VIDEO LECTURE

BACTERIOLOGY - CHAPTER  FOURTEEN  

SPIROCHETES AND NEISSERIA  

Dr Alvin Fox

Reading: Murray 6th edition Chapter 42 (Spirochetes)
Chapter  29 (Neisseria)

KEY WORDS
Spirochete 
Axial filament
Treponema pallidum
Syphilis 
Chancre 
Primary Lesion
Darkfield microscopy 
Secondary Lesion
Tertiary Lesion 
Anti-cardiolipin antibodies
Borrelia burgdorferi
Lyme disease
Relapsing fever
Leptospira (leptospirosis) 
Neisseria
Thayer Martin agar
Oxidase test
N. gonorrhoeae
Gonorrhea
N. meningitidis
Meningitis

  Figure 1. Congenital syphilis, primary and secondary syphilis rates, by year -- United States, 1992-1998. Epidemiology, surveillance. CDC/Dr. Demetri Vacalis  tdv0@cdc.gov 

  Figure 2 Histopathology showing Treponema pallidum spirochetes in testis of experimentally infected rabbit. Modified Steiner silver stain. CDC/Dr. Edwin P. Ewing, Jr.  epe1@cdc.gov 

SPIROCHETES

The most important genera of spirochetes are Treponema, Borrelia and Leptospira. These are are Gram negative bacteria that are long, thin, helical and motile. Axial filaments (a form of flagella) found between the peptidoglycan layer and outer membrane and running parallel to them, are the locomotory organelles.

Treponema pallidum

Syphilis

T. pallidum is the causative agent of syphilis, a common sexually-transmitted disease found world-wide (figure 1). It is  generally transmitted by genital/genital contact. Transmission in utero or during birth can also occur. Syphilis, chronic and slowly progressive, is the third most common sexually transmitted disease. After initial infection, a primary chancre (an area of ulceration/inflammation) is seen in genital areas (figure 4 and 6) or elsewhere (figure 3) within 10-60 days. The organism, meantime, has penetrated and systemically spread. The patient has flu-like symptoms with secondary lesions particularly affecting the skin (figure 5). These occur 2-10 weeks later. The final stage (if untreated) is tertiary syphilis (several years later). In primary and secondary syphilis organisms are often present in large numbers. However, as the disease progresses immunity controls bacterial replication and fewer organisms are seen. It is extremely difficult to detect spirochetes in tertiary syphilis. The systemic lesions of skin, central nervous system and elsewhere are suggestive of a delayed hypersensitivity reaction.

The organism cannot be cultured from clinical specimens. Thus, experimentally, syphilis is commonly studied in animal models. Also microscopic and serological methods are the only means of clinical diagnosis.

In primary syphilis (before immunity develops), the organisms are often present in sufficient numbers in exudates to be detected by dark field microscopy. In conventional light microscopy, the light shines through the sample and thin treponemes cannot be visualized. In dark field microscopy, the light shines at an angle and when reflected from the organism will enter the objective lens. The actively motile organisms appears brightly lit against the dark backdrop. Alternatively fluorescent antibody staining is used.

In secondary and tertiary syphilis, serological methods are usually used to detect syphilis. Screening methods are based on detecting serum antibodies to cardiolipin in patients (including VDRL test). The antibodies result from tissue injury, with autoimmunity developing to self components. Thus, there are many other diseases that result in anti-cardiolipin antibodies and false positives are common. However, these are cheap screening tests. More definitive diagnosis is achieved by detecting the presence of "specific" serum antibodies against treponemal antigens. These tests are more expensive and usually performed (as a definitive diagnosis) on sera previously shown to be positive after first detecting antibodies to cardiolipin.

No vaccine exists, but antibiotic therapy (usually penicillin G) is usually highly effective.

Bejel

Rare (in the US) diseases caused by organisms related to T. pallidum are bejel, yaws (figure 7) and pinta.

Bejel, also known as endemic syphilis, is not transmitted sexually but via contact, for example hands to broken skin. The disease can also be spread by sharing eating utensils. It is thus a disease of low income groups with poor hygiene. Depending on the route of transmission, skin or mucous membranes are the first to be infected but the bacterium can spread deeper to the bones. Thus, one sees sores in the mouth, throat and the nasal passages and the infected lesions can penetrate deep into the tissue causing major malformations of the face and limbs. This results in severe bone pain and there is also swelling of the lymph nodes. The T. pallidum organisms can be found in swabs of the sores. 

Treatment of bejel, which can be completely curative, is similar to syphilis, that is penicillin G or tetracycline. Bejel is found in the Middle-East, Africa, Australia and central Asia.

Pinta

Pinta is another non-venereal, treponematous disease which is caused by T. carateum. It occurs in the New World, particularly the Caribbean, central America and northern South America. Pinta is the Spanish for "painted". Again, it is a disease of poor regions with sub-standard hygiene and is spread by personal contact through cuts in the skin. This results in scaly red lesions (hence the name) which form a lump at the site of the primary infection. Small satellite lesions form around the primary lesion and lymph node swelling is also seen. Some months after the primary infection, the patient experiences more scaly red lesions that are now flat and tend to itch. These are the pintids and occur around or distant from the site of the primary infection. The color of the pintids changes to  blue black with time and then can lose pigmentation. Unlike bejel, the disease does not spread deep into the tissues and bones. Detection is is via serology or direct examination of lesion specimens under the light microscope.

Treatment of pinta is again curative and can be accomplished by a single injection of penicillin G.

Yaws

Yaws (figure 7) is another chronic treponematous disease of poor hygiene. It can be very disfiguring. It strikes mainly children in Africa, south Asia and northern South America. The causative agent is T. pertenue. As with pinta and bejel, spread is via direct contact through skin lesions. About a month after the infection, a papule forms at the infection site which transformsinto a crusted ulcer that takes months to heal. Painful swelling of the lymph nodes occurs. Later, soft growths appear on the face, buttocks and limbs. They can also occur in the bottoms of the feet causing the infected person to have a very characteristic walk which gives rise to the name of "crab yaws". Further formation of tumors and ulcers on the face can cause bone malformation and can be disfiguring. Microscopy (of samples from the lymph nodes) is diagnostic and there are various serological tests. 

Treatment of yaws is also a single penicillin G injection which can be completely curative

Figure 5 Secondary syphilis - mouth mucosa Bristol Biomedical Archive © University of Bristol. Used with permission

Figure 7 Yaws is a crippling and disfiguring disease affecting some 50 million people in the world   © WHO
 

WEB RESOURCES
Some facts about Syphilis
CDC

Some facts about Lyme disease 
CDC

Borrelia burgdorferi and Lyme disease

Lyme disease is caused by Borrelia burgdorferi  (figure 8 and 13) and is a relatively newly recognized disease. It is found widely in the United States (figure 9 and 10).  Although clinically first described in 1975, the role of a tick-borne spirochete was not proven until 1983. These ticks (figure 12 and 14) infect a large array of wild life, particularly white footed mice. A tick bite leads to transmission of B. burgdorferi causing an erythematous skin rash (figure 11) in a few days along with a transient bacteremia leading to (weeks or months later) severe neurologic symptoms or polyarthritis. Cardiac problems may occur in a minority of cases. If antibiotic therapy is initiated early, a cure is usually achieved. However, late antibiotic administration (penicillin or tetracycline) is often ineffective.

Figure 9 Reported cases of Lyme disease in the United States 1998 CDC

  Figure 10 Lyme disease risk by region of United States CDC

Figure 11 Lyme disease rash CDC

  Figure 12 Ixodes scapularis, tick vector for Lyme disease. Also known as Ixodes dammini. CDC
 

Figure 13  Morphology of Borrelia burgdorferi. Dark field image

Figure 14 Life cycle of Lyme disease ticks CDC

Diagnosis

The organism is highly fastidious, growing extremely slowly in tissue culture (not bacteriological) media. The vast majority of body fluid or tissue samples from patients with Lyme disease do not yield spirochetes on culture. Lyme disease is thus usually diagnosed by detection of serum antibodies to B. burgdorferi. However, acutely antibodies may not occur in detectable titer, making early diagnosis difficult. Whilst late diagnosis, (as mentioned above) may lead to ineffective treatment. Many patients are unaware of having had a tick bite or a rash.

Etiology

The chronic arthritis clinically resembles rheumatoid arthritis. Live agent is almost never cultivated from the joint (in common with other forms of reactive arthritis such as Reiter's syndrome and rheumatic fever). However, small numbers of persistent spirochetes and borrelial antigens have been detected histologically in human tissues. Whether the organism persists in a viable form or not remains to be determined. Thus, there is no clear explanation for the immunopathologic stimulus for chronic tissue injury in Lyme arthritis.

 

Relapsing fever

There are less than 100 cases of relapsing fever per year in US. Relapsing fever (with associated bacteremia) is caused by other species of Borrelia which are transmitted by tick (B. hermsii, rodent host) and lice (B. recurrentis, human host) bites. The term relapsing fever is derived from the following repeating cycle. As an immune response develops the disease relapses. However, the antigens expressed change and the disease reappears. The organism is extremely difficult to culture and there is no serological test. The organism is generally detected by blood smear.

Figure 15 Scanning electron micrograph of Leptospira interrogans strain RGA. Two spirochetes bound to a 0.2 µm filter. Strain RGA was isolated in 1915 by Uhlenhuth and Fromme from the blood of a soldier in Belgium. CDC/NCID/Rob Weyant  rsw2@cdc.gov 

Figure 16 Leptospirosis in the kidney  Bristol Biomedical Archive © University of Bristol. Used with permission

There are less than 100 cases per year in US. This flu-like or severe systemic disease is also a zoonotic infection. Leptospira (figure 15) are transmitted in water contaminated with infected urine from wild animals (including rodents) and farm animals and can be taken in through broken skin (e.g. bathing). Leptospira particularly infect the kidney (figure 16), brain and eye. They are the most readily culturable of the pathogenic spirochetes; but this is not routine and diagnosis is usually by serology.

Neisseria are Gram negative diplococci (pairs of cocci). These bacteria grow best on chocolate agar (so-called because it contains heated blood, brown in color); a modified (selective) chocolate agar commonly used is Thayer Martin. The colonies are oxidase positive (i.e. produce cytochrome oxidase) which is demonstrated by flooding the plate with a dye which on oxidation changes color.

N. gonorrhoeae (the "gonococcus")

N. gonorrhoeae (figure 20 and 21), found only in man, is the causative agent of gonorrhea, the second most common venereal disease. The organism often causes an effusion of polymorphonuclear cells. A smear (figure 17, 18, 19) may show the presence of Gram negative cocci present in cells. However, culture is essential for definitive diagnosis.

A common feature of disseminated gonoccocal disease is arthritis. Although commonly considered a form of septic arthritis, in many cases gonococci cannot be isolated from the joint (i.e. they are "reactive" in nature). Dermatitis is also common.

Penicillin therapy is still usually effective. However, resistant strains producing beta lactamases are sufficiently common that alternatives are recommended for all gonococcal infections; this includes ceftriaxone (a beta lactamase-resistant cephalosporin). There is no vaccine since strains are highly variable in their external antigens (both outer membrane and pili). Both are involved in the initial adhesion of the organism to genital epithelium.

IgA proteases (also produced by N. meningitidis) are involved in successful colonization. As for many other bacterial infections, a role for both the lipopolysaccharide and peptidoglycan in tissue injury have been suggested. Exotoxins are not believed to be of importance in pathogenesis.

WEB RESOURCES

Some facts about gonorrhea 
CDC

Diagnosis of Neisseria gonorrhoeae 
CDC Division of AIDS, STD, and TB Laboratory Research

Figure 20 Scanning electorn micrograph of  Neisseria gonorrheae © Margaret Ketterer, University of Iowa, Iowa City, Iowa USA and The MicrobeLibrary

Figure 22 Neisseria meningitidis, group C, in spinal fluid. CDC/Dr. M.S. Mitchell

  Figure 23 Neisseria meningitidis - coccoid prokaryote (dividing); causes meningitis and Waterhouse-Friderichson syndrome (a fulminating meningococcal infection occurring mainly in children under ten years old)  © Dennis Kunkel Microscopy, Inc.  Used with permission

N. meningitidis (the "meningococcus")

This organism (figure 22 and 23) resides only in man. The majority of cases are sporadic cases most commonly seen among young children. Outbreaks occur usually among adults living in confined and crowded conditions (e.g. army barracks). Initial infection of the upper respiratory tract (involving binding by pili) leads to invasion into the bloodstream and from there to the brain. Indeed, it is the second most common cause of meningitis (pneumococcus is the most common). It is usually fatal if untreated but responds well to antibiotic therapy. Thus, rapid diagnosis is important. The organism is often detectable in spinal fluid (Gram negative diplococci within polymorphonuclear cells) or antigenically. Culture on Thayer Martin (or similar) agar is essential for definitive diagnosis. Penicillin is the drug of choice.

Meningococci vary antigenically and can be serogrouped with anti-capsular antibodies. The capsule is an important pathogenesis factor allowing inhibition of phagocytosis. A vaccine against these capsular antigens is available. However, effective immunization against the most common group B has not been achieved.

Non-pathogenic species morphologically resembling Neisseria are found in the normal flora of the oropharynx but can be differentiated from the pathogenic Neisseria readily. These occasionally cause opportunistic human disease (including pneumonia).

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