisCo should still go through rounds of purification before use, but even with those precautions, this new method has bacteriophage scientists ready to dance the night away.

Link to the original post: Rand EA, Quinones-Olvera N, Jean KDC, Hernandez-Perez C, Owen SV, Baym M. 2025. Phage DisCo: targeted discovery of bacteriophages by co-culture. mSystems 10:e01644-24.

Additional references:

Clokie MR, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage. 2011 Jan;1(1):31-45. doi: 10.4161/bact.1.1.14942. PMID: 21687533; PMCID: PMC3109452.

Abedon ST, Danis-Wlodarczyk KM, Wozniak DJ. Phage Cocktail Development for Bacteriophage Therapy: Toward Improving Spectrum of Activity Breadth and Depth. Pharmaceuticals (Basel). 2021 Oct 3;14(10):1019. doi: 10.3390/ph14101019. PMID: 34681243; PMCID: PMC8541335.

Mushegian AR. Are There 1031 Virus Particles on Earth, or More, or Fewer? J Bacteriol. 2020 Apr 9;202(9):e00052-20. doi: 10.1128/JB.00052-20. PMID: 32071093; PMCID: PMC7148134.

Snapp EL. Fluorescent proteins: a cell biologist’s user guide. Trends Cell Biol. 2009 Nov;19(11):649-55. doi: 10.1016/j.tcb.2009.08.002. Epub 2009 Oct 8. PMID: 19819147; PMCID: PMC2784028.

Featured image: Made by author

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