0 Symbiotic Spirochetes In Animal Models

Recently, I noticed this intriguing item in the news that is of interest to many medical professionals around the world who are struggling to care for patients' difficult to eradicate C. difficile infections: A recent murine study[1] provided evidence that a simple mixture of six phylogenetically diverse intestinal bacteria - including novel species - can re-establish a health-associated microbiota and clear C. difficile infection from mice.

With this outcome, it's possible that medical professionals will no longer apply the treatment of last resort - fecal transplants - to save human patients' lives. Instead, all patients with raging C. difficile infections will have to do is swallow a pill or consume food that contains the strains of bacteria needed to reverse dysbiosis and rebalance the microbiota in their stomachs so that C. difficile is no longer a threat.

Over time, researchers are uncovering the complicated dynamic between different microorganisms which live inside the human gut. Also, they are refining their understanding of the dynamic between different microorganisms inside animals with increasing speed as more invasive studies can be completed in animals than can be easily completed in humans. Among these studies are those on the relationship between spirochetes and other microorganisms found in ruminants such as cattle and sheep, termites, and molluscs.

Symbiotic Spirochetes In Termites
 

Microscopic image of Mixotricha paradoxa
covered with thousands of Treponema spirochetes

One interesting and complex symbiotic relationship involving spirochetes is found within termites' guts. Spirochetes are one of the most abundant bacteria present in the gut fluid of termites, and the symbiotic relationship between various microbes in termites of all kinds actually predates the evolution of termites from their wood-feeding roach ancestors over 120 million years ago [2].

Most termites have spirochetes which are free-living in the gut fluid, but they have also been found as ectosymbionts attached to protists inside termite guts. Mixotricha paradoxa is protozoa found inside the gut of the Australian termite species, Mastotermes darwiniensis. It was originally thought the long tiny hair-like structures covering the length of its body were short cilia - outgrowths from the protozoa itself. However, upon closer examination years later, scientists Cleveland and Grimstone[3] discovered these were not cilia but a dense carpet of Treponema spirochetes - spirochetes which help propel Mixotricha paradoxa forward while it uses its own anterior flagella to steer in the right direction. How it coordinates this movement is unknown - it is surmised that they automatically synchronize due to their proximity.

These spirochetes not only help Mixotricha paradoxa move forward, though. One thing Mixotricha paradoxa does to help its host, the termite, do is help break down cellulose into sugars and then hydrogen, acetate, and carbon dioxide from the wood it eats. From there, what the Treponema spirochetes are predicted to do is oxidize the acetate which was produced and use it to support 100% of the termite's respiration requirements.[4]

What makes this symbiosis even more complex is that it doesn't stop there. No, not only does Mixotricha paradoxa have a Treponema spirochete helping it survive - but it also has three more bacterial species onboard: A lot of rod-shaped bacteria related to Bacteroides live on its surface, and is suspected to help breakdown cellulose as it sits alongside the Treponema spirochetes; a spherical form of bacteria lives inside Mixotricha which is hypothesized to act as mitochondria for the protozoa (as Mixotricha does not have its own mitochondria); a large spirochete attributed to the genus Canaleparolina.[5]

Not much is known about these three bacterial species' lives in Mixotricha paradoxa, and more research is needed. The most recent research on Mixotricha paradoxa adds to this complex symbiotic dynamic, as it has been discovered that not all glycolytic activities in Mixotricha paradoxa are produced by its microorganisms - cellulases have been detected in the salivary glands of Mastotermes darwiniensis - the termite itself.[6]

Symbiotic Spirochetes In Ruminants

The bovine or cow stomach has a wide variety of organisms inside it - such as fungi, bacteria, archaea, protista, and viruses. All these organisms help break down food, especially plant matter and in particular, cellulose. And like the Mixotricha paradoxa inside the Australian termite, Mastotermes darwiniensis, the organisms are all dependent on each other to some degree and use the byproducts of one another for their own benefit.

Cows - unlike people - have four stomach compartments to digest their food: the rumen, the reticulum, the omasum, and the abomasum. The rumen is the largest compartment, and it contains a huge number of different microbes. The reticulum is responsible for creating cud and trapping indigestible substances like rocks or nails - and unfortunately, can be subject to more injury than the other compartments. The omasum sends large substances to the rumen and reticulum while allowing smaller substances to pass on to the abomasum. And the abomasum is very similar to a human stomach, as it produces stomach acids and enzymes to break down proteins before sending the result to the small intestine.

While the most common bacteria in the bovine stomach are gram-positive cocci and rods, a smaller percentage of their population are spirochetes which play a role in ruminant digestion. Organisms such as Treponema bryantii, a saccharolytic spirochete, enhances the breakdown of cellulose while cellulolytic bacteria of different species break down plant cell walls into soluble sugars.

Two interesting passages from the publication, Interspecies bacterial interactions in biofilms, by James, Beaudette, and Costerton[7], highlight the relationship between Treponema bryantii and other microbes studied in vitro from bovine rumen:

"Observations of biofilms on cellulose particles from the rumen revealed cellulolytic as well as noncellulolytic bacteria enmeshed in the exopolysaccharide matrix of the biofilm. Addition of a noncellulolytic species, Treponema bryantii, to cultures of a cellulolytic species, Fibrobacter succinogenes or Ruminococcus albus, resulted in an enhanced rate of cellulose degradation. Presumably, T. bryantii utilized the hydrolytic products (eg, glucose or cellobiose) from the cellulolytic bacteria which may repress and/or inhibit the cellulolytic enzymes."

"...Microscopy of biofilms formed during protocooperative cellulose digestion by R. flavefaciens and T. bryantii revealed that cellulolytic R. flavefaciens cells were attached directly to cellulose particles, while the spirochete, T. bryantii, was located in the upper biofilm layers. This spatial arrangement and the mobility of spirochetes in viscous environments suggest that this organism may move through the biofilm, scavenging the products of the cellulolytic bacteria."

The first study of Treponema bryantii in 1980, Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria[8], offers more specifics in its abstract as to its biological requirements:

"...When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulose-containing agar medium the spirochete enhanced cellulose breakdown by the Bacteroides succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO2 atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO3, the spirochete grew to a final density of 1.9 X 10(9) cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO2 (HCO3-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete."

Spirochete Symbiosis In Molluscs

While so far there is no evidence molluscs harbor spirochetes which have symbiotic relationships with its host or other microorganisms, spirochetes which coexist peacefully within their host are worth noting.

Spirochetes from the genus Cristispira have been found inside more than 50 species of 22 families of marine bivalves and 3 freshwater bivalves. It has been shown to be a commensal organism living within molluscs and has not been shown to provide any benefit or disadvantage to molluscs such as Prince Edward Island oysters.

This past May, an interesting paper was published, Spirochetes in gastropods from Lake Baikal and North American freshwaters: new multi-family, multi-habitat host records[9].

The abstract states:

"We describe the first records of spirochetes in the gut of fourteen species of continental gastropods from a range of habitats and representing six families (Amnicolidae, Baicaliidae, Bithyniidae, Pyrgulidae, Lithoglyphidae and Benedictiidae). The bacteria were mainly found in the crystalline style sac, as has been reported in marine bivalves. The surveyed habitats include water bodies in North America and Eurasia, including deep water hydrothermal vent and gas hydrate zones in Lake Baikal. Spirochetes were present both in mature and young snails, but were not detected in embryos before hatching, indicating lateral transfer. The surveyed gastropods range in trophic strategy, including phyto-, detrito- and bacteriophagous grazers and filter feeders. Our results indicate that spirochetes are commensal in the surveyed gastropods with potential limited benefit and no detriment to the host animal. We suggest that the specialized internal habitat of the crystalline style sac in molluscs is likely to reveal unrecognized spirochete diversity that will shed new light on gastropod trophic ecology and spirochete diversity."

More research is needed to determine which limited benefits different spirochetes may provide for their hosts.

Looking at the symbiotic and commensal relationships between animals and spirochetes - or between spirochetes and other microbes - one has to wonder what kind of relationships different spirochetes have with us and microbes within us. Humans already play host to spirochetes which are considered commensal Treponema and unfortunate hosts to spirochetes which are pathogenic such as those which cause syphilis and Lyme disease (Borreliosis). But is there more to this story than is often told? Do these bacteria have deeper relationships?

References:

1) Trevor D. Lawley, Simon Clare, Alan W. Walker, Mark D. Stares, Thomas R. Connor, Claire Raisen, David Goulding, Roland Rad, Fernanda Schreiber, Cordelia Brandt, Laura J. Deakin, Derek J. Pickard, Sylvia H. Duncan, Harry J. Flint, Taane G. Clark, Julian Parkhill, Gordon Dougan. Targeted Restoration of the Intestinal Microbiota with a Simple, Defined Bacteriotherapy Resolves Relapsing Clostridium difficile Disease in Mice. PLoS Pathogens, 2012; 8 (10): e1002995 DOI: 10.1371/journal.ppat.1002995
2) Grimaldi, D. and Engel, MS. Evolution of the insects. 2005. Cambridge University Press, NewYork, NY.
3) Cleveland, L.R., and A.V. Grimstone. The fine structure of the flagellate Mixotricha paraodoxa and its associated micro-organisms. 1964. Society 159:668-686.
4) Leadbetter, J.R. Acotgenesis from H2 Plus CO2 by Spirochetes from Termite Guts. 1999. Science 283:686-689.
5) Brugerolle G. Devescovinid features, a remarkable surface cytoskeleton, and epibiotic bacteria revisited in Mixotricha paradoxa, a parabasalid flagellate. Protoplasma. 2004 Oct;224(1-2):49-59.
6) Konig, H., Li Li, Wenzel, M, Frohlich, J.  Bacterial Ectosymbionts which Confer Motility. p. 86 Molecular Basis of Symbiosis. 2006. Springer-Verlag.
7) G A James, L Beaudette and J W Costerton. Interspecies bacterial interactions in biofilms. Journal of Industrial Microbiology & Biotechnology. Volume 15, Number 4 (1995), 257-262, DOI: 10.1007/BF01569978
8) Stanton TB, and Canale-Parola E. Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria. Arch Microbiol. 1980 Sep;127(2):145-56.
9) Tatiana Sitnikova,Ellinor Michel, Yulia Tulupova, Igor Khanaev, Valentina Parfenova, Larisa Prozorova. Spirochetes in gastropods from Lake Baikal and North American freshwaters: new multi-family, multi-habitat host records. Symbiosis. May 2012, Volume 56, Issue 3, pp 103-110.

Additional Reading:
Xinning Zhang and Jared R. Leadbetter. Evidence for Cascades of Perturbation and Adaptation in the Metabolic Genes of Higher Termite Gut Symbionts. mBio vol 3. no.4 e00223-12. http://mbio.asm.org/content/3/4/e00223-12.full
Nordhoff M, Wieler LH.Berl Munch Tierarztl Wochenschr. 2005 Jan-Feb;118(1-2):24-36 .[Incidence and significance of treponemes in animals].[Article in German]

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