The Old New Method for Probiotics Enumeration

FISH, a method that relies on direct observation and immediateness, visually identifies live microbes, and discriminates bacteria to the species level.

The best discovery of the 10th Microbiome Congress that took place end of May 2022 at The Hague, for me, came from Jiri Snaidr, founder and CEO of Germany-based small company?vermicon.?Nothing to do with worms, vermicon stands for engineering (Verfahrenstechnik), microbiology, and consulting in German.

The problem

Jiri deplores that most academic and industry scientists report their findings on bacteria and the microbiome by relying on data.

“Modern technologies like -omics allow breakthrough outcomes, but sometimes miss out on the reality. Our field looks at 100 trillion small bacteria. Not just data, but Life. Pure Microbial Ecology.” — Jiri Snaidr, CEO of vermicon.

Probiotics manufacturers currently express the number of bacteria in their products in colony-forming units (CFU), based on culturing methods on Petri plates dating back to the end of the 19th?century or, the most modern ones, based on flow cytometry, a more immediate and reliable method based on analyzing each cell in a sample with a laser. This method, in comparison to plate count, enables a better discriminative power to identify the number of living, damaged, and dead cells in a sample. For more information about the two methods and their differences, you can refer to?this article.

Plate count is limited in many ways. The method doesn’t allow to grow all bacteria – indeed, in environmental samples?up to 99,9 % of all bacteria can not be cultivated; in microbiome research, it is expected to be about 75 % - and it is tricky for anaerobes and for novel species with specific nutritional requirements. Plate count is slow — before you see the dots on your Petri plate, you need to wait for several days (or sometimes weeks). It lacks precision because several separate bacteria can combine to create a single colony, leading to underestimating the real quantity of bacteria, which comes at a cost for manufacturers. It doesn’t account for dead bacteria or even those viable but non-culturable, although it is now clear that inanimate bacteria can have an effect.

A key limitation of flow cytometry is that the method is unable to identify bacteria, let alone different species in a sample. If microorganisms present a similar morphology, they will appear on the same cloud, indiscriminately, without much clue to their identity.

What FISH brings

Vermicon offers to close the gap on the microbiome puzzle thanks to a direct method of observation, enumeration, and identification — excluding bias from growing, sequencing, and computing.

The FISH technology (Fluorescence In Situ Hybridisation) is a molecular technology developed in the 80s based on the work of Carl Woese, the historic discoverer of Archaebacteria. Jiri and his colleagues at the Technical University of Munich applied it successfully to bacteria back in the early nineties, developed it further and were the first to apply it for the visualization of high genetic diversity in microbial communities. FISH allows the highly-specific detection of bacteria directly in the sample, including ex vivo, by fluorescence. It relies on the following pillars:

1. Phylogenetic analyses
2. Development of specific, fluorescently-labeled probes and reagents
3. Sample where the probes are applied
4. Observation of result

How does it work?

For every bacterial target (species, genus, or group) probes are designed to bind to specific sites that are unique to the target.

To shine a light on live bacteria, probes are designed to bind to the rRNA located in the ribosome, while the flashing of dead bacteria can be done with DNA binding. Why? Because rRNA is unstable so when the bacteria die, this ribosomal RNA quickly disappears. DNA being more stable, it will remain for a long time (think Jurassic Park).

The procedure starts with a fixation step, where the sample containing cells is treated with an alcoholic solution, which makes the cells porous and enables the fluorescently-labeled probes to penetrate the membrane. This kills the cells, but the status - the information on whether the cell was dead or living - will remain. With the subsequent hybridization procedure, the probes enter all cells, but only in the bacteria where they find their specific target sites are they able to bind and can not be washed away. After excitation with high-energy light, the bacteria start to shine. If red-labeled probes were designed for species A, all bacteria of species A would begin to shine in red. If there were green-labeled probes for species B, all bacteria of species B begin to shine green. This offers a real-time picture of the sample and the cells within it as well as their status - dead or alive - at the time of treatment.?

What are the benefits compared to the other methods currently in use?

Compared to plate count, FISH is able to retrieve any given bacterium in a sample, dead or alive, in a few hours, and gives information about the loci where they thrive.

Compared to flow cytometry, it can identify the bacteria, including different species in different colors in any given sample.

Compared to qPCR, it can discriminate live from dead cells, it is more reliable, more adaptable (the probes can be developed for a given level of taxonomic precision for each project), can pick up even small quantities of bacteria like tracked contaminants, and does not require highly educated personnel nor sterile rooms. Contamination is not a problem because there is no amplification step.

What is delivered to customers?

Vermicon proposes to support its customers with either a full-service model, proceeding to analyses themselves, including consulting and interpretation if needed, or the toolkit and training to support customers in becoming their own analysts. This comprises all the reagents required, the probes for the target bacteria and sites, the hardware to analyze the samples, and the training to use all of that.

What probes are readily available?

The company started in the sector of wastewater, then food and beverages, and recently approached the microbiome and probiotic supplements sector. It already proposes VIT?(for vermicon identification technology)?test kits for wastewater contaminants, drinking water, food and beverages, including lactic acid bacteria such as?Lactobacillus,?Pediococcus,?Leuconostoc,?Lactococcus,?Enterococcus?faecalis, and?Escherichia?coli.?Firms in the Live Biotherapeutic Products sector are?already testifying?of the value brought by their partnership with vermicon in R&D, manufacturing, characterization, and quality assurance for their products, like SymbioPharm for?E. coli?and OxThera for?Oxalobacter formigenes.

For species not listed in the catalog, the team is happy to develop the detection kits or services on demand.

Jiri Snaidr and his colleagues are proud to “make bacteria shine” and I wanted to share this bacterial light with you today.