The Antipathogenic Effects of HMOs against Clostridioides difficile

November 16, 2022 9 min read

HMO fight with Clostridioides difficile

The human gut is a world within us inhabited by populations of bacteria that call it home, some good and some bad. But what defines good or bad? To answer this, we need to delve into the world of gut flora.

Gut flora is a term used for all of the microorganisms that inhabit the gut. From the moment you arrive on the planet, there is an assortment of billions of bacteria vying for residency within you. If there is a penthouse suite in the gut, you’ll want friendly neighbours in there, neighbours that will protect you should an undesirable come calling.  

In general, your bacterial colonies can be good or friendly bacteria or bad (pathogenic) bacteria. Good bacteria like Bifidobacteria or Lactobacilli,amongst others, are needed in large numbers because they keep the gut in order and offer much-needed protection. They achieve this because, in large numbers, they take up space, eat undigested food sources, like fibre, change the intestinal pH, and release pathogenic inhibiting or killing metabolites. Collectively, these factors keep the number of potentially harmful pathogens like Clostridioides difficile (C. Difficile) (formerly Clostridium difficile) down, which is key to control within the gut[i].

In this article, we will explore the antipathogenic effects of two human milk oligosaccharides (HMOs), 2’-O-fucosyllactose (2’FL) and lacto-N-neotetraose (LNnT), against Clostridioides difficile. Using a study conducted by Vigsnaes et al.(2021) entitled 2’FL and LNnT Exert Antipathogenic Effects against C. difficile ATCC 9689 In Vitro, Coinciding with Increased Levels of Bifidobacteriaceae and/or Secondary Bile Acids, we will look at how this is achieved, and what results are being produced[ii].   

What is Clostridioides difficile?

You’re probably already familiar with this pathogen because, in 2016, it went through a name change from Clostridium difficile to Clostridioides difficile.The reason for this is that the species was transferred from the genus Clostridiumto Clostridioides because of the slight taxonomic differences. Although that may seem confusing, thankfully, Clostridioides still begins with a C, so the pathogen can still be referred to as C. difficileor C. difffor short.

  1. difficileis a normal member of the gut microbiome, but its growth is suppressed by other dominant bacteria. However, when it has the opportunity to flourish and multiply, it can become very disruptive and cause C. Difficileinfection (CDI).
  2. Difficileis a Gram-positive, spore-forming anaerobe. The spore-forming characteristic of this bacterium is what causes CDI, which advances in stages. These are:
  • transmission
  • germination
  • toxin production

Overall, these stages, particularly the last one, will affect the colonic epithelium to varying degrees, meaning the effects of the infection can range from slight diarrhoea to perforation of the colon and even death[iii].

One of the key reasons for the development of CDI is dysbiosis. Dysbiosis is when the gut flora becomes imbalanced, such as after a course of antibiotics. In a bid to oust a bacterium that has caused health issues, the antibiotic wipes out our good colonies, too[iv].

This allows numbers of undesirable characters such as C. diff that have, over time, developed antibiotic resistance to multiply into health-threatening numbers and can even lead to potentially life-threatening colitis. So, there is a real need to find alternative ways of addressing such diseases[v].

Symptoms of C. difficileinfection

  • mild to severe diarrhoea

  • high temperature (fever)

  • loss of appetite

  • feeling sick (nausea)

  • stomach ache[vi]

Complications of C. difficile

Due to the antibiotic-resistant capabilities of C. Difficile, there is a high chance of recurrence of CDI. This means that treatments once considered successful for ridding the body of the infection, like antibiotics, now carry substantial risks[vii].

Antibiotic resistance means that bacteria can, in response to the use of these medicines, become resistant to their effects. In other words, infections like C. diffcan still survive and thrive even if you are prescribed antibiotics which could once kill off the infection.

It’s a common myth that humans become antibiotic-resistant. We don’t. It’s the bacteria themselves that become antibiotic-resistant.

Health benefits of HMOs

Human milk oligosaccharides (HMOs) are very diverse and complex structures. They are non-digestible carbohydrates that are represented by over 200 structures. Because they are non-digestible, they can travel through the gastrointestinal tract and arrive at the required location intact, ready to deliver their prebiotic goodness to your commensal gut bacteria[viii].

HMOs have a composition of 5 monosaccharides; galactose (Gal), N-acetylglucosamine (GlcNAc), and glucose (Glc) are the foundations, and fucose (Fuc) or sialic acid (Sia) residues mean they can either fucosylated or sialylated.

Once in the colon, the HMOs enhance the growth of the beneficial Bifidobacteriagenus. These bifidogenic effects have been demonstrated in breastfed babies as well as adults following the consumption of HMOs and have been associated with numerous health advantages[ix]. For example, previous studies into irritable bowel syndrome (IBS) have provided positive results after the use of HMOs, such as the reduction of debilitating symptoms[x].

HMOs are responsible for the modulation of the gut microbiota, having been shown to support the development and response of the immune system as well as improve the function of the gut barrier. Alongside this, HMOs have been seen to affect the cell responses within the intestine and prevent pathogens from adhering to the epithelial cells. HMOs achieve this by binding to both epithelial receptors and pathogenic toxins preventing attachment to the mucosal surface by pathogens[xi][xii][xiii].

What was the research by Vigsnaes et al. about?

Vigsnaes et al. (2010) investigated the antipathogenic effects of human milk oligosaccharides, specifically 2’FL and LNnT, against Clostidioides difficile.The study allowed researchers to investigate the effects of these HMOs against CDI as well as their effects on microbial activity during antibiotic treatment.

How did Vigsnaes et al. conduct their research?

The antipathogenic effects of the HMOs were investigated using a combination of two in vitro gut models.

A reference sample strain of C. Difficilewas obtained that had been isolated from the faeces of an asymptomatic neonate. The strain was known to produce toxins TcdA & TcdB. After a series of processes to grow the sample, a subsample was taken and subjected to DNA extraction. The strain was confirmed by searching for the sequence this process produced against the RDP 16S rRNA gene database. Another subsample was taken, from which a spore stock was created after incubation for test 2.

Test 1 measured the impact of a single dose of HMOs on the levels of C. diff. The HMO test products, 2’FL, LNnT and 4:1 mixture of 2’FL and LNnT mix, were each added to faecal slurries prepared from faecal donations from three healthy volunteers. The donors had no prior history of gastrointestinal disorders and had not received antibiotics in the prior three-month period. These were then strongly diluted to represent dysbiosis and allow C. difficileto flourish.

Three experiments were then performed, one for each sample, where they were mixed with the HMOs and incubated for 48hrs. The HMOs were 2’FL, LNnT and a combination mix which was 4 parts 2’FL and 1 part LNnT (MIX). A blank sample was also collected throughout for each of the donors, where no HMOs were added. Samples of which were taken at 0/24/48 hrs.

During the second test, which was 11 weeks long, the long-term Pathogut™ model was used. It is a model that simulates the human intestinal microbial ecosystem. Treatments used were Vancomycin (antibiotic) and the HMO 2’FL and MIX. The model allows the C. difficilecycle to be mimicked and dysbiosis induced through clindamycin treatment, leading to infection with C. diff.Vancomycin, a type of antibiotic, was administered, which is known to treat the infection but is associated with recurrent infection. The test was used to assess the impact of the HMOs on microbial activity and composition during and after vancomycin treatment.

What were the results?

Overall, the results showed that all the HMO products used in the study showed strong antimicrobial effects against C. difficileduring the 48-hour incubations (test one). The researchers noted a significant increase of Bifidobacteriaceaeat 24 hours for LNnT and 48 hours for 2’FL, suggesting that this bacterial family may be involved in the fermentation of HMOs and possess the antimicrobial powers needed against CDI.

This was supported further by the increased levels of acetate at 48 hours. Acetate is an important short-chain fatty acid (SCFA) and an important cross-feeding metabolite for butyrate producers. Bifidobacteriaare also known to be potent producers of acetate. Therefore, these results suggest that the antipathogenic effects of HMOs may be exerted through their ability to boost the abundance of Bifidobacteriaceaespecies.  

In test 2, the Pathogut™ model, clindamycin (antibiotics) had been used to induce dysbiosis and CDI. The infection was then treated with vancomycin (antibiotic), a standard treatment for C. difficileinfection. Supplementation of 2’FL with vancomycin avoided the recurrence of the infection during the 3-week washout period but this couldn’t be attributed to the increase of the Bifidobacteriaceaefamily because they were eradicated following treatment with clindamycin.  

Instead, it is believed that the antipathogenic effects exerted against C. difficileduring the second test are likely because of increased production of the secondary bile acid, deoxycholic acid (DCA). DCA was exclusively produced following the co-supplementation of 2’FL.

Further research has shown that DCA, one of the most abundant secondary bile acids, can inhibit the germination and growth of C. difficile[xiv].In this study, a bacterial species related to Lachnoclostridium likely produced DCA in response to an increased presence of 2’FL.

Supplementation using the 2’FL and MIX showed a boost in microbial activity as well as acetate, propionate and butyrate levels. These HMOs also showed an increase in the abundance of Bacteroidetes,Firmicutes,and Verrucomicrobia. There was no recurrence of CDI with the 2’FL treatment, which could have been due to increased secondary bile acid production.

It was concluded that HMOs have the capability to exert antipathogenic effects against C. difficileinfection. The Pathogut™ study also demonstrated that during antibiotic treatment, HMOs increased microbial activity, leading to increased production of SCFAs and secondary bile acids. The study also highlighted that HMOs could combat CDI recurrence with the supplementation of 2’FL and could bring a positive reverse to the adverse effects of antibiotic treatments.   


Due to the extensive use of antibiotics over the past decades, there has been a developing resistance growing in potentially life-threatening pathogens. Because of this, there is a need for alternative treatments to combat this threat.

Human milk oligosaccharides, in particular, 2’FL and LNnT, have shown very encouraging results in this in vitro study by Vigsnaeset al . (2010).

As the study concluded, HMO supplementation produces antipathogenic effects against C. difficile. Although the antipathogenic mechanisms of action may not be complete, it also showed that some HMOs could halt CDI recurrence after antibiotic treatments.

It is exciting to see what future research will show as the potential of human milk oligosaccharides is explored further.

Want to help keep your gut microbiome in check and unleash the bifidogenic effects of HMOs? Try our range of PureHMOs.


Written by: Leanne Edermaniger, M.Sc. Leanne is a professional science writer who specializes in human health and enjoys writing about all things related to the gut microbiome. 


[i] Zhang YJ, Li S, Gan RY, Zhou T, Xu DP, Li HB. Impacts of gut bacteria on human health and diseases. Int J Mol Sci. 2015 Apr 2;16(4):7493-519. doi: 10.3390/ijms16047493. PMID: 25849657; PMCID: PMC4425030.

[ii] pathogens-10-00927-v2[3131].pdf

[iii] Gilca R, Frenette C, Thériault N, Fortin É, Villeneuve J. Attributing cause of death for patients with Clostridium difficile infection. Emerg Infect Dis. 2012 Oct;18(10):1707-8. doi: 10.3201/eid1810.120202. PMID: 23017180; PMCID: PMC3471622. 

[iv] Zhang S, Chen DC. Facing a new challenge: the adverse effects of antibiotics on gut microbiota and host immunity. Chin Med J (Engl). 2019 May 20;132(10):1135-1138. doi: 10.1097/CM9.0000000000000245. PMID: 30973451; PMCID: PMC6511407.

[v] Spigaglia P, Mastrantonio P, Barbanti F. Antibiotic Resistances of Clostridium difficile. Adv Exp Med Biol. 2018;1050:137-159. doi: 10.1007/978-3-319-72799-8_9. PMID: 29383668.


[vii] Song JH, Kim YS. Recurrent Clostridium difficile Infection: Risk Factors, Treatment, and Prevention. Gut Liver. 2019 Jan 15;13(1):16-24. doi: 10.5009/gnl18071. PMID: 30400734; PMCID: PMC6346998.

[viii] Bering SB. Human Milk Oligosaccharides to Prevent Gut Dysfunction and Necrotizing Enterocolitis in Preterm Neonates. Nutrients. 2018 Oct 8;10(10):1461. doi: 10.3390/nu10101461. PMID: 30297668; PMCID: PMC6213229.

[ix] Bode, L. Human Milk Oligosaccharides: Prebiotics and Beyond. Nutr. Rev. 2009, 67 (Suppl. 2), S183–S191.

[x] Iribarren, C.; Törnblom, H.; Aziz, I.; Magnusson, M.K.; Sundin, J.; Vigsnaes, L.K.; Amundsen, I.D.; McConnell, B.; Seitzberg, D.; Öhman, L.; et al. Human Milk Oligosaccharide Supplementation in Irritable Bowel Syndrome Patients: A Parallel, Randomized, Double-Blind, Placebo-Controlled Study. Neurogastroenterol. Motil. 2020, 32, e13920.

[xi] Šuligoj, T.; Vigsnæs, L.K.; den Abbeele, P.V.; Apostolou, A.; Karalis, K.; Savva, G.M.; McConnell, B.; Juge, N. Effects of Human Milk Oligosaccharides on the Adult Gut Microbiota and Barrier Function. Nutrients 2020, 12, 2808.

[xii] Donovan, S.M.; Comstock, S.S. Human Milk Oligosaccharides Influence Neonatal Mucosal and Systemic Immunity. Ann. Nutr. Metab. 2016, 69 (Suppl. 2), 42–51.

[xiii] Triantis, V.; Bode, L.; van Neerven, R.J.J. Immunological Effects of Human Milk Oligosaccharides. Front. Pediatr. 2018, 6, 190.

[xiv] Usui Y, Ayibieke A, Kamiichi Y, Okugawa S, Moriya K, Tohda S, Saito R. Impact of deoxycholate on Clostridioides difficile growth, toxin production, and sporulation. Heliyon. 2020 Apr 13;6(4):e03717. doi: 10.1016/j.heliyon.2020.e03717. PMID: 32322715; PMCID: PMC7160582.

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