Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
duncan
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by duncan » Sat 28 May 2016 0:43

I don't know, Henry...A potential Congressional investigation...Don't you usually maintain the source matters? Is any one petitioning for Congress to investigate ILADS?

No, I think that the ALDF is one of the organizations noted with that, er, distinction.

But if you are ok with that, you are certainly welcome to believe whatever you wish.

BTW, who authored that piece you linked to that struggles to refute the significance of the three distinct and highly regarded persister studies?

Henry
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by Henry » Sat 28 May 2016 15:40

More distracting statements to avoid dealing with the content of the piece, which you don't understand. That it emanates from the ALDF is sufficient for you to conclude that it must be bad.

I will close my discussion of this issue by making one last point. Those "round bodies" that appear when bacteria are treated with antibiotics are nothing more than cell wall or membrane deficient forms that arise when cell wall or cell membrane synthesis is inhibited by an antibiotic. Although Borrelia lack a true cell wall, their outer membrane is sufficiently rigid to give it the shape that it has as a spirochetes. When the outer membrane is weakened by exposure to an antibiotic, the cytoplasm then expands by pushing its way through the weakened membrane; this results in what is described by some as "blebs" -- or round bodies when the "blebs' separate from such cells. Why does this happen? It's is due to a process called reverse osmosis in which water molecules move from the outside to the inside of the cytoplasm where the salt concentration is greater. A weakened cell membrane structure enables this to occur.

Since the reaction of antibiotic with the 30S ribosomal subunit (leading to inhibition of protein synthesis) is reversible, as the ALDF article states, so is the formation of these cell wall or membrane deficient forms -- within a certain period of time after exposure to antibiotic and provided the osmomolarity is controlled. Otherwise, they will die in due time, without the need for another type of antibiotic to speed up the process.

The take home message is that these cell wall or membrane deficient forms are not cysts, nor are they intermediate stages in the natural life cycle of Borrelia. They are not unique to Borrelia. They are produced when all bacteria are treated with an antibiotic, and have no special relevance re: the poorly defined condition called chronic Lyme disease. Consequently, conducting further in vitro such as those described is not likely to be fruitful. It should be painfully obvious that they in no way approximate what is occurring in vivo. where the host immune system plays a major role.

Note: My objective here is not to respond to your irrelevant and distracting statements and prejudices. Rather, it is an effort to communicate with those who really want to understand what is going on and what can be learned or concluded from these in vitro studies. If you are incapable of engaging in such a discourse, then "buzz off".

duncan
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by duncan » Sat 28 May 2016 16:29

I think the polymorphic nature of Borrelia has been amply demonstrated over the years by many, Henry. I would hope you would agree this should not be the subject of any serious discussion any longer. :)

The relevance of this nature to the persistence of Bb in the 20% (or more?) of Lyme patients in whom historic therapy fails may be a subject for discussion in some shrinking circles. Regardless, that so many remain sick lends grim testimony to the need for more research.

Treatment implications such as cyst forms in late stage Lyme apparently may be further complicated by persister cells. The existence of Bb persister cells post-treatment has been elegantly demonstrated by Drs Lewis, Zhang and Embers in discreet efforts.

The take home message would seem to be that there are many reasons for the existence of an estimated 60,000 or so Lyme patients who are unwillingly added to the roles of the chronically ill year after year after year. Each of those reasons needs to be acted upon.

Accordingly, it is essential that new, robust, and well-funded research into universally effective treatments be initiated without further delay.

hv808ct
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by hv808ct » Sat 28 May 2016 17:00

Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters
Post by duncan » Sat 28 May 2016 0:43

I don't know, Henry...A potential Congressional investigation...Don't you usually maintain the source matters? Is any one petitioning for Congress to investigate ILADS?


What are the odds that Congress would “investigate” a private website about a vector-borne disease? These guys can’t even get together to recognize actual public health threats never mind the silly rants of the delusional denizens of the Internet.

duncan
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by duncan » Sat 28 May 2016 18:16

Concerned, hv808ct?

I'd put the odds at far better than they were before the petition.

Henry
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by Henry » Sat 28 May 2016 18:32

Duncan: All dying bacteria exhibit the various polymorphic forms of which you speak, regardless of where they are grown. When looking at millions of bacteria cells, one is bound to see a few of those. But, they are not to be construed as stages in the life cycle of that bacterial species. Rather, they are disintegrating, dying cells. If you consult a textbook on bacteriology and look at some of the pictures (circa 1950s?) of antibiotic-treated bacterial cells -- or even very old cultures-- you will lots of these polymorphic forms. They are not cysts.

I must say that I agree with your last point. However, such robust research should involve other treatment approaches and not focus solely on new and/or better antibiotics given in combination etc. After all, it has not been established --beyond the shadow of a doubt -- that what you and other call "chronic Lyme disease" is really caused by a persistent infection. Why not consider other treatment options -- just to be on the safe side ?

Although "persisters" have been isolated and cultured from Borrelia treat with antibiotics in vitro , they appear to be no different from the parent culture, i.e., they are not antibiotic resistant mutant strains of Borrelia. That has been established as noted in the ALDF article. However, that has not been the case for the in vivo studies. Although small numbers of intact Borrelia have been found in infected animals after treatment with antibiotics, the clinical relevance of such cells remains to be established. Such cells have not been cultured and have not be shown to be viable, let alone shown to produce disease. They could just as well be dead intact bacterial cells.

duncan
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by duncan » Sat 28 May 2016 18:44

a) I am not talking about dying bacteria.

b) I did not mention chronic Lyme. I said Lyme; that we are likely speaking to late stage Lyme should be understood.

c) There is a difference between persisters and antibiotic resistant cells.

d) It's good to know what one is trying to develop treatment for before you try to develop treatment for it. I am advocating for better treatments of Lyme disease - treatments that incorporate the most recent findings into their development. If I understand their efforts correctly, drs like Zhang at Johns Hopkins seem to agree.

e) I am sure that we both can agree that it's important to stay as current as possible with Lyme developments, and not get mired down in past misconceptions.

dlf
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by dlf » Sat 28 May 2016 20:07

Henry wrote:
Although small numbers of intact Borrelia have been found in infected animals after treatment with antibiotics, the clinical relevance of such cells remains to be established. Such cells have not been cultured and have not be shown to be viable, let alone shown to produce disease. They could just as well be dead intact bacterial cells.
Henry, would you please explain to me then, how it is that Borrelia could resurge after 12 months post treatment?

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): e86907. doi:10.1371/journal.pone.0086907

http://journals.plos.org/plosone/articl ... 086907.PDF

"However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues."

Here is a little more analysis from Hodzic.......

Lyme Borreliosis: is there a preexisting (natural)
variation in antimicrobial susceptibility among Borrelia
burgdorferi strains?
Emir Hodzic*
http://www.bjbms.org/ojs/index.php/bjbm ... e/view/594

Henry also seems to misunderstand the development and nature of round body forms and the differences between them and other morphological variations. Henry maybe this reference will help you out with that:

(This study is in German. The photography is very clear and there is a partial translation just below the link.) It does take a while for the pdf to open. It is a very large file. This was posted before on LymeNet Europe, but it could take more time for me to locate it to give credit to the person who did. Thank you to whomever did originally post this.

Rößle,B., Electron microscopy studies of Borrelia: ultrastructure and immune-cytology, Diss., Department of Biology, University Munich(2001).
Available from: http://edoc.ub.uni-muenchen.de/63/1/Roessle_Birgit.pdf
cell division: pl. 7 coccoid morphotypes, viable but not cultivable: pl. 8 (live staining), 9, 10, 11, 12, 13, 29, fig. 6 vital staining: pl. 8, 16 A, 17 A, 18 A, 19 A and C, 20 A and C, 21 A and C, 25 A and C, 26 A and C rolled spirochetes: pl. 9, 29 astrocytes and Bb (afzelii, garinii): pl. 30–37

Depending on the culture age Borrelia burgdorferi shows strong structural changes that can be examined by scanning electron microscopy. During the log phase the number of coil turns is increasing, and thus also the length of the Borreliae. Towards the end of the log phase they lose their typical helical shape. In the light microscope (dark field) one can simultaneously observe a loss of mobility. However, the loss of mobility does not simultaneously mean the death of Borrelia, because of the formation of moving helical Borrelia after seeding into fresh culture medium.

Furthermore, through this work a method was developed to produce coccoid morphotypes of the Borrelia cell. By incubation of Borrelia in distilled water (aqua destillata) it is possible to produce these types of Borrelia within a few minutes, in a reproducible manner. By vital staining it could be shown that the coccoid morphotypes are living form variants of the Borrelia cell. This form variants can not be cultured. Transferred into culture medium only helical Borrelia cells can be observed after 4-5 days.

The coccoid morphotypes are spherical swellings of the outer membrane, in which the protoplasmic cylinder is furling in tight turns. The space surrounded by the coiled protoplasmic cylinder is largely featureless. The flagella are located on the side facing away from the outer side of the membrane of the protoplasmic cylinder. The reconstruction of serial ultrathin sections revealed that these coccoid morphotypes are formed from a single Borrelia cell each; protoplasmic cylinder, cell wall and outer membrane remain intact.

Moreover, it was shown, as example Osp17, Osp35, and OspC, that the surface proteins of the helical Borrelia cell are also located in the coccoid morphotypes on the surface. These three proteins are also distributed evenly over the entire surface of the the coccoid morphotypes. However, through the formation of the coccoid morphotypes a significant reduction in the surface is observed, compared to the helical Borrelia cells. Thus, this coccoid form variants have only a reduced contact surface for the antibodies of the host. They probably represent forms that enable the microorganism to evade the immune system of the host.

Moreover, the adhesion between two different species of Borrelia burgdorferi has been studied in human astrocytes.

For this purpose the B. garinii strain PBi was used besides the aforementioned B. afzelii strain of PKo. This is an isolate of the CSF of a patient. Carried out as a 24-hour trial of co-incubation it was shown by light microscopy that both strains adhere to the astrocytes. However, the Borrelia afzelii strain PKo adheres far more often than those of B. garinii strain PBi overall. The scanning electron microscopic examination showed that a plurality of the two strains of Borrelia burgdorferi are in contact with the branches at the edge of the astrocytes and on their surface. The ability of Borrelia to adhere to the astrocytes may play a role moving from the bloodstream into the brain.

A clear reaction of the astrocytes to the contact with the microorganism in form of surface changes cannot be seen. Using the scanning electron microscopy both strains show Borrelia penetrating into cells. This penetration can be confirmed by ultra-thin sections of the co-incubation preparations in TEM (transmission electron microscopy). Here Borrelia could be found both in vesicles and free in the cytoplasm of the astrocytes. The intracellularly located Borrelia were still intact even after 24 hours. There are clearly no degeneration forms recognized. By penetrating into the astrocytes the Borrelia may succeed to survive a long time in the host.


Whoever wrote the ALDF piece needs to rethink it in light of conflicting scientific evidence.

Henry
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by Henry » Sat 28 May 2016 21:15

DLF: Since DNA has been found to persist in the tissues of infected animals for long periods of time after the infection has been cured by antibiotics, the work of Hodzic et al. does not necessarily prove the presence of viable Borrelia cells. Only that there is sufficient DNA in the specimens involved to code for flaB. Nothing more than that can or should be inferred. Also, I find it astounding that the copy numbers were almost identical to those for saline treated animals. Makes one wonder whether the antibiotic treatment was adequate to begin with, wouldn't you think?

Some of the examples cited by the German group involve "coccoid morphotypes" that are not cultivatible, "aged" cultures, and "coccoid" types that are artifacts produced when Borrelia are placed in distilled water (not good for these cells). I see nothing here that would alter my stated views, especially when there is no evidence to indicate that these forms are clinically relevant, i.e., that they produce disease. But, some people are fascinated by such pictures and are quite imaginative in interpreting what they mean.

duncan
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Re: Drug Combination Active against Amoxicillin-Induced Round Bodies of In Vitro B. burgdorferi Persisters

Post by duncan » Sat 28 May 2016 22:05

There may be merit to suggesting two broad camps within the Lyme clinician/research community have emerged, and they are not necessarily defined by IDSA vs ILADS.

One camp dismisses not only evidence that Bb persists despite conventional attempts at abx therapy, but it also minimizes or discounts patients' reports that their Lyme symptoms persist.

The other camp accepts patient reports, and acknowledges evidence that supports persistent infection after conventional treatment recommendations, as explanatory of those patient reports.

So, one camp puts patient reports first and foremost, the other not so much. Or at least, I suspect this is how history will relay the events years from now.

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