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Can Human Milk Oligosaccharides Neutralize Clostridium difficile Toxins?

August 21, 2022 10 min read

HMO & C. Difficile Toxins Biding

Human bacterial infections are common, and a healthy human immune system is equipped with the tools needed to fight off most infections. However, some bacterial strains can be deadly, evade the immune system, or resist antibiotic treatment.

Bacterial infections are transmitted by eating or drinking something contaminated, from person to person, or from the environment. To combat them, antibiotics are often prescribed which work by attacking and killing the invading bacteria. Unfortunately, broad-spectrum antibiotics, the type that targets many types of bacteria, also kill off the good bacteria present within the gastrointestinal (GI) tract. Because of this, there is a risk of giving bad bacteria the opportunity to grow in numbers and become pathogenic (disease-causing).

If these antibiotics are given when they are not needed it can result in the creation of antibiotic-resistant bacteria. So, if antibiotics are administered, the invading bacteria, who have grown wise to the antibiotic, will be able to continue flourishing while the beneficial microbes are killed off. In light of this, there is a need for alternative therapeutic approaches to tackling bacterial infections[i].

One bacterial infection which has developed new, highly toxic and drug-resistant strains is Clostridium difficile. In this article, we will be looking at a research study conducted by El-Hawiet et al(2011)[ii] which investigated the use of human milk oligosaccharides (HMOs) as potential inhibitors of C. difficile to suppress its disease-causing capabilities.

What is Clostridium difficile?

First isolated in the mid-1930s, C. difficile (originally Bacillus difficilis) is a Gram-positive, strictly anaerobic, and spore-forming bacterium. That means it can exist in harsh environments in an unresponsive state until the environment becomes suitable[iii].

It is naturally found in the large intestine and is present in many people. C. difficile is usually kept in check and in small numbers by other harmless bacteria, but when these other harmless or commensal bacteria are absent or low in numbers, typically after antibiotics, it can grow in large numbers and become pathogenic. As a result, C. difficile infection can lead to several mediated diseases from diarrhoea to pseudomembranous colitis, where the lining of the large intestine becomes severely inflamed due to the toxins C. difficile produces[iv].

In fact, C. difficile infection is the leading cause of hospital-acquired diarrhoea in the United States and Europe. It is also a re-emerging pathogen which can cause serious infection and its new more virulent strains are affecting even low-risk populations[v]. Hence why new therapeutic approaches are required.

The implications of C. difficile infection in humans

By the late 1970s, C. difficile had been identified as the main cause of pseudomembranous colitis and most incidences of antibiotic-associated diarrhoea[vi]. This discovery sparked several studies to be conducted analysing the connection between C. difficile and antibiotic-resistant diarrhoea. Collectively, these showed that C. difficile is a pathogenic bacterium that is able to cause severe disease in the gastrointestinal tract, especially in individuals undergoing antibiotic treatment[vii] [viii] [ix] [x] [xi] [xii] [xiii] [xiv].

However, the rise of a new and more virulent strain of the bacterium has led to an increase in morbidity (disease) and mortality (death) because of its ability to enhance the amount of toxins it can produce, contributing to a more serious diseaseii. Although the infection has previously been treated relatively successfully with antibiotics such as metronidazole and vancomycin (examples of broad-spectrum antibiotics), the new, powerful, and toxic strains render these treatments ineffective. Equally, recurrent C. difficile infections are common because of the continued disruption of the gut microbiome through antibiotic treatment.

C. difficile is responsible for producing six types of toxins (specific poisons) that can lead to toxin-mediated gastrointestinal diseases. There are two particular exotoxins (a polypeptide toxin released by a living bacteria) that are known to be lethal. These are known as C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB)[xv].

Both exotoxins belong to a wider clostridial glucosylating toxin family which use a molecular mechanism that’s able to target eukaryotic cells (cells containing hereditary information) using bacterial protein toxins and effectors. Both exotoxins share 47% amino acid sequencing but have different cytotoxic mechanisms (way to cause cell injury) because of differences in substrate specificity and structurei.       

Both Tcd A and Tcd B are large polypeptides with structures that can catalyse the transfer of glucose to the eukaryotic cell via their highly repetitive receptor bindings, this process leads to a disrupted cytoskeleton and ultimately kills the host cell[xvi].

Because of the emergence of these new toxic C. difficile strains and the imbalance caused to the gut microbiome following antibiotics, new treatments are needed to prevent C. difficile from colonising in the GI tract and to neutralise the cytotoxic effects of TcdA and TcdB.

So, it was proposed that host receptor cell analogues (similar cells) in different forms could prevent the TcdA and TcdB exotoxins from binding to the epithelial cells of the intestines. Human milk oligosaccharides (HMOs) have been shown to prevent the attachment of various bacterial and viral infections in human cells, like Streptococcus pneumoniae and Haemophilus influenzae.So, could they help to prevent C.difficile infection too?

Why was the study by El Hawiet conducted?

HMOs are known to protect newborn babies from various diseases, but they have been shown to block the attachment of infectious diseases to host cells[xvii] [xviii]. Plus, fucosylated (modified) oligosaccharides from human milk protect the young from a toxin produced by Escherichia coli and prevent Campylobacter jejuni from binding to the human epithelial cells[xix] [xx] [xxi].

HMOs act like prebiotics, sources of food that help to nourish the beneficial bacteria in your gut and have a selective advantage towards these healthy or good bacteria over pathogenic types. Equally, another part of their protective armoury is their similarity to the glycans pathogenic bacteria utilise to attach to the host’s epithelial cells, promoting the growth of beneficial gut bacteria and aiding in the prevention of pathogens binding to the host and causing disease[xxii].

As a result of previous research findings, this study looked at the binding of 21 HMOs (the most abundant types found in human milk) to fragments of both TcdA and TcdB. This was investigated by using direct electrospray ionization mass spectrometry (ES-MS) assay[xxiii]. Simulations were then carried out and cytotoxicity neutralization was performed to see the potential of the HMOs inhibiting the binding of TcdA and TcdB.

The results

The 21 HMOs (L1-L21) were each attached to specific fragments of each exotoxin; the A2 fragment of TcdA (TcdA-A2) and the B1 fragment of TcdB (TcdB-B1). Their affinities were quantified at 25 degrees and at pH 7. HMO L8, due to specific attributes, was chosen to undertake detailed tests using a referenced protein method, to examine nonspecific binding[xxiv].

The results showed that both the A2 and B1 fragments of the toxins bind to several of the investigated HMOs. The B1 fragment has a single carbohydrate binding site and A2 showed two equivalent carbohydrate binding sites[xxv] and are able to bind specifically to HMOs of varying sizes.

The ES-MS results point towards both A2 and B1 binding to several of the HMOs that were investigated. Of the 21 HMOs that were tested, the A2 fragment has a measurable affinity for eight of them (five neutral and 3 acidic HMOs), while B1 binds to 11 HMOs (all the neutral types except two and two acidic ones). However, neither of these fragments had a measurable affinity for lactose.

The results also showed that five of the neutral HMOs were recognised by both A2 and B1 which means there could be some structural similarity in the ligand binding sites of the two toxins. It also means there could be some common natural human receptors that are recognised by both A2 and B1.

The study found that the binding of HMOs to the fragments was weak across the board. Ultimately, the study concluded that HMOs did not inhibit the effects of the cytotoxins of TcdA and TcdB. This was due to the weakness of the intrinsic affinities that the toxins show towards HMOs.

Since the study by El-Hawiet et al(2011) more recent studies have been conducted, investigating the relationship between TcdA and TcdB and HMOs, and the possibility of using HMOs to inhibit the binding of the toxins from pathogen C. difficile.

  • A study conducted by Nguyen et al (2016), also found that individual purified HMOs weakly bound to the binding site of TcdA. However, they did find that binding an HMO mixture to the TcdA binding site was more efficient than a single purified HMO.
  • A further study by Vigsnaes et al(2022) assessed the antipathogenic effects of human milk oligosaccharides, specifically 2’-O-fucosyllactose (2’FL), lacto-N-neotetraose (LNnT), and a mix of both, against difficile.The study showed that HMOs may have the potential to prevent the recurrence of C. difficile infections, modulate the gut microbiome following antibiotic therapy, and ultimately lessen the adverse effects that antibiotics can cause[xxvi]

Conclusion

Clostridium difficile is the leading cause of hospital-acquired diarrhoea. It’s usually treated with broad-spectrum antibiotics which target many types of bacteria including those that provide you with health benefits. This has led to an increase in antibiotic-resistant bacterial strains as well as some that are hypervirulent and unresponsive to antibiotic therapy.

C. difficile is an example of a bacterium that has evolved new, more potent strains. Hence the need for a different therapeutic approach. The study by El-Hawiet et al(2011) looked at the potential of HMOs as C. difficile inhibitors using 21 of the most abundant HMOs found in human milk.

Overall, the study found that the binding of the toxin fragments included in the study to the HMOs was weak. It was also noted that HMOs do not significantly prevent the cytotoxic effects of TcdA and TcdB. However, later studies have shown that an HMO mixture may be more efficiently bound to the toxins compared to a single purified HMO, but more research needs to be conducted.

 

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. 

References

[i] Wollein Waldetoft K, Brown SP. Alternative therapeutics for self-limiting infections-An indirect approach to the antibiotic resistance challenge. PLoS Biol. 2017 Dec 28;15(12):e2003533. doi: 10.1371/journal.pbio.2003533. PMID: 29283999; PMCID: PMC5746204.

[ii] El-Hawiet A, Kitova EN, Kitov PI, Eugenio L, Ng KK, Mulvey GL, Dingle TC, Szpacenko A, Armstrong GD, Klassen JS. Binding of Clostridium difficile toxins to human milk oligosaccharides. Glycobiology. 2011 Sep;21(9):1217-27. doi: 10.1093/glycob/cwr055. Epub 2011 May 24. PMID: 21610194. 

[iii] Barer M, Irving W, Swann A, Perera N. Medical microbiology.

[iv] Salen P, Stankewicz HA. Pseudomembranous Colitis. [Updated 2021 Nov 15]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK470319/

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[vii] Aronsson, B., R. Mollby, and C. E. Nord. 1981. Occurrence of toxin-producing Clostridium difficile in antibiotic-associated diarrhea in Sweden. Med. Microbiol. Immunol. (Berlin) 170:27-35.

[viii] Bartlett, J. G., F. J. Tedesco, S. Shull, B. Lowe, and T. Chang. 1980. Symptomatic relapse after oral vancomycin therapy of antibiotic-associated pseudomembranous colitis. Gastroenterology 78:431-434.

[ix] Burdon, D. W., R. H. George, G. A. Mogg, Y. Arabi, H. Thompson, M. Johnson, J. Alexander-Williams, and M. R. Keighley. 1981. Faecal toxin and severity of antibiotic-associated pseudomembranous colitis. J. Clin. Pathol. 34:548-551. 

[x] Don, G. J., and A. E. Davis. 1981. The association between antibiotic- associated diarrhoea and C. difficile toxin in children. Aust. N. Z. J. Med. 11:433-434.

[xi] George, W. L., R. D. Rolfe, G. K. Harding, R. Klein, C. W. Putnam, and S. M. Finegold. 1982. Clostridium difficile and cytotoxin in feces of patients with antimicrobial agent-associated pseudomembranous colitis. Infection 10:205-208.

[xii] Tedesco, F. J. 1981. Antibiotic-associated colitis-an abating enigma. J. Clin. Gastroenterol. 3:221-224. 

[xiii] Meyers, S., L. Mayer, E. Bottone, E. Desmond, and H. D. Janowitz. 1981. Occurrence of Clostridium difficile toxin during the course of inflammatory bowel disease. Gastroenterology 80:697-670.

[xiv] Willey, S. H., and J. G. Bartlett. 1979. Cultures for Clostridium difficile in stools containing a cytotoxin neutralized by Clostridium sordellii antitoxin. J. Clin. Microbiol. 10:880-884.

[xv] Carter GP, Chakravorty A, Pham Nguyen TA, Mileto S, Schreiber F, Li L, Howarth P, Clare S, Cunningham B, Sambol SP, Cheknis A, Figueroa I, Johnson S, Gerding D, Rood JI, Dougan G, Lawley TD, Lyras D. Defining the Roles of TcdA and TcdB in Localized Gastrointestinal Disease, Systemic Organ Damage, and the Host Response during Clostridium difficile Infections. mBio. 2015 Jun 2;6(3):e00551. doi: 10.1128/mBio.00551-15. PMID: 26037121; PMCID: PMC4453007.

[xvi] Voth DE, Ballard JD. 2005. Clostridium difficile toxins: Mechanism of action and role in disease. Clin Microbiol Rev. 18:247–263.

[xvii] Andersson B, Porras O, Hanson B, Lagergard T, Svanborgeden C. 1986. Inhibition of attachment of Streptococcus pneumoniae and Haemophilus influenzae by human milk and receptor oligosaccharides. J Infect Dis. 153:232–237.

[xviii] Newburg DS, Ruiz-Palacios GM, Morrow AL. 2005. Human milk glycans protect infants against enteric pathogens. Annu Rev Nutr. 25:37–58

[xix] Cravioto A, Tello A, Villafan H, Ruiz J, Delvedovo S, Neeser JR. 1991. Inhibition of localized adhesion of enteropathogenic Escherichia coli to Hep-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast-milk. J Infect Dis. 163:1247–1255.

[xx] Ruiz-Palacios GM, Cervantes LE, Ramos P, Chavez-Munguia B, Newburg DS. 2003. Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection. J Biol Chem. 278:14112–14120.

[xxi] Korpela K, Salonen A, Hickman B, Kunz C, Sprenger N, Kukkonen K, Savilahti E, Kuitunen M, de Vos WM. Fucosylated oligosaccharides in mother's milk alleviate the effects of caesarean birth on infant gut microbiota. Sci Rep. 2018 Sep 13;8(1):13757. doi: 10.1038/s41598-018-32037-6. PMID: 30214024; PMCID: PMC6137148.

[xxii] Walsh C, Lane JA, van Sinderen D, Hickey RM. Human milk oligosaccharides: Shaping the infant gut microbiota and supporting health. J Funct Foods. 2020 Sep;72:104074. doi: 10.1016/j.jff.2020.104074. Epub 2020 Jul 3. PMID: 32834834; PMCID: PMC7332462.

[xxiii] Rizzarelli P, Zampino D, Ferreri L, Impallomeni G. Direct electrospray ionization mass spectrometry quantitative analysis of sebacic and terephthalic acids in biodegradable polymers. Anal Chem. 2011 Feb 1;83(3):654-60. doi: 10.1021/ac102579q. Epub 2011 Jan 4. PMID: 21204561.

[xxiv] Sun JX, Kitova EN, Wang WJ, Klassen JS. 2006. Method for distinguishing specific from nonspecific protein–ligand complexes in nanoelectrospray ionization mass spectrometry. Anal Chem. 78:3010–3018.

[xxv] Dingle T, Wee S, Mulvey GL, Greco A, Kitova EN, Sun JX, Lin SJ, Klassen JS, Palcic MM, Ng KKS, et al. 2008. Functional properties of the carboxyterminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile. Glycobiology. 18:698–706.

[xxvi] Vigsnaes LK, Ghyselinck J, Van den Abbeele P, McConnell B, Moens F, Marzorati M, Bajic D. 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. Pathogens. 2021 Jul 22;10(8):927. doi: 10.3390/pathogens10080927. PMID: 34451391; PMCID: PMC8402123.


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