What is a strain in microbiology and why does it matter?

By Prof. Colin Hill, Microbiology Department and APC Microbiome Ireland, University College Cork, Ireland

At the recent ISAPP meeting in Sitges we had an excellent debate on the topic of ‘All probiotic effects must be considered strain-specific’. Notwithstanding which side of the debate prevailed, it does raise the question: what exactly is a strain? As a card-carrying microbiologist I should probably be able to simply define the term and give you a convincing answer, but I find that it is a surprisingly difficult concept to capture. It is unfortunately a little technical as a topic for a light-hearted blog, but here goes. Let me start by saying that the term ‘strain’ is important largely because we like to name things and then use those names when we share information, but that the concept of ‘strain’ may have no logical basis in nature where mutations and changes to a bacterial genome are constantly occurring events.

Let’s suppose I have a culture of Lactobacillus acidophilus growing in a test-tube, grown from a single colony. This clonal population is obviously a single strain that I will name strain Lb. acidophilus ISAPP2022. That was easy! I am aware of course that within this population there will almost certainly be a small number of individual cells with mutations (single nucleotide polymorphisms, or SNPs), cells that may have lost a plasmid, or cells that have undergone small genomic rearrangements. Nonetheless, because this genetic heterogeneity is unavoidable, I still consider this to be a pure strain. If I isolate an antibiotic resistant version of this strain by plating the strain on agar containing streptomycin and selecting a resistant colony I will now have an alternative clonal population all sharing a SNP (almost certainly in a gene encoding a ribosomal subunit). Even though there is a potentially very important genotypic and phenotypic difference I would not consider this to be a new strain, but rather it is a variant of Lb. acidophilus ISAPP2022. To help people in the lab or collaborators I might call this variant ISAPP2022SmR, or ISAPP2022-1. In my view, I could continue to make changes to ISAPP2022 and all of those individual clonal populations will still be variants of the original strain. So, the variant concept is that any change in the genome, no matter how small, creates a new variant. When I grow ISAPP2022 in my lab for many years, or share it with others around the word, it is my view that we are all working with the same strain, despite the fact that different variants will inevitably emerge over time and in different labs.

Where the strain concept becomes more difficult is when I isolate a bacterium from a novel source and I want to determine if it is the same strain as ISAPP2022. If the whole genome sequence (WGS) is a perfect match (100% average nucleotide identity or ANI) then both isolates are the same strain and both can be called ISAPP2022. If they have only a few SNPs then they are variants of the same strain. If the two isolates only share 95% ANI then they are obviously not the same strain and cannot even be considered as members of the same species (I am using a species ANI cut-off of 96% that I adopted from a recent paper in IJSEM.

Where it gets really tricky is when the ANI lies between 96% (so that we know that the isolates are both members of the same species) and 100% (where they are unequivocally the same strain). Where should we place the cut-off to define a strain? At what point is a threshold crossed and an isolate goes from being a variant to becoming a new strain? Should this be a mathematical decision based solely on ANI, or do we have to consider the functionality of the changes? If it is mathematical then we could simply choose a specific value, say 99.95% or 99.99% ANI, and declare anything below that value is a new strain. Remember that the 2Mb genome size of Lb. acidophilus would mean that two isolates sharing 99.99% ANI could differ by up to 200 SNPs. This could lead to a situation where an isolate with 199 SNPs compared to ISAPP2022 is considered a variant, but an isolate with 201 SNPs is a new strain (even though it only differs from the variant with 199 SNPs by two additional SNPs). This feels very unsatisfactory. But what about an isolate with only 50 SNPs, but one that has a very different phenotype to ISAPP2022 because the SNPs are located in important genes? Or what about an isolate with an additional plasmid, or missing a plasmid, or with a chromosomal deletion or insertion? I would argue we should not have a hard and fast cut-off based on SNPs alone, but we should continue to call all of these variants, and not define them as new strains.

So, by how much do two isolates have to differ before we no longer consider them as variants of one another, but as new strains? I will leave that question to taxonomists and philosophers since for me it falls into the territory of ‘how many angels can dance on the head of pin?’

All this may seem somewhat esoteric, but there are practical implications. Can we translate the findings from a clinical trial done with a specific variant of a strain to all other variants of the same strain? If Lactobacillus acidophilus ISAPP2022 has been shown to deliver a health benefit (and is therefore a probiotic), can we assume that Lb acidophilus ISAP2022-1 or any other variant will have the same effect? What if a variant has only one mutation, but that mutation eliminates an important phenotype required for the functionality of the original strain? I am afraid that at the end of all this verbiage I have simply rephrased the original debate topic from ‘All probiotic effects must be considered strain-specific’ to ‘All probiotic effects must be considered variant-specific’. Looks like we might be heading back to the debate stage in 2023!

Bulgarian yogurt: An old tradition, alive and well

By Mariya Petrova, PhD, Microbiome insights and Probiotics Consultancy, Karlovo, Bulgaria

Family and family traditions are very important to me. Some of you may have seen my previous blog post on fermented food and my father’s tradition of making fermented cabbage and vegetables every autumn. Of course, this is not limited to my family – in Bulgaria, it is our culture and our country’s tradition. But despite the fact that I wrote about fermented vegetables first, Bulgarians are much more proud of another fermented product – yogurt.

I still remember waking up every morning when I was a kid and having a healthy homemade yogurt to start the day. I still do when I am back at home, because my father continues to make yogurt at home. Here, I’ll take you on a new adventure and tell you all about Bulgarian yogurt, an old tradition still alive in every home.

Élie Metchnikoff and his work are well familiar to anyone involved in probiotic research. In short, Metchnikoff observed in 1907 that Bulgarian peasants lived longer lives and he attributed this to their daily consumption of yogurt.

Thanks to Metchnikoff, research on Bulgaria and Bulgarian yogurt was put on the map because of our healthy way of living and eating fermented foods. You may know this part of the story. Still, few actually know that Metchnikoff was intrigued by the work of the Bulgarian researcher Stamen Grigorov a few years earlier. In fact, it was because of Stamen Grigorov’s work that we now know ‘who’ (i.e. which microbes) live in our yogurt and how essential those tiny bacteria are. In 1905 Stamen Grigorov actually discovered and isolated for the first time Lactobacillus bulgaricus (now known as Lactobacillus delbrueckii subsp. bulgaricus) from homemade yogurt. That’s why we are so proud of Bulgarian yogurt. Not only do we love to eat it, but the probiotic research was partially initiated in our country, and an entire Lactobacillus species is named after our country. There is even a small museum dedicated to Bulgarian yogurt and to the work of Stamen Grigorov, located in the house where he was born. In the museum, if you are visiting Bulgaria, you can learn how to make yogurt at home and a bit more about the history of Grigorov’s discoveries.

We are so proud of our yogurt that many Bulgarians will tell you that ancient Bulgarian tribes were the ones who discovered yogurt by accident. Since Bulgarian tribes were nomadic, they carried the milk in animal skins, which created an environment for bacteria to grow and produce yogurt. This is indeed the way people learned to make yogurt, but it most likely happened in many places independently. Of course, I know many countries make yogurt but I remain proud of all the discoveries that happened in my country (I am saying this because at times I have been judged when I tried to say how important we find the yogurt in Bulgaria and how proud we are).

Yogurt is a tradition in Bulgaria. I don’t know a Bulgarian who does not eat yogurt on a daily basis, up to a few pots per day. And I am not talking about those sweet yogurt products that are made by adding jam or vanilla. I am talking about real, natural yogurt, slightly sourer than most of the products that can be found in the Western world. We add yogurt to almost everything, it is just the perfect addition. It is even the basis of a traditional Bulgarian cold summer soup called “tarator,” made of yogurt, water, cucumber, garlic, and dill. We also make a salad with it called “snezhanka”, and it contains yogurt, cucumbers, garlic, and walnuts. (Recipes can be found below if you want to try something new during the lockdown.) In fact, I am so “addicted” to our yogurt that in every country I go to, the first thing I have do is to find a good yogurt. It took me years to find a good one in Belgium when I lived there (even though one product was labelled ‘Bulgarian yogurt’, it was not the same for sure). In Canada, it was somehow easier. After trying a few different products, it was even faster to find something that I like in the Netherlands, but they have many kinds of milk products. Yet none of them are truly comparable with what you can find in Bulgarian shops. Even the smallest shops have at least 3 to 4 different types because we have a lot of yogurt factories. Every product is different, it has a unique taste and can be made of different kinds of milk.

But honestly, nothing is the same as the homemade yogurt. Many people still make yogurt at home, including my father. I don’t quite remember a time when there was no homemade yogurt on the table at home. It was initially my grandmother making the yogurt and the white Bulgarian cheese (it is nothing to do with Feta but that’s the closest way to explain what it is). So it was somehow logical that my father started making yogurt as well. He knows the technique from his grandmother and grew up with fresh homemade yogurt. My grandparents had a lot of cows, sheep, and goats, so we always had plenty of milk to ferment. Making yogurt at home is so very simple that more and more young people dare to do it. In fact, making yogurt is so easy, I wonder why I am not doing it myself during the lockdown.

How to make it, you may ask?

So you need fresh milk, which my family in Bulgaria currently gets from a local farm. The milk is carefully boiled, and while it is still warm, transferred to a preferable container where you want to make the yogurt. We use old yogurt jars that were very popular before. For some time, my father also used Tupperware, so you can choose anything that you find handy. Before transferring the milk, my father also separates the cream from the milk in a separate jar and uses it to make homemade butter by constantly shaking the jar for around 10 minutes (it is an intensive workout, I tried it a few times!). The biggest problem these days is having a good starter culture so you can begin the milk fermentation. As a starter culture, most of the people, including my father, use a spoon or two of the previous batch of yogurt. So my father never finishes all the yogurt; he always makes sure that there are some leftovers so he can start a new fermentation. He usually adds one tablespoon of the old yogurt to 500 ml warm milk (around 45 C). Of course if the milk is too hot, the bacteria present in the starter culture will die, and nothing will happen. There is also the case that the milk is too cold, and then it will most likely still ferment, but it will have a strange consistency, something between milk and yogurt. If my father is out of old yogurt to start a new fermentation, he usually buys his favorite yogurt from the shop and uses this as starter. Once the jars are filled, he packs blankets all around them to keep the environment warm so the fermentation will begin. From here, you need around 4h to 5h to have a nice homemade yogurt. Simple and straightforward. The next morning you can have a great family breakfast, remembering the old traditions, talking about old memories, passing on the torch to the new generation, and enjoying a healthy start to the day.

The next time you have yogurt, I hope you enjoy it and remember the Bulgarian traditions!


Tarator soup recipe:

What you need: 1 cucumber, 250 -300 g yogurt, 1-2 cloves crushed garlic, salt, oil, water, fresh chopped dill. (Most of the ingredients depend on your taste so feel free to add more or less of certain ingredients. Some people also add parsley and walnuts, but it is up to your taste.)

How to make it: Peel and cut the cucumbers into cubes and put them in a preferred bowl; add the crushed garlic, and the minced dill. Beat the yogurt until it turns to liquid and mix it with the rest of the ingredients. Add salt and oil to taste. Add water to make the soup as liquid as you like. Put into the refrigerator to cool it. You can also make it with cold yogurt and cold water. It is perfect for the hot summer days.

Snezhanka (which means “Snow White” in Bulgarian) salad recipe:

What you need: 1 cucumber, 500 g yogurt, 1-2 cloves crushed garlic, 2-3 spoons ground walnuts, salt, oil, fresh chopped dill. (Again, it depends on your taste, if you like more cucumber or yogurt just add more.)

How to make it: First strain the yogurt for a couple of hours, so that all unnecessary water is drained away. Peel and cut the cucumbers into cubes and put them in the bowl. Add the strained yogurt. Add the fresh dill, salt and oil to taste. Sprinkle the walnuts on top of the salad. Perfect for all seasons. If you don’t have a fresh cucumber, you can also use pickles — the final result is also very delicious.