Bacteriophage Therapy

Bacteriophage are viruses that infect bacteria. They are the hitch hikers in fecal microbial transplantation. Some research suggests that these hitch hikers are just, if not more, important than the actual fecal bacteria.

What is Fecal transplantation?

Fecal transplantation has been all the rage in humans suffering from intestinal issues. There are now fecal transplants for dogs. These links are general information. In leading up to why fecal transplantation for dogs, Pilla (2020) discuss the use of carbohyrates in the production of “kibbles” that make up 95% of the dog food market. They also discuss the gut as the source of most of the serotonin in a dog. Some the dog makes from the amino acid precursor tryptophan and the balance produced by intestinal bacteria. Short chain fatty acid production by bacteria and the health of the dog was also discussed in the Pilla (23020) review. The review also mentioned the use of fecal transplantation in dogs to replace pathogenic microorganisms. Socała and coauthors (2021) wrote a nice review on the role of intestinal bacteria in the operation of the brains of we humans in health and disease. This image is part of Figure 1 and sums up a large portion of the discussion of rodent recipients of feces from humans with neurological issues.

This review also went into serotonin, short chain fatty acids as well.

  • Pilla, R., & Suchodolski, J. S. (2020). The Role of the Canine Gut Microbiome and Metabolome in Health and Gastrointestinal Disease. Frontiers in veterinary science, 6, 498. PMC free article
  • Socała K, Doboszewska U, Szopa A, Serefko A, Włodarczyk M, Zielińska A, Poleszak E, Fichna J, Wlaź P. The role of microbiota-gut-brain axis in neuropsychiatric and neurological disorders. Pharmacol Res. 2021 Oct;172:105840. free article

What is Bacteriophage therapy?

What is a bacteriophage is a good place to start. As mentioned previously, bacteriophage is a virus that infects bacteria.

Finding the perfect review article to sum up our resident bacteriophage is difficult. Lawerence (2019) is a good place to start.

The Lawrence review as a source of references

“Variation between phageomes of individuals has been shown to be even greater than that of bacteria. Even with this extreme variability, the phageome (as genes are to the genome, individual phage are to an enormous population) appears to be highly stable, perhaps more so than the associated bacteria in the gut, even in the presence of strong modifications to the microbiota, such as fecal microbiota transplant. Given their ability to evolve rapidly, the human gut likely contains dozens of “novel” phage species within a single individual. However, even with this high degree of mutation and evolution, it appears a “core virome” exists in the gut, providing temporal stability by interacting with the core bacteria found across humans.” This statement may explain why pre- and probiotics do not always work. The Lawrence (2019) review covers the virome/phageome of the skin, mouth, bladder, and vagina in addition to the GI tract. Three references are particularly interesting when it comes to

Broaker (2017) Hitch hiking phage are the stable members of an FMT

Broecker (2017), followed a patient who was cured with a FMT in two weeks after a prolonged Clostridium dificile chronic infection.

Some highlights of figure1 Broecker (2017) Pfam is a database of protein families. The data for individual phage parts are not shown.

Do these data really suggest that bacteriophage hitch hiking in the FMT are more stable than he erratic interchange of families of intestinal bacteria? At the end of 4.5 years, the former former Clostridium difficile patient resembled the donor. In the first 7 months, the phage characteristics were always close. Is this because no matter what phage one has, the sequence for the capsid, integrase, tail, and so on are always close?

Minot (2012) Hyper-variable loci

A locus, plural loci, is the physical site of a gene on a chromosome. Minot and coauthors isolated phage out of 0.5g feces from 12 healthy, non obese, donors. They used a low speed centrifugation step and the 0.22 micron filter that we plan to use. Minot et al added an additional cesium chloride centrifugation step because they were isolating nucleic acids for sequencing. This step set a limit on the size of the phage. They used a sequencer to read large numbers of circular and linear pieces of DNA and then pieced together sequences based on overlapping reads. Here is part of their Fig. 1

Most of Figure 1 from Minot (2012)

An example of a hyper variable region comes from our own immunoglobin genes that code for the antigen binding regions of our antibodies. An open reading frame (ORF) is simply the protein coding part from the start translation into protein) to stop codon. Recall that a codon is the three nucleotides that code for an amino acid of a protein.

Here is a quote: “Protein functions were inferred by comparing the conceptual translation of predicted ORFs to a curated database of protein families. A broad range of viral functions were identified in the encoded proteins (Fig. 1B), …. On average, 72% of the open reading frames (ORFs) did not resemble any recognizable protein family, emphasizing the immense diversity of novel genes in gut viral populations.” It is very encouraging that Minot et al only found one human virus in these 12 donors: human papilloma virus type 6b. These investigators found hyper variable regions that only vaguely resembled known proteins

  • Six encoded proteins that have similar sequences to cadherins, invasins, and fibronectins. These contain Ig-superfamily β-sandwich domains.
  • Three hypervariable ORF has leucine rich repeat structures. One of these had a type C lectin fold in the hyper variable region. Lectins are proteins that bind carbohydrates.

After two years of Covid-19 spike protein mutations, we get the idea of hyper variable in mysterious fecal bacteriophage proteins could have an impact on our GI flora.

What is a healthy Phage-ome anyhow? Manrique (2016)

Here is n image from this six year old deep sequencing publication of phage from three different locations.

from Manrique 2018

What types of bacteria does Virus 375 like to infect compared to Virus 873? How does this play out in predator/prey ecological models? How does diet determine whether these phage are dormant in the host bacteria versus under the right environmental stress to cause lysis? We may presume that a Western diet probably is most available to these 64 donors, each of which would probably have access to a more Asian diet in their respective cities.

  • Lawrence, D., Baldridge, M. T., & Handley, S. A. (2019). Phages and Human Health: More Than Idle Hitchhikers. Viruses, 11(7), 587. PMC free article
  • Broecker F., Russo G., Klumpp J., Moelling K. (2017) Stable core virome despite variable microbiome after fecal transfer. Gut Microb. 2017;8:214–220. PMC free article
  • Minot S., Grunberg S., Wu G.D., Lewis J.D., Bushman F.D. Hypervariable loci in the human gut virome. Proc. Natl. Acad. Sci. USA. 2012;109:3962–3966. doi: 10.1073/pnas.1119061109. PMC free article
  • Manrique P., Bolduc B., Walk S.T., van der Oost J., de Vos W.M., Young M.J. Healthy human gut phageome. Proc. Natl. Acad. Sci. USA. 2016;113:10400–10405. PMC free article

Onward to a 2021 global view

A team from Lawrence Berkeley National Laboratory (Nayfach 2021) assembled the Metagenomic Gut Virus catalogue that comprises 189,680 viral genomes from 11,810 publicly available human stool metagenomes. Double stranded DNA phage represented over 75% of genomes that infect members of the Bacteroidia and Clostridia classes. The authors identified 54,118 candidate viral species, 92% of which were not found in existing databases. For those who need a refresher on phylogenetic trees, this Wikipedia link offers a nice refresher. In this diagram we are looking at an “unrooted tree” in which no common ancestor is inferred. The distance between branch points is taken as distance in the degree of relatedness.

from Nayfach 2021

Nayfach (2021) devoted a lot of their discussion to error prone “reverse transcriptases” as means of introducing new mutations in the everlasting war on bacteria. Reverse transcriptases take RNA to double stranded DNA. This post is not going to get into how this happens in phage with DNA as the genetic carrier.

  • Nayfach, S., Páez-Espino, D., Call, L., Low, S. J., Sberro, H., Ivanova, N. N., Proal, A. D., Fischbach, M. A., Bhatt, A. S., Hugenholtz, P., & Kyrpides, N. C. (2021). Metagenomic compendium of 189,680 DNA viruses from the human gut microbiome. Nature microbiology, 6(7), 960–970. PMC free article
  • Camarillo-Guerrero, L. F., Almeida, A., Rangel-Pineros, G., Finn, R. D., & Lawley, T. D. (2021). Massive expansion of human gut bacteriophage diversity. Cell, 184(4), 1098–1109.e9. PMC free article

Prime time phage therapy

Luong and coworkers have published a method for enriching bacteriophage with targeted bacterial hosts. This seems kind of complicated to try on a dog who has messy feces and has been described as being “bipolar” by human fosters. Nayfach divided their phage into three groups according to their preferences in human intestinal bacteria. The dog situation could be different. Perhaps a tailored down version of Luong selection protocol could be utilized.

Luong, T., Salabarria, AC., Edwards, R.A. et al. Standardized bacteriophage purification for personalized phage therapy. Nat Protoc 15, 2867–2890 (2020). https://doi.org/10.1038/s41596-020-0346-0

Colonic Delivery