Friday, April 22, 2011

0 Phage Therapy and Borrelia burgdorferi

EDITED February 27, 2012 to include information on specific phages of B. burgdoferi.

Earlier this week, we discussed the use of phage therapy - the medical use of viruses found in nature that kill bacteria.

Phage therapy has been a part of regular medical treatment in Eastern Europe for over 85 years, but most of the research published has been in the Russian and Georgian languages since the primary former Soviet institution for the research and collection of a huge phage library has been in Tblisi, Georgia.

Eliava Institute of Tblisi, Georgia -
major bacteriophage research center
Those familiar with the Georgian language have stated that detailed documentation for double-blind controls was lacking in research mentioned, so the work as a whole wasn't taken seriously once translated. However, if research came from patient case studies, then documentation wouldn't require blind controls and simply record individual patients' responses. Either way, it is unknown to me how much of the research has been translated or has been made available for translation, given many people do not speak Georgian and because part of the research was written in the era of censorship in Soviet Georgia, some research may not have been published at all - even in a Russian translation.

Tbilisi's Eliava Institute, however, is not the only place in Eastern Europe that has conducted phage research - the Polish Academy of Science has a special institute that is also involved in phage research and therapy. You can learn about their current research here:
The Ludwik Hirszfeld Institute of Immunology and Experimental Therapy (Polish Academy of Science) and read specific research papers in English right here: Evergreen College Guide to Polish Phage Research.

Both of these institutions have had success in treating local patients as well as visitors from abroad. And with growing antibiotic resistance worldwide, one has to wonder why is it phage therapy isn't being used in the west to treat more patients? Why isn't it being used to treat Borrelia burgdorferi, the bacteria which causes Lyme disease?

These are two different questions, one of history and politics, and one of science. To explore them both requires a bit more backstory and examination of the FDA's regulations regarding the adoption of new medical therapies.

In the 1990's, entrepreneurs from the US and Canada traveled to the Eliava Institute to investigate their use of phage therapy and see if they could use the same medical treatment to help patients in the United States. Due to the FDA's regulatory system on all new therapies - especially combination or "cocktail" drug therapies - the use of phage therapy on patients in the United States would be a long way off, and any company investing in phage therapy would be using it for other purposes first.

As a result, in the United States, phage therapy is being used as a spray to protect all kinds of food (the FDA approved of treating cheese first, then other foods) from developing Listeria monocytogenes, bacteria that can lead to severe infection and sometimes even be fatal in vulnerable populations. There have also been treatments developed for veterinary healthcare, such as ear drops for dogs to treat ear infections (otitis media), and the most recent application of phages is using them on surgical equipment and clinic surfaces.

The road to adopting phage therapy for use on treating people in the western hemisphere has been a somewhat rocky one, given that the first entrepeneurs who went to Tblisi and came back to form a phage therapy research startup company had a bit of a falling out: The main financial backer for the company, Canadian Caisey Harlingten, was rumored to have had arguments over who would receive patent rights on work created with the company's new CEO, Richard Honour, and Honour decided to shut down work being done at the Eliava Institute and develop genetically modified phages in the US.

After this, personnel which had been recruited from Tblisi to go work in the United States for Harlingten's company were not happy with this arrangement, jumped ship, and went on to form their own startup, Intralytix. Intralytix - unlike other pioneering phage startups - decided to focus on phage treatments for animals and general products instead of human therapy.

After three years of operating at a loss, Caisey Harlingten resigned from his company, Phage Therapeutics - as did Richard Honour and the chief financial officer.

Last I read, Phage Therapeutics was supposed to have a particular phage that kills 93% of a broad spectrum of over 1,000 of S. aureus and S. epidermidis strains that were isolated from patients in the US, Canada, and South America. This phage was supposed to have been in preclinical trials and was supposed to enter clinical trials against eye infections.

But somewhere along the line, Phage Therapeutics changed hands, their stock devalued, and I discovered that as of February 22, 2008, Phage Therapeutics International Inc. was acquired by Surge Solutions Group, Inc. in a reverse merger. SSGI, Inc., through its subsidiary, Surge Solutions Group, Inc., provides construction and environmental services in Florida. Nothing to do with phage technology. What happened to the above mentioned broad spectrum phage mix?

Where one company falls, others spring up to take their place. There are a growing number of startups in the phage business, but mostly doing business like Novophage, which specializes in using phages to remove biofilms from industrial equipment.

The first clinical trials using phage therapy were conducted in Europe and America. One clinical trial involved a cocktail of eight bacteriophages (five against Pseudomonas aeruginosa, two against Staphlococcus aureus, and one against Escherichia coli) on leg ulcers in 2008 at The Wound Care Center in Lubbock, Texas.  Following that trial, the Southwest Regional Wound Care Center used bacteriophages along with other methods to treat antibiotic-resistant infections under a limited study. Further information on this study has not been published to date.

Bacteriophages are being studied in fighting against E. coli infections in Bangladesh, and phase 2a clinical trials in the UK have been conducted for using phage therapy on chronic inner ear infections caused by Pseudomonas aeruginosa at the Royal National Throat, Nose, and Ear Hosptial in London. Very positive results on clinical and bacteriological efficiency and safety concerns have been reported on this latter trial.

In 2010, a nebulizer treatment using bacteriophages of Burkholderia cepacia complex (full text) to treat cystic fibrosis was developed, and earlier study was completed on the development of an inhaler to treat Staphylococcus aureus or Pseudomonas aeruginosa. So far, the inhalers have yet to be tested on people.

There is an international conference on bacteriophages that is held in Olympia, Washington, and hosted by Evergreen College. Dr. Elizabeth Kutter, professor of microbiology at the college took a keen interest in bacteriophage therapy years earlier, and had traveled to Tbilisi herself to investigate the treatment and their results. Since then, she has been actively pursuing research into bacteriophages and promoting it for use in medicine. The college has its own special phage projects page you can look at to see research conducted on phage therapy around the world.

Even though there is interest in bacteriophages, few clinical evaluations have been published on them because the data available are at a very early stage, making it difficult to attract further funding - and as mentioned earlier, the use of phage often involves a "cocktail" of more than one virus to treat a patient and this challenges the FDA's regulatory standpoint on cocktail treatments.

Also, using phage therapy in Eastern Europe focused mainly on treatment for wounds and intestinal infections - conditions which could be treated using phages topically in ointments, sprays, and dressings or capsules and enemas. Intravenous therapy (IV) - while used on occasion - did not make up the majority of treatments given, so little has been known about their effectiveness.

There is some evidence that phage therapy can work in IV therapy, but it was suggested that in this form it is more likely to come with a drawback: just as Lyme disease patients experience a Herxheimer reaction from antibiotic therapy, patients receiving phage therapy can also have a Herxheimer reaction from phage therapy. One veterinary study, though, has shown that no notable negative reactions or effects were noted (Soothill, 2004).

As as a commenter on my previous post mentioned, there are shortcomings as well as benefits to the use of phage. But overall, the risks of using phage therapy seem lower than those of antibiotics so far because the antibiotic resistance issue and risk of C. difficile are gone (someone is even working on phage therapy for C. difficile).

Despite the growing evidence that phage therapy can be safe and effective, there are some challenges that even people who are most unfamiliar with phage therapy have pointed out at least one of them:
  • We don't know much about how phages interact with gut flora. Suspicions are most are benign if not helpful because we already have bacteriophages living in our stomachs and intestines all the time.
  • Some research has shown one kind of phage - T-even bacteriophage - show inhibition of lysis in low-oxygen environments. 
  • Both carbohydrates and bile salts can interfere with bacteriophages ability to replicate in the stomach. 
  • If a bacteriophage that was lytic becomes lysogenic, it will integrate with its host, enabling it to transfer bacterial virulence genes into other bacteria. This is why therapeutic phages must be entirely lytic and cannot carry toxic or housekeeping genes associated with lysogeny.
Even though these drawbacks exist, research is underway to find solutions that address them because the risk of not having phage therapy can be worse for some patients with very deadly infections which are becoming increasingly antibiotic resistant.

Can phage therapy work on killing Borrelia burgdorferi?

So far I have not seen any phage therapy research for Borrelia burgdorferi - however, the Phage Therapy Center for patients in Tblisi, Georgia claims they have phage therapy to treat Lyme disease coinfections.

In terms of phage therapy for Lyme disease itself, though - the best answer I can give at this writing is a theoretical maybe someday.

This is based on the idea that there is a phage for every bacteria out there if we were only to look for it and find it. It's also based on the idea that we have the technology available to potentially modify Lyme disease's known phages in order to change its behavior - or perhaps create a delivery system which could lyse Borrelia in a manner that phage does.

But so far - unlike Staphloccocus and other bacteria - few phages which attack and kill Borrelia have been documented. Publications on virulent phages of Borrelia are sparse, and there is only a little more documentation on phages in spirochetes as a whole.

B3-like morphology
phage on spirochete
In 1982, Hayes, Burgdorfer, and Barbour recorded their observations of a phage attacking Borrelia burgdorferi in vivo and took photographs to record the event. The images captured are of a B3-like bacteriophage, described by the researchers as having a "40- to 50-nm elongated head and a tail 50 to 70 nm in length. It appears devoid of collars or kite-tail structure".

There are two aspects of these images below which are  compelling: One is that they give us a rare glimpse of a phage which can actually kill Borrelia burgdorferi. (Wouldn't it be fabulous if we could somehow find a way to harness this as a treatment method, and find phages for all strains of Borrelia?) The second is that we have a photo of gemmae - a form of Borrelia which is not mentioned much in today's genomic oriented Borrelia research.

  •  (a) Section profile of a gemma with its attendant membrane bound granules or spherical bodies. Arrows indicate cross-section profiles of bacteriophage heads. (b) Internal attachment of bacteriophage to outer membrane material after plasmolysis of the spirochete. Arrows indicate remnants of plasma membrane.
A passage within the text, "Bacteriophage in the Ixodes dammini Spirochete, Etiological Agent of Lyme Disease", sheds some light on what is known about this phage and its relationship to Borrelia burgdorferi:
"Thus far, only those spirochetes showing left-handed coiling have been found to be phage infected. Figure ld shows phages that are associated with a spirochete with left-handed coiling. Bacteriophage heads in longitudinal and cross-sectional profiles were also observed within granules located within the aneurysmic blebs (Fig. 2a).

Completely assembled phages were more clearly seen in rarely occurring plasmolysed cells (Fig. le and 2b). In negatively stained preparations of spirochetes, they have only been detected internally (Fig. 2c). Bacteriophages previously reported to infect other spirochetes (15-17) are described as polyhedral and tailed (7) or cubic (5) in symmetry."
It appears that only those spirochetes which coil in a counterclockwise direction had phages. Why didn't any spirochetes with a clockwise coil have phages? Is there some inherent difference in their surface which makes it harder for phage to adhere to them?

In 1993, Neubert et al wrote about finding phage which were induced while introducing the antibiotic, ciprofloxacin, to Borrelia spirochetes. These A-1 and B-1 type phages were not virulent phages such as Hayes et al's B3-like phage.

The ultimate Borrelia book, "Borrelia: Molecular Biology, Host Interaction and Pathogenesis", has some passing mention of phages of Borrelia as well as a map of known and possible prophages in its plasmids. It also mentions a more recent discovery than Hayes, Burgdorfer, and Barbour's B3-like phage.

phiBB-1, prophage of
Borrelia burgdorferi
In 2001, Eggers et al published their discovery of a phage of Borrelia burgdorferi (Bb) named phiBB-1 (also written as φBB-1). It is not the best candidate for use in bacteriophage therapy because it is a prophage - also known as a temperate phage or lysogenic phage.

Lysogenic phages remain inactive as viruses when they are prophages, and only replicate together with the host genome unless mobilized. In contrast, virulent phages, having replicated and assembled into complete virions, cause rapid lysis and death of the bacterial cell, with release of 10–100 virions per phage; these virions then find more prey and die out when they cannot find any more bacteria.

Every time Borrelia burgdorferi divides, the viruses internalized in its plasmids divide with it. The viruses are an integral part of the plasmids and contribute to the functionality and antigenic variation of the spirochete - they have become part of the bacteria. In technical terms: The phiBB-1 prophage is capable of transducing a cp32 (circular plasmid) between cells of the same isolate and between different Bb isolates (gene transfer between different Borrelia spirochetes). This means this prophage could play a role in the genetic diversity of different Bb isolates.

Lytic-Lysogenic Phage Cycles
image by Suly12, Wikipedia
See the image to the left. If a bacteriophage is virulent, it will deposit its genes into bacteria so that it replicates and kills the bacteria from inside by lysing its membrane. The viruses then continue in search of more of the same bacteria to feast on. This is called the lytic cycle.

But if a bacteriophage is temperate or lysogenic, though - a prophage - then it will deposit its genes into bacteria so that they mix with the bacteria's own genes and divide with them each time the bacteria divides. This is called the lysogenic cycle.

Borrelia burgdorferi's plasmids contain virus genes which are locked into the lysogenic cycle.


Hypotheses Of Altering Phages To Lyse Borrelia

In order to put phiBB-1 to work at killing Bb, someone would have to genetically engineer it or introduce some agent which turns it into a virulent phage that kills Bb rather than adding its own DNA to its plasmids. Or, maybe phiBB-1 could be modified in a different way: don't bother changing its prophage nature, just program it to turn off DNA replication and gene expression in the bacteria's plasmids.

Another thing that could be done is to have someone extract the lysing proteins that work with phiBB-1 and find a method of delivery to Bb so those proteins could go to work on killing Bb outside in - maybe attach it to a non-pathogenic adenovirus that is programmed for such an adventure. There are such delivery systems being experimented with in general right now - but nothing yet for Borrelia.

These are wild hypotheses about how an existing phage we know about could be used to kill Bb, but it is not proven this would work. People are thinking of the biotech applications of phiBB-1 - but so far, I have seen only one patent application referring to its use.

The best option, obviously, would be to find naturally occurring phages which lyse Borrelia burgdorferi (as well as other strains) and find a method for using them to treat patients - though there are likely to be technical challenges in applying this as well.


References:
Wired magazine: http://www.intralytix.com/Intral_News_Wired.htm
A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic-resistant Pseudomonas aeruginosa; a preliminary report of efficacy. Wright A, Hawkins CH, Anggård EE, Harper DR. Clin Otolaryngol. 2009 Aug;34(4):349-57.
Viruses Vs. Superbugs: A Solution to the Antibiotics Crisis? By Thomas Häusler
Soothill, J.S. Hawkins, C. Anggard, E.A. & Harper, D.R. (2004) Therapeutic use of bacteriophages. Lancet Inf. Dis. 4, 544-545.
Microbiologist, the magazine of the Society for Applied Microbiology (June 2009, Vol.10 No.2)
Bacteriophage Therapy: Exploiting Smaller Fleas. Stan Deresinski. Clin Infect Dis. (2009) 48 (8): 1096-1101. doi: 10.1086/597405 link: http://cid.oxfordjournals.org/content/48/8/1096.full
Bacteriophage in the Ixodes dammini Spirochete, Etiological Agent of Lyme Disease. Stanley F. Hayers, Willy Burgdorfer, Alan G. Barbour. Journal of Bacteriology, June 1983, p. 1436-1439. link: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC217620/pdf/jbacter00247-0414.pdf
Demonstration of Cotranscription and 1-Methyl-3-Nitroso-Nitroguanidine Induction of a 30-Gene Operon of Borrelia burgdorferi: Evidence that the 32-Kilobase Circular Plasmids Are Prophages. Hongming Zhang and Richard T. Marconi. Journal of Bacteriology. December 2005, Vol. 187, No. 23 p. 7985-7995.
Bacteriophages induced by ciprofloxacin in a Borrelia burgdorferi skin isolate. Neubert U, Schaller M, Januschke E, Stolz W, Schmieger H. Zentralbl Bakteriol. 1993 Aug;279(3):307-15. link: http://www.ncbi.nlm.nih.gov/pubmed/8219501 Bacteriophage-like particles associated with a spirochete. Berthiaume L, Elazhary Y, Alain R, Ackermann HW. Can J Microbiol. 1979 Jan;25(1):114-6.
link: http://www.ncbi.nlm.nih.gov/pubmed/427652
http://en.wikipedia.org/wiki/Lysogenic_cycle


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