0 Symbiotic Spirochetes In Animal Models

Recently, I noticed this intriguing item in the news that is of interest to many medical professionals around the world who are struggling to care for patients' difficult to eradicate C. difficile infections: A recent murine study[1] provided evidence that a simple mixture of six phylogenetically diverse intestinal bacteria - including novel species - can re-establish a health-associated microbiota and clear C. difficile infection from mice.

With this outcome, it's possible that medical professionals will no longer apply the treatment of last resort - fecal transplants - to save human patients' lives. Instead, all patients with raging C. difficile infections will have to do is swallow a pill or consume food that contains the strains of bacteria needed to reverse dysbiosis and rebalance the microbiota in their stomachs so that C. difficile is no longer a threat.

Over time, researchers are uncovering the complicated dynamic between different microorganisms which live inside the human gut. Also, they are refining their understanding of the dynamic between different microorganisms inside animals with increasing speed as more invasive studies can be completed in animals than can be easily completed in humans. Among these studies are those on the relationship between spirochetes and other microorganisms found in ruminants such as cattle and sheep, termites, and molluscs.

Symbiotic Spirochetes In Termites
 

Microscopic image of Mixotricha paradoxa
covered with thousands of Treponema spirochetes

One interesting and complex symbiotic relationship involving spirochetes is found within termites' guts. Spirochetes are one of the most abundant bacteria present in the gut fluid of termites, and the symbiotic relationship between various microbes in termites of all kinds actually predates the evolution of termites from their wood-feeding roach ancestors over 120 million years ago [2].

Most termites have spirochetes which are free-living in the gut fluid, but they have also been found as ectosymbionts attached to protists inside termite guts. Mixotricha paradoxa is protozoa found inside the gut of the Australian termite species, Mastotermes darwiniensis. It was originally thought the long tiny hair-like structures covering the length of its body were short cilia - outgrowths from the protozoa itself. However, upon closer examination years later, scientists Cleveland and Grimstone[3] discovered these were not cilia but a dense carpet of Treponema spirochetes - spirochetes which help propel Mixotricha paradoxa forward while it uses its own anterior flagella to steer in the right direction. How it coordinates this movement is unknown - it is surmised that they automatically synchronize due to their proximity.

These spirochetes not only help Mixotricha paradoxa move forward, though. One thing Mixotricha paradoxa does to help its host, the termite, do is help break down cellulose into sugars and then hydrogen, acetate, and carbon dioxide from the wood it eats. From there, what the Treponema spirochetes are predicted to do is oxidize the acetate which was produced and use it to support 100% of the termite's respiration requirements.[4]

What makes this symbiosis even more complex is that it doesn't stop there. No, not only does Mixotricha paradoxa have a Treponema spirochete helping it survive - but it also has three more bacterial species onboard: A lot of rod-shaped bacteria related to Bacteroides live on its surface, and is suspected to help breakdown cellulose as it sits alongside the Treponema spirochetes; a spherical form of bacteria lives inside Mixotricha which is hypothesized to act as mitochondria for the protozoa (as Mixotricha does not have its own mitochondria); a large spirochete attributed to the genus Canaleparolina.[5]

Not much is known about these three bacterial species' lives in Mixotricha paradoxa, and more research is needed. The most recent research on Mixotricha paradoxa adds to this complex symbiotic dynamic, as it has been discovered that not all glycolytic activities in Mixotricha paradoxa are produced by its microorganisms - cellulases have been detected in the salivary glands of Mastotermes darwiniensis - the termite itself.[6]

Symbiotic Spirochetes In Ruminants

The bovine or cow stomach has a wide variety of organisms inside it - such as fungi, bacteria, archaea, protista, and viruses. All these organisms help break down food, especially plant matter and in particular, cellulose. And like the Mixotricha paradoxa inside the Australian termite, Mastotermes darwiniensis, the organisms are all dependent on each other to some degree and use the byproducts of one another for their own benefit.

Cows - unlike people - have four stomach compartments to digest their food: the rumen, the reticulum, the omasum, and the abomasum. The rumen is the largest compartment, and it contains a huge number of different microbes. The reticulum is responsible for creating cud and trapping indigestible substances like rocks or nails - and unfortunately, can be subject to more injury than the other compartments. The omasum sends large substances to the rumen and reticulum while allowing smaller substances to pass on to the abomasum. And the abomasum is very similar to a human stomach, as it produces stomach acids and enzymes to break down proteins before sending the result to the small intestine.

While the most common bacteria in the bovine stomach are gram-positive cocci and rods, a smaller percentage of their population are spirochetes which play a role in ruminant digestion. Organisms such as Treponema bryantii, a saccharolytic spirochete, enhances the breakdown of cellulose while cellulolytic bacteria of different species break down plant cell walls into soluble sugars.

Two interesting passages from the publication, Interspecies bacterial interactions in biofilms, by James, Beaudette, and Costerton[7], highlight the relationship between Treponema bryantii and other microbes studied in vitro from bovine rumen:

"Observations of biofilms on cellulose particles from the rumen revealed cellulolytic as well as noncellulolytic bacteria enmeshed in the exopolysaccharide matrix of the biofilm. Addition of a noncellulolytic species, Treponema bryantii, to cultures of a cellulolytic species, Fibrobacter succinogenes or Ruminococcus albus, resulted in an enhanced rate of cellulose degradation. Presumably, T. bryantii utilized the hydrolytic products (eg, glucose or cellobiose) from the cellulolytic bacteria which may repress and/or inhibit the cellulolytic enzymes."

"...Microscopy of biofilms formed during protocooperative cellulose digestion by R. flavefaciens and T. bryantii revealed that cellulolytic R. flavefaciens cells were attached directly to cellulose particles, while the spirochete, T. bryantii, was located in the upper biofilm layers. This spatial arrangement and the mobility of spirochetes in viscous environments suggest that this organism may move through the biofilm, scavenging the products of the cellulolytic bacteria."

The first study of Treponema bryantii in 1980, Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria[8], offers more specifics in its abstract as to its biological requirements:

"...When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulose-containing agar medium the spirochete enhanced cellulose breakdown by the Bacteroides succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO2 atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO3, the spirochete grew to a final density of 1.9 X 10(9) cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO2 (HCO3-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete."

Spirochete Symbiosis In Molluscs

While so far there is no evidence molluscs harbor spirochetes which have symbiotic relationships with its host or other microorganisms, spirochetes which coexist peacefully within their host are worth noting.

Spirochetes from the genus Cristispira have been found inside more than 50 species of 22 families of marine bivalves and 3 freshwater bivalves. It has been shown to be a commensal organism living within molluscs and has not been shown to provide any benefit or disadvantage to molluscs such as Prince Edward Island oysters.

This past May, an interesting paper was published, Spirochetes in gastropods from Lake Baikal and North American freshwaters: new multi-family, multi-habitat host records[9].

The abstract states:

"We describe the first records of spirochetes in the gut of fourteen species of continental gastropods from a range of habitats and representing six families (Amnicolidae, Baicaliidae, Bithyniidae, Pyrgulidae, Lithoglyphidae and Benedictiidae). The bacteria were mainly found in the crystalline style sac, as has been reported in marine bivalves. The surveyed habitats include water bodies in North America and Eurasia, including deep water hydrothermal vent and gas hydrate zones in Lake Baikal. Spirochetes were present both in mature and young snails, but were not detected in embryos before hatching, indicating lateral transfer. The surveyed gastropods range in trophic strategy, including phyto-, detrito- and bacteriophagous grazers and filter feeders. Our results indicate that spirochetes are commensal in the surveyed gastropods with potential limited benefit and no detriment to the host animal. We suggest that the specialized internal habitat of the crystalline style sac in molluscs is likely to reveal unrecognized spirochete diversity that will shed new light on gastropod trophic ecology and spirochete diversity."

More research is needed to determine which limited benefits different spirochetes may provide for their hosts.

Looking at the symbiotic and commensal relationships between animals and spirochetes - or between spirochetes and other microbes - one has to wonder what kind of relationships different spirochetes have with us and microbes within us. Humans already play host to spirochetes which are considered commensal Treponema and unfortunate hosts to spirochetes which are pathogenic such as those which cause syphilis and Lyme disease (Borreliosis). But is there more to this story than is often told? Do these bacteria have deeper relationships?

References:

1) Trevor D. Lawley, Simon Clare, Alan W. Walker, Mark D. Stares, Thomas R. Connor, Claire Raisen, David Goulding, Roland Rad, Fernanda Schreiber, Cordelia Brandt, Laura J. Deakin, Derek J. Pickard, Sylvia H. Duncan, Harry J. Flint, Taane G. Clark, Julian Parkhill, Gordon Dougan. Targeted Restoration of the Intestinal Microbiota with a Simple, Defined Bacteriotherapy Resolves Relapsing Clostridium difficile Disease in Mice. PLoS Pathogens, 2012; 8 (10): e1002995 DOI: 10.1371/journal.ppat.1002995
2) Grimaldi, D. and Engel, MS. Evolution of the insects. 2005. Cambridge University Press, NewYork, NY.
3) Cleveland, L.R., and A.V. Grimstone. The fine structure of the flagellate Mixotricha paraodoxa and its associated micro-organisms. 1964. Society 159:668-686.
4) Leadbetter, J.R. Acotgenesis from H2 Plus CO2 by Spirochetes from Termite Guts. 1999. Science 283:686-689.
5) Brugerolle G. Devescovinid features, a remarkable surface cytoskeleton, and epibiotic bacteria revisited in Mixotricha paradoxa, a parabasalid flagellate. Protoplasma. 2004 Oct;224(1-2):49-59.
6) Konig, H., Li Li, Wenzel, M, Frohlich, J.  Bacterial Ectosymbionts which Confer Motility. p. 86 Molecular Basis of Symbiosis. 2006. Springer-Verlag.
7) G A James, L Beaudette and J W Costerton. Interspecies bacterial interactions in biofilms. Journal of Industrial Microbiology & Biotechnology. Volume 15, Number 4 (1995), 257-262, DOI: 10.1007/BF01569978
8) Stanton TB, and Canale-Parola E. Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria. Arch Microbiol. 1980 Sep;127(2):145-56.
9) Tatiana Sitnikova,Ellinor Michel, Yulia Tulupova, Igor Khanaev, Valentina Parfenova, Larisa Prozorova. Spirochetes in gastropods from Lake Baikal and North American freshwaters: new multi-family, multi-habitat host records. Symbiosis. May 2012, Volume 56, Issue 3, pp 103-110.

Additional Reading:
Xinning Zhang and Jared R. Leadbetter. Evidence for Cascades of Perturbation and Adaptation in the Metabolic Genes of Higher Termite Gut Symbionts. mBio vol 3. no.4 e00223-12. http://mbio.asm.org/content/3/4/e00223-12.full
Nordhoff M, Wieler LH.Berl Munch Tierarztl Wochenschr. 2005 Jan-Feb;118(1-2):24-36 .[Incidence and significance of treponemes in animals].[Article in German]

This work by Camp Other is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License.

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3 NOTES: House Subcommittee Hearing On Lyme Disease

Here are some very rough notes from today's hearing on Lyme disease in Washington, DC. Please be aware that notes on about 5 minutes of testimony are missing towards the end of the day's hearing. If the webcast is archived, viewers may wish to refer to it to make their own notes.

House Committee on Foreign Affairs, Subcommittee on Africa, Global Health, & Human Rights hearing

2PM EDT
Tuesday, July 17, 2012
2172 Rayburn HOB
Washington, DC, USA

"Global Challenges in Diagnosing and Managing Lyme Disease - Closing Knowledge Gaps"

OPENING REMARKS

CHRIS SMITH - CHAIR - NJ Rep

We have a missed decade on Lyme disease research
Chris mentions chronic Lyme disease controversy
Mentions bill to establish tickborne disease committee and take fresh look at all the scientific approaches to Lyme disease
Mentions some history about Lyme disease including Borreliosis in Europe and discovery in Lyme, CT
2010 - 20,000 cases reported, but actual number of maybe 300,000 cases in US in 2010
North America Borrelia burgdorferi s.s.
Different species = different manifestations of disease
Clinical manifestations come in three stages
Mentions two distinct views of Lyme and research conflicting
Hard to catch, easy to cure vs. easy to catch, hard to cure
IDSA states short course of abx ok - anything else is risky
ILADS says the science is unsettled
One or more cause of chronic Lyme disease symptoms is possible, including persistent infection
Persistent symptoms could be due to a combination of persistent infection and immune problems

Three areas we need to took at:
1) diagnostics
2) post treatment symptoms
3) available treatments in light of science

CDC: 2 tier serological testing, but should be used for surveillance not diagnosis -unfortunately it is used for diagnosis by some doctors instead of clinical diagnosis
Europe: 8 immunoblots were shown to have a wide range of sensitivity and specificity
Bolen/NIAID: Lyme multistage disease is difficult to diagnose at any stage
Dr. Eshoo: Has exciting info on new diagnostic tools

Persistence: IDSA says Lyme disease cannot be persistent
Chris Smith says there is plenty of evidence that it can persist past antibiotic treatment
Mentions Barthold's studies and persistence in animal model studies
Mentions possible mechanisms of persistence studies: morphological changes (cell wall deficient, biofilms, etc)

Counterargument by IDSA: there are no convincing arguments that post treatment persistent Lyme disease infection is possible.
Chronic Lyme disease is a misnomer according to IDSA.
IDSA treatment guidelines should be based on best science - Smith argues they don't take into consideration possibility of persistent infection and deny patients needed treatment.

Chris Smith reports that the IDSA, NIH, and CDC were invited to the hearing today. The IDSA representative had scheduling conflict, and they were requested to testify at a very near future date before the subcommittee.

MS. BASS

Mentions the serious concern of missing early diagnosis
States few cases of LD in CA - MS. Bass says she thinks Lyme disease is under reported in CA especially in Central Valley
"Deer ticks" aptly named, as deer can carry 1000 ticks on their back on average.
But white footed mice are now known to be major carriers of the disease.
Other animals are now suspected including birds.
Need new solutions and explorations.
Changes in temperature, weather, and precipitation and their role in spread of Lyme disease should be examined.
Improved detection of early disease is contributing to higher reported numbers to some degree.
Local health departments are increasing awareness in spring and summer.
How can we improve awareness with limited budgets and resources?
Work with WHO to prioritize the disease esp in emerging areas such as Asia.

Rep. GIBSON

This is constituent driven. Major issue in upstate NY. (After he retired from army, so many people are suffering from LD and are confused and medical community is divided over its treatment. A Lyme disease task force would resolve this. Need more awareness about disease and diagnosis. Need to make sure money goes to right place. Money has gone into similar place before and the results were the same.

Most encouraging is research to be published in next year about role of confections in persistent symptoms.

WITNESS TESTIMONY

DR. STEPHEN BARTHOLD, UC DAVIS

Borrelia persists normally in immune competent host
i.e. mice rats hamsters gerbils guinea pigs dogs nonhuman primates
antibiotics are likely to fail under some circumstances if not many circumstances
finds self in contentious field - is somewhat of a pariah
we know in pre-immune stage animals can be cured
in immune stage, animals are not easily cured by antibiotics
Only a few places are really studying post antibiotic treatment Lyme - my group, Finland, NY, Louisiana... [?]
Ceftriaxone, Tigecycline, Amoxicllin, Zithromax: spirochetes are shown to survive these antibiotics in animal models
clonal population of Lyme disease spirochetes -> given antibiotic treatment -> results in non cultivable but living spirochetes
Transcribing RNA = metabolically alive spirochetes
exoplant -> carries infection from Bb infected animal to naive animal

Completed study, hope to publish it soon: 12 month post treatment resurgence of spirochetes found in mice.
what is significance of these spirochetes?
viruses can re-ignite and cause disease - question is: can spirochetes?
The answer is not known yet; something unique is going on with Bb and needs further study

DR. RAPHAEL STRICKER
San Francisco, CA - LLMD

speciality in internal medicine, ILADS Vice President
has 2,000 LD patients
number of patients has grown exponentially in past 15 yrs
patients all over the world Canada Brunei Costa Rica UK even NJ
reflects an increasing rate of LD around the world
Lyme disease is the most common TBD

patients develop muscle, joint, neurological, and cardiac symptoms
despite common disease, doctors are ignorant of how to diagnose and treat:
a bulls eye rash may be absent (50% not seen)
patient may be unaware of tick bite (some bitten by poppy seed size tick)
patients have a wide range of symptoms - doctors are often unaware of this wide range

testing remains problematic (not standardized and insensitive)
treatment has evolved in haphazard fashion (IDSA guidelines only address acute infection; standard guidelines ignore more chronic and severe form)
LD has become international medical disaster
thousands of patients suffer from undiagnosed and untreated Lyme disease
and even though it is all over media
the IDSA sits by and does nothing

State of California is grateful to state health board for forming a state Lyme disease advisory board
for requiring mandatory laboratory reporting of Lyme disease to Department of Health just like system for reporting STIs
California has a physician protection law which allows doctors to treat Lyme disease patients as they deem appropriate
Stricker thinks a model for a Lyme disease national advisory board could stem from CA state model

We need CDC and NIH to abandon failed Lyme disease research programs
need them to have targeted research for better tests just as they did for AIDS
need to have more research on treatment of chronic Lyme disease
need to get them to look at evidence and discard dogma about what chronic Lyme disease is
we need for them to listen to patients and how they are affected

Almost 2 decades ago Dr. Joe Burrascano testified before the Senate. He said, "The very existence of 100s of LD support groups, etc underscores many problems which exist in real world of LD." Two decades later and it's the same story.

DR. MARK ESHOO
IBIS Biosciences, Abbott Labs

Need to develop better diagnostic tests
number cases steadily increasing
Lyme disease is severely under reported
Babesiosis is also important
Babesiosis can be mistaken for malaria (Babesia looks similar to malaria under the microscope, too)
Other TBDs are also present

Lyme spread by ticks to mice, and then ticks infect more mice
mice become chronically infected with Lyme disease
bacteria evolved to evade immune system, especially immune privileged sites (e.g. skin, joints)
humans can have long lasting or chronic infection
those infected develop neurological and joint problems
best time to treat is early in infection
most typical early symptoms are bulls eye rash, flu-like, fatigue, aches

CDC 2 tier test - involves indirect detection of antibodies; has 3 problems:
1) can take Lyme patient more than 3 wks for immune system to detect infection
2) interpretation of 2 tier tests can be subjective and change outcome
3) even after treatment, patient can remain positive… controversy over how long to treat patient (weeks? months? years?)

Abbott Labs is looking directly at presence of DNA of pathogens
Sensitive direct assay of organism is historically very difficult because spirochetes are present in small numbers
Abbott made an assay with 8 independent tests to detect the presence of bacteria in blood
they use large volume of blood
and find way to amplify the presence of small numbers of bacteria (bacterial DNA) in blood

Eshoo et al did a study which could find organism very early in infection in doctor's office (refer to abstract) in 62% of patients

Another area of research Eshoo is working on:
variations of strains may determine type and severity of disease
need to study 100 different strains of Bb and what makes them different in terms of impact of disease

We need more government research and funding
- sensitive test for direct detection early in infection before dissemination (monitor responses to treatment
- find out cause of PTLDS. A direct diagnostic tool would be useful
- need to increase research into diff Borrelia strains differences and their role in human infection

PAT SMITH, LDA USA

LD called yuppie or housewife disease
patients have been referred to by some doctors as being paranoid, hysterical, hypochondriac, etc. without any evidence and without looking that something else could be wrong
many advocacy organizations in the world have been victimized in peer reviewed publications
Many patients confide they'd rather have cancer than Lyme disease due to the misunderstanding and controversy over the disease
Patients want studies which solve their dilemmas (such as doctors don't believe they're sick, answer the question "Why isn't the government doing anything?")
The outcome of small clinical trials/studies put a coffin the nail for treatment of chronic Lyme disease and for a number of reasons, these studies were inadequate.
The conclusion was that no treatment is effective for long term Lyme.

Lyme in the south - there are many myths. Myths that:
No lyme disease can be found in south or west
No reservoir hosts in the south
Deer ticks on lizards prevent Lyme disease in ticks all throughout the south
Deer ticks in south do not bite people (?!)
These claims are not scientifically backed.

Patients are overburdened with medical problems.
There are cutbacks in public health depts. so number of cases are unknown.
Pharmacists who won't fill prescriptions for Lyme disease patients in some places.
Munchausens by proxy charges are made toward mom's who treat kids with Lyme disease with long term antibiotics.

Guidelines that are written by researchers and not clinicians are problematic
IDSA is against any sort of treatment for CLD - either antibiotics, alternative treatment, or the use of supplements
CDC surveillance criteria for Lyme disease has formed basis of IDSA guidelines and this is problematic
patients can die of Lyme disease: study of 114 patients who had Lyme disease who died. After they died, most terminal events for which LD was known as the underlying cause have listed on their death certificate a reported cause of death which researchers stated were thought to be unrelated to Lyme disease. Only one patient was said to have died of complications of Lyme disease directly. Question that.

Has seen recorded 22 point IQ drop in kids with Lyme disease due to infection affecting brain
kids have killed themselves due to Lyme disease - due to pain and due to disbelief by peers and others of their having the disease

Pat Smith has had 2 daughters affected by Lyme disease

CDC, NIH, IDSA were absent at hearing and she thinks they are avoiding responsibility when they were invited to be part of the process to help patients.

EVAN WHITE, NYC
Patient - Had Chronic Lyme Disease

(wife just had baby, he is on SKYPE  - his life is now normal being part of point of his testimony)

20 yr advocate borne out of very tragic case of his own Lyme disease due to being given limited antibiotic treatment
at end he is now well because of conscientious doctors
today is father and practicing attorney and employer.

he says his case is an illustration: if not for long term treatment, none of this would be possible

so many people do not have benefits he had
if they had access to treatment, they would be contributing members of society
his story has happy ending
he is fighting for fellow Lyme patients to have same access to treatment
he is advocating for change in limited guidelines

his case study is that short term treatment did not work
long term treatment helped him recovered
he was ill at 11 yrs old
missed school due to flu-like symptoms
doctor did diagnose Lyme disease and he was given 1-2 wks of antibiotics
after 2 weeks he did not get better
the response to not recovering after 2 wks was that PT and psych therapy was needed after those 2 weeks
daily his condition deteriorated
he was a 12 yr old who trusted his doctor
but 6 months later so bad could not do anything
could not go to school, had trouble getting out of bed
blood test showed Lyme disease and confections were very much present months later

here is devastating result of un (or under treated) Lyme disease
6 months of no treatment sent him into tailspin
he vastly deteriorated, went from active athletic child to one who couldn't care for himself
had muscle atrophy, neurological problems
60 lbs at age 13, called a vegetable, and doctors were confused by his state
doctors put him in children's rehabilitation care
did brain scan
it revealed Lyme disease's affect in its passing the BBB: hypoperfusion
he had trouble reading and talking
and was surrounded by doctors who had no idea why he was in condition he was in
2 yrs bounced from hospital to hospital
6 months in children's hospital

went home and parents arranged for appt with LLMD
the LLMD "got it" and had their own personal experience with the disease - not just treating other patients
he had long road ahead for recovery but this was his turning point
he was on long term antibiotics coupled with supplements
2 year crawl to get out of that place
even if it were 10 yr crawl that would have been ok with him
stopped using wheelchair
got out of bed
began to take care of self
began to read again
began to be able to communicate again
got him on trajectory to become person he is today and fully recovered

hoping through this testimony that patients who are affected can get treatment they need to recover from chronic Lyme disease
the net effect of current guidelines restricting treatment deprives so many if not all from having health care option to seek long care treatment that does work for many patients so that they can recover and live long healthy lives

Rep. SMITH

intro Stella in UK

STELLA HUYSHE-SHIRES
Lyme Disease Action (UK)

UK doctors not taking patients seriously
Department of Health accreditation of Lyme Disease Action, Lyme Disease Action is now considered an unbiased source of information on Lyme disease in the UK
papers say Lyme Disease is overdiagnosed
public say it is underdiagnosed
what is the evidence?
we don't know incidence of Lyme disease in the UK
One GP practice finds it 20x greater than numbers which are reported
1300 cases found in one year may mean there are really more like 26,000 cases in UK

survey:
23% patients found ot have Lyme disease but the rest with similar symptoms were diagnosed with CFS
there is concern CFS patients are misdiagnosed and have Lyme disease
on the flip side maybe
100 people year in clinics in UK may be misdiagnosed with Lyme disease
But in a CFS clinic - 40% patients were misdiagnosed with CFS

Why is LD difficult to diagnose and what can be done about it?
we need unequivocal tests and clear guidelines
none exist in UK
most doctors haven't seen Lyme enough and rely on blood tests for diagnosis in UK

unreliable info on internet, certain labs, etc only part of story even if there is an element of truth in it
European challenge of more than one strain of Bb adds to complexity of test issue
Scotland uses more bands in its lab than other locations - leading to different line drawn for positive test results and access to treatment

Most treatment recommendations based on opinion not evidence
need other stakeholders to investigate Lyme disease
Lyme Disease Action (UK) is working with James Lind Alliance to engage doctors in more awareness of LD in patients and in general

The biggest challenge globally is recognition of unknowns in Lyme Disease
All across Europe there is a polarization of opinions along IDSA/ILADS poles
and there may be reluctance to climb out of one's entrenched view of Lyme disease

In Northern Europe doctors rely heavy on test results - similar issues found there.
In Central Europe, doctors have more experience: Lyme is a big problem, doctors say the tests are not good enough; doctors say they don't know how to effectively treat all patients.

Politics are a problem.
Uncertainty of the science is a problem.
Politics prevents recognition of the uncertainties.

QUESTION AND ANSWER SESSION (Rep Smith/Rep Gibson ask questions)

HUYSHE-SHIRES

A: HPA guidelines come from IDSA
Worse thing when patients are told symptoms are in their head
IDSA only recognizes visible arthritis then patient may get further tx
HPA does follow IDSA guidelines
indiv doctors sometimes make indiv clinical decision
case studies London school of hygiene (4-5 yr period) - some patients did not recover after initial course of abx, then some not after second, then some had a third. Between each treatment, patients were believed and found rising antibody levels.
Doctors will say adequate tx occurred, but it's adequate in terms of meeting guidelines but not in terms of effectively treating patient

BARTHOLD

Q: Are proposals being rejected for research at NIH?

A: Peer review is an issue. Peers are divided just are anyone else in Ld community
have direct exp in prejudicial statements in grant application reviews - peer view of applications does not get over the barrier
NIH is struggling to fund investigators
young people are not entering science, old people are leaving
in that environment combination of things - anything controversial having difficulty being funded
made NIH call for application on research on persistence after antibiotic treatment
only suggestion is his
we scientists are always looking for money. Follow the money.
NIH invests in biodefense then people gravitate towards biodefense research.

STRICKER

Q: Rep.brings up conflicts of interest and suppression of data in IDSA guidelines review.

A: IDSA hearing was organized by IDSA and no treating physicians were on the committee
Even though guidelines were flawed they were ruled acceptable.
Stricker encouraged by Dr Eshoo's research and development of advanced early testing.

["To date no antibiotic treatment treats biofilms." - attribution?]

Q: Rep comments to Barthold: issue of "mopping up" after antibiotics
host immune system must mop up remaining spirochetes... Explain.

BARTHOLD

A: using biofilm analogy: there is a population of microorganisms, some of which are dormant
dormant non-dividing bacteria are universally tolerant of antibiotics and are not dividing or active metabolically
Borrelia: we know it is dividing and disseminating and susceptible to antibiotics early on but during the immune phase in animals there is a 10 fold reduction in population (not necessarily in biofilms) and what is found are non-dividing spirochetes; they are dormant and antibiotics are not touching them
What is unique is they grow out but they cannot be cultured - they may be attenuated.


STRICKER

A: Borrelia has molecular machinery to make biofilms according to Dr. Stricker.
Stricker states cell wall deficient form evades antibiotics and it needs to be researched more.

Q: Rep. Asks Dr. Eshoo: How close are you to coming up w new test and why is Big Pharma not getting further involved?

ESHOO

A: It's a small market according to BP and takes lots of money and time to invest.
Lot of people in medical community say current test is good enough.

Eshoo thinks sensitivity needs to be improved and tests need to be improved to end the controversy.

Who wants to be infected for 3 weeks or more untreated waiting for a positive blood test? Nobody.

Rep. Q to Stricker:

Are there people outside the IDSA guidelines panel who notice there's a problem [with testing]?
Does Dr. Francis Collins (NIH director) say "What is wrong here? Why is this a persistent bone of contention?"

STRICKER

A: Blumenthal investigation found there are 14 people in the IDSA who control guidelines, testing, and diagnostic guidelines of Lyme Disease. The rest of the IDSA (8,000 people) defer to this group.

PAT SMITH, Lyme Disease Association

LDA has Scientific and professional review board
It is voluntary board
If issues need to be addressed or LDA is considering funding research, the board is asked to comment on it using their expertise
CALDA, LRA, etc also rely on this board

STRICKER

Q: Rep. [?]: you have 2,000 patients. What is your takeaway from this huge patient number and how are they when they find you?

A: Number of patients exceeds CDC reporting. That's one thing it tells me. Number of those affected may be 10 fold higher.
Many come after yrs of misdiagnosis and no treatment.
70% of patients get better. He finds this gratifying and he turns a deaf ear to the controversy because of outcome.
Uses long term antibiotic treatment.
Published study last year on neurological patients needing 6-12 months of antibiotic treatment to improve.

[5-6 minutes of testimony notes missing]


COMMENTS NEAR END

8.75 million dollar research fund Chris Smith says has been put forward. Chronic Lyme is supposed to be included in this research. How should the wording for the law be improved and how can there be better oversight to get money to the right place?

Barthold said: Follow the money.
If you enlarge the pot and spend it on research that's already been done, we get nowhere.
If we and NIH recognize persistence after treatment as an issue, then new research would be done on this issue.
A more narrowly focused call for applications would help if NIH would agree with that - research on chronic symptoms and the biology/pathology of the organism.

Dr. Eshoo said the field needs support to get off the ground and that RFAs must be specifically targeted toward solving narrowly defined problems.

PAT Smith has concern research money goes to post Lyme disease syndrome and not chronic Lyme disease - which is a different condition.
Patient perspectives on issues of the disease is important and knowing how it's affecting them is important.
Advocates, treating physicians, and patients need to be involved in the process of determining what needs to be researched.

Q: Rep. Gibson: How do you expand Lyme literacy among old and new doctors?

STRICKER

A: ILADS has preceptorship program. Can learn about diagnosis and treatment of Lyme disease. Program is funded privately. Mentor doctors who want to get involved sign up.

Stricker has trouble finding physicians willing to get involved due to controversy. This has had a chilling effect on mentorship.


Q: Rep Gibson: is that because state med board may censor them?

A: Yes. Though state has protection law, the risk censoring by board and other doctors may still go on.

Has 27 studies in table in written testimony (table 2) showing persistence in humans after IDSA guidelines-based treatment.

BARTHOLD

We need to know what is happening in humans but animals allow us to extrapolate models. Many people are using animals but looking only at acute early phase of infection. Not many people are looking at chronic persistent infection in animals. Fewer than five labs worldwide are studying this but it is the most important aspect of the disease.

HUYSHE-SHIRES

Borrelia has been isolated in patients after initial treatment - there are some cases recorded in published papers.
Some people improve after longer treatment
We need more investigation to determine how to better diagnose and treat Lyme disease

IDSA guidelines are accepted in the UK
Summary of recommendations for treatment by European Federation of Neurology Specialists (organization name needs fact checking) - make the point that in neurological Lyme there are no good trials of more than 28 days antibiotic treatment for neuroborreliosis.
They base this on opinion because there is no trial in Europe for longer term treatment of neuroborreliosis.

There are some trials which show good recovery, but at most 60-70% patients experience a good response. A patient does not consider 7 of 10 people responding to treatment as being a rate that is excellent.

PAT SMITH

Children are most affected by Lyme. They have more complications and are greatly impacted by their peers and teachers and what they are saying about them.

It is appalling what comments are being made about students who are ill with Lyme who cannot make it to school because they are too sick. No one wants to stay at home with their mother from school for four years.

One problem is inadequate early antibiotic treatment may lead to a poor antibody response and negative tests, which then put child at further risk for being disbelieved for having Lyme disease.

Some family services will take kids away from parents if those parents treat one child with antibiotics for chronic Lyme disease. This is serious and kids are psychologically damaged by the disease as well as the response from society and their community towards their illness.

We have the knowledge and tools in this country to stop this.

HUYSHE-SHIRES

1-3 people in UK are believed to have expertise on Lyme in UK, and those 1-3 apply IDSA guidelines and support them
NHS did not make any needed changes to their guidelines even though they should be made to suit the UK patient population

ETHAN WHITE

If sick, seek out people who will lead you to knowledgeable doctors who will offer treatment for Lyme disease.

SOME CLOSING COMMENTS

Barthold thinks people on both sides are good people, and he thinks we need to move past contentiousness and work together to help those affected by Lyme disease.

We need to work to get out of entrenched positions and get to the bottom of what's happening using science.

It's time for people to get together and show their cards and be willing to act in the best interest of patients and work past this contentiousness.

---

Comments:

Will be adding links relevant to this testimony soon.

If anyone who gave testimony at the hearing reads these notes and sees a correction that needs to be made, please request correction in comments and I will revise this post.

UPDATE - July 17:

Original testimonies are now available for download including additional materials from each of the witnesses. Scroll down this page for pdf files: http://foreignaffairs.house.gov/hearings/view/?1455

UPDATE - July 19:

Ustream has two streaming video archives of the hearing available at the following links:

http://www.ustream.tv/recorded/24058724

http://www.ustream.tv/recorded/24060689

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0 Blog Log: Spirochetes Unwound On Persisters

Well, our favorite spirochete blogger has posted about the magic of antibiotic tolerance today - an apropos choice for this evening's post to follow on the heels of the discussion about Embers et al paper, "Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection".

While this entry on persisters in Spirochetes Unwound is not specifically about Borrelia, it is an interesting read for those who wish to learn more about the mechanisms related to antibiotic tolerance and how some bacteria can be dormant and survive its wrath.

Excerpt:

"Bactericidal antibiotics are effective at killing proliferating bacteria as long as they don't carry mutated or acquired genes that encode resistance to the antibiotics. Unfortunately even antibiotic-sensitive bacteria can tolerate antibiotics under some circumstances. Bacteria that are in a nondividing "dormant" state often survive antibiotic exposure. When the antibiotic is removed and growth resumes, the bacteria regain susceptibility to antibiotics.

At first glance antibiotic tolerance appears to be a passive process in which nondividing cells survive simply because the target of the antibiotic is inactive. However, this is not correct. Antibiotic tolerance requires an active response by the bacteria. The nondividing bacteria that survive antibiotic treatment are called persisters. Persisters may account for infections that are difficult to eradicate with antibiotics..."

Read more here: http://spirochetesunwound.blogspot.com/2012/02/magic-of-antibiotic-tolerance.html

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0 Dr. Alan MacDonald Discussing Spirochetal Biofilms on LNE

There are a series of discussions going on right now on Lymenet Europe I want to point out.

It looks like Dr. Alan Macdonald is having an involved exchange about biofilms in different spirochetes with someone (Henry) who has identified as a microbiologist in previous entries.

You might want to check out this thread now:

Biofilms of still yet spirochetal type - Treponema:
http://www.lymeneteurope.org/forum/viewtopic.php?f=5&t=3607

Also follow the following related threads:

Structure of Biofilms of Borrelia Lecture link:
http://www.lymeneteurope.org/forum/viewtopic.php?f=5&t=3602

Biofilms of yet another spirochetal species - Leptospira:
http://www.lymeneteurope.org/forum/viewtopic.php?f=5&t=3606

I would like to see more doctors and microbiologists engage in discussion about Borrelia and other spirochetes more often - especially if the implications have an impact on translational medicine and clinical outcome. Obviously some of this discussion is going to be purely speculative, but it is interesting to hear different points of view.

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0 Review Of The 2011 Lyme and Tick-Borne Diseases National Conference

In October 2011, a national conference on Lyme and tick-borne diseases was held in Philadelphia by Columbia University and the Lyme Disease Association.

Here is a brief overview of the topics presented:

• Dr. J. William Costerton’s riveting talk on “The Role of Biofilms in Chronic Bacterial Infections” reviewed the history of the discovery of biofilms, demonstrating that these biofilms enable micro-organisms to resist host defenses and antibiotics, enabling infections to become chronic.
• Dr. Eva Sapi’s talk on “Killing Borrelia – an impossible job?” addressed various mechanisms associated with Borrelia burgdorferi that may help it to survive despite antibiotic treatment.
• Dr. Jason A. Carlyon’s talk focused on Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis (HGA). This emerging tick-borne pathogen demonstrates stealth trickery, enabling it to avoid and even subvert immune cells.
• Dr. Richard Marconi’s talk on “C-Di-GMP” described research demonstrating that the cyclic nucleotide, c-di-GMP, plays a critical role in regulating several important cellular processes.
• Dr. Chris Earnhart’s talk described work developing a novel next-generation Lyme disease vaccine based on outer surface protein C. Osp C is expressed by all Bb species and strains and is expressed in the human host for several weeks before being down-regulated.
• Dr. Robert S. Lane gave a brief overview of his research team's long-term studies of the ecology and epidemiology of Lyme disease in California, and then summarized some exciting recent findings regarding the genospecies and genotypes of Borrelia burgdorferi s. l. that infect the western black-legged tick and humans in this region.
• Dr. Karen Newell Rogers presented a talk about novel ways to target chronic inflammation and chronic immune activation among patients with chronic Lyme disease. The primary controversy with Lyme disease has been whether the disease is the result of long-lasting bacterial infection or whether long-term symptoms result from a post-infectious, uncontrolled autoimmune response.
• Dr. Robert Yolken’s talk on “Infections and Human Neuropsychiatric Diseases” focused on the Stanley Center’s work at Hopkins which has examined infectious triggers of psychosis.
• Dr. Josep Dalmau’s talk on “The Clinical Spectrum and Cellular Mechanisms of Autoimmunity in NMDA and other synaptic receptors”. His pioneering work studying anti-NMDA receptor encephalitis shows how an immune response triggered by a tumor (e.g., ovarian teratoma) or perhaps an infectious process, results in antibodies that can attack critical receptors and synaptic proteins in the Central Nervous System involved in memory, behavior, cognition, and psychosis.
• Dr. John Aucott’s talk on “Early Lyme disease” reported from the SLICE prospective cohort and his Maryland studies.
• Dr. Reinhard K. Straubinger's talk on “Canine and Equine Lyme Borreliosis” focused on Lyme borreliosis in animals, especially in dogs and horses.
• Dr. James Moeller presented a talk on “Immunologic aspects of neuropsychiatric illness: Lyme disease as model”.
• Dr. Brian Fallon presented a talk on “Models of Chronic Lyme Disease”. The talk started with a review of the terms that refer to chronic symptoms and recommendations on how the the IDSA’s definition of Post-treatment Lyme Syndrome could be improved. This talk reviewed the evidence regarding models of persistent infection and/or persistent immune activation.
• Dr. Andrew Walter reported on Ehrlichiosis and Hemophagocytic Lymphohistiocytosis (HLH) in cases of children diagnosed in Delaware.
• Dr. Andrea Gaito provided an update on the clinical evaluation and treatment of Lyme Arthritis from an autoimmune perspective. Lyme arthritis occurs in sixty percent of patients with untreated Lyme disease.
• Dr. Ingeborg Dziedzic presented an interesting (and at times entertaining) overview of how Lyme disease impacts the eye, emphasizing that the eye is in part like the skin and in part like the brain.
• Dr. Vijay Thadani presented an overview of seizures and non-epileptic seizures, showing videos of both. Brain infections such as Lyme disease can lead to the development of epilepsy.
  
• Dr. Steve Bock addressed complementary and integrative medicine approaches to the treatment of chronic Lyme disease.
• Dr. Elizabeth Maloney addressed studies of antibiotic treatment of Lyme disease, providing a thoughtful and critical review of the literature to identify lessons, gaps, and future research needs.

READ MORE - Full Presentation Information Here:
2011 Lyme and Tick Borne-Diseases National Conference Summary Report

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9 Abstract: Evaluation of in-vitro antibiotic susceptibility of Bb

I was recently asked about this study in comments, since there has been much discussion about this research in the Lyme disease patient community online:

Evaluation of in-vitro antibiotic susceptibility of different morphological forms of Borrelia burgdorferi

My opinion of it thus far is one of waiting and seeing - I'm not sure what to think yet and I'm waiting to hear more information... I'm guessing this is an initial publication of findings and a more detailed paper similar to Dr. Sapi's cancer research papers will be published in the near future.

Tinidazole

I have a number of questions and thoughts on it, and prior to this publication, I had the impression that tinidazole has helped a number of Lyme disease patients - but there has been limited research on it (See: Brorsons).

The Brorsons stated in their 2003 paper, An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole:

"Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that, when the concentration of TZ (Tinidazole) was ≥ MBC, the contents of the cysts were partly degraded, core structures did not develop inside the young cysts, and the amount of RNA in these cysts decreased significantly. When cysts were exposed to TZ, both the spirochetal structures and core structures inside the cysts dissolved, and the production of blebs was significantly reduced."

But I digress...

To take each question and thought about Dr. Sapi's latest paper by point:

"Three morphological forms of B. burgdorferi (spirochetes, round bodies, and biofilm-like colonies) were generated using novel culture methods."

Is there a description of these novel culture methods? Have they been through a process of verification and validation? How are they superior to other methods?

If different media is used other than BSK/BSK-H, it would be good to know if that affects the results in some fashion.

"Minimum inhibitory concentration and minimum bactericidal concentration of five antimicrobial agents (doxycycline, amoxicillin, tigecycline, metronidazole, and tinidazole) against spirochetal forms of B. burgdorferi were evaluated using the standard published microdilution technique."

What exactly were the concentrations?

It's important to know, and compare against other studies which have been done in vitro. Of all the literature I've read on this, standardization of these documented concentrations is lacking.

And to note: This was an in vitro test, not in vivo. Given everything that is known about Bb's behavior in vivo, in vitro tests will only give us part of the picture. More on that in a minute, below...

"The susceptibility of spirochetal and round body forms to the antibiotics was then tested using fluorescent microscopy (BacLight™ viability staining) and dark field microscopy (direct cell counting), and these results were compared with the microdilution technique. Qualitative and quantitative effects of the antibiotics against biofilm-like colonies were assessed using fluorescent microscopy and dark field microscopy, respectively."

How do these tests compare to one another relative to results found for each? How do the novel culture methods affect outcome relative to standard culture methods?

How is something biofilm-like, versus a biofilm?

"Doxycycline reduced spirochetal structures ~90% but increased the number of round body forms about twofold. Amoxicillin reduced spirochetal forms by ~85%–90% and round body forms by ~68%, while treatment with metronidazole led to reduction of spirochetal structures by ~90% and round body forms by ~80%. Tigecycline and tinidazole treatment reduced both spirochetal and round body forms by ~80%–90%."

This is interesting... Some of these results match earlier research findings on specific antibiotics used to treat Bb.

In 2008, Xiaohua Yang, Andrew Nguyen, Dan Qiu, and Ben Luft did some research on the effectiveness of tigecycline and doxycycline on Borrelia burgdorferi in vitro in In vitro activity of tigecycline against multiple strains of Borrelia burgdorferi: http://jac.oxfordjournals.org/content/63/4/709.short

In this abstract it was stated:

"Tigecycline inhibited the growth of and killed the organism more rapidly than doxycycline. Tigecycline was able to kill B. burgdorferi within 24 h at clinically achievable concentrations (> 1 mg/L). In contrast, doxycycline was bacteriostatic and required 48–72 h to achieve its maximal inhibitory effect. The anti-Borrelia activity of the antibiotics was tested against 20 different isolates from three species. Tigecycline was 16- to 1000-fold more active than doxycycline at immobilizing Borrelia for the 20 isolates tested."

The MIC versus the MBC is important to know, and it affects outcomes on a timeline.

Comparing Tigecycline to doxycycline sounds like comparing an axe to a thousand papercuts - the former is just going to be more immediately effective than the latter... And if Bb happens to disseminate rather quickly in an individual patient, there's a chance the doxycycline prescribed may not be enough to adequately treat the patient if you're going by this MIC - especially if one of Bb's strategies is to evade the immune system. (Let's not even get into how ineffective doxycycline is for prophylaxis for now...)

More recently, in 2010, Louis Ates, Christa Hanssen-Hübner, Douglas E. Norris, Dania Richter, Peter Kraiczy and Klaus-Peter Hunfeld conducted the study, Comparison of in vitro activities of tigecycline, doxycycline, and tetracycline against the spirochete Borrelia burgdorferi.

In their abstract they stated:

"The overall rank order of MIC90s was tigecycline (≤0.016 mg/L) > ceftriaxone (0.03 mg/L) > cefotaxime (≤0.125 mg/L) > doxycycline (0.25 mg/L) > tetracycline (0.25 mg/L). The rank order of MBC90s was tigecycline (0.5 mg/L) > ceftriaxone (2 mg/L) > tetracycline (16 mg/L) > doxycycline (16 mg/L) > cefotaxime (>16 mg/L).

High in vitro activity of the new glycylcycline against Borrelia was further substantiated by time-kill experiments performed with B. afzelii isolate EB1. Parallel testing of tigecycline and ceftriaxone demonstrated a bacteriostatic effect for 0.016 mg/L of tigecycline and for 0.03 mg/L for ceftriaxone after 72 h of incubation. Moreover, tigecycline was bactericidal at a concentration of 0.25 mg/L showing a > 3 log₁₀ unit reduction of the initial inoculum, whereas for ceftriaxone a concentration of 2 mg/L was needed."

So as you can see in this earlier research, tigecycline is highly effective in vitro, and doxycycline is ranked much further down the list of effectiveness.

Stepping into our time machine again, and going back to 2004 to Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Rebecca Rödel, Georg Acker, Volker Brade, and Peter Kraiczy's study, Comparison of In Vitro Activities of Ketolides, Macrolides, and an Azalide against the Spirochete Borrelia burgdorferi: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310164/

Now this was measuring an entirely different group of antibiotics, with the following results:

"The ketolides were the most potent against borrelial isolates on a micrograms-per-milliliter basis. For all agents except cethromycin and telithromycin, the MIC at which 90% of isolates were inhibited (MIC90) and the MBC at which 90% of the isolates were killed were ≥0.01 μg/ml and > 0.25 μg/ml, respectively."

and

"In our study, the rank order of activity by classical macrolides and azalides against borreliae clearly corresponds to the effectiveness of these agents as revealed by in vitro susceptibility studies and clinical treatment trials to date (2, 4, 5, 7, 8, 9, 11, 23, 24), demonstrating higher in vitro effectiveness for azithromycin (MIC90, 0.0156 μg/ml) than for erythromycin (MIC90, 0.0625 μg/ml), roxitromycin (MIC90, 0.0625 μg/ml), and clarithromycin (MIC90, 0.0312 μg/ml). Median MICs of the different substances, however, tended to vary over a 10-fold range between individual strains, with the B. garinii isolate PSth and the B. afzelii isolate EB1 showing the highest MICs for both the classical macrolides and the ketolides."

So in this study, of the antibiotics looked at, ketolides were more effective than macrolides, and azithromycin was more effective than other macrolides.

We also see that MIC's are different for different European Bb isolates. More on that below, in another cited study.

And then they said this about treatment failure:

"Classical macrolides and azalides frequently fail in the therapy of early LD (7, 14, 17, 26), and clinical relapse has been observed following conclusion of treatment (14, 17, 26). Moreover, it has been speculated that resistance may develop in borreliae preexposed to erythromycin owing to resistant subpopulations (25). Based upon our findings, however, the ketolides were superior in vitro on a micrograms-per-milliliter basis when tested alongside classical macrolides under identical test conditions in BSK."

There are a number of studies out there showing that Borrelia burgdorferi can be antibiotic resistant, with some being erythromycin resistant. Because of this, it is critical for doctors to weigh the use of erythromycin in patients - especially pregnant women with Lyme disease - against the risks of another antibiotic which is more effective.

The surprising thing about Dr. Sapi's study, to me, is the part about amoxicillin being that effective at reducing both spirochetal and round body forms. I say this, because I thought earlier research points towards amoxicillin being a less effective treatment than doxycycline.

Did I miss something, though? I thought earlier research did not look at antibiotic impact on round body forms in general, though - other than the Brorsons' metronidazole and tinidazole research.

Image taken from Brosons' research, An in vitro study of the
susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole

But earlier research is out there which confirms the effectiveness of amoxicillin on Borrelia burgdorferi, and found in a 2003 publication, In Vitro Susceptibility Testing of Four Antibiotics against Borrelia burgdorferi: a Comparison of Results for the Three Genospecies Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto.

What's fascinating to note here, again, is that different isolates of Bb respond very differently to different antibiotics!

"In 7 out of 12 comparative evaluations (P  > 0.05), MBCs were significantly different among the three genospecies. B. garinii seemed to be especially susceptible to azithromycin, while amoxicillin had a significantly greater effect on B. burgdorferi sensu stricto compared to the other genospecies. Ceftriaxone had the lowest MBC with B. afzelii and increasingly higher MBCs with B. garinii and B. burgdorferi sensu stricto. Doxycycline did not show any remarkable differences in its effects on the three genospecies."

So amoxicillin apparently doesn't suck when it comes to treating Borrelia burgdorferi, but it's not as effective on the other genospecies. (C'mon, Dr. Luft, please get that test working so we know which Bb we have to treat it the most effectively right off the bat.)

If it is found in vivo that doxycycline creates round bodies that contribute to the spirochete's survival, then doxycycline - what is typically given to patients diagnosed early with Lyme disease - would be contraindicated.

Whether the round bodies are as relevant as a potential "stasis" of metabolism in Borrelia burgdorferi remains to be seen. Either way, in vivo findings are different from in vitro findings.

In vitro, tigecycline was said to be many times more powerful than other antibiotics in killing Borrelia burgdorferi...Yet as we can see from Barthold's experiments on mice, viable spirochetes are found after tigecycline treatment in vivo, and viable enough that they can be picked up by ticks and transmitted to a new host.

Is there any treatment which can be used that would ensure the destruction of these remaining spirochetes, and would their demise lead to the end of persisting symptoms in patients who have them - or would there be an ongoing immune dysregulation which was triggered by their existence which continues after they are all dead?

This is something I'd really like to see studied.

Getting back to the last bit of Dr. Sapi's paper...

"When quantitative effects on biofilm-like colonies were evaluated, the five antibiotics reduced formation of these colonies by only 30%–55%. In terms of qualitative effects, only tinidazole reduced viable organisms by ~90%. Following treatment with the other antibiotics, viable organisms were detected in 70%–85% of the biofilm-like colonies."

I'd like to see analysis of how each of the five antibiotics fared relative to one another within biofilm-like colonies, rather than a range. It'd be good to do a direct comparison of each antibiotic against each form in vitro including biofilm-like colonies.

So... On the whole, I'd say take note of the study, with the message that independent confirmation and reproducibility of the methods chosen and these findings are important - and it's good to make note of these findings in relationship to other research already completed.

If anyone heads to the University of New Haven on the 21st to see the presentation on this, I'd love to get a report from you in comments about what was said.

In the end, I'll leave you with this closing thought from a paper from 2005, In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy :

"... similar to failures of chemotherapy for Treponema pallidum in syphilis (24), clinical treatment failures have been reported to occur in early LB cases for almost every suitable antimicrobial agent (10, 12, 28, 38, 42). Furthermore, the currently available diagnostic techniques do not reliably discriminate among possible reinfection, true endogenous relapse, and coinfection with other tick-borne pathogens (12). These drawbacks together with the phenomenon of resistance to therapy in individual patients undoubtedly contribute to the inconsistencies surrounding the optimal treatment regimens for LB and are often misinterpreted and misused to support prolonged antibiotic treatment regimens. However, relatively few cases of culture-proven treatment failure have been published (19, 22, 28, 29, 37, 38, 39), and the underlying mechanisms of antimicrobial resistance in B. burgdorferi sensu lato remain unresolved."

And there you have it. This pretty much characterizes the scientific reasons contributing to the ongoing controversy, five years later: Yes, there are treatment failures; yes, they are hard to diagnose and distinguish from coinfection and reinfection; yes, there is antimicrobial resistance; yes, scientists state these issues contribute to what is viewed as a misuse of prolonged antibiotic treatment.

But if treatment is necessary - whether it is a relapse or a new infection - then treatment is necessary.

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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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