Various features of Bb pleomorphic forms

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
RitaA
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Various features of Bb pleomorphic forms

Post by RitaA » Fri 9 Jan 2015 23:38

http://mic.sgmjournals.org/content/earl ... 0027.short
Morphological and biochemical features of Borrelia burgdorferi pleomorphic forms

Leena Meriläinen1,
Anni Herranen2,
Armin Schwarzbach3 and
Leona Gilbert4

Author Affiliations

1 University of Jyväskylä
2 University of Jyväskylä
3 Borreliose Centrum Augsburg
4 University of Jyväskylä

Received 11 November 2014.
Revised 16 December 2014.
Accepted 27 December 2014.

Abstract

Spirochete bacteria 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 bacteria to change its morphology to round bodies. 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 round bodies had an intact and flexible cell envelope demonstrating that they are not cell wall deficient, or degenerative as previously implied. However, the round bodies displayed lower metabolic activity compared to spirochetes. 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.

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ChronicLyme19
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Re: Various features of Bb pleomorphic forms

Post by ChronicLyme19 » Tue 13 Jan 2015 18:41

Thanks for posting. I find this part a bit scary:
Here, there is evidence that biofilm-like colonies establish even at early exponential phase of growth (Fig. 2f)
suggesting that high density is not the only factor enhancing biofilm formation in suspension
Some nice conclusions:
We demonstrated here that collagen is present on EPS of B. burgdorferi biofilm-like colonies in suspension (Fig. 7) supporting the previous findings that suspended biofilms are biofilms, not just cell aggregates.
Interestingly, transformation of spirochetes to RBs increased remarkably when growth conditions changed to media with HS, nutrient poor mammalian RPMI culture medium or H2O (Fig. 2).
The treatment with HS and distilled H2O induced morphologically similar RBs, indicating that the transformation does not occur only because of the osmotic stress.
Remarkably, we found that RBs have lower metabolic activity (Fig. 3b); however, they have the ability to revert back to spirochete form and regain their ATP activity.
We provide a step-by-step model, which is in harmony with the findings of others (Al-Robaiy et al., 2010; Murgia & Cinco, 2004), that the outer membrane of B. burgdorferi is flexible allowing it to expand during RB transformation when the protoplasmic cylinder is folding within the envelope (Fig. 4, Fig. 5a, Movie S1). Furthermore, the flagella, located in the periplasmic space in spirochetes (Barbour & Hayes, 1986; Zhao et al., 2013), were present in RBs indicating that these forms can maintain the motility and skeletal components and then recruit them again during reversion back to spirochetes (Fig. 6).
Now, my question is this is maybe how it reacts in blood, but is this representative of say in the spinal fluid or brain fluid? I'd still love to see someone follow up on the comment from the other study on what happens to round body forms when exposed to TNF-alpha inhibitors.
Half of what you are taught is incorrect, but which half? What if there's another half missing?

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ChronicLyme19
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Re: Various features of Bb pleomorphic forms

Post by ChronicLyme19 » Tue 13 Jan 2015 19:14

Oh wow, look at this. All the steps from spirochete to dormant round body form.
Figure 4 reduced.jpg
Figure 4 reduced.jpg (126.69 KiB) Viewed 2200 times
Fig. 4. Round body formation evolved through the expansion of the outer membrane and folding of the protoplasmic cylinder. (a) Stepwise demonstration of RB development using transmission electron microscopy (TEM) images of H2O and human serum RBs. From left to right parental spirochete, spirochete with initial membrane expansion, bleb where folding of the protoplasmic cylinder inside the outer membrane is initiated, and the bleb transitioning to the RB formation with folded protoplasmic cylinder under the expanded outer membrane are presented. (b) Cross- sections of completed RBs and the organization of folded protoplasmic cylinder are visualized with TEM micrographs. RB is displayed from the side, from the front and from the top, respectively.
Half of what you are taught is incorrect, but which half? What if there's another half missing?

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ChronicLyme19
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Re: Various features of Bb pleomorphic forms

Post by ChronicLyme19 » Tue 13 Jan 2015 19:56

Plus, when you combine this study with this one, you get some really good evidence towards a possible persistence mechanism. Doxycycline was shown to increase the round body form:
http://www.ncbi.nlm.nih.gov/pubmed/21753890

My guess is it doesn't like the doxy and curls up and then just lies dormant until you stop the antibiotics. Pretty scary considering this is the antibiotic of choice normally given to people with tick bites.
Half of what you are taught is incorrect, but which half? What if there's another half missing?

RitaA
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Re: Various features of Bb pleomorphic forms

Post by RitaA » Tue 13 Jan 2015 22:19

ChronicLyme19 wrote:Plus, when you combine this study with this one, you get some really good evidence towards a possible persistence mechanism. Doxycycline was shown to increase the round body form:
http://www.ncbi.nlm.nih.gov/pubmed/21753890

My guess is it doesn't like the doxy and curls up and then just lies dormant until you stop the antibiotics. Pretty scary considering this is the antibiotic of choice normally given to people with tick bites.
Thanks for the links and images, ChronicLyme19.

From the link you provided above:
RESULTS:

Doxycycline reduced spirochetal structures ∼90% but increased the number of round body forms about twofold. Amoxicillin reduced spirochetal forms by ∼85%-90% and round body forms by ∼68%, while treatment with metronidazole led to reduction of spirochetal structures by ∼90% and round body forms by ∼80%. Tigecycline and tinidazole treatment reduced both spirochetal and round body forms by ∼80%-90%. When quantitative effects on biofilm-like colonies were evaluated, the five antibiotics reduced formation of these colonies by only 30%-55%. In terms of qualitative effects, only tinidazole reduced viable organisms by ∼90%. Following treatment with the other antibiotics, viable organisms were detected in 70%-85% of the biofilm-like colonies.
This may help to explain why some doctors add metronidazole (aka Flagyl) to doxycycline when treating Lyme disease patients. I don't recall hearing or reading about any doctor relying exclusively on metronidazole, tigecycline or tinidazole, but all that really means is that I don't remember hearing or reading about it.

One thing I do anticipate is that one or more members will discount/dispute these research findings because:

1) they are in vitro studies, and
2) the research was carried out by Sapi and others at the Lyme Disease Research Group, Department of Biology and Environmental Sciences at the University of New Haven in Connecticut and Dr. Stricker (of ILADS)

That said, who else is studying the true effects of antibiotics on Lyme disease in humans?

From the last sentence of the full article:
Our in-vitro methodology will facilitate the design of experiments that mimic tissue-based in-vivo conditions in order to optimize the antibiotic treatment of Lyme disease.

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ChronicLyme19
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Re: Various features of Bb pleomorphic forms

Post by ChronicLyme19 » Wed 14 Jan 2015 2:52

These guys have done some, this is my next set of papers to work my way through.
http://www.ncbi.nlm.nih.gov/pubmed/?ter ... BAuthor%5D

Brorson Ø, Brorson SH
Department of Microbiology, Sentralsykehuset i Vestfold HF, N-3116 Tonsberg, Norway

They've hit some of the major favorites like:
tigecycline
tinidazole
metronidazole
hydroxychloroquine
grapefruit seed extract

And also others I haven't heard of ever being used:
telithromycin
ranitidine bismuth citrate
Half of what you are taught is incorrect, but which half? What if there's another half missing?

hv808ct
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Re: Various features of Bb pleomorphic forms

Post by hv808ct » Wed 14 Jan 2015 4:49

Re: Various features of Bb pleomorphic forms
Post by ChronicLyme19 » Tue 13 Jan 2015 19:56

Plus, when you combine this study with this one, you get some really good evidence towards a possible persistence mechanism. Doxycycline was shown to increase the round body form: http://www.ncbi.nlm.nih.gov/pubmed/21753890
Maybe you should read Anni Herranen’s thesis, which provided much of the data for the paper. It’s a PDF available here: https://jyx.jyu.fi/dspace/bitstream/han ... 203319.pdf
My guess is it doesn't like the doxy and curls up and then just lies dormant until you stop the antibiotics.
That’s why we have an immune system. Absent a functioning one, all the antibiotics in the world aren’t going to save you from a cut finger.

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Re: Various features of Bb pleomorphic forms

Post by X-member » Wed 14 Jan 2015 14:28

hv808ct wrote:
That’s why we have an immune system. Absent a functioning one, all the antibiotics in the world aren’t going to save you from a cut finger.
I also (together with late Lyme borreliosis) have immunodeficiency (CVID).

Do you think that our immune system always can "take care of" bacteria that are not killed by a short course of Doxycycline?

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inmacdonald
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Re: Various features of Bb pleomorphic forms

Post by inmacdonald » Wed 14 Jan 2015 20:56

REMINISCENCES of Henry's submissions to this forum...

Who spoke authoritatively
On this forum

About the absolute IMPOSSIBILITY of the very existence
OF Borrelia Cystic forms ( aka ROUND BODY forms )

Continuing Education turns naysayers into ardent believers..

The first publication on Cystic Borrelia was authored by me
In year 1988 in the Annals of the New York Academy of Sciences..
And the image evidence came from microscopic examination
Of an Alzheimer's autopsy brain !


Alan B. MacDonald, MD, FCAP
JANUARY 14 ,2015

Lorima
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Re: Various features of Bb pleomorphic forms

Post by Lorima » Thu 15 Jan 2015 1:42

This reminds me, we previously discussed another thesis with good pictures:

http://www.lymeneteurope.org/forum/view ... f=5&t=4206

Here is the opening post:
panda wrote:Rößle, Birgit (2001): Elektronenmikroskopische Untersuchungen an Borrelien: Ultrastruktur und Immuncytologie. Dissertation, LMU München: Fakultät für Biologie

http://edoc.ub.uni-muenchen.de/63/ [abstract, German]
http://edoc.ub.uni-muenchen.de/63/1/Roessle_Birgit.pdf [full text, German, 84.4 MB, fascinating pictures]

Rößle, B., Electron microscopy studies of Borrelia: ultrastructure and immunocytology, Diss., Department of Biology, University Munich (2001)

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

viable but not cultivable: http://www.springerlink.com/content/92u623g2335xj21g/
astrocytes: http://en.wikipedia.org/wiki/Astrocytes

_______________
Automatic translation of the summary, with some revisions by Panda:

Lyme borreliosis is the most common tick-borne infectious disease of humans in the northern hemisphere. The pathogen Borrelia burgdorferi sl is divided into three human pathogenic species, B. burgdorferi ss, B. garinii and B. afzelii.

In this work, the structure of the example of Borrelia afzelii strain PKo, the skin isolate of a patient, using electron microscopy methods were investigated. Scanning and transmission electron microscopy studies of the Borrelia B. afzelii strain PKo were performed to investigate the ultrastructure of Borrelia cells under in vitro culture conditions.

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.

The studies on the ultrastructure of the Borrelia cell were performed on Borrelia from the log phase of the culture. Borrelia cells are at this time 10-20 microns in length and have 3-9 helical turns. Ultrathin sections show that the spirochetes consist of a protoplasmic cylinder, which is bounded by a 4 nm thin membrane. At a distance of 5 nm the cell wall joins to it. Borrelia cells are surrounded by an outer membrane that surrounds the periplasmic space. This membrane is bulged to a tunnel in which the endoflagella are lying. At each end of the cell are located 7-9 insertions of flagella arranged in the longitudinal axis of Borrelia cell. Each of the flagella is inserted only at one end of the cell. In the central region of Borrelia cell there is an overlap of the flagella of the cell ends. The overlap region is only vaguely distinguished. Based on these investigations a detailed scale model of a Borrelia cell can be reconstructed.

Another aim of the study was to solve the localization of important immunodominant proteins. With the aid of immuno-gold markers it was shown by high-resolution scanning electron microscopy that the proteins p17 (Osp17), p35 (Osp35) and p58 (Osp58) are localized on the surface of the outer membrane of the Borrelia cell. By optimizing the labeling method the signal could be increased to such an extent that the uniform distribution of these proteins could be presented over the entire cell surface. Due to their location on the surface of the Borrelia cell these proteins are vaccine candidates.

The localization of type-specific and conserved epitopes of the two immunologically heterogeneous surface proteins OspA and OspC was detected by immuno-gold markers using type-specific and broadly reactive monoclonal antibodies. Type-specific and conserved epitopes of both proteins are located on the surface of the outer membrane. The localization of broadly reactive epitopes of OspA and OspC on the surface of the Borrelia cell seems promising that these proteins could be used as vaccine antigens also in Europe.

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. Transfered 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 can not 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.

Best,
Panda


Do take a look; the pictures are gorgeous.
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