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.
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