Periodic Reporting for period 2 - Lacto-Be (Advancing Lactobacillus’ beneficial potential)
Reporting period: 2021-09-01 to 2023-02-28
Lactobacilli have a strong, but underexplored potential as sustainable bio-based solutions for many food and health-related problems. Since Nobel-laureate Eli Metchnikoff hypothesized that lactic acid bacteria can promote human health in the gut, the research on lactobacilli and probiotics has mainly focused on the human gut and fermented dairy foods. However, a major knowledge gap exists on the beneficial potential of Lactobacillus species in other human body sites (vagina, skin, upper respiratory tract), animals (e.g. chickens, honey bees), plants, crops, and even on abiotic surfaces. In addition, lactobacilli play a key role in many plant- and vegetable-based fermentations, where they promote the shelf life and nutritional value of food and feed. Yet, why and how Lactobacillaceae species can be beneficial in such a wide variety of habitats is currently underexplored.
Therefore, the core aim of this project is a systematic and integrated analysis of the evolutionary history, ecology, and beneficial functions of Lactobacillus species. We aim to follow an unconventional approach situated at the intersections of molecular microbiology (focusing on a single microbe), molecular ecology (focusing on microbial communities) and comparative genomics with an evolutionary perspective on niche adaptation of lactobacilli.
In the first phase of the project, we have set up the research lines on the ecology and functionality of lactobacilli in the female reproductive tract, embedded in a unique Citizen Science project named Isala (https://isala.be/en). This approach provided us with a unique dataset on the dominance and prevalence of lactobacilli in the vagina and a unique biobank of vaginal lactobacilli. With the genome sequence of these novel lactobacilli isolates, we can reconstruct the evolutionary history. The vaginal lactobacilli isolates are screened in various assays for their antimicrobial capacity against urogenital pathogens. In parallel to lactobacilli in the vagina, we have also set up a second major research line on lactobacilli in fermented vegetables for which we also want to build up a unique collection of microbiome profiles and isolates. To better understand how these lactobacilli can exert beneficial activities in these habits, novel genetic tools such as prime editing are now explored to validate the function of various genes with predicted interesting functions.
By looking deeper into Lactobacillus biology, we hope to make a paradigm shift in probiotic research on lactobacilli, by shifting from a classical ad hoc base to a unique knowledge-based framework for strain selection and analysis of fitness and performance.
Therefore, the core aim of this project is a systematic and integrated analysis of the evolutionary history, ecology, and beneficial functions of Lactobacillus species. We aim to follow an unconventional approach situated at the intersections of molecular microbiology (focusing on a single microbe), molecular ecology (focusing on microbial communities) and comparative genomics with an evolutionary perspective on niche adaptation of lactobacilli.
In the first phase of the project, we have set up the research lines on the ecology and functionality of lactobacilli in the female reproductive tract, embedded in a unique Citizen Science project named Isala (https://isala.be/en). This approach provided us with a unique dataset on the dominance and prevalence of lactobacilli in the vagina and a unique biobank of vaginal lactobacilli. With the genome sequence of these novel lactobacilli isolates, we can reconstruct the evolutionary history. The vaginal lactobacilli isolates are screened in various assays for their antimicrobial capacity against urogenital pathogens. In parallel to lactobacilli in the vagina, we have also set up a second major research line on lactobacilli in fermented vegetables for which we also want to build up a unique collection of microbiome profiles and isolates. To better understand how these lactobacilli can exert beneficial activities in these habits, novel genetic tools such as prime editing are now explored to validate the function of various genes with predicted interesting functions.
By looking deeper into Lactobacillus biology, we hope to make a paradigm shift in probiotic research on lactobacilli, by shifting from a classical ad hoc base to a unique knowledge-based framework for strain selection and analysis of fitness and performance.
In the first phase of the Lacto-Be project, we have set up the research lines on the ecology and functionality of lactobacilli in the female reproductive tract (or vagina) and have embedded this in a unique Citizen Science project named Isala (https://isala.be/en). During the stringent international COVID-19 lockdown, which co-incidenced with the start of our participant recruitment, we have managed to convince > 5500 participants to express their willingness to provide vaginal swab samples and fill out an extensive questionnaire on many microbiome-modulating factors . This was filled out by more than 85% of the registered participants, providing us with a unique set of epidemiological data . The questionnaire was organized via the GDPR-proof, online platform Qualtrics. Subsequently, we managed to convince >3300 women to actually donate two vaginal swabs: one for 16S amplicon sequencing and one for culture. This first sampling campaign then provided us with a unique dataset on the dominance and prevalence of lactobacilli in the vagina (3326 microbial profiles) and a unique biobank of vaginal lactobacilli (currently at 245 isolates and still expanding).
The 3326 Isala vaginal microbiome profiles with data on the dominance and prevalence of lactobacilli were analyzed at several levels. We investigated the composition of the microbiome space with the application of a novel embedding on 16S data. This approach displayed the variation in microbiome composition across all samples, revealing a continuum of samples between the classically described vaginal community state types. Further, we studied the correlation structure between the bacteria in the vaginal microbiome . Associations between the responses to over 100 survey questions and the bacterial abundances and microbiome space were also already analyzed and this at three different levels. We have found unprecedent effects of lifestyle and environmental factors at all three levels.
The Isala isolates obtained so far have already been screened for interesting phenotypes and dominance in the vaginal niche and these screenings will be continued with new isolates. These screenings include various in vitro assays for antimicrobial capacity against key pathogens, including spot assays, radial diffusion assays, a time course and biofilm analysis. These tests have already revealed a variety of interesting strains with strong antimicrobial activity against vaginal (opportunistic) pathogens. Part of these isolates have also already been whole-genome sequenced. With these genome sequences, we can now reconstruct the evolutionary history and delve into the conserved gene content, while also screen for putative genes that might give the lactobacilli an advantage in the vaginal niche such as those encoding putative antimicrobial compounds. Attempts have also been made to produce gene disruption mutants in the most promising vaginal isolates to validate the function of particular genes and molecules. A homologous recombination approach proved insufficient but currently, prime editing is being optimized for use in lactobacilli and already seems promising. If successful, this will be used to create a whole new set of gene disruption mutants. Finally for the vaginal lactobacilli research line, it is worth mentioning that the > 3300 Isala swabs taken for culture analysis have also been explored for their potential for metabolomics analyses. Important metabolites such as L-, D-lactic acid and glycogen have already been screened for to look for associations with certain microbiome profiles.
In parallel to lactobacilli in the vagina, we have set up a second major research line on lactobacilli in fermented vegetables. The microbial composition of many different vegetable fermentations, including sauerkraut, beetroot, carrot juice, and kimchi has already been determined by 16S amplicon sequencing to gain new insights in the dominance of particular lactobacilli such as Lactiplantibacillus and Leuconostoc taxa in various vegetable fermentations.
The 3326 Isala vaginal microbiome profiles with data on the dominance and prevalence of lactobacilli were analyzed at several levels. We investigated the composition of the microbiome space with the application of a novel embedding on 16S data. This approach displayed the variation in microbiome composition across all samples, revealing a continuum of samples between the classically described vaginal community state types. Further, we studied the correlation structure between the bacteria in the vaginal microbiome . Associations between the responses to over 100 survey questions and the bacterial abundances and microbiome space were also already analyzed and this at three different levels. We have found unprecedent effects of lifestyle and environmental factors at all three levels.
The Isala isolates obtained so far have already been screened for interesting phenotypes and dominance in the vaginal niche and these screenings will be continued with new isolates. These screenings include various in vitro assays for antimicrobial capacity against key pathogens, including spot assays, radial diffusion assays, a time course and biofilm analysis. These tests have already revealed a variety of interesting strains with strong antimicrobial activity against vaginal (opportunistic) pathogens. Part of these isolates have also already been whole-genome sequenced. With these genome sequences, we can now reconstruct the evolutionary history and delve into the conserved gene content, while also screen for putative genes that might give the lactobacilli an advantage in the vaginal niche such as those encoding putative antimicrobial compounds. Attempts have also been made to produce gene disruption mutants in the most promising vaginal isolates to validate the function of particular genes and molecules. A homologous recombination approach proved insufficient but currently, prime editing is being optimized for use in lactobacilli and already seems promising. If successful, this will be used to create a whole new set of gene disruption mutants. Finally for the vaginal lactobacilli research line, it is worth mentioning that the > 3300 Isala swabs taken for culture analysis have also been explored for their potential for metabolomics analyses. Important metabolites such as L-, D-lactic acid and glycogen have already been screened for to look for associations with certain microbiome profiles.
In parallel to lactobacilli in the vagina, we have set up a second major research line on lactobacilli in fermented vegetables. The microbial composition of many different vegetable fermentations, including sauerkraut, beetroot, carrot juice, and kimchi has already been determined by 16S amplicon sequencing to gain new insights in the dominance of particular lactobacilli such as Lactiplantibacillus and Leuconostoc taxa in various vegetable fermentations.
We have set-up a unique citizen science approach (https://isala.be/en) to engage women to donate vaginal swabs that allowed us to build up a unique collection of vaginal lactobacilli and vaginal microbiome profiles .
We have now unique biobank of lactobacilli (> 10000 isolates) from different habitats and environments (mostly from vagina and fermented vegetables) . We have genetic access to several of these wild lactobacilli from vagina and fermented vegetables via electrotransformation. In addition, we are implementing a new prime editing approach in lactic acid bacteria.
We have already generated new insights in primary and secondary metabolite synthesis in lactic acid bacteria.
We have now unique biobank of lactobacilli (> 10000 isolates) from different habitats and environments (mostly from vagina and fermented vegetables) . We have genetic access to several of these wild lactobacilli from vagina and fermented vegetables via electrotransformation. In addition, we are implementing a new prime editing approach in lactic acid bacteria.
We have already generated new insights in primary and secondary metabolite synthesis in lactic acid bacteria.