Subgingival Microbes

In Atlas of Oral Microbiology, 2015

4.2.9 Treponema

Spirochetes are a type of slender, curved spiral, highly motile gram-negative bacteria. Common genera of spirochetes are: Spirochaeta, Cristispira, Treponema , and Borrelia.

4.2.9.1 Treponema

Treponema is a genus of commonly found oral bacteria that are closely related to periodontitis and the etiology of implant periarthritis. Species commonly detected in the oral cavity are Treponema denticola, Treponema scaliodontum, Treponema macrodentium, Treponema oralis, Treponema intermedia, Treponema maltophilum, Treponema socranskii, and Treponema vincentii. In addition, the gastrointestinal tract and the vagina are also major colonization sites for bacteria of this genus in humans.

Clinical samples of Treponema are ideally observed with a dark field or a phase contrast microscope. Treponema cells are gram-negative, but most of the strains do not take up stain easily by Gram staining or Giemsa staining. Silver impregnation stain and Ryu's stain are better for the observation of Treponema cells. Presently, we commonly use the Congo red negative stain method, as it is not only economic and simple but the helical cells are also very easy to observe (Figure 4.22(A)). The morphology of the bacterial cells can be seen under SEM (Figure 4.22(B) and (C)).

Figure 4.22. (A) Plaque samples of Treponema cells (Congo red negative staining). (B) Plaque samples of large, medium, and small Treponema cells (SEM). (C) Plaque samples of Treponema and Borrelia cells (SEM). Treponema cells measure 0.1–0.4   μm   ×   5–20 μm. They form a spiral rod and can be either a tight, regular, or irregular spiral. Cells have one or more axial flagella inserted into the plasma membrane at opposite ends of the rod. In old cultures, huge bubbles and spiral spheres can be observed. Large, medium, and small spirochetes can be observed in clinical samples. The spiral-shaped cells measure 0.2–0.5   μm   ×   3–20   μm, and form regular or irregular loose spirals. The cells stain gram-negative.

The outer membrane of Treponema cells is similar to the outer membrane of gram-negative bacteria cells. The content includes lipids, proteins, and carbohydrates. The lipids are mainly made up of phospholipids and glycolipids, while the cell wall contains muramic acid, glucosamine, and ornithine. Peptidoglycan accounts for 10% of the dry weight of the cell.

The genus Treponema consists of obligate anaerobes, but those species that are pathogenic to humans may be microaerophiles. Treponema are heterotrophic bacteria that mainly metabolize through fermentation. They can use a variety of carbohydrates and amino acid as their carbon source and energy. Treponema species are difficult to grow in artificial culture media, the growth of some species requires long chain fatty acids from serum, and other species require branched-chain fatty acids. Treponema denticola, Treponema vincentii, and Treponema scaliodontum require cocarboxylase in serum.

The genomic GC content of most Treponema species ranges from 25% to 54% (by Tm). The type species is T. pallidum.

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Spirochetes

John R. ColeJr., in Diagnostic Procedure in Veterinary Bacteriology and Mycology (Fifth Edition), 1990

Laboratory Examinations

Direct Examination

Treponema hyodysenteriae can be observed in the mucosal lesions of the large intestine by darkfield or phase-contrast microscopy using the following procedure (14).

1.

Rinse or lightly scrape a portion of the affected mucosa to remove debris.

2.

Suspend a portion of a deep scraping from the mucosa in a drop of saline or water on a microscope slide.

3.

Examine by darkfield or phase-contrast microscopy at a magnification of 400–1000.

4.

Observe three to five spirochetes per high-power field.

It is important to differentiate T. hyodysenteriae, which is 7–8 μm long, loosely coiled, motile by flexing movements, and tapered at the ends, from the smaller, tightly coiled spirochetes normally found in swine. Mucosal or fecal smears may be stained with crystal violet, carbolfuchsin, or Victoria blue 4-R stains, although wet mount preparations to observe motility are preferred. These spirochetes may be observed in histologic sections of the colonic mucosa stained with the Warthin–Starry, Goodpasture's, or Victoria blue 4-R stains (Fig. 5-3).

Figure 5-3. Treponema hyodysenteriae in a section of swine intestine. Warthin-Starry stain, × ∼1000.

Rectal swabs or feces may be utilized for examination from a live animal, but numbers of organisms in the feces may be small and the spirochetes not readily detectable (15).

Isolation Procedures

Treponema hyodysenteriae can be isolated from the intestinal mucosa using the following procedure (16, 17).

1.

Take 6–8 in of spiral colon from an acutely affected animal. The sample may be held for 2–3 days at 4°C before isolation attempts. Do not freeze.

2.

Open the colon longitudinally and remove the mucosa with a sterile swab.

3.

Alternatively, prepare a 1:10 suspension of the colonic mucosa in saline.

4.

Centrifuge slowly for 10 min to remove the large particulate matter.

5.

Pass the supernatant through a series of cellulose acetate filters: prefilter, 8.0 μm, 3.0 μm, 1.2 μm, 0.8 μm, 0.65 μm, and 0.45 μm.

6.

Streak the mucosal swab or the material from the filtrate that passed the 0.8 μm, 0.65 μm, and 0.45 μm filters onto freshly prepared or prereduced trypticase soy agar (TSA) containing 5% defibrinated bovine or equine blood. The addition of 400 μg/ml of spectinomycin (The Upjohn Company, Kalamazoo, Michigan) and 1% sodium ribonucleate (United States Biochemical Corp., Cleveland, Ohio) to the medium suppresses most of the contaminating flora, does not adversely affect the isolation of T. hyodysenteriae, and enhances the degree of hemolysis

A medium (BJ) which further suppresses fecal contamination and enhances growth of T. hyodysenteriae has been described (18). In addition to the basic medium (TSA) and 5% blood, it contains five antibiotics (spiramycin, rifampin, vancomycin, colistin, and spectinomycin), and a 5% pig feces extract.

7.

Incubate the plates at 42°C in an anaerobic container. A vented Gas-Pak® jar with cold palladium catalyst (BBL, Cockeysville, MD) may be used to obtain a hydrogen and carbon dioxide atmosphere of 80:20 H2–CO2 by evacuation and refilling. A H2–CO2 generator envelope (GasPak-BBL) is also acceptable.

Cultural Characteristics and Identification

Growth of T. hyodysenteriae on blood agar is evidenced by a zone of clear (β) hemolysis, which may contain small, white translucent colonies. This is in contrast to Treponema innocens, which is weakly β-hemolytic and considered nonpathogenic (14). Treponema hyodysenteriae is gram-negative but is more readily observed using the stains listed under Direct Examination. Treponema hyodysenteriae is 6–8.5 μm long, 0.32–0.38 μm in diameter, loosely coiled, motile, cytochrome oxidase negative, catalase negative, stimulated by hydrogen, and anaerobic.

Treponema paraluis–cuniculi is pathogenic for animals and the cause of rabbit syphilis. Diagnosis is based on lesions around the genitalia and demonstration of the organisms in these lesions by staining or darkfield microscopy.

Treponema suis has been observed in washings of ulcerated preputial diverticula in pigs (19).

Treponema succinifaciens, a small anaerobic spirochete, has been isolated from the colon of a pig and is considered nonpathogenic (17).

Serologic Procedures

Tests that have been adapted for use in the diagnosis of swine dysentery or for detection of carrier animals are the tray agglutination test (20), microtitration agglutination test (21), and ELISA (22). The ELISA procedure appears to be the most sensitive of these tests, and it may be useful for determining the status of a herd by diagnostic laboratories that have personnel and equipment necessary to perform this test.

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Laboratory and Pet Rodents, and Lagomorphs

Molly Varga , Tim Morris , in Veterinary Reproduction and Obstetrics (Tenth Edition), 2019

Treponema paraluis cuniculi

Treponema paraluis cuniculi is a spirochaete organism, which causes venereal syphilis in rabbits (DiGiacomo et al. 1984). It can be transmitted either by mating with an affected individual or via direct contact of kits with an affected doe; asymptomatic carriers/shedders exist within the population. Treponema infection manifests as papules, vesicles, ulcers, and crusting around the external genital organs and, occasionally, the eyes/nose and perineum; diagnosis can be made on clinical signs and confirmed by serology (Saito et al. 2003). Diagnosis can be aided by an ELISA test, which is available commercially, dark field microscopical examination of scabs and exudate demonstrating helical organisms, or histopathology. Treponematosis can be associated with an unwillingness to mate and abortions in affected does. Treatment is three times a week doses of procaine/benzathine penicillin. Response to treatment is rapid and is often noted after the first dose of medication; differential diagnoses include atypical myxomatosis and autoimmune skin disease.

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Non-sporing anaerobes

R.P. Allaker , in Medical Microbiology (Eighteenth Edition), 2012

Spiral-shaped motile organisms

Several Treponema species are found in the mouth and elsewhere in the body (see Table 36.1). They are thought to be an important component of the mixed anaerobic infection associated with acute necrotizing ulcerative gingivitis along with fusobacteria and Prev. intermedia, and may also contribute to other forms of periodontal disease. The proportion of motile spiral organisms seen by dark-ground microscopy in samples from the gingival pocket increases markedly when there is evidence of periodontal destruction.

Motile, spiral-shaped, Gram-negative anaerobes of the genus Anaerobiospirillum have been isolated from patients with diarrhoea and from bacteraemia. Although comparatively rarely isolated from man, they can cause serious infections. The distribution and normal habitat of this and other morphologically similar organisms are not well understood. In some cases the source of infection may be domestic animals and pets.

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Leptospira, Borrelia and Treponema

Antonio Sampedro , Francisco de Asís Ramirez , in Encyclopedia of Infection and Immunity, 2022

Abstract

Leptospira, Borrelia and Treponema are pathogenic members of phylum Spirochaetes. Spirochetes are gram-negative, motile, spiral bacteria. Bacteria have an outer membrane and an inner membrane, with axial filaments called endoflagella. The flagella run in periplasmic space between membranes and these cause a twisting motion which allows the spirochaete to move about.

Leptospira species, causes leptospirosis, one of the widespread zoonoses and is found in tropical and temperate areas of the world. Leptospirosis is carried out with a wide variety of symptoms that can lead to confusion with other diseases such as aseptic meningitis, dengue fever, influenza or hepatic disease. Untreated leptospirosis can lead to meningitis, liver failure, respiratory distress, and even death.

Genospecies B. afzelii, B. burgdorferi and B. garinii are the causative agent of Lyme borreliosis, a bacterial disease which is transmitted through bites from ticks of the genus Ixodes and is characterized by a variety of clinical symptoms. Lyme borreliosis can manifest itself dermatologically, neurologically or through internal disorders. B. recurrentis, are important human pathogens that cause relapsing fever.

T. pallidum subspecies pallidum is the causative agent of syphilis, a sexually transmitted disease and according to the WHO, in 2012 there were 5.6 million new cases of syphilis worldwide. Other members of the genus Treponema are responsible for diseases such bejel, yaws, and pinta and play important role in periodontal diseases.

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Treponema and borrelia

A. Cockayne , in Medical Microbiology (Eighteenth Edition), 2012

Key points

•

The genus Treponema includes the agents of syphilis (T. pallidum), yaws (T. pertenue), bejel (T. endemicum) and pinta (T. carateum). All are essentially morphologically and antigenically identical spirochaetes, which cannot be cultivated in vitro.

•

These diseases, if untreated, characteristically progress to chronic disease through distinct early and late stages and pathologies whose appearance is separated by latent periods of variable length.

•

Diagnosis of syphilis, which is primarily sexually transmitted, is made by clinical observation and confirmed serologically.

•

All of these organisms are sensitive to benzylpenicillin, which can be used to treat the early stages of disease.

•

The genus Borrelia includes agents of Lyme disease (B. burgdorferi sensu lato) and relapsing fevers (B. recurrentis and others), all of which are transmitted to man by ticks or lice.

•

Lyme disease, if untreated, may progress through distinct clinical phases (stages 1–3), resulting in later pathology (e.g. arthritis, neurological damage) in some individuals.

•

Relapsing fevers are characterized by recurring periods of fever and remission associated with antigenic variation of the borreliae. Infection may be fatal.

•

Lyme disease is diagnosed clinically and confirmed serologically. Doxycycline may be used to treat the early stages.

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The Microbiome in Health and Disease

Matthew Silbergleit , ... Ikuko Kato , in Progress in Molecular Biology and Translational Science, 2020

3.2 Microbiological characteristics of dentilisin synthesis

Dentilisin is produced primarily by mammalian oral Treponema species, 83,87,88 represented by T. denticola (Td), a key pathogen of periodontitis, which are Gram-negative obligate anaerobic bacteria, spirochaetes uniquely lacking LPS. It is important to note that substantial inter-strain variability exist in this enzymatic activity, which is not entirely explained by the presence or sequence variabilities of encoding genes. 83,87,88 Typically, the dentilisin complex is encoded by a three-gene operon, consisting of prcB, prcA and prtP, with a putative promoter upstream of prcB. 85,86 These gene sequences are recently annotated, 83 demonstrating that prcB is the most conserved (89% out of 202 amino acids identical among the strains). prcA encoding 639 amino acids was 77% identical among the strains, and much of the variability was in three regions (151–180, 328–362 and 512–545). In prtP that transcribes 766 amino acids, the predicted catalytic triad (Asp203, His258, Ser447) was conserved in all strains, and most prtP inter-strain variations were concentrated in the C-terminal 270 residues (up to 20%). 83 However, variability in proteolytic activities was not correlated with overall homology of prtP, suggesting that other unidentified sensing and signaling mechanisms modulate expression of these operons.

In Td, the dentilisin complex is located on bacterial outer membrane and extracellularly. 89 However, the secretion system by which the three subunits are transported through inner and outer membranes has not been well understood, Yet, studies have demonstrated that this complex is co-localized with another major Td virulence factor, Msp (major outer sheath protein), 83,85 encoded by msp and playing a major role in host adhesion. 90 MSP locates within the outer membrane, but is predominantly periplasmic, with only limited surface exposure. 91 Importantly, there is close interplay between PrtP and outer membrane segment of Msp, 92 and, as a result, msp mutants lack CTLP activity while prtP mutants show aberrant Msp expression. 93 In addition to cell surface expression, a recent mass-spectrometry-based study revealed that the three dentilisin subunits were enriched in outer membrane vesicles, 89 which enable remote delivery of the toxin.

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Syphilis

Lorenzo Giacani , ... Arturo Centurion-Lara , in Vaccines for Biodefense and Emerging and Neglected Diseases, 2009

Classification

T. pallidum is one of four recognized human pathogens within the genus Treponema : T. pallidum subsp. pallidum (syphilis), T. pallidum subsp. pertenue (yaws), T. pallidum subsp. endemicum (bejel), and T. carateum (pinta). The infections caused by these organisms are similar in that they appear in distinct clinical stages, are chronic, and can cause serious destructive late sequelae. The Treponema also include oral treponemes that are associated with periodontal disease, Treponema paraluiscuniculi which causes a venereal disease in rabbits, a group of organisms (including Treponema brennaborense) associated with papillomatous digital dermatitis and ulcerative mammary dermatitis in cattle and sheep (Stamm et al., 2002), and symbionts of the termite hindgut (Lilburn et al., 2001).

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OSTEOLOGICAL & DENTAL PATHOLOGY

Tim D. White , Pieter A. Folkens , in The Human Bone Manual, 2005

17.2.4 Treponemal Infections

Skeletally significant diseases caused by a microorganism known as a spirochaete (in the genus Treponema ) are yaws and endemic and venereal syphilis. These diseases have a worldwide distribution today, but considerable controversy exists over their origins and distribution several hundred years ago. Debate centers on whether syphilis (misdiagnosed as leprosy) was present in the Old World before Columbus returned from the New World. The controversy is well-reviewed by both Steinbock (1976) and Ortner and Putschar (1981), with a more recent update by Baker and Armelagos (1988) .

In syphilis, the microorganisms enter the body through the skin or mucous membrane sites. Tertiary syphilitic skeletal lesions occur progressively, usually beginning between 2 and 10 years after infection. These can be complex, but there is usually an osteological focus of the disease in the frontal and parietals, the facial skeleton, and the tibia. Individual lesions may not be distinguishable from some cancers, tuberculosis, or other infectious changes. Steinbock (1976), Ortner and Putschar (1981), and Buckley and Dias (2002) provide further details.

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Nitric Oxide and Other Small Signalling Molecules

Jeffrey A. Cole , in Advances in Microbial Physiology, 2018

9.3 Hybrid Cluster Proteins

Hcps are widely distributed from extremely thermophilic archea such as Pyrococcus furiosus to Spirochaetes, Treponema , and the photosynthetic Rhodobacter capsulatus (Cabello et al., 2004; Overeijnder, Hagen, & Hagedoorn, 2009). It is present in most γ, δ, and ɛ proteobacteria, but absent from methanogens and most denitrifying bacteria.

The high-resolution structure of the Hcp from D. vulgaris (Hildenborough) revealed three domains: an N-terminal mainly α-helical domain and two similar domains comprising a central β-sheet flanked by α-helices. There are two iron–sulphur clusters located within 11   Å of each other. The conventional N-terminal [4Fe–4S] cubane cluster is close to an indentation on the surface of the protein and can also be approached on the opposite side by a long solvent channel. The hybrid [4Fe–2S–2O] cluster is buried in the central hydrophobic region. A large hydrophobic cavity in domain 3 is consistent with it functioning as a channel for NO diffusion. There is a second more hydrophilic channel that links the hybrid cluster to the outside of the protein. If like the E. coli protein the Desulfovibrio Hcp is a high-affinity NO reductase, one can speculate that the surface cavity might be a binding site for an electron donor protein or a protein that protects the [4Fe–4S] centre from substrate inactivation. The hydrophilic channel might function as the electron transfer pathway to the hybrid cluster, and the hydrophobic channel would provide access to the site of NO reduction by the hybrid cluster.

D. vulgaris Hildenborough is especially interesting because it encodes two copies of both ROO and Hcp. One set of genes, hcp1 and roo1, is located on a 47   kb mobile genetic element that can be lost spontaneously. The hcp1 and roo1 genes are adjacent to each other, but are they cotranscribed? The second copies of the genes are distributed elsewhere on the chromosome (Johnston et al., 2009). The rate of growth in a normal lactate plus sulphate medium is lower when the genomic island is present than when it has been lost, but the island provides protection against both oxidative stress and nitrosative stress induced by the addition of 2   mM nitrite. Yurkiw et al. (2012) then showed that the genomic island is required for electron transfer from lactate to nitrite, but not for electron transfer from hydrogen to nitrite. They also confirmed that the genomic island (ROO1?) and ROO2 confer increased resistance to oxygen and proposed that both Hcp1 and Hcp2 contribute to resistance to nitrite stress. Subsequently, Figueiredo et al. (2013) demonstrated that mutants defective in all four hcp and roo genes lost viability when challenged with macrophages. The hcp2 gene is most highly induced in response to NO released from 100   μM dipropylenetriamine NONOate, and the hcp2 mutant was least able to survive nitrosative stress.

Many questions remain unanswered. Although growth inhibition by nitrite was originally attributed to the chemical formation of toxic polysulphide, in view of the reassigned function for ROO, it is more likely due to NO generated from nitrite. This assumption has not been checked experimentally. It would be interesting to determine whether growth in the presence of nitrite can be restored by adding PTIO, which stoichiometrically destroys NO, to the growth medium (see, for example, Wang et al., 2016, for details). Expression of the hcp2 gene was reported to be induced 100-fold only in the presence of the genomic island, and nitrite addition induced both hcp1 and hcp2 expression. Further experiments are now required to determine whether one or both Hcp1 and Hcp2 can function as NO reductases. It is also unknown whether the genomic island generates an induction signal or encodes a transcription factor required for increased hcp2 expression. Even if NO is the inducer to which hcp1 and hcp2 respond, it is unknown how NO is generated from nitrite in a strain that lacks both NrfA and genes for any nitrate reductase.

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