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

[nnecker]

I think history will show how the Lyme problem was solved using preventive methods,not with treatment methods.

[duncan]

And for all those who were sickened before those preventative measures?

Or those clinicians and researchers who turned a deaf ear to patients' cries? Or worse, made conditions harder for the sick through calumny, or took aggressive measures to impede progress?

History has a funny way of judging.

[nnecker]

Do you hear that Henry and hv808ct.History,duncan, and his Lyme kook gang are going to judge you.Do you think you are going to lose any sleep tonight?

[Henry]

I think those LLMDs who make lots of money by convincing naive and uninformed people that they have Lyme disease -- when they don't-- so that they can peddles all sort of unproven remedies have more to worry about the judgement of history than I do. Duncan ought to give some thought to whether he is helping the quacks or their victims who appear to be destined to receive even more antibiotics in combination for longer periods of time -- maybe forever?-- until all those nasty persisting "round bodies" are gone. By that time, fungi might start growing out of their ears and noses of these patients.

[dlf]

Henry, you wrote:

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?

Henry, you seem to have missed one point of the Hodzic study completely. Based upon flaB DNA qPCR, only a few tissue sites were positive at 2, 4 or 8 months in antibiotic-treated mice (Table 2). In contrast to the first 3 intervals, all antibiotic treated mice and all saline-treated mice at the 12 month interval were test positive for flaB DNA qPCR at multiple tissue sites, and in most cases, all 3 triplicate samples were flaB DNA qPCR-positive (Table 2). In addition, flaB DNA copy numbers in all tissues of antibiotic-treated mice were nearly equivalent to flaB DNA copy numbers in respective tissues of saline-treated mice at 12 months.

For the mice that were necropsied 4 months later something must have changed to have increased the positive testing results so dramatically. Dead bacterial DNA remnants don't just increase in numbers and don't suddenly appear in multiple tissues when they weren't found there previously......... Do they???

You did get my second point though. Yes, I did also wonder whether the antibiotic treatment was adequate to begin with because it sure looked like all 8 mice seemed to be recovering at eight months after treatment and all 8 mice at 12 months looked like they had treatment failure.

This is where it gets really interesting.........for this study mice were administered 16 mg/kg ceftriaxone in 500 ml 0.9% normal saline intraperitoneally twice daily for 5 days and then once daily for 25 days. This dosage regimen was utilized as a model for induction and analysis of persisting non-cultivable B. burgdorferi, since it has been previously shown to result in persistence of non-cultivable B. burgdorferi when administered at 3–4 weeks or 3–4 months of B. burgdorferi N40 infection. According to this paper, peak serum ceftriaxone levels exceeded the B. burgdorferi cN40 MIC and MBC.

An earlier study:

Effectiveness of Antimicrobial Treatment against Borrelia burgdorferi Infection in Mice
KATHLEEN D. MOODY, RUTH L. ADAMS, AND STEPHEN W. BARTHOLD*
Section of Comparative Medicine, Yale University School of Medicine,
Received 15 October 1993/Returned for modification 7 March 1994/Accepted 27 April 1994
http://www.ncbi.nlm.nih.gov/pmc/article ... 1-0123.pdf

"The efficacy of various dosages of eight antibiotics used for borreliosis treatment was evaluated for C3H/HeNCrlBR mice, which reproducibly develop persistent infection, arthritis, and carditis when inoculated with Borrelia burgdorfieri. Amoxicillin-clavulanic acid, ceftriaxone, and high-dose penicillin G effectively eliminated infection and disease. Oxytetracycline, doxycycline, chloramphenicol, erythromycin, and azithromycin failed to cure infected mice. There was a correlation between peak serum antibiotic concentrations in mice, as determined by agar well diffusion bioassays, and therapeutic levels in humans. When experimentally inoculated mice were treated at 1 week postinfection with ceftriaxone (16 mg/kg of body weight twice daily for 5 days) and monitored for up to 90 days, all treated mice were free of spirochetes and had no gross or histologic lesions. This antibiotic regimen at days 7 to 11 postinoculation eliminated the spirochetes so that there were no relapses during the 90-day observation period. For experimentally inoculated mice treated with ceftriaxone at 7 or 14 days postinfection, arthritis, carditis, and infection were eliminated. When treatment began at 30 and 90 days after inoculation, infection and active cardiac and arthritic lesions were eradicated; however, residual mild synovitis and vasculitis persisted in some mice. In comparison with inoculated, untreated mice, ceftriaxone therapy at 7, 14, 30, and 90 days postinfection abrogated the development of antibody titers against B. burgdorferi. Having the potential to determine the presence of the spirochete through culture and histologic lesions makes the B. burgdorferi-inoculated C3H mouse model a valuable adjunct in evaluating chemotherapeutic options for Lyme disease."

<snip>

"In the present study, the beta-lactam antibiotics (amoxicillinclavulanic acid, ceftriaxone, and high-dose penicillin G) were most effective at eliminating infection, carditis, and arthritis in experimentally inoculated mice. All three of these antibiotics achieved peak serum concentrations greater than or equivalent to human treatment values (29, 35). Once-daily penicillin therapy for 5 or 7 days failed to eliminate infection in experimentally infected hamsters and gerbils (22, 24, 33); however, similar doses given four times daily were effective in the mice of this study and in gerbils treated three times daily for 10 days (21), indicating that frequency of administration can be a significant variable in animal studies. Although the tetracyclines are commonly prescribed for human Lyme disease patients, their efficacy in infected mice was poor. Treatment for up to 14 days with doxycycline resulted in abatement of gross joint enlargement; however, there was no significant effect in abrogating development of histologic arthritis or carditis................ Mice treated with doxycycline had serum antibiotic concentrations comparable to those of humans at 15 min. This expanded-spectrum tetracycline has greater lipid solubility than other tetracyclines, which may account for more-efficient absorption after oral administration (46, 48). Although we demonstrated systemic absorption of doxycycline, clearance of infection and disease in inoculated mice did not occur."

According to this study, when treatment consisting of ceftriaxone (16 mg/kg of body weight twice daily for 5 days) began at 30 and 90 days after inoculation, infection and active cardiac and arthritic lesions were eradicated; however, residual mild synovitis and vasculitis persisted in some mice. Apparently that dosage of ceftriaxone achieved peak serum concentrations greater than or equivalent to human treatment values. The Hodzic study used exactly this dosage for their study initially and then followed it with another 25 days of 16 mg/kg ceftriaxone once a day.

So, to answer your question Henry, yes it did and does make me wonder whether the antibiotic treatment was adequate to begin with. My general sense of this is no, the treatment was likely not adequate. Since the Moody study was attempting to evaluate the variety of most often used antibiotics (for treating humans) in mice to the equivalent treatment levels recommended for and used in humans, their study findings are downright disturbing. Wouldn't you agree???

Henry, you also wrote:

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.

Sometimes pictures are far more explanatory than simple words. I was subtly suggesting that those pictures demonstrate certain features and properties of these round body forms. I guess I needed to be more direct. Here is another study that is more graphic despite having fewer pictures.

http://mic.microbiologyresearch.org/con ... 00027#tab2

Morphological and biochemical features of Borrelia burgdorferi pleomorphic forms

Authors: Leena Meriläinen1, Anni Herranen1, Armin Schwarzbach2, Leona Gilbert1

Microbiology, March 2015 161: 516-527, doi: 10.1099/mic.0.000027
Subject: Cell and Molecular Biology of Microbes
Received: 11/11/2014 Accepted: 27/12/2014 Published Online: 01/03/2015

The spirochaete bacterium Borrelia burgdorferi sensu lato is the causative agent of Lyme disease, the most common tick-borne infection in the northern hemisphere. There is a long-standing debate regarding the role of pleomorphic forms in Lyme disease pathogenesis, while very little is known about the characteristics of these morphological variants. Here, we present a comprehensive analysis of B. burgdorferi pleomorphic formation in different culturing conditions at physiological temperature. Interestingly, human serum induced the bacterium to change its morphology to round bodies (RBs). In addition, biofilm-like colonies in suspension were found to be part of B. burgdorferi’s normal in vitro growth. Further studies provided evidence that spherical RBs had an intact and flexible cell envelope, demonstrating that they are not cell wall deficient, or degenerative as previously implied. However, the RBs displayed lower metabolic activity compared with spirochaetes. Furthermore, our results indicated that the different pleomorphic variants were distinguishable by having unique biochemical signatures. Consequently, pleomorphic B. burgdorferi should be taken into consideration as being clinically relevant and influence the development of novel diagnostics and treatment protocols.

snip

Here we confirmed for the first time that RBs actually have an intact cell envelope with a peptidoglycan layer ( Figs 5 and 7 ), indicating that they do not fulfil clearly the definitions of spheroplasts, CWD or encysted forms although there are some modifications in the cell envelope and cell wall architecture. Furthermore, the intact cell envelope of RBs (Fig. 5 ), similar to the spirochaete, provides evidence for the previous suggestion ( Alban et al., 2000 ) that RBs are not degrading cells. To avoid confusing terminology, we suggest that B. burgdorferi spherical shapes are termed ‘round bodies’ to describe these forms better.

This is also really interesting........it seems that you don't need distilled water or antibiotics to alter Bb spirochetes to dormant round forms. Just having them disseminate into human serum can do this all on its own. I wonder then, do some of the spirochetes start to change their morphology to round forms right in the tick, during the blood meal?

[Henry]

DLF: With respect to your last point, such "forms" also appear with time and with aging. They represent senescent dying cells. No big deal, unless someone can demonstrate that they have clinical relevance, i.e., that they can produce disease. That has not happen as yet.

[duncan]

Ignore the studies; ignore the images; ignore the patients.

That's an odd recipe for helping the sick.

Henry, I'm still waiting on the name(s) of the author(s) of the ALDF article.

[nnecker]

Ignore the kooks.Ignore duncan.

[Henry]

Good advice, nnecker. I will do that. Thank you.

[dlf]

Duncan, I used to think when Henry came on and posted a comment that directed us to a new ALDF paper, that possibly he might be the author. Maybe that is true for some of them, but I really don't believe he could have written this one. This one is very carefully written and is well-crafted to manipulate the thinking of its target audience.

The first paragraph is the typical stuff that we have all seen and read before coming from IDSA supporters touting their Lyme ideology and policies. The material in the middle is basic information that any physician would know and understand, and seems to be inserted to act as something of a 'red herring' designed to let readers feel that they should be comfortable in accepting the article's conclusions. It is the last two paragraphs that provide food for thought.

From the ALDF draft article:

Some investigators have reported the presence of intact bacterial cells in the
tissues of animals treated with what appears to be adequate amounts of antibiotics
after infection with Borrelia burgdorferi. However, these may just be intact nonviable
cells; unlike the “persisters” found in in vitro studies, these intact cells have
not yet been isolated, re-cultured, and then shown to produce disease (12-14).

Although there is no evidence to indicate that “persisters” play a significant role in
the pathogenesis of Lyme disease in humans, the complete elimination of infection
is seldom used as the benchmark for success in the treatment of other infectious
diseases; the resolution of illness and lack of relapse, rather than microbiologic
cure, are of primary concern. At this time, there is no information to indicate that
small numbers of Borrelia might be found in some antibiotic-treated
immunocompetent patients who are asymptomatic.

Notice the difference between what is in the first of these paragraphs from the article (above) and in Henry's response to my post, even after he edited it. (below). The paragraph above is careful not to directly state that these cells represent anything in particular. Whoever wrote that paragraph used the qualifying phraseology "may just be" and "have not yet been". It would seem the author, but not Henry, is hedging his bets.

Henry wrote:

DLF: With respect to your last point, such "forms" also appear with time and with aging. They represent senescent dying cells. No big deal, unless someone can demonstrate that they have clinical relevance, i.e., that they can produce disease. That has not happen as yet.

In the concluding paragraph of the ALDF draft article the writer seems to already be trying to wiggle their way out of being proven wrong when studies ultimately do show that persister cells are clinically relevant. Of course, it starts with the old, 'there is no evidence', which to lay people implies that exhaustive well-designed research studies have been done and nothing was found. But, which people in the know have come to learn should be interpreted as meaning whatever limited studies have been done were most likely designed to not find anything. The rest of this sentence, "........the complete elimination of infection is seldom used as the benchmark for success in the treatment of other infectious diseases; the resolution of illness and lack of relapse, rather than microbiologic cure, are of primary concern." , is something everyone reading this can understand and relate to.

The closing sentence, while true, just seems to have been stuck in there completely out of context and out of the blue. Besides that, who the heck would care if, "small numbers of Borrelia might be found in some antibiotic-treated immunocompetent patients who are asymptomatic"?

But then, of course the ALDF writer would never willingly admit that far too many real-life patients do NOT have resolution of illness and lack of relapse.

In some respects, I think this article and the decision to write it points to a growing insecurity in maintaining their stance in light of evidence that is being generated mostly without their participation.