How the Félix d’Hérelle Center manages its phages

Issue 87 | August 7, 2020
19 min read
Capsid and Tail

Dr. Sylvain Moineau and Denise Tremblay have a nice chat in the lab (Source: @universitelaval)

Since 2003, Denise Tremblay has managed the phage collection at the Félix d’Hérelle Reference Center for Bacterial Viruses at the Université Laval in Quebec City, Canada. We interviewed Denise about how the center collects, stores and distributes its phages, and why COVID-19 researchers are requesting their phages.

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What’s New

The Mayo Clinic has invested $1.75 million in Adaptive Phage Therapeutics, which will allow APT to initiate a clinical trial to address periprosthetic joint infections (an estimated commercial market of $900 million per year). The trial is set to start in the fall of 2020, with anticipated commercial availability in 2023.

Biotech newsClinical TrialFunding newsPhage Therapy

Rijul Kochhar (MIT) published a new article on the pre-d’Hérelle history of phages, and the many meanings of phages today in the space of this ‘pre-discovery’. It’s a story of science, religion and late 19th century colonial healthcare with 21st century consequences. This work is the result of Rijul’s direct fieldwork, done through MIT’s Doctoral Program in History, Anthropology, Science, Technology, and Society.

HistoryPhage Therapy

Jimmy Trinh (Texas A&M University) and colleagues published a new paper in Nature Communications looking at lambda phage infecting single host cells using live-cell and in-situ fluorescence imaging. They found that different viral DNAs establish separate subcellular compartments within cells, which results in heterogeneous viral developments within single cells.

Phage-host interactionsResearch paper

Holger Loessner (Paul-Ehrlich-Institut, Germany) and colleagues published a new paper in Antibiotics on the use of kinetic fingerprinting to explore the dynamics of specific phage-host pairs, with the goal of designing effective phage therapies. For a Klebsiella pneumoniae phage-host pair, they saw that a reduction in bacterial concentration required a high multiplicity of infection, complete bacterial clearance was hard to accomplish, and binding affinity was one of the most crucial factors for bacterial reduction.

Phage-host interactionsResearch paper

Karim Abdelkader (Ghent University) and colleagues published a new paper in Applied and Environmental Microbiology showing that an Acinetobacter baumannii phage lysin has a turgor pressure-dependent intrinsic antibacterial activity and can revert colistin resistance.

LysinsResearch paper

Vito Adrian Cantu (San Diego State University) and colleagues published a new preprint on Phage Artificial Neural Networks (PhANNs), a fast and accurate machine learning tool and web server that can classify phage proteins into ten major classes of structural proteins. This tool should help understand the functions and environmental contributions of phage-encoded structural proteins, many of which are still function-unknown.

Bioinformatics Tool

Huijun Geng (Dalian University of Technology, China) and colleagues published a paper looking into phage therapy as a treatment for Staphylococcus aureus-induced mastitis using a mouse model. Mice with induced mastitis showed improved mastitis pathology and a reduction in bacterial counts after the administration of phage.

Animal modelPhage TherapyResearch paper

Latest Jobs

Rob Edwards is hiring three postdocs to run the new Flinders Accelerator for Microbiome Exploration (FAME): a research associate in bioinformatics, a research associate in modelling marine microbes and a FAME liaison. Message Rob on Twitter for details.
David Pride at UC San Diego is seeking a Postdoctoral Fellow to investigate the role of phages as members of the human microbiome and to assist in the efforts to identify and characterize phages. The work will be in conjunction with the Center for Innovative Phage Applications and Therapeutics (IPATH).
The Wilke Lab and the Barrick Lab at UT Austin are hiring a postdoc in the area of systems and synthetic biology of phages.
Nancy Tawil at PhageLux, a phage therapy company in Montreal, Canada, is hiring a postdoc to work on phage delivery systems.

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Seeking MS2 phage

Hello Everyone ! I’m in need of the MS2 bacteriophage and its host E. coli strain in India. Other than ATCC, I find no suppliers in India. Could anyone please share them or direct me to a supplier in India please? Thanks much ! [email protected]

Seeking phages

Bacteriophage Forum: A series of ecology/evolution phage talks

Kavli Institute, UC Santa Barbara

The Kavli Institute for Theoretical Physics at UC Santa Barbara is hosting a series of phage talks (particularly from the eco-evolutionary perspective) called Bacteriophage Forum.

The dates are: August 11,18, 25 and September 1, 8, 2020 at 12:00-1:15, 1:30-2:45 pm PDT.

Tentative line-up of the talks:
Aug 11: Matthew Sullivan (OSU) and Forest Rohwer (SDSU)
Aug 18: Graham Hatfull (Pittsburgh/HHMI) and Joshua Weitz (Georgia Tech)
Aug 25: Luciano Marrafini (Rockefeller/HHMI) and Paul Turner (Yale)
Sept 1: Alison Buchan (UTK) and Britt Koskella (UCB)

Register here.

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SBRT Webinar: Phages for Global Health

Society for Bacteriophage Research and Therapy (SBRT)

The Indian Society for Bacteriophage Research and Therapy (SBRT) is a platform where researchers working on phages and their applications share expertise & resources.

SBRT is pleased to announce a webinar on “Using phages to combat AMR: Scientific capacity building through laboratory training workshops” by Dr. Tobi Nagel, Founder & President, Phages for Global Health; Prof. Martha Clokie, Professor, University of Leicester, UK & Editor-in-Chief of PHAGE: Therapy, Applications, and Research, and Dr. Janet Nale, Research Associate, University of Leicester. The mission of Phages for Global Health, a US non-profit, is to facilitate the application of phage technology and empower scientists to develop phage products that will be both technically effective and socially accepted within the local cultural contexts of the trainees.

Please join us on 14 August (Friday) at 8.30 pm IST/8 am PDT/4 pm BST. Register here.

Virtual Event

PHAVES 5: AMA with Dr. Evelien Adriaenssens on Aug 18

Phage Directory

Thanks to all who attended PHAVES 4 this week! We had a great turnout!

Next up will be an Ask Me Anything (AMA) with Dr. Evelien Adriaenssens on August 18 at 11:00 AM Eastern (GMT-4) / 4:00 PM UK (GMT+1). Evelien will answer your questions about phage taxonomy, viromics, and her transcontinental career progression from PhD student to postdoc to new PI, so bring your questions! Register here!

TaxonomyViromicsVirtual Event

How the Félix d’Hérelle Center manages its phages

Profile Image
Phage microbiologist and co-founder of Phage Directory
Co-founder
Phage Directory, Atlanta, GA, United States

Jessica Sacher is a co-founder of Phage Directory and has a Ph.D in Microbiology and Biotechnology from the University of Alberta.

For Phage Directory, she takes care of the science, writing, communications, and business aspects.

Profile Image
Lab Manager
Université Laval, Quebec City, Canada
Felix d'Herelle Center, Quebec City, Canada
Skills

Molecular Biology, Phage isolation, Phage-host interactions

  • Day-to-day tasks of the Felix d’Herelle collection
  • Specialized in Streptococcus thermophilus and Lactococcus lactis phages

The Félix d’Hérelle Reference Center for Bacterial Viruses is located at the Université Laval in Québec, Canada. Read more on their website, or here in Capsid & Tail.

Jessica: How did you get started at the Félix d’Hérelle Center?

Denise: After graduating with a BSc in Microbiology, I started in the summer of 1996 as a master’s student in Prof. Sylvain Moineau’s laboratory. I did my master’s on phages of Streptococcus thermophilus. The plan was to isolate phages from a cheese factory, and then sequence the genome of one of them. At that time, sequencing was very expensive, so when we sequenced the first phage genome of the lab, I think it cost the lab $40,000. It was $40 for a Sanger sequencing run. And it was $65 per primer. And so that’s what I did for my master’s, I isolated a few phages and we sequenced and characterized one.

After my master’s, I started to work as a research assistant with Sylvain. We were mainly working on Streptococcus thermophilus and Lactococcus lactis phages at the time. Then in late 2002, Prof. Hans Ackermann retired from the Université Laval and he kindly donated his phage collection to our lab. I started to manage the collection in January 2003. Prof. Ackermann started the Félix d’Hérelle Reference Center for Bacterial Viruses in 1982.

How does a phage go from being isolated to being added to your collection?

The vast majority of the phages we have in the collection were not isolated by us. We receive them from elsewhere, because it’s a long process to characterize them. When the paperwork (MTA) is done, I will receive phage(s) from a researcher. Then, I will always start from a plaque, because sometimes there can be many variants of the phage in the lysate. I make a lawn, pick a plaque, amplify it, do quality control assays to confirm the phage, and that becomes the master phage that I will put in the collection. As soon as I have a stock in the freezer, I add the phage to the website so people can order the phage.

Do researchers usually approach you and ask if they can send you a phage, or do you typically ask them for their phages?

We go to them; it’s really rare that someone wants to deposit the phage by themselves in the collection. Unless they are retiring and want to make sure that their phages are not lost! Usually we contact researchers and say, for instance, “we are looking through the ICTV classification and we’re trying to obtain a sample of each phage genus.” And lately, since we’ve been working a lot more at home (COVID-19) and ICTV is introducing several new phage genera, we’ve made a list of phages that we’d like a sample of, and we’ll be asking researchers for these samples soon.

So the Félix d’Hérelle Center aims to collect phages according to taxonomic diversity?

Yes, the goal is to only have reference phages, so we’re not trying to have all the T4 mutants, for instance. Although if you have mutated a gene and want to deposit the mutant phage for storage purposes, we’ll accept it. But we’re not looking for those phages; we’re looking to represent taxonomic diversity.

How do you store your phages?

We’ll freeze stocks of the lysate in 50% glycerol. I also always keep a fresh lysate in the fridge, so it’s easier to access if someone asks me for a phage. We also lyophilize them. And what we do sometimes is to freeze-dry the phage-infected bacterial host!

Can you talk more about your freeze-drying process?

Freeze-drying is something I do mostly for phages that are sensitive to long-term storage at minus 80. Freeze-drying phage lysates doesn’t always work well, so I will freeze-dry these phages inside the bacteria, which offer an extra protection to the phage genome. I grow a bacterial host, infect it with the phage, and after 10-15 minutes I spin the infected cells down, freeze them in milk, which also helps protect the bacteria, and then I freeze-dry that.

How do you make sure phages are ready to ship, in the event that you get a request?

The worst situation is when you try to amplify a phage and it’s not working! Some phages are really tricky and you have to play a bit with conditions and media to get a high enough titer. There are phages that are really easy to work with, like the T phages infecting E. coli. We keep these ready; I can just take a sample of the lysate from the fridge, titer it, and if the titer is high enough I will just send it. For some phages, like phi6, their titer will drop relatively rapidly in the fridge at four degrees, so you always have to amplify it every time someone orders it. That’s why we started to freeze-dry these kinds of phages inside their host bacteria, so we can just ship the ampoules directly to the researchers. Overall this saves a lot of time, even if it takes time to prepare the freeze-dried ampoules.

I’ve heard that your lab has a comprehensive cleaning process. Do you have any tips for keeping a phage lab clean and free of contamination?

We entirely clean the research and collection labs three times a year. At the beginning of every university semester, so for us January, May and September. And when we do that, we wash and disinfect everything, even the walls, shelves and bottles. It’s also a good time to look at your stocks and say, okay this one is old, get rid of that. It takes the entire day to do it.

I know some labs are afraid of phages, particularly E. coli phages, because if your lab does some cloning in E. coli and you have someone working with T1, well, you might not be happy about that. But we’re cloning a lot in E. coli in our research labs, and we haven’t had any phage issues with our cloning. Fingers crossed!

One piece of advice to avoid contamination is to disinfect the pipettes, and also to use filter pipette tips. We also throw out our phage buffer solutions very often, because if you contaminate your phage buffer, everything else will be contaminated. We fill several small bottles of phage buffer, and every time I work with one I just throw it away and take a new one. And we each wash our benches every time we start an experiment, and after every experiment. It’s even worse now with COVID-19, of course… now we wash the doors, handles and everything all the time!

Are the people that send you phages typically excited about sharing them with others?

Yes, people who are willing to share phages with us are usually also willing to share them with everyone. For some phages in the collection, we need to ask the owner for permission before we send the phage to someone. So if someone asks for a phage that is protected in this way, I will tell that person to write to the owner of the phage. This is only for a few phages though.

Do you ever send phages to companies?

Yes we will also send phages to companies, though usually it’s for research purposes only. But sometimes we have companies that will ask for commercial use of the phage. If it’s for a phage that was isolated by another lab, which is most of the time, the Université Laval will contact that lab/University to see if they want the company to commercialize it. Sometimes the lab will say “yes but I will take care of that with the company”, and then we are no longer part of the process. And sometimes the lab will say, “okay, it will be a tripartite agreement, because the Félix d’Hérelle collection at the Université Laval has maintained the phage, so they should also receive royalties.” In that case it becomes an agreement between all three parties.

How does the MTA (material transfer agreement) process typically go?

When I need to send a phage to a research laboratory, we use a short, basic MTA. But when it’s for a company, we use an extended MTA. When I receive a request for a phage, I just send it to our university’s IP department, they get everything signed, then they send me the file and I ship the phage.

Is your university supportive of this work? Do they like the fact that you run this center?

Yes, they now support us through funding, which is used to buy materials and maintain some equipment. All the invoicing is also done by the University’s financial department; I just give them the information and they do the invoicing. I think they see it as a way to increase the University’s visibility in the research community.

How is the center funded?

We were previously funded by NSERC (Canada’s main scientific research funding agency), as part of a specific program that funded research facilities across Canada. But that funding program ended abruptly around 2011 and it no longer exists. This was a big blow to the entire Canadian research community, including us. Now our university provides some funds, and we are also part of a few research networks in the Province of Quebec that provide additional funding. But these sources do not cover all our expenses. This is why we charge fees when we send phages to a lab.

Do you keep track of how many times each phage is ordered?

Yes, I do and this is fun! I have a list of my 20 most-sent phages, and every month we have a meeting, and I show that list. For now the most-requested phage is 2972, it’s a Streptococcus thermophilus phage. We published a paper a few years ago on the use of the CRISPR-Cas system as a teaching tool in undergraduate microbiology labs. It has become quite popular, so there’s a lot of universities ordering this phage. And I think the second-most ordered one is phage T4, because, well, it’s T4…

What has it been like for your lab during the COVID-19 pandemic so far?

During the lockdown, many researchers were asking us for phages as surrogates for SARS-CoV-2. Phages Phi6, MS2, PR772, and phiX174 were requested many times, because they’re tailless and have been used previously as surrogates for other eukaryotic viruses. So we asked permission from the university to go back to the lab to prepare them for shipping. We decided to freeze-dry the four phages, and the four bacterial hosts, so we’d be able to ship them rapidly. It took us some time to prepare them, but it was worth it because we have shipped many of them.

What was the last phage you sent?

This morning I sent a phage for COVID-19 research, but I sent another one at the same time that was not a COVID-19 phage. It was funny because it was the first time I sent that phage to a lab since 2003. It’s a phage infecting Hafnia, I don’t even know where it came from! I ship a lot of phages these days, because people are going back to their labs now, and they want to get back to working with phages.

Resources


Stephanie Lynch helped us produce this week’s article by helping us summarize articles for the What’s New section. Thanks Stephanie!!

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