Saturday, May 14, 2016

Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatment

As a follow up to my previous post, I would like to say something about several mouse studies from Stephen Barthold's group.  These papers are often cited by those who believe that retreatment is needed in patients who continue to experience symptoms following treatment of Lyme disease with conventional antibiotic regimens.  The assumption is that post-treatment symptoms stem from spirochetes surviving the initial antibiotic therapy.

In the 2008 and 2010 studies (described in detail here and here), Barthold's group gave doxycycline, ceftriaxone, or tigecycline to mice with disseminated Borrelia burgdorferi infection.  As expected, all tissues were culture negative up to three months following antibiotic therapy.  Tissues from untreated mice were culture positive.  However, B. burgdorferi DNA and mRNA were detected by PCR in up to half the treated mice, and microscopy revealed a few intact spirochetes in collagen-rich tissues from these mice.  Ticks allowed to feed on the treated mice even transmitted the spirochetes to other mice (albeit immune deficient ones), where B. burgdorferi DNA was detected by PCR.  Clearly, the spirochetes that survived antibiotic treatment were alive despite being unculturable.

Although live spirochetes remained following antibiotic therapy, there was no evidence that they were capable of causing disease.  Lyme disease is driven by inflammation, but no inflammatory response in the form of infiltrating immune cells were seen in tissues harboring the spirochetes.  A critic of the work also pointed out that the number of spirochetes declined during the 3 months following treatment, implying that any lingering spirochetes would eventually disappear.  It seemed unlikely that a similar phenomenon was responsible for persisting symptoms following treatment of Lyme disease in human patients, who may suffer with disabling symptoms for years.

In 2014 Barthold's group came out with another paper, which I'm discussing here for the first time.  Again, mice with disseminated B. burgdorferi infections were treated with antibiotics, ceftriaxone in this case.  But this time, the mice were left for up to a year before their tissues were examined for the presence of B. burgdorferi.  Control mice were mock treated with saline and examined along with the treated mice.

There weren't any surprises when tissues were tested by culture.  Most of the control mice were culture positive at all time points (2, 4, 8, and 12 months) with both tissues tested, the urinary bladder and the skin where B. burgdorferi was inoculated to initiate infection.  None of the treated mice were culture positive at either site at any time point.

PCR testing for B. burgdorferi DNA was done with tissue obtained from six sites in the mice.  Ticks allowed to feed on the mice were also tested for the presence B. burgdorferi DNA by PCR in a method called xenodiagnosis.  All saline-treated mice were PCR positive in most tissues tested, and most tested positive by xenodiagnosis.

The results with the mice treated with ceftriaxone are shown in the table below.  Each row represents a single mouse.  Note that each tissue homogenate was tested three times.

Table 2 from Hodzic et al., 2014.  "Interval" = time after completion of treatment; "Inoc" = skin from inoculation site; "HB" = heart base; "VM" = ventricular muscle; "QM" = quadriceps muscle; "Tt" = tibiotarsus; "XenoDx" = xenodiagnostic ticks (# ticks testing positive/# ticks placed on mouse).

They saw something remarkable with the mice left for 12 months.  Although few tissues were positive at earlier time points, most tissues extracted from mice a year after treatment tested positive.  6 of the 8 mice also tested positive by xenodiagnosis.  So, instead of eventually disappearing, the spirochetes proliferated starting at some point after 8 months elapsed following treatment.  This resurgence occurred even though the spirochetes remained unculturable.

Barthold's group also looked for evidence of inflammation.  Despite the resurgence of spirochetes, they did not see much evidence of inflammation by microscopy of the tissues 12 months  following antibiotic treatment.  However, the researchers pointed out that no conclusions can be drawn about the ability of the persisting spirochetes to cause disease since inflammation was minimal even in saline-treated mice, which harbored culturable spirochetes.

The researchers next looked for molecular evidence of inflammation.  They measured transcript levels of 18 cytokines in the base of the heart, heart muscle, quadriceps muscle, and leg joint 12 months after treatment with ceftriaxone or saline.  The levels of cytokine transcripts in the two groups were compared to those in age-matched uninfected mice.  Not surprisingly, saline-treated mice had what the authors deemed a "proinflammatory" cytokine profile, most likely due to their ongoing infection.  Antibiotic-treated mice also had a proinflammatory cytokine profile, although it differed from that of the saline-treated mice.  This observation is the first to suggest that the mice were responding to persisting spirochetes that survived antibiotic treatment.

In conclusion, the evidence is convincing that B. burgdorferi persists in mice for a long time after antibiotic treatment.  They don't eventually disappear and may even proliferate.  Whether these unculturable spirochetes are capable of generating an inflammatory condition necessary for disease is less clear, though mice do appear to generate a unique cytokine profile in response to the persisting spirochetes.

Barthold's group caution readers from applying the findings too broadly:
Because of the controversial nature of these findings, they should not be over-interpreted and certainly not translated directly into clinical management of human Lyme borreliosis.

So is there any relevance of these findings to post-treatment symptoms in humans?  I will touch upon this issue in a future post.

Reference

Hodzic E, Imai D, Feng S, & Barthold SW (2014). Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice. PLOS One, 9 (1) PMID: 24466286

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17 comments:

  1. Very interesting. Thank you for sharing your knowledge and thoughts on these animal studies concerning borrelia persistence and spirochetal viability after treatment.

    I'm wondering if at some point doing autopsy work on humans who have been treated with antibiotics but still had persisting symptoms might not reveal some answers in terms of the viability of spirochetes post antibiotic exposure. The organism is highly tropic, so it would be interesting to see what tissues like brain autopsies might show, there have been some very small scale studies but not much follow up in this arena (to my knowledge).

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    1. Autopsies aren't necessary just yet. One group is recruiting volunteers with post-treatment symptoms to see if Lyme Borrelia can be detected by tick xenodiagnosis (https://clinicaltrials.gov/ct2/show/NCT02446626). The first phase of this study has been completed (https://clinicaltrials.gov/ct2/show/NCT01143558).

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    2. Very interesting blog You have.

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    3. Great thanks for the head's up!

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  2. P.S. what are the results of this phase 1 ?

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    1. Results of the first phase were published in this paper: http://www.ncbi.nlm.nih.gov/pubmed/24523212. The main goal of this pilot study was to see if it is safe to do xenodiagnosis on Lyme disease patients. I'll say something about this study in a future post.

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    2. so in Your opinion, taking under consideration current knowledge, what is most probable reason for PTLSD ?

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  3. "Whether these unculturable spirochetes are capable of generating an inflammatory condition necessary for disease is less clear, though mice do appear to generate a unique cytokine profile in response to the persisting spirochetes." why would not they cause disease ? are they damaged somehow ?

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    1. Maybe I should have said that there is still little evidence that they cause disease. They may be capable of causing disease, but the limitations of Barthold's mouse model prevented them from answering the question (the limitation being the inability of his mice to infiltrate infected tissues with inflammatory cells even when left untreated).

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    2. Apparently the spirochetal forms are very hard to find in a host, the spirochetes convert fast into L - forms and we are dealing with Borrelia not spirochetes, as concerning the names... The same thing is in syphilis, 3rd stage of syphilis is that spirochetes are almost impossible to be isolated...Why would that be ? Are You familiar with Lida Mattman stealth pathogens book ?

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    4. Are You familiar with this movie ? https://youtu.be/WozrCFW0mRM Please pay attention how laboratory tried to culture spirochetes and could NOT, untill they called Lida Mattman

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    5. Here is medium that Lida Mattman and Phillips used to grow spirochetes from blood borellia but also Multiple Sclerosis patients... http://lymerick.net/MPM-2001-medium-Bb.html She says there is nothing easier in the world that not growing them if You dont do it right

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    6. I've been very interested in Mattman's work, I know she studied under Gabriel Steiner who was the spirochetal expert of his time. Grier wrote an interesting bit about her: http://www.lymeneteurope.org/info/cell-wall-deficient-bacteria

      I think she was finding both borrelia and another spirochetal organism in quite a few MS patients in a study she ran but have been unable to track down the paper so I have no idea on the specifics.

      This was a rather intriguing release in the last couple days from Dr MacDonald's work: http://www.prnewswire.com/news-releases/lyme-bacteria-hides-inside-parasitic-worms-causing-chronic-brain-diseases-300270742.html Need more research but it is interesting.

      There is also a nice video lecture by Mattman on youtube, but other than that one lecture it is very hard to find much information on her. I'll likely invest in getting her book at some point.

      I think the million dollar question is how common is the borrelia organism in the human host? how often does it cause disease? could it be part of the natural human microbial environment? are there other more important factors at play like immune system genetics that complicate the ability to handle this pathogen? I also imagine the wide genetic diversity of the borrelioses plays a factor in pathogenicity and illness manifestations as well, along with the other pathogens harbored in the human host. Immune system could reach a tipping point where it might no longer be able to keep a lid on the pathogens causing the system to go haywire. Guesses.

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    7. I have this book, i could give You the references to the MS studies on borrelia cultures from the chapter on spirochetes

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    8. that would be great, thank you so much!

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