Author: Alice Cadman

Editing the genomes of our gut bacteria will “create a whole new field of biology” in the coming decades, a Nobel prize-winning geneticist has said at the opening of a London summit on the future of human genetic engineering.

Jennifer Doudna, from the University of California Berkeley, said that tweaking the DNA of the bacteria that live in our guts has the potential to help us understand and combat many diseases in humans.

Conditions from Alzheimer’s to asthma have been linked to the composition of our microbiome but the population of our gut is difficult to study and treatments designed to alter its composition so far are imprecise and based on taking faecal transplants.

Yet, said Doudna, there was a clear medical and scientific need to control the microbiome. “Microbiomes are increasingly indicated in all sorts of connections to human disease,” she said. “So for example, we know that there’s an important role of the human gut microbiome in diseases that include infections, and even neurodegeneration.”

Doudna helped create the precision editing technology known as Crispr, which has revolutionised the field of genetics, and could, she said, now be applied here too.

Crispr is going to enable not only some exciting individual applications, but really I think it will create a whole new field of biology because it’s going to open the door to understanding how these microbiomes behave in a way that has not previously been possible,” she said.

And not just in human guts. She argued that the technology might also have applications in animals, for instance by creating cows that produce less methane, a greenhouse gas. “In livestock, there is a powerful connection between the microbiome found in the cow rumen and the generation of methane. And surprisingly much of it is actually exhaled from these cows. It leads to about 30 per cent of the global annual emissions of methane,” Doudna said.

The possibilities for understanding and controlling gut bacteria offered by Crispr editing meant we should take it seriously. It was plausible, though, that it would first see genuine success in a different animal. “Having the ability to manipulate the cow rumen microbiome using Crispr would be an extraordinary advance, because we could actually change this in a calf, that would have an altered microbiome over the course of its entire lifetime,” she said.

Preliminary work suggested this was feasible, and had some surprising additional benefits, she said. “It reduces the methane emissions, which is very important. But also, it’s attractive to farmers because it means that there’s more efficient conversion of feed into food.”

Doudna was speaking in an opening video message for the 2023 Human Genome Editing Summit at the Francis Crick Institute in London. Delegates from around the world will consider the scientific, technical and ethical implications of their work. The field has rapidly matured in the past decade, thanks in a large part to Doudna’s technology.

Crispr works like molecular scissors, giving scientists the ability to seek out a strand of DNA, chop it out and replace it. Although editing of genomes was possible before, it was vastly more cumbersome.

Since its development in 2012, Crispr has become a standard laboratory tool, as well as a source of promising therapies. Among the other topics under consideration were the success of Crispr-based treatments to tackle sickle cell anaemia, a crippling genetic blood disorder. Approaches based on gene editing have produced significant success but the cost is prohibitive for much of the world, which is important because the condition is historically most prevalent in equatorial populations.

Agri-TechE’s REAP conference took place on November 8th 2022 to review how the meeting of real and virtual worlds is creating a new generation of technologies that are ‘making sense of agriculture’ and that can provide the intelligence needed to take the best action on-farm. Every year Agri-Tech E handpicks some of the most exciting early-stage agri-tech companies to take part in the REAP Start-up Showcase.

This year’s Start-up Showcase included FOLIUM Science’s carve out investment opportunity FLOURISH, a new type of crop protection and seed treatment that will improve yields and boost productivity.

FLOURISH makes ‘bad’ bacteria self-destruct to create a healthy microbiome

Bacterial crop diseases cause devasting losses. Some are controlled by antibiotics, which results in damage to beneficial organisms in the microbiome and the risk of resistance. FLOURISH offers an alternative plant protection strategy. By disrupting the metabolism of the pathogen, it enables natural competition to create a healthy growing environment, which boosts yield.

Ed Fuchs is a Co-founder of FLOURISH, a carve-out from FOLIUM Science, which has developed a Guided Biotics® technology for animal health and now has a number of product candidates ready for commercialisation via multinational feed additives distributors, following successful safety assessments by regulators.

Ed comments: “Plants extract nutrition from their environment using complex interactions with microorganisms in the soil. The importance of this microbiome is increasingly recognised in human and animal health.

“Our technology uses either bacteria or phages (virus particles) that are highly selective for a particular type of bacteria, such as Pseudomonas, to share a specific DNA sequence with the target that redirects its immune system, causing the pathogen to self-destruct. As the sequence is very specific and targeted to the particular bacteria it will not damage organisms that are vital for plant growth. This process therefore gives the beneficial organisms a competitive advantage.”

In trials with tomatoes the use of Guided Biotics® was shown to reduce Pseudomonas syringae and Botrytis cinerea by more than 99.9% and double the weight of the tomato yield.

Ed explains:“Our first product candidate uses an epiphyte (a naturally occurring bacteria from the tomato plant), to share the DNA sequence which gets rid of the Pseudomonas Syringae. The epiphyte also naturally produces molecules to remove Botrytis (fungus) and the combination enables the yield improvement.”

Over 12 million hectares of tomatoes are grown globally and they are at risk of bacterial canker which is caused by Pseudomonas syringae. Canker is also a serious disease of stone fruits such as cherries and peaches, showing the technology has a significant global market across many disease targets.

The use of phage particles is already regarded as safe by major territories and is being increasingly used in diagnostics and medication. The ‘reprogramming’ uses the gene editing technology CRISPR, which is recognised as distinct from genetic modification as it does not introduce alien DNA into the organism. Regulatory reform already underway is likely to support the wider use of this technology.

Ed continues: “FOLIUM has benefited from funding and due diligence from Innovate UK; with the necessary funding it has the potential to be spun-out as a plant-focussed company addressing a global crop protection market valued at £50bn.

“FOLIUM benefited greatly from the profile gained at the REAP 2018 Start-Up Showcase and I hope to repeat this success with FLOURISH.”

Dr Belinda Clarke, Director of Agri-TechE, comments: “Antibiotic resistance is a global ‘One Health’ problem and FLOURISH is offering an innovative alternative. I am sure that it will attract significant interest from the agri-tech innovation ecosystem. REAP has provided profile for many exciting early-stage businesses with collaborators, end users and investors. 14 of the companies previously featured have collectively raised over £92 Million in the last three years.”

FOLIUM Science, alongside other leading UK science and technology businesses is proud to be part of an initiative that showcases the UK’s world-class science and research capabilities to address global challenges and build a future which is greener, safer, and healthier.

The brochure has been produced by the Department for International Trade (DIT) and is to be launched at the World One Health Congress in Singapore and IHF World Hospital Congress in Dubai.

The aim of the initiative is to attract investors and collaborators from around the world. The UK offers one of the strongest and most productive health, life, agricultural and environmental science sectors in the world with a high concentration of world leading talent, reference laboratories, innovative research programmes, and ample opportunities for value creation through partnering, deal-making or investment.

FOLIUM Science is well placed to tackle two of the global challenges
highlighted by the ONE HEALTH initiative. Antimicrobial Resistance (AMR) and climate change are both enormous challenges for animal farming. The animal’s gut bacteria are central to both gas emissions and an unhealthy microbiome.

FOLIUM Science’s patented technology, “Guided Biotics®”
improves gut health by removing unwanted bacteria and regulating its metabolism. Guided Biotics® reduce the need to use antibiotics, reduce GHG emissions and improve productivity for our farming community.

Bristol based biotech business FOLIUM Science has completed an important milestone in its commercialisation phase as it continues to develop its ground-breaking technology platform that selectively removes unwanted bacteria from an animal’s gut.

This patented technology, called Guided Biotics®, will remove the need to use antibiotics in farmed animals. In a world where antimicrobial resistance is a global problem, where antibiotics are increasingly ineffective, unacceptable to consumers and restricted in use, FOLIUM Science’s revolutionary biotechnology helps to solve these problems by supporting positive animal nutrition and sustainable animal farming.

In collaboration with a leading multinational animal nutrition partner, FOLIUM Science can now build on promising in-vivo trial work and begin the process of bringing the first product to market.

Using the Guided Biotics® technology, trials in poultry flocks have shown incredibly promising results in reducing Salmonella. Food poisoning continues to be a problem across the world, with salmonellosis cases now increasing in many countries. Non-typhoidal salmonellosis is reported to cause over one million infections, 19,000 hospitalizations and over 400 deaths annually in the US, with some Salmonella strains showing antibiotic resistance.

Salmonella in the gut of a chicken is difficult to control, however unlike the action of antibiotics that will kill the good bacteria in the gut as well as the bad, Guided Biotics® selectively remove only the undesirable bacteria, leaving the beneficial bacteria intact. This supports a positive gut microbiome by allowing these beneficial bacteria to thrive.

Guided Biotics® technology represents a new category in animal feed additives and functional nutrition, that can directly benefit animal well-being by supporting a healthy microbiome.

The development of modelling techniques to quickly assess the effectiveness of new products is an innovative tool that allows the FOLIUM Science team to screen alternatives and identify the versions that are most likely to be successful. This creates an efficient product delivery process that can be taken forward into live trials.

Creating strong and focussed teams in each development area has also proved fruitful where technical specialists and support staff work closely together to share ideas and improve expertise across the business.

The new partnership and investment will enable FOLIUM Science to move to the next stage of commercialisation and not only develop the application of Guided Biotics® technology in poultry but continue the development of future programmes that include applications in cattle, swine and aquaculture. Development platforms in the pipeline also include products to modulate an animal’s microbiome to give the friendly bacteria beneficial advantages over pathogens in the gut.

FOLIUM Science CEO Ed Fuchs says “I am delighted to have achieved this breakthrough which was part of the strategic roadmap created 4 years ago when the business was founded.  Our goals and focus on the Guided Biotic Platform are fully aligned to the multinational partner’s animal nutrition business unit. This validates the market opportunity and unlocks well established capability to deliver Guided Biotic products in market. 

It is a credit to the FOLIUM Science team how they have evolved the Guided Biotic platform to application in the most challenging real-world environment.  We can also celebrate our own expansion of capacity in the move to new laboratory facilities at Science Creates in Bristol.  These facilities enable us to achieve our vision and expand into modulating gut microbiomes to reduce waste and improve productivity.

The founders are also excited to be working on similar technology for food processing, plant health and human dietary requirements.  These applications are a mere walk in the park!”   

The joint development agreement involves an undisclosed sum for a multi-year investment and commercialisation rights to BiomElix One®, a feed additive for poultry and other species that targets all Salmonella serotypes.

The benefits of working with a major multinational with a very strong research ethic  are clear. The collaboration between both teams of scientists on product development alongside shared contacts and expertise will bring advantages to all stakeholders. The manufacturing facilities and regulatory know-how offered by the partnership will also have a significant impact on the speed with which products can be brought to market and facilitate the advancement of new platforms.

The FOLIUM Science team of scientists will continue to operate from the dedicated research base in Bristol UK enabling the business to further expand the capabilities of Guided Biotics® across wide range of pathogenic, wastage and spoilage bacteria.

TRISTAN COGAN, HOLGER KNEUPER, HADDEN GRAHAM and MARTIN WOODWARD present a trial for a new CRISPR-based patented technology introduced into a vector Escherichia coli probiotic designed to selectively remove all Salmonella serovars from the bird gut.

Over the past few decades the meat, egg and milk sectors have faced the need to reduce the routine use of antibiotics in animal production, and the high incidence of food poisoning  associated with animal product consumption. Approximately 130,000 tonnes of antibiotics were used in 2013 worldwide, with 75% of this in animals. Up to 90% of these antibiotics can be excreted into the environment via urine and feces, and approximately 400 resistance markers against 25 antibiotics can be found in chick caecal bacteria. Globally, around 700,000 human deaths per annum are attributed to antibiotic resistance and this is predicted by the FAO to increase to 10 million by 2050. With rising concern about the development of antibiotic resistance in human health, regulators, consumers and retailers have led the drive to reduce the sub-therapeutic use of antibiotics in animal feeds to zero. Endemic disease is re-emerging, adding costs to animal production systems and driving the need for alternative non-antibiotic interventions.

Food poisoning continues to be a problem across the world, with salmonellosis cases now increasing in many countries. Non-typhoidal
salmonellosis is reported to cause over one million infections, 19,000 hospitalizations and over 400 deaths annually in the US, with some Salmonella serovars in food showing antibiotic resistance. Although salmonellosis incidents are traditionally relatively low in Australia, recent egg-associated outbreaks have brought this back to the attention of the regulators and consumers. It is now possible to cause a targeted bacterium to self-destruct through the use of CRISPR, the biological sequences that make up the bacterial immune system. This technology is extremely precise, such that it can target a specific bacterium or a defined range of bacteria. This means that, unlike many antibiotics, it can be used to remove only the unwanted bacteria in the animal gut microbiome and leave beneficial gut flora unchanged, potentially enhancing the well-being of the animal. One way to induce bacteria in the animal gut to self-destruct is to introduce a suitable plasmid into the target organism(s) through conjugation via a probiotic included in the feed or drinking water. The
current trial looks at the ability of this technology, named Guided Biotics, to reduce Salmonella colonization in challenged broilers.

A non-pathogenic Escherichia coli strain was used as the vector in this trial, and was loaded with a plasmid including a CAS sequence and 3 target sequences specific to all Salmonella serovars. Ross 308 as-hatched birds (165) were obtained on day of hatch and housed under controlled biosecure conditions, with access to water and standard commercial
rations ad libitum.

Birds were dosed continually from day 1 with either:
1) No addition to water (45 birds)
2) Unmodified E. coli vector at 108
cfu/mL drinking water (45 birds)
3) Anti-Salmonella Guided Biotics
at 108 cfu/mL drinking water (45
birds)

In parallel, a group of 30 birds was dosed orally with 0.5 ml 105 CFU/ mL Salmonella Enteritidis strain FS26 on day 1. Birds were checked for Salmonella colonization at day 3 by
cloacal swab (ISO 6579-1:2017).
On day 5, three verified Salmonella[1]colonized birds (seeder birds, with 105 CFU/g in swabs) were marked and added to each of the test groups. Fifteen non-seeder birds from each group were euthanazed on day 12 (7 days post-mixing with seeder birds) and caecal contents were counted for Salmonella using both direct and enhanced methods. Caecal samples were serially diluted in PBS before plating onto XLD agar for direct counts, whilst for enhanced  counts the samples were first incubated in Selenite Cystine broth for 18 hrs at 41o C before plating and counting (ISO 6579-1:2017). For the purpose of data transformation, samples
negative in either method were allocated a count of 1 CFU/g, while those negative in direct counts but positive in the enhanced method were allocated 500 CFU/g. Body weights of the remaining birds were monitored at day 42. Counts and weights were log transformed and statistical analysis conducted using GraphPad Prism. Data were assessed for normality of distribution using a D’Agostino and Pearson omnibus normality test and non-normal were analysed using a Kruskall-Wallis test with Dunn’s multiple comparison test post hoc.  Differences were analyzed using Fisher’s exact test.
Results
All birds in the seeder group showed cloacal Salmonella counts of 105 CFU/g by day 3. By day 12 (7 days post introduction of seeder birds to test groups) all birds in the Control and E. coli vector-only groups were positive using the enhanced counts method, exhibiting caecal counts of 500-4,000,000 CFU/g.
Twenty two of these 30 birds were also positive with direct count. However, when the anti-Salmonella Guided Biotics was added to the drinking water, Salmonella was not detected in any birds with the direct method, and only 8 of the 15 birds tested were positive with enhanced counts. The Guided Biotics treatment reduced (p < 0.001) mean Salmonella counts by approximately log-3 (from log 4.12 to log 1.26, equivalent to 14,200 CFU/g to 18 CFU/g) and also improved 42-day liveweight by 15% (p = 0.02; Figure 1). Discussion The challenge method employed in this study is consistent with that often use in Salmonella vaccine tests and may be regarded as severe. All seeder birds were infected when introduced into the test pens, and the Salmonella shed to in-contact birds would be expected to be highly infective. This was confirmed by the universally high caecal counts in all Control birds 7 days after seeded-bird introduction. Conversely, the Guided Biotics, delivered by conjugation in the digestive tract, was able to stop Salmonella colonization in 8 out of 15 (53%) of the test birds. The average Salmonella count in caecal digesta was also reduced by approximately log-3 (thousand[1]fold) and the maximum Salmonella count lowered from 4 million CFU/g in Control birds to 500 CFU/g in Guided Biotics treated. The 15% increase in liveweight of birds fed the Guided Biotics relative to the Control birds further indicates the severity of the Salmonella challenge employed in this trial. The lack of any effect of the E. coli vector on colonization confirms that the Guided Biotics plasmid was essential for Salmonella reduction. This initial trial establishes the capability of Guided Biotics technology to specifically remove unwanted bacteria, in this case a single Salmonella serovar. The tested Guided Biotics is designed to target all known 2,400 Salmonella serovars, and laboratory trials have established efficacy across the main serovars involved in human food poisoning. Ongoing laboratory tests have also indicated that solutions for other unwanted bacteria, such as Clostridium perfringens and Avian Pathogenic E. coli, are feasible.
Furthermore, because the design of the targeting is specific, tests have confirmed that off-target killing of desirable or commensal bacteria can be avoided.
Conclusion
It is clear that this Guided Biotics technology has the potential to make a substantial contribution to the replacement of antibiotics in poultry production, reduce zoonosis incidents
and maintain bird performance in antibiotic-free diets.