The story behind Latilactobacillus fragifolii, a new bacterial species isolated in our lab.

A new bacterial species? Well, new to us, as it was never ‘seen’ or described by humans before. 

For me, there is a very personal story attached to this new species. It started in 2017. I moved to central Portugal together with a friend. A place where time moves at a slower pace. We moved to a garden that was filled with olive, citrus, fig, plum and apple trees as well as lavender, rosemary and much more. Our goal was to create a food producing garden that needed little input. With input, I mean, mechanical input such as tilling, mowing and leaf blowing, and chemical input such as fertilizers and pesticides. Reducing input also entails sacrificing some output, harvest. But overall, this management should lead to a more sustainable ecosystem. This idea also lies at the basis of many agriculture movements you might have heard of, such as agroforestry and permaculture. Although it is debatable whether these low-input agricultural systems are a solution for our global food production, many ideas from low-input systems can be of great value for intensive agriculture. Searching for these sustainable solutions was an important inspiration for starting my PhD.

Now, when does this tiny new bacterium come into the picture? After I started my PhD at the Lebeerlab, I planned on returning to Portugal for a change of scenery while writing a review on biocontrol in the phyllosphere [1]. To make things a bit more official, Prof. Sarah Lebeer said with a smile “OK, if you bring some phyllosphere samples with you to isolate bacteria from”. And so I returned with a bag full of icepack-chilled tubes filled with leaves. Airport security looked at the bag, made a little joke about the danger of cold leaves, and let me pass. Arriving late at Brussels airport, I rushed back to the lab and started processing the samples. The next days, colleague Leen Van Ham continued culturing the bacteria that had grown on the agar plates. The isolates were identified by sequencing a marker gene, the 16S rRNA gene, and comparing it to a database. I was thrilled because many isolates were lactic acid bacteria, uncommon and understudied inhabitants of plant leaves. However, I was still unaware of the real identity of some these isolates. The database comparison had shown a good match to already known species and didn’t reveal any novelty. 

A few months later, my colleague Tom Eilers had news. He had been looking into the full genomes of these isolates and said that some matched only for 88% with their closest relatives. While it is known that bacteria from the same species have at least 93% identical DNA in their shared genomic regions. A similarity of 88% meant that the isolates in our freezer belonged to an unknown species!

So how is this possible, given that the 16S gene had not seemed so novel? It appears that in some cases, the 16S rRNA gene mutates very slowly, making relatively distant bacteria seem closely related. Alternatively, the gene may have been very recently exchanged between the new species and species whose 16S sequences are in the reference database. Using the entire genome avoids these pitfalls of single-gene analysis and allowed us to conclusively identify the isolates as a new bacterial species. On a side note, for mammals this “species threshold” is much higher. Did you know that we share 98.8% of our DNA with chimpanzees?

Next, we conducted a series of experiments to characterize the new species and to distinguish it from its closest relatives. You can read all about it in this manuscript [2]. Some interesting observations were that the new species produces catalase, an enzyme protecting it against oxidative stress. It is able to consume typical plant-associated sugars, arabinose and sucrose. And the bacterium is yellow, as a result of the production of carotenoids, a pigment that protects the cells against UV stress. You could say these carotenoids act like sunscreen, quite useful when you are residing on a leaf during summer in Portugal 😎.

All these observations make us think that this species might be well equipped to survive on leaf surfaces, and that it could be used for applications in agriculture. Maybe one day, this bacterium, discovered thanks to our efforts in creating a sustainable forest garden, will contribute in solutions for crop production on a larger scale?

What’s in a name?

fragifolii, composed of the latin words fragum, meaning strawberry plant, and folium, leaf. Put together this results in fragifolii, “of a strawberry leaf”. Also Latilactobacillus is a new term, introduced after splitting the Lactobacillus genus, meaning “widespread milk rodlet”[3].
  1. Legein M, Smets W, Vandenheuvel D, Eilers T, Muyshondt B, et al. Modes of Action of Microbial Biocontrol in the Phyllosphere. Frontiers in Microbiology 2020;11:1619.
  2. Legein M, Wittouck S, Lebeer S. Latilactobacillus fragifolii sp. nov., isolated from leaves of a strawberry plant (Fragaria x ananassa). Int J Syst Evol Microbiol 2022;72:005193.
  3. Zheng J, Wittouck S, Salvetti E, Franz CMAP, Harris HMB, et al. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol 2020;70:2782–2858.

Starting the sisterhood on vaginal health: now in Peru

Introducing Laura

Almost a year ago, I wrote a blog about vaginal microbiomes and ethnicity. As a Isala team member, born and raised in Peru, I am now super excited that, thanks to the global minds starting grant, we could now extend Isala to Peru, where “Isala” becomes “Laura”, named after Esther Rodríguez Dulanto (1872-1919), the first female physician in Peru.

The Laura project aims to map the vaginal microbiome of healthy women of reproductive age from two geographically contrasting regions in Peru, the coast (Lima city) and the Amazon (Iquitos city). Moreover, this project aims to raise awareness on vaginal health among the public and build capacities (e.g. training on molecular and microbiology techniques) in Peruvian researchers.

Each region has distinct environmental and ethnic differences. Lima, unlike Iquitos, is an urbanized city and, sadly, one of the most polluted cities in Latin america1. Another difference between these regions is eating habits. For instance, in Iquitos, fish consumption is higher than Lima2; corn and cereal consumption is also markedly different with 63% and 38% for Lima and Iquitos, respectively3. All this information, along with other habits, was gathered in the questionnaire and will help us understand the possible factors influencing the difference in vaginal microbiota composition, if any.


Peruvian network on vaginal microbiome research

To investigate the vaginal microbiome in Peru, in the context of Isala and Laura projects, we have established a research network with two Peruvian universities Universidad Peruana Cayetano Heredia – UPCH (Lima) and Universidad Nacional de la Amazonia Peruana – UNAP (Iquitos).

Moreover, the year before Laura, we started an academic collaboration with another Peruvian university, Universidad Nacional San Agustín de Arequipa (UNSA), located in the highlands. The UNSA and Lebeerlab teams will map the vaginal microbiome of women of reproductive age and menopause. Of note, each research group is led by excellent and enthusiastic female researchers Theresa Ochoa (UPCH), Viviana Pinedo and Graciela Meza (UNAP) and Ada del Carpio (UNSA), all of them willing to contribute to women’s health!

This project also builds capacities in young Peruvian researchers and students on microbiome research. Specifically, they will learn microbiology and molecular biology techniques applied to vaginal microbiome research.

To achieve the aim to raise awareness on vaginal health in Peruvian women, volunteers will receive information from health care professionals as well as leaflets with infographics with content on vaginal microbiota facilitating science communication as well as education in vaginal health. Finally, we have launched the Isala web page in Spanish, so that volunteers and interested women can check on several topics related to vaginal health such as menstrual health, contraceptive methods among others.


Expected and unexpected challenges

I must admit that coordinating a research project from the other side of the Atlantic Ocean is challenging, with for instance having late-night meetings due to time zone differences (that was expected). In addition, the current pandemic has increased the complexity. Since a lot of lab materials were required for covid, purchasing reagents became really hard.
Nevertheless, some good news arrived recently; after several months or waiting, our project was approved by the Peruvian ethical committee. Finally, we were allowed to recruit volunteers! And we did it!

We are happy to announce that in total 112 volunteers participated in Laura! Stay tuned for future updates! 😊

References

  1. WHO. Ambient outdoor air pollution database, by country and city (xlsx file). Geneva: World Health Organization (WHO), Department of Public Health, Environmental and Social Determinants of Health; 2016.
  2. https://www.mimp.gob.pe/webs/mimp/sispod/pdf/63.pdf
  3. https://web.ins.gob.pe/sites/default/files/Archivos/cenan/van/sala_nutricional/sala_6/2016/01_Sala_Situacional_Alimentaria_Nutricional_2_Consumo_alimentario.pdf

Trans-Atlantic mentoring in NSURP 2021

Another pandemic-summer that wreaked havoc on opportunities of young scientists to not only enrich their learning but also provide real-world experience that enables them to put everything they have learned into action. Since many research institutions cancelled formal summer research experience programs, we were very excited to see that NSURP came back this year.

“NSURP is a community-driven initiative to create rewarding remote summer research opportunities for BIPOC (Black, Indigenous, People of Color) and Latinx undergraduate students in the microbial sciences.”

However, this year the core NSURP team of microbiologists (Dr. Michael D. L. Johnson, Dr. David A. Baltrus and Dr. Jennifer Gardy) that founded NSURP last year, managed to provide full-time paid internships through various sponsorships. And as you can read here, we truly enjoyed being part of the NSURP community last year. So we did not doubt for a second when we received the invitation to submit a project again and be matched with an enthusiastic mentee. And what a mentee? Jocelyn absolutely blew us away with her never-ending enthusiasm, out-of-the-box thinking and critical researcher-mindset.

Just like last year, we shaped the project together with the student based on the Lebeerlab research lines, my PhD and their interest. At the Lebeerlab, we truly believe that a hands-on approach to guiding students exponentially helps them to master technical and soft skills, gain essential knowledge and build a network. Together with partners-in-crime, post-doc Irina Spacova and prof. Sarah Lebeer we worked out the framework of the project and left plenty space for Jocelyn to be scientifically creative.

Again, I’m writing this blogpost as a super proud mentor!! Jocelyn truly gave it her all and delivered an exquisite end result. I genuinely enjoyed seeing her spark for microbiome research lit brighter and brighter each week. Just like last year, Jocelyn got a virtual “Welcome to our lab”-tour (inspired by MTV Cribs), met some lab members, gracefully presented her work in the Applied & Environmental Microbiology-section, and made a poster for upcoming conferences.  

Jocelyn, I’m wishing you very best for all your future endeavors and hope all your dreams come true. I also hope to see you soon in real-life, which totally gave me a déjà vu to saying the exact same thing last year. So maybe I should get to it and finally cross the pond 😊

Again a major thanks to the organizing committee of NSURP. I truly appreciate all the effort and organization that went into making this possible. #NoLabNoProblem


Name: Jocelyn De Paz
Affiliation: Suffolk University Boston
Title project: The Influence of Riboflavin-Producing Bacteria on Women’s Health

Jocelyn’s testimony

“This summer I was admitted into NSURP and was matched with my mentor Sarah Ahannach at the University of Antwerp. When I got my acceptance email and connected with Sarah, I realized I had obtained my dream job. The reason being that I was not only going to complete my work on the human microbiome, but I also was going to assist in destigmatizing vaginal health. Both my passion for science and women’s health was tied into one, it presented me with a new avenue on how I could continue my future studies.

Within the program I assisted a clinical study for the Isala Project, titled: ‘Assessing Lactobacilli Viability, and Proliferation within the Female Microbiome.’ Completing this project, I learned much about the workflow of intervention studies from the planning to the methods on how samples are collected and analyzed. I never thought that I was going to have the chance to create a clinical trial proposal so early on in my career. I truly appreciate all the opportunities Sarah and the Lebeer lab had to offer and the guidance they have given me. I am honored to continue to work with them on completing a mini literature review in collaboration with another student entering the lab.

Actively working on these two projects I gained so much knowledge about vaginal health and how studies on the female microbiome and female health are highly needed. These projects also exposed me to the ways I could grow my career in microbiology to continue expanding the knowledge on women’s health. I obtained amazing soft skills that I am eager to continue utilizing and expanding as I pursue my graduate school career. I will always be grateful for this program as it gave me the chance to grow as scientist and a mentee. I want to give a great thank you to everyone that made all this possible. A special thank you to prof. Sarah Lebeer, dr. Irina Spacova, and Sarah Ahannach for all their assistance, support, and great mentoring, I am eager to continue working with you all within the upcoming months!”

Eat, sleep, autoclave, repeat…

For several months now, my university is in code red due to the high cases of corona infections and deaths. In case you would have missed it, we’re in a global pandemic and that poses challenges for everyone. Code red means that theoretical courses cannot take place on campus and all students are following online classes. In this regard, I’m grateful that I still have the opportunity to get up every morning and go to the lab to perform my experiments. It is one of the few things giving me a routine and a sense of purpose in these strange times.

Though I really enjoy working in the lab, it is kind of odd, since there’s only half of the staff working. As a consequence, you can (not seldom) find us thesis students video calling our mentors to make sure we’re following the right protocol and we’re working with the correct tubes, boxes and machines etc. It is a bit of a weird sight, though! To limit the spreading of SARS-CoV-2, we are also not allowed to eat together or hold big meetings in the same room. Consequently, all co-workers are spread over the entire floor to follow the online meetings to discuss recent advances in our work and brainstorm about future possibilities.

A rare benefit of the current situation is that I can manage my course material better. Since I work a lot in the lab in Antwerp, and my courses would normally have been given in Leuven, there was no possibility to attend all the courses live and, in the meantime, perform all my planned experiments. Now, I can access all the recordings of the lessons online and process them at my own pace. So, in this regard, it is not all bad, I guess. Nevertheless, it can get quite lonely. Social isolation is real, for any age group and what I experience form my immediate environment it that it is taking its toll on students as well. All we really have to do is to perform at the moment when it is expected from us. And although we, as second masters, have enough maturity and study experience to make it work this way, without any form of relaxation, it gets pretty fast you, yourself and your books (plus your not easily transformable lactobacilli) for the rest of the year. As ‘proof of concept’, most of my classmates I have only recently met at the exam, which was the biggest social event this academic year since March 2020, thus that says a lot! Notwithstanding, health comes first, and we need to keep doing the same old until things get better and the virus loses its grip in the society.

All I want to say is that I’m looking forward to my vaccine and to more normal times, but isn’t that true for everyone?

Lactobacilli and Bioengineers, a relationship under tension

My last semester as a master student ‘Cellular and Genetic engineering’ has recently started. At this moment of my study, I have had countless lectures about how we scientists can genetically alter micro-organisms to not only study them and try to understand them, but also to implement them in all kinds of sustainable applications. Thus, theoretically I should now be capable of translating any bio-application you can think of into a complex working genetic circuit, eventually resulting in an organism that does exactly what it was intended for. So, bestowed with all this knowledge and a healthy confidence, I threw myself on my master thesis. The research objective of my project is the functional characterization of specific genes in different lactobacilli. These bacteria are the favorite bacteria of the Lebeer lab, living at mucosal surfaces of the human body, including our skin, respiratory tract, gut, urogenital tract etc. Lactobacilli are considered beneficial for normal human functioning and one way to acquire them is via food. The lactobacilli that I am currently investigating were previously isolated form either carrot juice fermentations during the ‘Ferme Pekes’ project or from healthy human vaginas in the ‘Isala’ study. In short, I need to figure out why these specific lactobacilli are so interesting for us and which genes make them so interesting.

Starting the project, I was already picturing all the possible real-life applications that could stem from my research, a new probiotic, an innovative bioactive compound, a cure for cancer or HIV, world peace … who knew? My enthusiasm quickly met the reality of the situation. Lactobacilli aren’t all keen to cooperate with me on this. In fact, they do their utmost best to resist all my efforts to introduce genetic constructs, while this is necessary to elucidate their special traits. So, with a profound reality check, it was time to get back to the basics of genetic engineering!

Genetic engineering altogether comprises an extensive set of techniques to modify DNA either randomly or at very specific sites. One thing that most of these techniques have in common, is that they depend on the introduction of little pieces of DNA along with some molecular cut/paste tools (1.). Once inside, these tiny tools help me to find my genes of interest and specifically alter them by changing their code. For example, by cutting some parts out or replacing them by a self-designed code, the gene becomes disrupted. Because of this, the bacterium cannot access it anymore what causes it to lose a specific characteristic. And so, a mutant is born! By then comparing this mutant with the unaltered bacteria (called ‘wild-type’) in all kinds of tests, I can check which trait my mutant has lost. This way, genetic engineering helps to link genes to bacterial characteristics, enabling us to understand them better. However, most bacteria are naturally not really inclined to take up any additional genetic information from the environment, such as my tools and constructs. This is more scientifically termed as ‘transformation resistant’. Since this is the first critical step to carry out any type of genetic modification, it forms a major bottleneck for their further investigation. Until I can get the constructs inside, I cannot modify them, and I cannot link their traits to their genes. Luckily, scientists have evolved ways to give nature a hand. But, while some bacteria only need a little push, others require complete treatments and still others will stubbornly continue to resist any type of transformation. Unfortunately for my colleagues and me, most lactobacilli belong to these last two categories, what definitely complicates the relationship with our beloved research object.

The most common way to get any type of large molecule into a bacterial cell is by electrotransformation1. In short this means that by giving bacteria a strong electropulse, we can make little holes in their cell walls3.  As long as these pores are present, the cells are made permeable and a passage for the uptake of DNA is available2. However, randomly firing some electrical pulses at a bacterial culture will most often not seal the deal. In fact, most bacteria need a thorough preparation by which their cell wall is temporarily weakened and more willingly to become electropermeable3. In this respect, the major challenge for lactobacilli is that these preparation protocols are quite variable between species, and even between strains3. Finding the right electrotranformation protocol is therefore often done via a time-consuming trial and error method. Nonetheless, it remains an extremely important step in successful genetic manipulation. Hence, a large part of my work this year at LAMB consists of the optimization of the electrotransformation protocol of the earlier mentioned fermentative and vaginal lactobacillus strains.  

Although this resistance of lactobacilli towards artificial transformation slows down our ability to elucidate their function in the human body and fermented foods, I also must highlight that this does not inherently mean something bad. Whereas it implies that under natural conditions these bacteria are also not prone to take up undesired or dangerous genetic information, such as antibiotic resistance genes or virulence genes. This genetic robustness makes them safer and favors their use in fermented foods and probiotics.

References:

  1. Börner, R. A., Kandasamy, V., Axelsen, A. M., Nielsen A. T., Bosma, E. (2019) ‘Genome editing of lactic acid bacteria: opportunities for food, feed, pharma and biotech’, FEMS Microbiology Letters, 336(1), doi.org/10.1093/femsle/fny291
  2. Luft, C. and Ketteler, R. (2015) ‘Electroporation Knows No boundearies: The Use of Electrostrimulation for siRNA Delivery in Cells and Tissues’, Journal of Biomolecular screening, 20(8), pp. 932-942. doi:10.1177/1087057115579638.
  3. Wang, C., Cui, Y. and Qu, X. (2020) ‘Optimisation of electrotransformation (ETF) conditions in lactic acid bacteria (LAB)’, Journal of Microbiological Methods, 174 (march), p. 105944. doi:10.1016/j.mimet.2020.105944.

NSURP 2020 #NoLabNoProblem

An often overlooked aspect of this pandemic is the unfortunate loss of unique opportunities for young scientists to cultivate a deeper love for science, develop new skills, connect with (inter)national mentors, grow their professional networks, and open doors to new opportunities. University labs, companies and other institutions normally organize summer projects to enrich young researchers’ learning.

“NSURP is a community-driven initiative to create rewarding remote summer research opportunities for BIPOC undergraduate students in the microbial sciences.”

However, due to COVID-19, most summer programs were cancelled and the impact of these disruptions are especially profound on BIPOC (Black, Indigenous, People of Color) students. The National Summer Undergraduate Research Project (NSURP) is a grassroots project started by a team of microbiologists (Dr. Michael D. L. Johnson, Dr. David A. Baltrus and Dr. Jennifer Gardy) who recognize the importance of summer research experiences and who want to support BIPOC students in finding their love of science in those projects. NSURP is designed to:

  1. Matchmake BIPOC undergraduate students on a (inter)national level to microbiology laboratory mentors who can provide a remote summer research project experience.
  2. Provide a BIPOC science and professional development seminar series for those students.
  3. Provide a platform for the matched students to present their research online in an official capacity.

Total matched NSURP students in the summer of 2020

I had the incredible opportunity to be matched with two amazing students (Vanessa and Gabriel). We first had an introductory meeting about the research done at the Lebeerlab, my PhD and their interests. The next step was to work out a feasible summer project in which they could flourish, learn about a different side of microbiology and be free to incorporate their own intake. A big thanks to our post-doc Irina Spacova is here at place for helping out with the framework of the projects and the co-supervision 😊.

Both students chose to work on a literature review about two entirely different subjects (cfr. testimonies of students below). We planned a Skype meeting every week to discuss the subject, their progress and generally just talk about our lives and the academic world. A funny anecdote is that we only had a few overlapping hours as our time zones were very different (Eastern Standard Time versus Central European Time) but we made it work. Next, we even organized a virtual “Welcome to our lab”-tour (inspired by MTV cribs) where I showed them around our lab with the occasional meet & greet with a lab member.

Granted, the remote guidance was challenging but over the course of a few months, I can fondly say that I am genuinely proud of the way they worked out their projects; the in-depth knowledge they generated; and the critical scientific attitude they built. At the end both, Vanessa and Gabriel, finished an excellent mini-review and presented their work gracefully for prof. Sarah Lebeer. I am wishing them a bright future and the very best for all their new ventures. Hope to see you both soon in real-life 😊.

A big thanks goes out to the organizing committee of NSURP. I truly appreciate all the effort and organization that went into making this possible.

Testimony NSURP students

Check out their presentations and abstracts

Name: Vanessa Ibrahim
Affiliation: Harvard College
Title project: Microbial fingerprinting of trace evidence in forensic research with a focus on sexual assault cases

“This summer I had the amazing opportunity to work under Sarah and learn more about her work with the Isala project. More specifically, my project was to conduct a literature review on the human microbiome and its potential in forensic casework, particularly in cases of sexual assault. Through the process of researching for and writing this literature review, I was able to take away a lot of key concepts including how prevalent cases of sexual assault are worldwide, how next-generation sequencing has paved the way for the field of microbial forensics, and how body-fluid identification using microbial fingerprinting could be a valuable tool in the prosecution of sexual assaults. Special thanks to prof. dr. Sarah Lebeer, dr. Irina Spacova, and Sarah Ahannach for all of their guidance over the last few months!”

Name: Gabriel Aborisade
Affiliation: University of Maryland
Title project: When a commensal becomes a pathogen: the role of microbe-microbe interactions with GBS as a model organism

“I participated in NSURP because I wanted to gain more experience writing literature reviews related to microbial research.  I also aimed to organize my research thoughts and transfer skills accrued for future use, such as graduate programs and fellowships/grants applications.

During the program, I completed a guided literature review titled “When a commensal becomes a pathogen: the role of microbe-microbe interactions with group B Streptococcus as a model organism.” I thoroughly enjoyed the experience; though it was daunting at times due to my workload outside the program, I persevered and pulled through due to my mentor, Sarah, incessantly following up and encouraging me. Constructive feedbacks I received were also quite helpful. I got to see where I stood in terms of writing a quality academic paper. I was also able to practice my time management skills and use my persistence and work ethic.

I was quite happy with my progression throughout the NSURP program. I got to see myself grow in knowledge and skill. For instance, at the beginning of the project, I found it difficult briskly going through a journal and deducing the main point and deciding if it would be useful for my project or not but over the weeks, I got better at that by day. I also got better at utilizing my primary resources: my mentor, Sarah, google scholar, and my university’s online library database.

Practically, the project provided me something different in terms of doing research. My undergraduate research experiences mostly focused on doing bench lab work and field sampling, but this was the first time I spent so much time on the internet digging for journals relevant to my research topic. Furthermore, I felt my participation also made me a more exciting candidate for my graduate school applications. Though not 100% in line with my graduate school focus, my review topic was intriguing, and faculty members I conversed with listened with interest, asked questions, and picked more interest in me. 

I am incredibly grateful for this opportunity. After further reflection, I realize that this experience has built on my molded interest in microbial research and asserts my desire to pursue an academic career. This experience may seem to be only valuable academically, but I find it incredibly valuable personally.”