In March of 2020, a paper written by a multinational group of scientists was published that rewrites the book on the genus Lactobacillus, amongst the most beloved organisms of fermented food producers. What had been a massive single genus containing over 250 species has now officially been broken down into 25 new, smaller genera. Bronwen Percival spoke with one of the lead authors of the paper, Professor Sarah Lebeer of the University of Antwerp in Belgium, about the project, her work on the role of lactobacilli in human and other microbiomes, including those of food, and how this knowledge might ultimately be applied.
How did this project to split up the genus Lactobacillus come about? Is it something that clearly needed to be done for a long time, or was it a more sudden decision prompted by a particular event or development?
A big group of scientists all knew that it had to be done… We were struggling with the correct taxonomy; [for example] a lot of microbes in the databases were called Lactobacillus casei but were actually paracasei. [As] molecular biologists, [we are] fans of using the genome sequences to make classifications— We are not a fan of doing multiple tedious phenotypic assays which are traditionally done in taxonomy. We started to collaborate with taxonomists, such as Giovanna Felis, Elisa Salvetti, Bruno Pot, Jinshui Zheng, Michael Gänzle, and others who were also trying to make reclassifications. They were doing similar things to us, but also different things. And there were other groups of scientists doing slightly different things…and [making] what we sometimes felt were somewhat arbitrary decisions and classifications. We all wanted to the same thing [to classify the bacteria appropriately and consistently] but we were all doing it in a slightly different way, so we decided it was better to collaborate and decide together how it should be done. We sent a letter to the editor of Applied and Environmental Microbiology (a peer-reviewed journal), a call to all scientists internationally, [saying] if you’re doing work in this area please let us know and maybe we can collaborate. We didn’t want to have one group propose a way, and then one month later have another group saying that it’s not how it should be done. [Ultimately the paper had 15 authors from institutions in Belgium, China, Ireland, Italy, Canada, Germany, and Taiwan.]
What do you call this wider group of related genera now? I keep wanting to call them Lactobacilli but that’s just a single genus within them now…
We also still struggle with this. [We consider] the name “lactobacilli” a popular name, which can still be used for species that were part of the old Lactobacillus genus, although it would be more scientifically correct to talk about the Lactobacillaceae [family]. Looking at the core genes that are similar to all of them: they all produce lactic acid, which is crucial to their biology, and most have a rod shape. We wanted to understand which parts are conserved in certain groups and why [and classify them accordingly].
You’ve identified 23 new genera; are all of these likely to have roles to play in food production/be found in food matrices? Or are we talking about just a few that are likely to be relevant?
I don’t think all, but many might have roles to play, particularly if we increase the scope of your question to include fermented animal feeds like silage and insects such as honeybees that play a role in honey and pollination of many crops we eat such as strawberries, blueberries, apples, broccoli, nuts, and many others. The prime example is Lactobacillus delbrueckii. It was the first Lactobacillus ever described; it is host-adapted, but it is also widely used for yoghurt. The same genus has other species—for example gasseri and acidophilus—also used for fermented milk, yoghurt and related probiotic products and what we call man-made food environments.
Would all these bacterial species have GRAS status in food production? Would all of them be acceptable to find in food?
There are a lot of very good data on safety upon consumption of large doses and a long history of safe use for this group of microbes, so there are many indications that they can be regarded as safe; we often do big screenings of their genomes and you can see that they lack (known) virulence properties. [But this is something] that we’re still exploring. In the end, any microbe can become a pathogen if it acquires virulence genes by taking up genes from the environment, so safety and pathogenicity should be addressed at strain level. One thing that could be a concern is if they take up antibiotic resistance markers; that’s not a desired property. You can also screen for that, and for their propensity to take up new genes and exchange genetic information. The ability to easily exchange genes can also be a good thing because it allows them to adapt more easily to new environments, but it’s also a potential risk factor.
Many food producers tend to classify microbes by their function rather than by their genetic relatedness; for example, cheesemakers are often taught that mesophilic cultures are the lactococci and leuconostocs, while thermophiles are lactobacilli (and Streptococcus thermophilus) but after reading this paper that understanding is clearly oversimplified. To what extent do these new genera encompass microbes that are likely to behave in similar ways, or have similar functionality within fermented food systems?
Yes, one of the ideas behind this project is that it makes it easier to describe the microbes’ functionalities, and to predict their role in food ecosystems. With this classification, we now have better tools to talk about bacteria in food, and better reflect their properties.
[However, on a day-to-day basis, users of commercial cultures or probiotics containing these reclassified species are not likely to notice a difference in the labelling, as the team took care to choose new names for the genera containing the most common members that also begin with the letter “L”. Thus, what was once Lactobacillus casei has become Lacticaseibacillus casei, and Lactobacillus plantarum has become Lactiplantibacillus plantarum, but on most culture labels, they will remain listed as L. casei and L. plantarum, respectively.]
It looks like a lot of the organisms within this group can be isolated from gastrointestinal tracts. Any thoughts on their role/s within that system?
A quick answer is that we don’t know. But based on the available data they seem to have a function as a keystone species[i], so [even though they are] not so abundant in these gastrointestinal environments, they have a role in keeping certain pathogens low, such as the Enterobacteriaceae (Gram-negative potential pathogens). They also have a role in immune modulation: they have a typical Gram-positive cell wall of which many molecules can interact with immune receptors. This is important for the immune system balancing that is happening in the gastrointestinal tract. The third key function they seem to have is communication, for example with the gut epithelium. This epithelium forms the barrier [of the intestine] in the GI tract. Its cells need to be in good shape and ‘close’ properly to avoid pathogens and toxins entering, but also allow nutrients to be taken up. There are a lot of nice examples of molecular interaction between lactobacilli and the barriers of the intestinal walls.
It seems like lots of claims are being made (and probiotics and fermented foods sold) without necessarily having strong evidence that they have a health benefit. What are your thoughts on the use of probiotics and probiotic-rich foods to enhance human health?
There is some evidence that probiotics have an impact on the gut microbial population, but that’s not necessarily what a probiotic should be taken for. You have so many microbes in your GI tract and it’s a misconception that they need to establish themselves in the gut before they can be helpful. Also, if you have 10^14 per gram of microorganisms in your gut and you introduce 10^9 probiotics or fermented food microorganisms, it’s very difficult to detect the introduced bacteria, [even at a level of 1,000,000,000 per gram] they are [still] outnumbered by 100,000 times. There is a recent paper in Nature Communications showing that the LAB found in fermented foods can be found as up to 1% of the total fecal population, and can be detected [more] as technology is improving.
But you have to ask the question whether they have a beneficial effect based on their number or their activity. There is a lot of data including clinical trials that they can have a beneficial effect on the immune system and have barrier enhancement effects. Also they can help reduce GI and respiratory infections, sometimes by direct anti-pathogenic effects and sometimes by stimulating the immune system. From the general public perspective, it’s not universal, not black and white, and it doesn’t work in all situations. If people are already healthy, it’s hard to do clinical trials to see whether something makes people more healthy, so that’s also a challenge. The best documentation is in cases of actual problems, like newborn babies with necrotizing enterocolitis.[ii]
As more related bacterial species are identified, do you think that they will all be able to fit happily within one of these newly-defined genera, or do you think that more might be needed?
[The new classification is] pretty comprehensive, but nothing is ever final. Especially now if we are doing studies in remote areas like parts of South America or Africa, if we find novel fermented foods that have not been well studied, they may have microbes that end up in a new category. Soon we’ll likely need an extension, but we believe that the criteria we described are valid, so we can use the same rationale to describe another genus.
Is there anything else you think is worth adding for our audience of fermentation enthusiasts?
I didn’t mention yet that my team is also working on signature genes—we look whether [members of] a newly proposed genus have a conserved set of genes that are unique for this group, to show signature genes that reflect common functions and evolutionary background. You can do this analysis without having to know the exact functions of these genes. The first results were already quite surprising: we didn’t expect these signature gene patterns to be so clear. Sometimes it was a group [of bacteria] that we couldn’t describe well but we could see that they have a very nice set of [shared] signature genes. The next step is for us and other scientists is to study their function so we will better understand their key properties, for example their adaptation to a particular lifestyle or habitat. For food fermentations, if we understand such key functions better, we can better direct the fermentation and the food that producers want to make.
One example is Lactobacillus casei: they are often wrongly identified as paracasei, but then we found that the real casei have a catalase gene, an enzyme which is needed to withstand oxidative stress. That helped us a lot: we were able to disentangle that if a strain of this newly proposed Lacticaseibacillus genus has this gene, it can better adapt to environments with oxidative stress. The ones without it probably prefer and are better adapted to anaerobic conditions.
It’s very inspiring for us as molecular microbiologists to speak with food professionals because it provides important information to help us mine all these gene sequences and genomes. It’s good not to forget the information that’s already out there in people’s practical experience. We need to know what people do and their practical experience with microbes such as in fermented foods and how you can direct them helps us understand the biology much better.
Thanks very much to Professor Sarah Lebeer for taking the time to talk with us. The full paper is free to access here.
[i] a species which has a disproportionately large effect on its natural environment relative to its abundance
[ii] a serious illness in which tissues in the intestine become inflamed and start to die