Thursday, November 8, 2012

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

According to the CDC, 10-20% of Lyme disease patients who have completed antibiotic therapy continue to suffer from symptoms such as joint, muscle, and neurological pain.  The following hypotheses are often presented as possible reasons for the lingering symptoms:  autoimmunity triggered by the infection, tissue damage inflicted by the spirochetes, and (depending on whom you ask) failure of antibiotics to kill all the spirochetes.  A new paper from Linda Bockenstedt's group at Yale proposes that antibiotic treatment of disseminated Borrelia burgdorferi infection leaves behind inflammatory pieces of dead spirochetes that are responsible for the persisting symptoms.

Bockenstedt's group used the mouse model of Lyme disease for the study.  To ensure that the tissues harbored enough B. burgdorferi spirochetes to be visible by intravital microscopy, the mice were genetically deficient in the intracellular signaling protein MyD88.  MyD88 links the recognition of microbial parts by most Toll-like receptors to activation of certain nuclear genes whose products are involved in the inflammatory process.  Mice lacking MyD88 are unable to control the proliferation of a number of bacterial pathogens, including B. burgdorferi.  The load of B. burgdorferi in tissues is about 100-fold higher in MyD88-deficient mice than in mice with a complete immune system.

The spirochetes were genetically altered to express green fluorescent protein (GFP).  The GFP+ B. burgdorferi was introduced into the MyD88-deficient mice by tick inoculation.  21 days later some of the mice were treated for one month with doxycycline, one of the antibiotics used to treat Lyme disease in humans.

The researchers next peered into the thin layer of skin covering the ear by intravital microscopy.  In the mice that were left untreated, they saw lots of spirochetes scurrying about in the dermis.


At the deepest depths of the dermis, they noticed immobile specks and patches of green material deposited near the cartilage.  They also saw the deposits in the doxycycline-treated mice.  The material was detected by immunofluorescence of ear sections with antibody against B. burgdorferi up to 10 weeks after antibiotic treatment was completed, indicating that the immune system was unable to clear the deposits.

There was no evidence that any spirochetes survived antibiotic treatment.  The researchers did not see any motile spirochetes in the skin by intravital microscopy.  In addition, tissues were culture negative, ticks that fed on the treated mice were culture negative (xenodiagnosis), and transplantation of skin from the treated mice failed to transmit the infection to recipient mice.  Based on these results, the authors concluded that the deposits were remnants of dead spirochetes.  As expected, untreated mice tested positive by these assays.

Since chronic infection can lead to Lyme arthritis, the investigators also examined the joints.  In another set of mice, the infection was allowed to proceed for four months.  The mice were then treated with the antibiotic ceftriaxone for 18 days.  When the researchers looked in the joints by intravital microscopy, they again saw the green material (see figure below).



Fig. 5 from Bockenstedt et al. showing the surface of the patella where it meets the tendon (enthesis).   Panel A, from mouse infected for 4 months, untreated.  Panel B, from mouse infected for 4 months and then treated with ceftriaxone for 18 days. Scale bar, 30 ┬Ám.

A critical issue to address is whether the amorphous material left behind following antibiotic treatment inflames the joints.   The authors could not answer this question directly because of the limitations of the mouse model. Histopathology is unlikely reveal joint inflammation, even in the untreated animals, because laboratory mice do not reliably exhibit joint inflammation so late (4-5 months) during B. burgdorferi infection.  Instead, the authors conducted a test tube experiment to see whether the deposits had inflammatory potential.  They ground up joint tissue from antibiotic-treated mice in buffer and applied the homogenate to cultured mouse macrophages.  The macrophages responded by producing TNF, a key cytokine that promotes inflammation.  The more tissue that was added, the more TNF that was produced by the macrophages.  In contrast, joint tissue from uninfected mice did not promote TNF production by the macrophages.  Therefore, the deposits had the potential to spark inflammation, even after motile spirochetes were eliminated by antibiotics.  The debris would continue to inflame the tissues even after antibiotics killed all live spirochetes, explaining why symptoms persist in ~10% of Lyme arthritis cases even after antibiotic treament.

The relevance of the deposits to Lyme disease in humans could be questioned because the MyD88-deficient mice did not have a complete immune system.  The authors addressed this concern in the Discussion by mentioning a recent study that described a TLR1 variant linked to severe inflammation and treatment failure in Lyme arthritis patients.  Although the gene encoding MyD88 has never been examined in Lyme disease patients, it is conceivable that the TLR1 variant or different forms of other immune genes lead to deposits of Borrelia antigen in the joint and other host tissues.

The authors also addressed the possibility that the deposits are really biofilms, which generally resist killing by antibiotics.  Biofilms are believed to be populated by persister cells, which are in a nondividing state that allows bacteria to tolerate antibiotics.  According to the authors, if the deposits had harbored persister cells, those cells should have resumed growing when conditions became favorable for growth again.  Because the skin and joints from the treated mice were culture negative and because the skin also tested negative by xenodiagnosis and transplantation assays, the authors quickly dismissed the biofilm hypothesis.

Stricly speaking, the authors are correct.  Persister cells should start multiplying again in fresh culture medium.  However, it's hard to dismiss the biofilm hypothesis completely given the known examples of culture-negative chronic infections associated with biofilms (see this review for one example).  Electron microscopy of the joint tissue could reveal whether these deposits are intact spirochetes or debris.

Regardless of their exact nature, deposits of antigen have never been detected within the joints of Lyme arthritis patients.  Allen Steere's group failed to find such deposits in pieces of synovial membrane removed from 26 patients with antibiotic-refractory Lyme arthritis.   The findings of Bockenstedt and colleagues, who detected the deposits in a location outside of the synovial membrane, suggest that Steere's group was looking in the wrong place.


Featured paper

Bockenstedt, L., Gonzalez, D., Haberman, A., & Belperron, A. (2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy Journal of Clinical Investigation, 122 (7), 2652-2660 DOI: 10.1172/JCI58813
 
Helpful references

Bolz DD, Sundsbak RS, Ma Y, Akira S, Kirschning CJ, Zachary JF, Weis JH, and Weis JJ (August 1, 2004).  MyD88 plays a unique role in host defense but not arthritis development in Lyme disease.  The Journal of Immunology 173(3):2003-2010.  Link

Strle K, Shin JJ, Glickstein LJ, and Steere AC (May 2012).  Association of a Toll-like Receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis.  Arthritis and Rheumatism 64(5):1497-1507.  DOI: 10.1002/art.34383

Bakaletz LO (October 2007).  Bacterial biofilms in otitis media, evidence and relevance.  The Pediatric Infectious Disease Journal 26(10):S17-S19.  Link

Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC (December 1999).  Lack of Borrelia burgdorferi DNA in synovial samples from patients with antibiotic treatment-resistant Lyme arthritis.  Arthritis and Rheumatism 42(12):2705-2709.  DOI: 10.1002/1529-0131(199912)42:12<2705::aid-anr29>3.0.CO;2-H


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7 comments:

  1. Dr Barthold - Barthold: dormant, non-dividing bacteria are tolerant of antibiotics. During persistent infection there is a 10-fold reduction in number of bacteria but they are not dividing, so antibiotics can't hurt them.

    100% of animals remain infected after antibiotics--- described as debris but it is often metabolically viable organisms.

    12 months after treatment we see widescale prevalence of spirochetes but it remains to be determined the role of this persistence --not everyone is showing symptoms.

    Biofilms - universal mechanism for bacteria, virus and fungus throughout the world for microbial communities. Borrelia - persistent infection --- reduction in host/animal ( with antibiotics?)- go dormant (not necessarily in bio film) - antibiotics can't touch.

    If you just enlarge the pot of funding, and you award the money to the same old school club of people who feed off Lyme disease, we won't get anywhere. If NIH is willing, they should put out a call for applications [for research] that is more focussed on biology. http://foreignaffairs.house.gov/hearings/view/?1455

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  2. Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.
    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0048277

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    Replies
    1. Thanks for posting the link to their study. They present some amazing images in their paper. I plan to write up a short post about their work.

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  3. Biofilms of borrelia burgdorferi In Vitro Observations
    http://molecularalzheimer.org/

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  4. "According to the CDC, 10-20% of Lyme disease patients who have completed antibiotic therapy continue to suffer from symptoms such as joint, muscle, and neurological pain. "

    Thank you for posting this article. As one of those "10-20%" who continues to suffer from persisting symptoms after initial infection, it is a step in the right direction to find potential evidence for what may be causing my pain and suffering. The symptoms are not just pain related, though there is that - there are also cognitive deficits such as memory and concentration problems, moderate to severe fatigue, pain after short term physical exertion, sleep disturbances, and more.

    I am not sure what causes my symptoms and have been reading whatever research I can to try to get a better understanding of my condition, and it has been frustrating because based on limited data as a patient there are few choices available for treatment and what we do is somewhat experimental in terms of trying longer term antibiotic courses, pain medication, heat and hydrotherapy, and so on.

    "Stricly speaking, the authors are correct. Persister cells should start multiplying again in fresh culture medium. However, it's hard to dismiss the biofilm hypothesis completely given the known examples of culture-negative chronic infections associated with biofilms (see this review for one example). Electron microscopy of the joint tissue could reveal whether these deposits are intact spirochetes or debris."

    This is what I want to know: How long can spirochetes persist, and how does this fit into a model of latency? Is Borrelia burgdorferi like Treponema pallidum, which can be asymptomatic and latent but later evolve into a fulminant third stage neurological infection years later? Does latency = persisters if this occurs?

    I agree that electron microscopy studies are in order. Studies are needed on the extracellular matrix to see if biofilms are present.

    "Regardless of their exact nature, deposits of antigen have never been detected within the joints of Lyme arthritis patients. Allen Steere's group failed to find such deposits in pieces of synovial membrane removed from 26 patients with antibiotic-refractory Lyme arthritis. The findings of Bockenstedt and colleagues, who detected the deposits in a location outside of the synovial membrane, suggest that Steere's group was looking in the wrong place."

    Self-reporting here: My pain has rarely been right in the joint. I've had swelling in the joints, but most of my pain is in the tendon right above the joint when I get it. That and I get burning muscle pain upon short periods of pushing myself phyiscally for a shorter period of time than the average person who is, say, engaging in heavy lifting or walking. Don't know - thought I'd share this as a possible (yet anecdotal) data point.

    How do you see this research as potentially tying into Tunev's and Barthold's? Care to speculate?

    I liked your earlier entry on Tunev et al's study and think this, too, is an interesting write-up from another field:

    http://memoryreactivaction.wordpress.com/2012/08/01/borrelia-burgdorferi-the-master-manipulator/

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    1. I'll comment about the relationship between the Barthold and Bockenstedt studies. The theme that unifies their findings is that antibiotic therapy of infected mice leaves behind spirochetes in some form, whether live (but attenuated) or dead debris. Why does the immune system fail to finish the job started by the antibiotics? I hesitate to place the blame on an aberrant antibody response (the Tunev study) since no one has actually shown that the antibodies produced during B. burgdorferi infections are of poor quality. Of course it wouldn't matter if we knew for sure that these remnants didn't cause harm, but Bockenstedt's work suggest that they may (possibly depending on the genetic background of the host). My guess is that Barthold's attenuated "persisters" would also have inflammatory properties under some condition yet to be discovered.

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  5. To reject the borrelia biofilm concept based on the following 4 declarations is to fail to understand the biology of biofilms
    as defined by Dr. William Costerton:
    These allegations by Bockenstedt include:
    1. Lack of observed Persister microbe regrowth in Planktonic microbiologic media
    2. Culture Negativity of tissues with biofilm in Planktonic microbiologic media.
    3. Skin transplant failure to transmit infection, and Xenodiagnosis
    negative microbe recovery results : without defining the
    depth of the putative "glob units of borrelia" from
    the skin surface and witha reconciliation of the
    depth of the Tick hypostome into the deep subcutis,
    as opposed to tick feeding on sperficial murine dermial
    tissues.
    4. Electron microscopic study - "indeterminant for "debris"
    - without presenting the raw data from the purported
    electron micrographs for peer review.
    --------------
    Collectively these 4 pitals call up the authors' failure to understand
    Biofilm biology and
    Merely weighs final judgment of 4 tests based on Planktonic expectations.
    Bockenstedt, L., Gonzalez, D., Haberman, A., & Belperron, A. (2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy Journal of Clinical Investigation, 122 (7), 2652-2660 DOI: 10.1172/JCI58813
    Objections to the Bockenstedt"Data" are as follows:

    1 Persister phenotype/genotypes have never been isolated in pure form
    precisely because Per-sisters Fail to grow with the kinetics
    of growth of Planktonic Microbes.
    2. Culture negativity is a Costerton cornerstone Concept
    for the behavior of True biofilms
    Therefore Culture negativity Adds to the specificity of
    the "globs of Bockenstedt" as being Biofilm communities
    3. Skin Tranplants have never been utilized to Judge biofilms
    of any microbial species. Xenodiagnosis has never been utilized
    to judge the status of any candidate biofilm community
    healthy or otherwise.
    4. Electron Microscopy Raw data was not presented for the reader to
    evaluate in Bockenstedt's Paper.
    ------------------
    By Sheer Density of borrelia profiles alone, the images of Bockenstedt demonstrate biofilm communities. Never before has any investigator of Borrelia ever published images of borrelia in such high density in the tissues a mammal. Biofilm communities attain
    densities of microbes which far exceed the density of Planktonic
    microbes in mammalian tissue. When the microbes actually physically
    overlap with one another, the microbes have reached a biofilm community density level. Organisms in biofilms are Specialized, and that specialization includes the generation of Novel DNA Sequences
    when compared with Planktonic forms, and novel proteins when compared with Planktonic forms. Horizontal DNA transfer occurs between members of the biofilm community and is responsible in part for the DNA In Flux within borrelia biofilm communities. Granular borrelia, Cystic Borrelia, Planar non coiled borrelia, and Cell wall deficient Borrelia are all evidence of specialization of biofilm borrelia community members, These morphologic specializations are clearly demonstrated and annotated in Bockenstedt's figure legends.
    Cell death of individual members of borrelia within a borrelia biofilm community is expected, since it is by cell death, that elements of protein, and DNA are deposited into the Extracellular matrix material which holds biofilm of borrelia together and which provides protective activities to the borrelia community.
    To summarize: Each of the "challenge" tests applied by Bockenstedt
    fails to eliminate borrelia biofilms from consideration, and in fact
    adds specificity to the biofilm mature of the great innumerable sheets of specialized borrelia in the Murine peri-articular soft tissues.
    Submitted August 15 2013
    Alan B. MacDonald MD FCAP FASCP

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