Efficacy of Phage Therapy for Adherent – Invasive Escherichia coliStudent: Shanelle Gingras (7700506)

Lecturer: Dr. D. Bay

Topic: Can bacteriophages be effectively used to treat adherent invasive E.coli infections (AIEC)? Briefly review AIEC infections, virulence and invasive factors associated AIEC. Describe the problem of chronic Crohn’s disease and ulcerative colitis. What are the benefits/ limitations of using bacteriophages to treat gastrointestinal tract infections and are they the best answer for in vivo AIEC treatment?

Introduction:Adherent-invasive Escherichia coli (AIEC) is a pathobiont that in healthy individuals is a

symbiont within our intestinal tract, however under certain circumstance the bacterium causes inflammation and ultimately illness1,2. AIEC is classified based on its ability to adhere and invade intestinal epithelial cells, and to survive and replicate within macrophages, all while not displaying any currently known or detectable pathogenic invasive determinants1,2. AIEC can be found in a multitude of humans including healthy individuals, patients with irritable bowel disease, all ethnicities and both children and adults1. Humans are not the only animal AIEC is found in however, it has also been found in dogs with granulomatous colitis, cattle with bovine mastitis, and birds with avian colibacillosis, therefore it does not have a narrow host range1.

Along with a high host diversity there is also high clonal diversity. Many strains can be found in the B2 phylogroup, which have the most virulence factors, however some strains can be found in less virulent subtypes, such as A, B1, and D1. AIEC strains have been found to have sequence similarities in both phylogeny and virulence factors with extraintestinal pathogenic Escherichia coli (ExPEC), and specifically resembles the virulence factors of disease within urinary tract infections, neonatal meningitis, and avian colibacillosis1. AIEC and Disease:

Within AIEC genomes there are many genes for type 6 secretion systems, which may facilitate survival through excluding other bacteria or by protecting itself and sibling cells1,3. Type 6 secretions systems function similarly to an inverted bacteriophage puncturing mechanism, as they are evolutionary, structurally, and functionally related3. As we can see in Figure 1, 13 subunits come together in a syringe-like apparatus and substances are injected into other cells by puncturing the target cell with its tail spike and driving the tail tube through the baseplate using the contraction of the tail sheath3. The substances excreted will vary depending on the function, for example, if the type 6 secretion system is targeting the human’s intestinal epithelial cell they may want to excrete a substance that aids in adhesion or invasion3. However, if the type 6 secretion system is aiming to exclude other resident intestinal bacteria it would excrete a substance that would promote cell death, such as peptidoglycan hydrolases3.

AIEC might not have any pathogenic determinants, but there are many virulence factors including a capsule, lipopolysaccharide expression, iron uptake, genes for biofilm formation, and adhesion and invasive genes as the name suggests1. A key factor in AIEC virulence is the ability to survive and replicate within phagolysosomes within macrophages1. Macrophages are able to kill bacteria within the phagolysosomes using acidity, oxidative stress, and various enzymes and antimicrobial compounds, but AIEC strains are able to resist these mechanisms with stress and RNA binding proteins, thiol-disulfide oxidoreductase, and FAD-dependent oxidoreductase, respectively1. Adherence and Invasion:

In order to invade intestinal epithelial cells, AIEC must first adhere to the cells. AIEC uses type I pili (FimH) expressed on the cell surface of the bacteria to bind to the cell adhesion molecule 6 (CEACAM6) receptor on the intestinal epithelial cells1. In CD patients the intestinal epithelial cells upregulate CEACAM6, which further progresses colonization and therefore disease1. LF82, the reference strain for AIEC, has also been found increase the expression of CEACAM6 on the cell surface, which is an important virulence factor allowing the bacteria to increase its colonization of the mucosa1.Irritable Bowel Disease

AIEC has been implicated as a factor for irritable bowel disease (IBD), which includes Crohn’s disease (CD) and ulcerative colitis (UC), however the connection isn’t as clear in the case of UC1. Patients with CD and UC both experience diarrhea, abdominal pain and cramping, fever, bloody stools, and inflammation of the skin, joints and eyes4. There are some differences between the diseases as CD can occur anywhere within the gastrointestinal tract within all layers of the bowel walls and UC is restricted to the colon and the superficial layer of the bowel wall4. Another difference between the diseases is the consistency of infected tissue, as continuous inflammation is seen within UC patients where patients with CD can have intermittent inflammation4. Both diseases are multifactorial, with genetics, environment, and bacteria all playing a role in disease onset and persistence.

It is clear that IBD is an inflammatory disease, however it is still unknown whether AIEC initially colonizes the intestinal epithelial cells and therefore draws the inflammatory cells to the tissue, or if the inflammation was present to begin with and AIEC exploits this phenotype to colonize the tissue1.

IBD is currently not curable and can severely debilitate a patient. The inflammation within the bowels can cause severe narrowing of the intestinal wall leading to blockages, which must be removed in surgery5. Chronic inflammation within the intestinal tract can also lead to open sores in the form of ulcers and fistulas that have a potential for serious infection and may also need to be corrected with surgery5. Patients with IBD need to be cautious about their diet as malnutrition can arise due to diarrhea, fistulas, intestinal inflammation, and iron sequestering from AIEC5. The diet is also important for IBD patients because adequate levels of vitamin D allow for homeostasis with the mucosa of the gastrointestinal tract, protecting patients from bacterial translocation1. Diet also appears to play a roll with biofilm formation of AIEC due to maltodextrin that is a starch present in higher amounts within Western diets, which may be partially responsible for the increased disease within Westerners1.Host Defects:

As mentioned above, AIEC is not exclusively found in diseased individuals and part of their classification is based on the absence of pathogenic invasive determinants, therefore it appears as if disease is at least partially due to host defect1. Multiple alleles have been identified to be risk alleles for Crohn’s Disease (CD) including nucleotide-binding oligomerization domain-containing-2 (NOD2) which codes for a protein that interacts with the peptidoglycan found in the cell membrane of bacteria2. It appears as if the leucine-rich repeat domain of NOD2 is important for the binding of NOD2 to its ligand, as many of the risk alleles for CD are located within this region2. There are also two risk alleles, ATG16L1 and IRGM, which both encode for a protein involved in autophagy2. NOD2 is also implicated in autophagy as it interacts with ATG16L1 during bacterial infections, therefore variants within that gene can impact two important antibacterial pathways1.Autophagy defects are important because it causes inflammation through the decrease in efficiency for the sensing and handling of intracellular bacteria1. Specifically during AIEC infection, autophagy is induced in order to hinder replication and control the infection, therefore variants that impair this function are going to contribute to disease1.Bacteriophage:

Bacteriophages are viruses that infect bacteria and are more prevalent worldwide than any other organism6. They have two main forms of infection within their lifecycle, lysogenic and lytic, and an intermediate stage that occasionally happens before the bacteriophage diverges into one of the two main infections7. The lysogenic lifestyle is characterized by dormant infection

where the bacteriophage will integrate into the bacteria’s genome or exist as a plasmid while allowing the bacteria to live normally7. This stage can persists for thousands of generations and can even alter the phenotype of the bacteria by expression genes of its own in a process called lysogenic conversion7. When bacteriophages are in the lytic phase of their life cycle they infect and kill bacteria quickly, leading to a rapid change in the bacterial population present7.

Bacteriophages can be found within the environment where their target bacteria is present, with 10 bacteriophages present for every one bacteria in both fresh and salt water7. In more complex environments researchers have had trouble distinguishing an accurate ratio, however if niche environments are similar in representation it would be expected that there are approximately 10 times as many bacteriophages present for every on bacteria7.

Bacteriophages have been isolated from sediments that are decades old and some lab cultures stored in fridges for more than 4 decades haven’t reduced in concentration, however there are lab cultures that reduce in titre weekly, therefore not every bacteriophage is similar in terms of persistence outside of its normal environment, and quite possibly within their normal environment as well7. Case Study:

Galtier et al. investigated the efficacy of using bacteriophages to target AIEC strain LF82 within murine and human intestinal cells, to evaluate whether phage therapy was a potential treatment for CD8. The researchers used a cocktail of three phages (LF82_P2, LF82_P6, LF82_P8) isolated from waste water that demonstrated complimentary host ranges8. Using phages with complimentary host ranges is important because of AIEC strains are in multiple phylogroups and serotypes and phages have a very narrow host range, therefore to treat an infection that has more than one strain involved a phage cocktail is ideal8.

Two strains of LF82 were used to test the efficacy of phage therapy, one unmodified (LF82) and one modified to be resistant to streptomycin and kanamycin (LF82SK). Each of these strains were incubated on LB agar and the titre of the phage was determined by spotting the cocktail onto the lawn of bacteria8.

The phages were then investigated for multiplication rate within the intestines using gut homogenates of ileal, colonic, caecal, and faecal samples8. As we can see in Figure 2, all three phages were able to replicate within all samples, however the replication within the ceacal and fecal samples was not as high, especially for LF82_P28.

With confirmation that the three phages chosen both kill AIEC and are able to replicate within the intestinal environment ex vivo, Galtier et al. used a cocktail made up of the three bacteriophages in equal amounts to treat AIEC in CEABAC transgenic mice8. CEABAC transgenic mice express human CEACAM receptors, which make them a good model for human infection as AIEC uses CEACAM to adhere to the intestinal epithelial cells. The mice who received phage therapy saw a significant reduction in AIEC within fecal samples whether the administration of phage was given 2, 3, or 5 days post infection, which is demonstrated in Figure 38.

Galtier et al also tested the levels of phage present within fecal samples of mice infected with AIEC and mice uninfected to determine whether the phage persists when its host is not present in the environment8. The mice without LF82 infection had a complete reduction of phage down to undetectable levels, where the mice with LF82 infection had a gradual reduction with phage dissipating as less and less bacteria were available within the environment due to host lysis (Figure 3)8.

The researchers then investigated the reduction of colitis symptoms using the streptomycin and kenamysin resistant strain, LF82SK, in conventional mice with induced colitis symptoms from administration of 2% dextran sodium sulphate (DSS)8. For this experiment one group of mice was used as a control with PBS administration, one group as a preventative treatment with 3 X 107 pfu administered 8 days post infection, and one curative treatment group with the same dose being administered on 10 days post infection8.

The control group saw an increase in disease activity index score (DAI), which is an assessment score that takes into account weight loss, stool consistency, and blood in stool, from 1 point on day 7 to approximately 4 points on day 25 (Figure 4B)8. The preventative treatment at day 10 saw a reduced DAI in comparison to the control and returned to baseline levels days 14, 22, and 258, where the curative mice on day 10 (day of phage administration) had a DAI higher than 2, which subsequently fell approximately 1 point on day 14 and remained low for both 22 and 25 days8. To directly measure the level of LF82SK present within the mice fecal levels were collected for days 7 to 258. The control saw a consistent level of LF82SK present within the stool, while the preventative mice saw a reduction by 4 times, and the curative mice saw a reduction of 43 times. At day 25 the mice were sacrificed and ileal and colonic samples were tested for LF82SK infection and confirmed the decolonization of both ileal and colonic tissues with LF82SK (Figure 4 C,D)8.

All experiments ex vivo an in vivo in mice had promising results, however the results must be confirmed in humans in order to be a viable treatment, therefore the researchers used ileal biopsies from CD patients to confirm their above findings8. 6 patient samples were infected with LF82SK and then treated with the phage cocktail to determine if the phage was able to actively replicate within this environment and effectively kill AIEC8. 5 hours after phage administration the mean n – fold replication was determined to be 9.43 X 103 ± 2.07 X 104, which increased at 24 hours to 3.22 X 104 ± 6.31 X 103, therefore the phage cocktail was in fact able to replicate and therefore kill AIEC similar to the previous experiment within the murine model8.Benefits of Phage Therapy:

From the case study above it appears as if phage therapy for AIEC is a potential therapy for humans suffering from CD, but what are the benefits of choosing phage therapy for a bacterial infection?

One of the main highlights of phage therapy is the narrow host range of phages, consisting of sometimes only a few strains of a single species9. This narrow host range significantly decreases the off target killing of the resident microflora. Antibiotics do not discriminate against bacteria that may be commensals for the human body. Instead they are designed to target a certain mechanism within a particular bacterium and it will target that mechanism whether the bacteria is the target or not. There are many cases of illness following treatment with antibiotics, including Clostridium difficile and Candida albicans9. C. difficile is able to take over the gastrointestinal tract after antibiotics wipe out the bacteria that competitively interferes with the over growth or entry of C. difficile. Once colonized the bacteria is extremely hard to get rid of and has dire consequences, sometimes even death. In the case of Candida albicans the repercussions are not as serious as C. difficile, as yeast infections are able to be treated quite readily, but with the growing antibiotic resistance this may not always be the case. C. albicans colonizes the vaginal tract in a similar fashion to C. difficile with the gastrointestinal tract, as it is able to dominate the vaginal microflora after antibiotic use because

the resident microflora, primarily Lactobacillus, is no longer present to produce lactic acid, which excludes certain bacteria from surviving.

This narrow range is also beneficial in terms of resistance. With the phage only infecting a select few strains it reduces the amount of bacteria exposed to the microbial agent, which therefore limits the amount of bacteria that will have a chance to adapt a resistance mechanism9. Phages also do not use the same mechanisms to kill bacteria that antibiotics do, therefore bacteria that are resistant to current antibiotic treatments are still susceptible to phage treatment9.

The worry that bacteria will evolve mechanisms to combat phage infection, similar to how they have with antibiotics, is much lower than that of antibiotic resistance10. Bacteria do evolve quickly to survive, including anti – phage mechanisms, however phage adapt just as rapidly10. Two of the main anti – phage mechanisms present within bacteria are restriction – modification systems (RM) and CRISPR/Cas adaptive immunity10.

RM functions by modifying its own DNA using methylation and degrading any DNA that is not methylated, as this signals it is foreign10. Phages have adapted multiple ways to combat this system including methylation via host’s enzyme or enzyme of its own, proteins that target the enzyme involved in foreign DNA degradation, or through palindromic avoidance where the phage will no longer be recognized by the restriction enzyme as it binds to symmetrical sequences10.

CRISPR/Cas functions by creating a double stranded break in foreign DNA or RNA and taking segments of that genome and integrating it into the bacteria’s genome within spacers10. This allows the bacteria to quickly recognize an intruder in the future and directly bind to the sequence in order to cleave the DNA or RNA, however phages have been found to evolve their genome so that the sequence acquired in the CRISPR/Cas mechanisms can no longer recognize and bind to the genome10.

Another benefit of using phage treatment is self – replication. After bacteriophages infect their host, they begin assembling more viruses to release to the environment upon lysis of the host cell. This replication is termed auto dosing, as the phage itself contributes to the overall dose for the patient10. Auto dosing is great for limiting the amount of times patients have to self-administer treatment, which is a benefit for compliancy issues10. One of the major reasons antibiotic resistance has been increasing substantially in the last few decades is due to patients taking antibiotics until they are feeling better, but not finishing the full course of treatment. The bacteria are reduced to a level where disease symptoms subside, however there are still bacteria present that have been exposed to the antibiotic and are therefore able to create mechanisms to resist antibiotics in the future. With auto dosing, this is circumvented by not relying on the patient to properly complete the prescribed treatment.

Auto dosing is also is also a benefit of phage therapy from an economic standpoint. With a lower initial dose needed to treat a patient there will be less cost in time and development of larger batches of phage treatment, however an optimal level must be identified so that the patient’s recovery is the first priority10.

As we saw in the case study above, the replication of the phage is correlated with the number of hosts present in the environment1. This is a benefit as the phage will not continue to replicate within the body, or remain at the same level the patient was originally dosed at after it has lysed all of the host cells; therefore it will not persist in the patient after the infection has been cleared.10

The combination of self – replication only occurring in the presence of its host and having a narrow host range vastly improves the environmental impact when compared to antibiotics9. Antibiotics are excreted from the patient’s body and eventually end up back in the environment and water systems, greatly affecting microbial populations and therefore reshaping ecosystems10. Phages are only present when their host is within the environment, and are only be able to infect the few strains it targets therefore it is unable to reshape a diverse ecosystem on its own9.

Phage is also a practical treatment because they are easy to collect, as they are commonly found within the environment, including waste water and soil9. Antibiotics take decades to go through development and take millions of dollars, but with phages readily available within the environment, the cost of developing phage treatments would be vastly lower than antibiotics and would take a fraction of the time, especially after years of stream lining.

The most important benefit of them all is the safety of using phage treatment. Phages are primarily proteins and nucleic acids, which confer a low toxicity, if any, and they already live within our bodies9. Phage therapy may currently be in the process of being rigorously tested in the Western world, however multiple countries, including Georgia and Bangladesh, have already been using phages to treat patients. In Bangladesh a study was published in 2017 that provided 120 children with oral phage treatment for their acute diarrhea and no adverse events were seen. Georgia has been using phage treatments since World War II and has had great success. Eliava Institute in Tbilsi, Georgia, will swab patients and do susceptibility tests with both antibiotics and phage preparations to determine which course of action is warranted11. If the premade phage solutions and antibiotics are unable to treat the infection there is a database available with an extended spectrum of not as commonly used phages in culture, and if susceptible to one of those the phage will be purified for the patient on an individual basis11.Negatives of Bacteriophages:

There are a few downsides to phage treatments that have to be considered. Not all phages are appropriate for treatment, including temperate phages that integrate into the host genome, but do not kill the bacteria, phages that code for toxins that would be harmful to a patients, and phages that have poor bactericidal efficiency9.

Another issue for phage treatment is one of the benefits mentioned earlier, narrow host range. When a patient presents with an unknown illness and doesn’t have time to wait for a diagnosis a broad range antimicrobial is needed to at least begin to treat the infection while further tests are being completed. The narrow host range of bacteriophages would not allow for a quick treatment of an unknown pathogen, however having a cocktail of phages prepared for common infections is a way to circumvent this problem, which Eliava Institute employs9,11.

One of the current largest negative with phage treatment is the lack of medical trials within the Western world9. From the evidence I have presented above, it appears as if phage therapy is a fantastic choice for the treatment of bacterial infections, especially with the rising antibiotic resistance, however the clinic trials must be performed to validate the information presented within in order for phage therapy to become a viable solution in the the Western world. Conclusion:

AIEC is a pathobiont that naturally lives within the gastrointestinal tract of animals, however under certain circumstance AIEC can contribute to IBD, specifically CD1. AIEC can be treated with bacteriophages, as Galtier et al. found a three phage cocktail to auto dose, decrease AIEC colonization of the intestinal tract and improve colitis symptoms8. Phage therapy has many

known benefits, and very little negatives, however more clinical trials are needed around the world to confirm the above findings.

Figures:

Figure 1. Type 6 secretion system present in Escherichia coli3. OM represents the outer membrane, PG represents the cell wall, and IM represents the inner membrane3.

Figure 2. Phage replication in ileal (il.), colonic (co.), caecal (ce.), and faecal (fe.) gut secretions within LF82SK – colonised mice over three days8. A multiplicity of infection (MOI) of 0.01 was individually added to an exponentially growing culture of LF82SK (Ct.) within lysogeny broth, and the supernatants incubated for 5 hours at 370C before counting the plaques formed8. n – fold multiplication is relative to initial number of phages added8.

Figure 3. AIEC strain LF82 reduction in fecal samples of transgenic CEABAC mice8. (B) Level of AIEC present in fecal sample. Red represents mice that were given PBS as control, blue represents mice given 2 doses of 3 X 107 pfu phage cocktail 7 hours apart, and the days represents how many days post infection the mice were given treatment or control8. (C) Level of

phage present in fecal sample7. Red represents mice that were uninfected with LF82, blue represents LF82 infected mice8.

Figure 4. Phage therapy’s impact on AIEC strain LF82SK infection and colitis symptoms8. Control (co.) received phosphate buffered saline (PBS), preventative (pr.) received 3 X 107 pfu phage cocktain on day 8, and curative (cu.) received 3 X 107 pfu phage cocktain on day 108. (B) Disease activity index. (C) Quantitative polymerase chain reaction data on Day 25 from ileal gut secretions. (D) Quantitative polymerase chain reaction data on Day 25 from colonic gut secretions.

References:

1. Palmela, C. et al. Adherent-invasive Escherichia coli in inflammatory bowel disease. Gut 67, 574–587 (2018).

2. Smith, E. J., Thompson, A. P., O‘Driscoll, A. & Clarke, D. J. Pathogenesis of adherent–invasive Escherichia coli. Future Microbiol. 8, 1289–1300 (2013).

3. Journet, L. & Cascales, E. The Type VI Secretion System in Escherichia coli and Related Species. EcoSal Plus 7, (2016).

4. UCLA Center for Inflammatory Bowel Diseases - Los Angeles, C. Ulcerative Colitis vs Crohn’s Disease. Available at: https://www.uclahealth.org/gastro/ibd/ulcerative-colitis-vs-crohns-disease. (Accessed: 24th January 2019)

5. Mayo Clinic. Crohn’s disease - Symptoms and causes - Mayo Clinic. (2018). Available at:

https://www.mayoclinic.org/diseases-conditions/crohns-disease/symptoms-causes/syc-20353304. (Accessed: 28th January 2019)

6. Speck, P. & Smithyman, A. Safety and efficacy of phage therapy via the intravenous route. FEMS Microbiol. Lett. 363, 242 (2016).

7. Clokie, M. R., Millard, A. D., Letarov, A. V & Heaphy, S. Phages in nature. Bacteriophage 1, 31–45 (2011).

8. Galtier, M. et al. Bacteriophages targeting adherent invasive Escherichia coli strains as a promising new treatment for Crohn’s disease. J. Crohn’s Colitis 11, jjw224 (2017).

9. Loc-Carrillo, C. & Abedon, S. T. Pros and cons of phage therapy. Bacteriophage 1, 111–114 (2011).

10. Stern, A. & Sorek, R. The phage-host arms race: shaping the evolution of microbes. Bioessays 33, 43–51 (2011).

11. Brüssow, H. Phage therapy for the treatment of human intestinal bacterial infections: soon to be a reality? Expert Rev. Gastroenterol. Hepatol. 11, 785–788 (2017).