Thursday, January 12, 2012

9 Paper: Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection

Lymenet Europe scooped this first, and it's important enough that I had to share it here...

Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection (2012)

Monica E. Embers, Stephen W. Barthold, Juan T. Borda, Lisa Bowers, Lara Doyle, Emir Hodzic, Mary B. Jacobs, Nicole R. Hasenkampf, Dale S. Martin, Sukanya Narasimhan, Kathrine M. Phillippi-Falkenstein, Jeanette E. Purcell, Marion S. Ratterree, Mario T. Philipp


Source Links:
http://www.plosone.org/article/fetchArticle?articleURI=info%3Adoi%2F10.1371%2Fjournal.pone.0029914


PLoS ONE 7(1): e29914. doi:10.1371/journal.pone.0029914

Abstract

The persistence of symptoms in Lyme disease patients following antibiotic therapy, and their causes, continue to be a matter of intense controversy. The studies presented here explore antibiotic efficacy using nonhuman primates.

Rhesus macaques were infected with B. burgdorferi and a portion received aggressive antibiotic therapy 4–6 months later.

Multiple methods were utilized for detection of residual organisms, including the feeding of lab-reared ticks on monkeys (xenodiagnosis), culture, immunofluorescence and PCR.

Antibody responses to the B. burgdorferi-specific C6 diagnostic peptide were measured longitudinally and declined in all treated animals.

B. burgdorferi antigen, DNA and RNA were detected in the tissues of treated animals.

Finally, small numbers of intact spirochetes were recovered by xenodiagnosis from treated monkeys.

These results demonstrate that B. burgdorferi can withstand antibiotic treatment, administered post-dissemination, in a primate host.

Though B. burgdorferi is not known to possess resistance mechanisms and is susceptible to the standard antibiotics (doxycycline, ceftriaxone) in vitro, it appears to become tolerant post-dissemination in the primate host. This finding raises important questions about the pathogenicity of antibiotic-tolerant persisters and whether or not they can contribute to symptoms post-treatment.



Comments:

A number of Lyme disease patients who initially read this abstract went wild over it, thinking, "Here is the Holy Grail - here is the research that firmly establishes that anyone with post-treatment persisting symptoms is suffering from a chronic bacterial infection".

While this paper is an important piece of research, it is not the final word that clinches chronic infection as the cause of post-treatment symptoms. It does lay the groundwork for specific research that will bring us closer to an understanding of what happens with Borrelia burgdorferi in vivo.

Taking a closer look, the following passages of the full text caught my attention:

This is just a small thing, but I like that the three primary hypotheses for persisting symptoms are mentioned in the introduction section:
"Signs and symptoms of putative failure of antibiotic treatment in late disease or ineffectiveness of repeated treatment in patients with PTLDS may be formally attributed to several causes, including: 1) spirochetes that persist in the tissues, likely in small numbers, inaccessible or impervious to antibiotic; 2) inflammatory responses to residual antigens from dead organisms; or 3) autoimmune responses, possibly elicited by antigenic mimicry [10]."
It's important to note these three hypotheses when reading the text to follow because the authors indicate how findings may relate to them.
"In the assessment of gross pathology, histology, and immunofluorescence, no gross lesions were observed in any of the animals. Fragments of heart and meninges were collected postmortem, and fixed, sectioned and stained for histology and immunofluorescence. Three animals, all of them treated, had moderate to severe inflammatory lesions in the heart."
Post treatment animals showed signs of moderate to severe lesions in the heart. At what rate would it take for these lesions to heal and how much they would affect the animals over time?
Does comparable damage occur in humans after the same antibiotic treatment?
"At 7 and 11 months PI, all monkeys were fed upon by Ixodes scapularis nymphs for the uptake of persisting spirochetes by xenodiagnosis. The number of nymphs that fed to repletion varied considerably between animals. For the first round, a total of 7, 8, and 11 ticks, respectively, fed on the treated animals, whereas only 5 ticks fed on each of the untreated animals (Table S2). Tick midguts were split into 3 parts for culture, direct fluorescence and for DNA extraction. The probability of recovering spirochetes would be higher from animals upon which more ticks feed. As such, intact spirochetes were detected from the cultured midgut contents (Figure 5A) or directly from tick midgut smears (Figure 5B) of two animals at 7 months PI, both of which had been treated. These animals (GB56 and GA59) also had the most xenodiagnostic ticks feed (Table S2)."

The use of xenodiagnosis in this situation can be used to support Koch's first and second postulates, but Koch's third and fourth postulates require more evidence. Intact spirochetes were found in ticks which had fed on antibiotic treated animals. In order to meet Koch's third and fourth postulates, another experiment would need to be conducted where the ticks feed on uninfected animals and disseminated spirochetes would have to be found in tissues in the newly infected animals.

(Additional postulates can be tested for on a molecular level. See Molecular Koch's Postulates. The below excerpt relates to this with evidence of transcription of a lp28-1 gene in a treated animal.)

"A few spirochetes grew in cultures of organ tissues collected post-mortem from each animal after > 9 weeks, but we were unable to subculture any spirochetes from either treated or untreated animals due to their slow growth. We therefore pelleted these cultures to confirm their identity and test their viability by DNA/RNA analysis. Transcription was detected in culture pellets and the tissues of treated animals, indicating that the bacteria were metabolically active (Figure 6C, D). Figure 6D shows ospA transcription detected directly in tissues harvested from treated and untreated animals. We also hypothesized that persistent spirochetes may lose linear plasmid 28-1 (lp28-1), which encodes the VlsE antigen bound by the anti-C6 antibody. Transcription of a lp28-1 gene (bbf26) was verified in organ tissue from both untreated animals and one treated animal (Figure 6D)."
In the 2009 paper, Antibiotic Treatment of Animals Infected with Borrelia burgdorferi, Gary Wormser asked, "What are causes of the attenuation of the spirochetes that persist posttreatment? Are they in the process of dying? Are they producing mRNA, and if so, which mRNA? Are they motile? Can they replicate? Are they genetically altered? Can they regain pathogenicity?"

In his own conclusion, he states, "The biological nature of these spirochetes is unclear," along with some caveats about the likelihood of their being pathogenic.

Now here we have evidence that points to Borrelia spirochetes which are metabolically active and transcribing OspA and an lp28-1 gene in antibiotic treated animals. So at least part of Wormser's questions are answered. But not all.

Now we examine the discussion portion of the paper...
"Our results indicate that disseminated spirochetes of two different B. burgdorferi strains can persist in the primate host following high dose, or long-lasting antibiotic therapy."
Many patients have made note of this particular passage, with the concern that surviving spirochetes are the source of persisting symptoms after high dose or long-lasting antibiotic therapy. A number of people have stated that this finding justifies the use of long-term high dose antibiotic therapy. However, it's not over from the researchers' perspective. Their work is still cut out for them - they still have to do additional research that links the presence of metabolically active spirochetes with reproduction and symptoms in the host.
"At the molecular level, B. burgdorferi DNA would indicate the presence of organisms, live or dead. The detection of RNA, however, should indicate that those present are metabolically active and thus alive. In Experiment 1, spirochetal DNA and RNA were detected in the tissues of a few animals, independent of treatment. This may reflect a low spirochetal burden, lack of flaB transcription [37], and/or seclusion in untested tissues."
This is probably one of the most important passages in the full text. While spirochetal RNA
was present in antibiotic treated animals and spirochetes were alive, the signficance of their presence has to be clearly determined. The worthwhile thing to note at this stage is that the spirochetes were metabolically active and alive after long-term high dose antibiotic therapy.
"[...] few slow-growing organisms were recovered by culture from each animal. We detected the ospA transcript in culture pellets from tissues of four animals and directly from at least one tissue from each animal. We chose ospA because this gene has been shown to be transcribed by host-adapted B. burgdorferi [38], in disseminated infection [39], and because of the induction of an anti-OspA response in patients [40] post-dissemination."
So much discussion in various publications has been leveled at the presence of anti-OspA response in patients and host animals, with the correlation being made between anti-OspA and the inflammatory response rather than the presence of continued infection. What if both are present? Given Barthold's studies on the immune system's response to Borrelia burgdorferi in lymph nodes, would a combination of infection and inflammatory response after antibiotic treatment be likely in treated human hosts?
"It has been postulated that the joint tissues provide a protective niche during antibiotic treatment [41]. Our studies and others [37], [42], however, have not demonstrated a specific predilection for spirochete presence in joints of treated animals."
Pointing this out for all Lyme disease patients: While Lyme arthritis is a well-known manifestation of Lyme disease, Borrelia spirochetes do not seem to congregate in joints on a massive scale.

(Personally, I have suspected nerve and tendon involvement all along, based on my own symptoms.)
"A “persister” phenotype may possibly be responsible for the recalcitrance of persisting spirochetes made evident by previous studies in mice and dogs [37], [42], [45], and by those presented in this report. Perhaps incomplete clearance of bacteria following antibiotic treatment is not a phenomenon unique to B. burgdorferi, but one that occurs with other bacterial infections as well. In this case, xenodiagnosis enables detection of otherwise inconspicuous live organisms through acquisition by the natural vector."
This is speculative, but something worth following up on in future studies: Is there a persister phenotype of Bb that survives high dose antibiotic treatment? Does this mean that Borrelia is antibiotic resistant - or more precisely, antibiotic tolerant? If so, how will long-term high dose antibiotic treatment kill these remaining spirochetes if it hasn't happened after the dosages and durations stated?
"Also, the C6 titers declined in some untreated animals over a long period of time, but not in others, though presence of spirochetes was indicated in C6-negative untreated animals by IFA or PCR/RT-PCR. This is likely due in part to genetic differences in outbred animals."
How do these data apply to untreated human hosts? Do people have different genetic backgrounds which can lead to negative tests even when spirochetes are present?

Let's read on...
"Possible explanations for the lack of correlation between C6 response and presence of spirochetes include: (1) the anti-C6 titer is an indicator of treatment efficacy and the infection is cleared despite the presence of spirochetal genetic material/antigen; (2) organisms persist and the anti-C6 titer does not reflect their presence, perhaps due to loss of plasmid lp28-1 (which encodes VlsE, the parent molecule of C6); or (3) anti-C6 titer declines with a significant reduction in spirochetal burden, but a low number of organisms reside in the host; if these organisms are dormant, then transcription of vlsE also may be negligible, minimizing re-stimulation with antigen.

The detection of intact organisms ruled out the first explanation and detection of transcript (bbf26) from lp28-1 disproves that explanation #2 may be operating exclusively. We therefore favor explanation #3 and seek to determine the level of transcriptional/metabolic activity, and pathogenicity of persistent organisms. If, for example, spirochetes that are recovered by xenodiagnosis from treated animals turned out to be non-pathogenic, this would validate the decline in C6 titer as a measure of successful treatment outcome."
Based on the above, the researchers have stated that:
  • Metabolically active and live spirochetes have been found in primates which were treated with long-term high dose antibiotics.
  • Whether or not these spirochetes can reproduce has not yet been established.
  • Whether or not these spirochetes are infectious has not yet been established.
"Due to the relatively small quantities of bacteria over a large amount of tissue, we were unable to reliably quantify DNA or transcript levels in nonhuman primates. Similarly, the recovery of few spirochetes by tissue culture is aptly a reflection of the rhesus model and not necessarily the treatment [48]."
This indicates that the authors of the paper are giving word of caution that the presence of a few spirochetes in non-human primates may be due to their being non-human primates and not because of the antibiotic treatment they received.
"The most pressing question in terms of human disease is whether or not spirochetes remain pathogenic after antimicrobial therapy. Similarly, do spirochetes persist long-term, or are they eventually cleared by the host? Clearly, the phenotype of persistent organisms needs to be elucidated. These studies support the use of the C6 test for diagnosis and measurement post-treatment; however, the absolute quantification of antibody levels may be essential in determining treatment efficacy for PTLDS patients, as low levels (yet above baseline) may indicate presence of residual spirochetes or antigen."
Yep, see my earlier bullet points.
"Finally, the use of variable and pulse-dosing regimens of antibiotics may improve efficacy [43] and this warrants testing in an appropriate model."
Now this is interesting... The suggestion is that pulsing antibiotics may work better as a treatment model for Borrelia/Lyme disease infection.
"Finally, in these studies we used an artificial mode of inoculation and spirochetal dose. The experimental results must be confirmed with tick-mediated infection, which is our intent. Our studies do however offer proof of the principle that intact spirochetes can persist in an incidental host comparable to humans, following antibiotic therapy. Additionally, our experiments uncover residual antigen associated with inflammatory foci. Whether persistent spirochetes or spirochetal antigen can cause PTLDS remains unanswered."
There you have it. This paper is a stepping stone to more research that will characterize the nature of Borrelia spirochetes after long term high dose antibiotic treatment. Research that I hope is coming soon.

9 comments:

  1. Wonderful news thanks for posting. reposted on Looking at Lyme Disease Facebook and Twitter.

    As Gill aid on Eurolyme 'The Holy Grail'
    I remember Ben Luft saying at IOM that Barthold would have some important research to share later in the proceedings about his Rhesus monkey work- Barthold never said a word and I was bitterly disappointed I thought he really sat on the fence. Perhaps he has been giving his 'chums' time to re group, or apply for their pensions LOL!

    ReplyDelete
  2. Joanne,

    Thanks for sharing news of this publication with others. I think it's an important paper to share, and important to read the full text and not just the abstract - though the abstract is definitely eye-catching.

    These particular passages in the research leapt out at me:

    "A few spirochetes grew in cultures of organ tissues collected post-mortem from each animal after >9 weeks, but we were unable to subculture any spirochetes from either treated or untreated animals due to their slow growth. We therefore pelleted these cultures to confirm their identity and test their viability by DNA/RNA analysis. Transcription was detected in culture pellets and the tissues of treated animals, indicating that the bacteria were metabolically active (Figure 6C, D). Figure 6D shows ospA transcription detected directly in tissues harvested from treated and untreated animals. "

    "At the molecular level, B. burgdorferi DNA would indicate the presence of organisms, live or dead. The detection of RNA, however, should indicate that those present are metabolically active and thus alive. In Experiment 1, spirochetal DNA and RNA were detected in the tissues of a few animals, independent of treatment. This may reflect a low spirochetal burden, lack of flaB transcription [37], and/or seclusion in untested tissues."

    "Antibiotic tolerance has been demonstrated in vitro with several bacterial species, both gram negative (E. coli) and gram positive (Staphylococcus spp.). The fact that organisms can persist in the presence of antibiotics such as penicillin and cephalosporins (ceftriaxone) that interfere with cell wall synthesis appears to stem from their ability to enter a dormant, non-dividing state [43], [44], thus avoiding the need for cell wall synthesis to continue growth. The other antibiotic that was used in these experiments is doxycycline. The tetracycline class of antibiotics corrupts translation at the ribosome; therefore, minimal gene expression from dormant organisms may be unaffected. A “persister” phenotype may possibly be responsible for the recalcitrance of persisting spirochetes made evident by previous studies in mice and dogs [37], [42], [45], and by those presented in this report. Perhaps incomplete clearance of bacteria following antibiotic treatment is not a phenomenon unique to B. burgdorferi, but one that occurs with other bacterial infections as well. In this case, xenodiagnosis enables detection of otherwise inconspicuous live organisms through acquisition by the natural vector."

    So this is all notable.

    Two things have to happen next, I think:

    1) Discover how long these remaining spirochetes can remain dormant and if they reproduce, at what rate do they reproduce?

    2) Provide evidence that host symptoms are directly attributed to the presence of the spirochetes and the damage they do.

    I don't think we are quite at the stage where this paper definitely states that a chronic infection is driving patients' symptoms. This is a step closer to that, though.

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    Replies
    1. I don't disagree with what you are saying Camp but we do now know that Lyme spirochetes can remain despite host immune system plus antibiotics clearance so it is a nonsense for our health agencies to continue to say that in disseminated cases that a couple of weeks antibiotics is all that is needed- they just do not know neither does anyone else.

      What I find interesting is that there were so few ticks feeding and yet they managed to pass on the spirochetes - does that not suggest that there must have been plenty circulating in the blood for it to be passed so easily? This is something many people have found in microscopy but this is frowned on by our health authorities as a way of detecting spirochetes.

      Delete
    2. I don't think you get time to follow Facebook so I will share Pam Weintraub's comments
      'of great importance to Lyme --the long-awaited persistence study out of Emory, in rhesus monkeys. This is the study that was commissioned to accompany the Klempner study on persistence in humans, by NIH. I have been waiting for these authors to buck the politics and publish for years.'
      'it is still going to be very complex. I know that Barthold does feel these spirochetes cause cytokine and chemokine cascades --normally drivers of autoimmune disease. But if infection is the driver ...there are probably different paths to treatment.'
      'Yup this is the big one'

      Delete
    3. Joanne,

      I agree that the evidence that spirochetes can survive antibiotic treatment must be factored in to making decisions about patient treatment. I think you're absolutely right and saying health agencies do not know enough about this either way - that's been a long standing problem.

      Standard light microscopy has not been considered by microbiologists to be the most accurate for determining the specific spirochete with which one is infected (is it Treponema denticola, or another non-pathogenic Treponeme, or is it something harmful like Borrelia burgdorferi)? What one needs to do for identifying specific pathogenic bacteria is use fluorescently labeled antibodies to bind to antigens on the bacteria and examine them using a fluorescent microscope. (See http://en.wikipedia.org/wiki/Fluorescence_microscope)

      Thanks for sharing Pam Weintraub's comments on this paper. I think that the work Barthold has been doing over the years has been very important and it needed to be shared right away. I also think that additional research is needed and we need to find out how much of patients' symptoms are caused by the infection relative to the immune/inflammatory response and genetic factors of both the bacteria and the patient. "There are probably different paths to treatment" - I'd have to agree with this.

      Delete
    4. I've covered all these questions in a paper I wrote titled: The Case For Chronic Infection: The Case For Chronic Infection: Evidential persistence of Borrelia species post antibiotic exposure in vivo and in vitro.

      All available studies on this specific topic are included in the paper, as well as bold print to emphasis crucial pieces of each study.

      The problem isn't a lack of evidence. There is an abundant array, over 1500 pages worth supporting Chronic Infection. I include only the abstracts.

      https://acrobat.com/app.html#d=sbb-EmpQrQTgrPoezLGreg

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    5. Michael,

      Welcome to Camp Other blog.

      I don't think there is any dispute that Borrelia spirochetes have been found after antibiotic exposure in both in vitro and in vivo studies. There are quite a number of these studies cited on this blog which I call to the reader's attention. So on this count, yes, there is plenty of evidence spirochetes are found after antibiotic treatment. But the issue for many researchers has been whether or not these remaining spirochetes were metabolically active or alive - as well as whether or not they could reproduce and be pathogenic. They want to satisfy not only Koch's original postulates but Koch's Molecular postulates as well. If the studies you cite have evidence to support Koch's Molecular postulates, it would be good to review them in full text.

      Delete
  3. Note to readers:

    This is really nagging on me:

    "Transcription of a lp28-1 gene (bbf26) was verified in organ tissue from both untreated animals and one treated animal (Figure 6D)"

    That plasmid confers infectivity and is supposed to be responsible for antigenic variation. I'd have to look up bbf26 to see its specific relevance, but there is evidence that if that plasmid is lost or damaged that mice can clear the Borrelia infection more easily. It loses its virulence.

    If transcription of an lp-28 gene occurs, this opens the door to a lot more questions that need answering. So far, a sample size of one animal is very small. Other people are going to point this out. One is all you need to provide evidence that yes, Bb plasmid gene transcription can occur even AFTER high dose antibiotic treatment. But the more examples of this which can be found, the stronger one's argument is for investigating it and potentially supporting the persistent infection model.

    Numbers matter. So does method. Barthold is meticulous and exacting, as someone else has stated on this blog earlier. But he will be expected to produce confirmation of his results and other reseachers will want to see more treated animals that result in this finding.

    ReplyDelete
  4. Let's just face the simple fact.

    Lyme bacteria is a lot more complex that ALL of the best experts and science can understand.

    Theory's are just that and patient experiences are not just subjective. After a well documented case of Lyme my wife developed two autoimmune diseases.

    The bottom line is all camps need to pull their heads out and develop more research and science to understand this highly complex - well traveled, easy to get bacteria.

    Then who ever discovers the real facts beginning to end should speak. The rest of the pundits should produce or bag the noise.

    In fact let's see if the IDSA docs can solve this problem - or do they just want to promote an agenda, not a solution.

    ReplyDelete

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