Culture of the Entire Mouse to Determine Persistance of c.BB

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
edbo
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Culture of the Entire Mouse to Determine Persistance of c.BB

Post by edbo » Sun 31 Aug 2014 21:30

http://www.ncbi.nlm.nih.gov/pubmed/25155590
Antimicrob Agents Chemother. 2014 Aug 25. pii: AAC.03751-14. [Epub ahead of print]

Culture of the Entire Mouse to Determine Whether Cultivable Borrelia Burgdorferi Persist in Infected Mice Treated with a Five Day Course of Ceftriaxone.

Pavia CS1, Wormser GP2.

Abstract

Although controversial, it has been suggested that antibiotic treatment of laboratory animals infected with Borrelia burgdorferi often leads to the persistence of residual spirochetes that are claimed to be viable but non-cultivable. If viable cells of B. burgdorferi do persist following antibiotic therapy, one possible explanation for the lack of cultivability is that too few organisms persist in any given tissue site that might be sampled and cultured. In this study, we treated SKH (hairless) mice, with B. burgdorferi infection of 3 months duration, with either ceftriaxone or saline for 5 days and then cultured a suspension extract of nearly the entire mouse using a combined in vivo/in vitro culture method. All of the saline-treated (control) mice were culture positive, compared with none of the antibiotic-treated mice. Our findings further document the effectiveness of antibiotic therapy in eradicating cultivable cells of B. burgdorferi, irrespective of tissue or organ site.
I find this a very interesting publication. What are your thoughts on this?

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LHCTom
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by LHCTom » Sun 31 Aug 2014 22:37

Reminds me of a high school biology experiment.

Study design was optimized to prove what he already belived. Good scientists think about why the might be wrong and design a study to try and uncover their mistakes. 30 days of treatment and only 5 days till the culture is worthless science.

A number of observations by other scientists plus a little common sense might have caused Dr Wormser to think about these things in his study design.

Its been a very common observation that once borrelia adapts to the host environment and is under immune system pressure plus an all out antibiotic assault is driven to a minimum surviving population. That population has been very hard to culture with variants of the original Barbour culture design. Is that culture medium and process really properly designed for spirochetes with a dramatically shifted gene expression (transcriptome) that may be in a persister surival mode with their metabolism shut down. Persisters will not culture in any medium until they revert. In one study the resurgence took a full year but were still uncultivible. So the evidence suggests if you want to learn something new, you would go back to culture design plus wait at least a year post treatment before even trying.

These scientists need to try and understand whether their standard culture techniques are at fault due to transcriptome changes. They also need to allow enough time for persisters to shift to normal and multiply. And do the restored persisters have a new transcriptome or even altered/missing genes such that their culture needs have changed. They should stop assuming a culture designed for tick spirochetes will really meet the needs of post immune/treatment in new hosts spirochetes. That's a bad assumption and bad science. And at a minimum they should do their terminate and culture of the treated mice on multiple new culture design variants done by independent culture experts without a stake. Then over a schedule of something like every month for at least a year. And even at that, they may not have gotten the culture right and should perform both xenodiagnosis before termination plus DNA and RNA PCR. If they did all that it would mean something. Good scientists also know its hard to prove a negative.

http://onlinelibrary.wiley.com/doi/10.1 ... 0200.x/pdf
Bacteria in the viable but nonculturable (VBNC) state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies, but are still alive (Oliver, 2000). Despite their typically low levels of metabolic activity, they are again culturable upon resuscitation. Since the pioneering study by Xu et al. (1982) over 25 years ago, a large body of literature has evolved from researchers worldwide documenting the existence of a VBNC state in a wide variety of bacteria. Most investigators believe it to be a survival strategy in response to harsh environmental conditions, and it is now clear that the VBNC state constitutes an important reservoir of pathogens in the environment (Lleòet al., 2007a). The medical implications of this fact are numerous (Sardessai, 2005). For example, it appears that the ‘latent’ or the ‘dormant’ phase of Mycobacterium tuberculosis infections represents the VBNC state in this pathogen (Shleeva et al., 2004; Young et al., 2009), and that the recurrence of tuberculosis years after a person was thought to be tuberculosis free is due to resuscitation of this pathogen from the VBNC state (Pai et al., 2000). The list of pathogenic bacteria that have adopted this lifestyle as a means of survival includes not only those that infect humans but also those that infect such diverse animals as fish (Magariños et al., 1994; Biosca et al., 1996; Rahman et al., 2001), corals (Banin et al., 2000; Israely et al., 2001) and sea urchins (Masuda et al., 2004). Many plant pathogenic bacteria entering this state have also been described (e.g. Grey & Steck, 2001; Imazaki & Nakaho, 2008; del Campo et al., 2009; Ordax et al., 2009). Indeed, the list of pathogens is ever increasing, and includes Campylobacter spp., Escherichia coli (including EHEC strains), Francisella tularensis, Helicobacter pylori, Legionella pneumophila, Listeria monocytogenes, M. tuberculosis, Pseudomonas aeruginosa, several Salmonella and Shigella spp. and numerous pathogenic Vibrio spp. A list (likely incomplete) of these is provided in Table 1; a more complete list, including nonpathogens, can be found in Oliver (2005a).
Last edited by LHCTom on Sun 31 Aug 2014 22:58, edited 1 time in total.
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

duncan
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by duncan » Sun 31 Aug 2014 22:48

I've a couple thoughts, edbo.

First, just curious: How were the mice infected with Bb?

That aside, ok, they cultured the entire mouse. The ENTIRE mouse. Whiskers and tail and all? So, how does using the same method that hasn't revealed cultivable Bb cells on most of the mouse in the past, make any more sense when using it on ALL of the mouse? Aren't the authors in effect, repeating the same claim, just in a higher pitch and louder?

More to the point, how does this study disprove the hypothesis that post-treatment viable Bb cells persist, and are simply not cultivable? I mean, isn't the conclusion of this abstract little more than the authors once again could not cultivate Bb cells post-treatment? In other words, are not the authors admitting to making the same mistakes over again in their quest to find viable uncultivable post-treatment Bb persisters?

If the idea is the remaining spirochetes are no longer cultivable through existing methods, but remain viable in a morphed form, then what was the purpose of this exercise?

Edited to add: LHCTom, looks like we posted almost at the same time, and with I think pretty much the same point in mind. Only you said it much better, and much more Sciencey. :)

edbo
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by edbo » Sun 31 Aug 2014 23:24

Thanks for your feedback, duncan and LHCTom. Your posts have reminded me of trying to stay skeptical about everything... :)

There are a few more excerpts on relative-risk's blog, which might help in the discussion.

http://rel-risk.blogspot.de/2014/08/can ... vists.html
Excerpts from:
Pavia CS, Wormser GP, Culture of the Entire Mouse to Determine Whether Cultivable Borrelia Burgdorferi Persist in Infected Mice Treated with a Five Day Course of Ceftriaxone. Antimicrob Agents Chemother. 2014 Aug 25.

If viable cells of B. burgdorferi do persist following antibiotic therapy, one possible explanation for the lack of cultivability is that too few organisms persist in any given tissue site that might be sampled and cultured. In this study, we treated six mice with single daily doses of the antibiotic ceftriaxone for just 5 days and then cultured a suspension extract of nearly the entire mouse using a combined in vivo/in vitro culture method. No positive cultures were obtained, although B. burgdorferi could be recovered on culture of every mouse in the control group of infected mice treated with saline.

Mice were infected by injection with a tuberculin syringe intradermally in the abdominal area with 100,000 cells of B. burgdorferi in a volume of 0.1 ml of BSK medium. Three months later, the mice were given a single daily dose of either saline (control) or ceftriaxone (Rocephin, Roche Laboratories, Nutley, NJ) at a dose of 50 mg/kg for 5 consecutive days…. The saline and ceftriaxone injections were given in a volume of 0.1 ml intramuscularly. Treatment was initiated at a time point at which infection with BL 206 is known to be widely disseminated in other mouse species (eg., C3H mice), on the basis of the results of prior investigations.

Whole body extracts of the euthanized mice were processed…. After excising and discarding the stomach and the entire intestinal tract (to eliminate possible contamination by the indigenous gastrointestinal flora), the remaining bodies of the mice were processed separately by mincing them finely with scissors and forceps in a volume of 2.0 ml of BSK medium. Included in the whole body extracts were the following organs/tissue sites: brain, heart, kidneys, liver, lungs, lymph nodes, multiple bones and joints, musculature, pancreas, reproductive organs, spleen and skin (from abdominal, back and tail areas). The liquid contents (about 1.0 ml) of the minced body parts were collected and then injected intraperitoneally into separate normal recipient SKH mice. Four weeks later, the recipient mice were euthanized and separate extracts of excised urinary bladders and ears were prepared, cultured, and examined microscopically as described above; positive cultures were then subcultured….

None of the 6 mice that received ceftriaxone were culture positive directly or after inoculation of tissue extracts into other mice. Male and female mice were equally susceptible to infection and curable with antibiotic treatment. None of the cultures from ceftriaxone treated mice were unevaluable due to contamination with other microorganisms.

Our findings demonstrate that B. burgdorferi cannot be cultured from experimentally infected mice following just 5 daily doses of ceftriaxone treatment at a dosage of 50 mg/kg administered intramuscularly even when nearly the entire mouse is cultured. Given the short half-life of ceftriaxone in mice, residual ceftriaxone would not have been expected at the 7 to 10 day time point post-treatment when the mice were cultured. The ceftriaxone treatment regimen we chose was based on the uniformly successful results with this dosage and duration of ceftriaxone in a prior study of C3H mice that were also infected with the BL206 strain of B. burgdorferi, in which treatment success was judged based on the absence of a positive culture. Therefore, our findings may not be generalizable to other antibiotic treatment regimens.

In fact, it is clear that, after exposure to levels of antibiotics in vitro that are below the minimum bactericidal concentrations, that B. burgdorferi can still be cultured in BSK medium. It is also clear in several animal systems that recovery of B. burgdorferi in culture is correlated with the dosage of antibiotic administered with a much greater likelihood of positive cultures found in those animals receiving the lowest drug doses. However, based primarily on certain mouse studies reported by one group of investigators, it has been concluded that antibiotics, independent of drug class, never fully eradicate viable B. burgdorferi and instead result in the persistence of replication competent cells that have become non-cultivable and remain non-cultivable for at least 12 months following completion of antibiotic treatment. No mechanism has been discovered to explain this conclusion. These putative residual spirochetes do not appear to cause inflammation even when introduced into SCID mice (i.e., immunodeficient mice which typically develop histologic evidence of inflammation when infected with B. burgdorferi), nor do they apparently elicit a serologic response in the mice which were initially infected despite the reported observation that they may eventually markedly increase in numbers in certain tissue sites approaching the quantity of borrelial cells in untreated mice.

The results of our study indicate that if antibiotic therapy should induce a viable but non-cultivable state for B. burgdorferi in mice, the non-cultivability is not related to insufficient tissue sampling and is probably unrelated to simply having only a very small number of cultivable cells left in the mouse.

velvetmagnetta
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by velvetmagnetta » Mon 1 Sep 2014 5:21

What, does Dr. Wormser have only one friend left? Don't these papers usually have an "et al." to them?

I am relieved to see that Lyme does not persist when the host is drowned in antibiotics. I'm not so sure we want to apply this methodology to humans.

I applaud him doing an actual experiment this time, though.

The strange I-told-you-so tone of this article sounded so defensive that I had to go look up the paper those "certain" other scientists published:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900665/

This one, you can actually read the whole article. It comes to a conclusion I don't like, but I must admit that, unlike this Pavia/Wormser experiment, it is good science. If I can admit that, why can't Dr. Wormser? I have a lot more to lose if spirochetes persist than he does!

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ChronicLyme19
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by ChronicLyme19 » Tue 2 Sep 2014 19:47

Amen. LHCTom and Duncan. Studies like this are really doing a disservice to the public and will likely lead to more people with chronic infections. Plus what a waste of study money, why can't you put that money toward good use of figuring out why the bacteria get hunkered down in some people? What about our bodies makes it such a nice place for the Lyme to live?

Hopefully since this journal has a lower impact factor it won't cause as much of a problem.
Half of what you are taught is incorrect, but which half? What if there's another half missing?

velvetmagnetta
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by velvetmagnetta » Tue 2 Sep 2014 20:48

LOL, Duncan -
The ENTIRE mouse. Whiskers and tail and all?
The ENTIRE mouse. So there.

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LHCTom
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by LHCTom » Wed 3 Sep 2014 20:31

Has anyone access to the actual paper. I refuse to pay Wormser $25 on principle. The abstract and RR excerpt only say "culture positive" but don't menation what that means? Did they:

Use a monoclonal antibody stain and look through a darkfield microscope? or
Use PCR?
The greater the ignorance, the greater the dogmatism.

Attributed to William Osler, 1902

lou
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by lou » Thu 4 Sep 2014 15:52

Yes, what a joke this paper is: non-cultivable bacteria cannot be cultured. Five days post treatment.

hv808ct
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Re: Culture of the Entire Mouse to Determine Persistance of

Post by hv808ct » Thu 4 Sep 2014 17:16

Regardless of the role that the VBNC [viable but not culturable] state plays, it is clear that a large number of nonspore-forming bacteria, most notably a large number of human pathogens, are capable of entering this state, maintaining cellular structure and biology and continuing significant gene expression while otherwise non-culturable by ‘standard’ laboratory methods. That they can exit from this state, and become culturable again, is also undeniable.

The passage of VBNC through an appropriate animal host will induce return of culturability. Even these VBNC bacteria retain their pathogenicity and may trigger life in vivo and thus cause severe disease (Sardessai, 2005).

Any cell which has the capacity to be cultured in the future but does not grow under standard conditions for that organism is better termed ‘not immediately culturable’, as it remains culturable in an ultimate sense.

Check out Barer and Harwood, 1999, and Barer, 1997.

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