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 Bacterial identification 
  in the diagnostic 
  laboratory versus 
  taxonomy


SPIROCHETES AND 
  NEISSERIA  

 
SPIROCHETES  
      Treponema, 
      Borrelia and 
      Leptospira) 
 
NEISSERIA
    
N. gonorrhoeae 
       N. meningitidis 

BACTERIOPHAGE  
EXCHANGE OF  
  GENETIC 
  INFORMATION

Enterobacteriaceae,
  Vibrio, Campylobacter
  and Helicobacter

Streptococci  
Streptococcus  
  pneumoniae
and 
  Staphylococci  
 

ANAEROBES AND 
  PSEUDOMONAS - 
  OPPORTUNISTIC 
  INFECTIONS 

MYCOBACTERIA,  
  CORYNEBACTERIA
  AND LEGIONELLA  

BORDETELLA AND 
  HAEMOPHILUS  

RICKETTSIA, 
  EHRLICHIA, COXIELLA 
  AND BARTONELLA
  

ZOONOSES  
  LISTERIA, 
  FRANCISELLA,   
  BRUCELLA, BACILLUS  
  AND YERSINIA 





















 

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  SPIROCHETES AND NEISSERIA        

Dr Alvin Fox

Medical Microbiology, MBIM 650/720  Lecture: 40

Reading: Murray third edition Chapter 41 (Spirochetes) 28 (Neisseria).

SPIROCHETES (Treponema, Borrelia and Leptospira)

Spirochetes 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 and syphilis

Some facts about Syphilis from CDC

syphilis.jpg (35933 bytes)  Congenital syphilis, primary and secondary syphilis rates, by year -- United States, 1992-1998. Epidemiology, surveillance. CDC/Dr. Demetri Vacalis  tdv0@cdc.gov 

trepo.jpg (105351 bytes)  Histopathology showing Treponema pallidum spirochetes in testis of experimentally infected rabbit. Modified Steiner silver stain. CDC/Dr. Edwin P. Ewing, Jr.  epe1@cdc.gov 



T. pallidum 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 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 (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.

primary-syphil.jpg (499791 bytes) Primary Syphilis Bristol Biomedical Archive © University of Bristol. Used with permission

secy-syphilis.jpg (454719 bytes) Secondary syphilis - mouth mucosa Bristol Biomedical Archive © University of Bristol. Used with permission

syph1.jpg (10377 bytes) Primary syphilis. Primary chancre on the glans  The University of Texas Medical Branch syph3.jpg (19520 bytes)  Primary syphilis. A vulvar chancre and condylomata acuminata  The University of Texas Medical Branch 

For further clinical images, go here

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.

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

yaws.jpg (9509 bytes) Yaws is a crippling and disfiguring disease affecting some 50 million people in the world   © WHO

 

Borrelia burgdorferi and Lyme disease

Some facts about Lyme disease from CDC

borrelia.jpg (70291 bytes) Histopathology showing Borrelia burgdorferi spirochetes in Lyme disease. Dieterle silver stain. CDC/Dr. Edwin P. Ewing, Jr. epe1@cdc.gov 

lyme epid.gif (37479 bytes) Reported cases of Lyme disease in the United States 1998 CDC

 

lymerisk.gif (20885 bytes) Lyme disease risk by region of United States CDC

 

Lyme disease is a relatively newly recognized disease. Although clinically first described in 1975, the role of a tick-borne spirochete was not proven until 1983. These ticks 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 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.

lymerash.jpg (104804 bytes) Lyme disease rash CDC lyme.jpg (98863 bytes) Ixodes scapularis, tick vector for Lyme disease. Also known as Ixodes dammini. CDC

lymecycle.gif (42095 bytes) 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.

 

Leptospirosis

leptospira.jpg (56195 bytes) 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 

lepto-kidney.gif (53248 bytes) 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 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, 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

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")

Some facts about gonorrhea from CDC

neis-dk.jpg (11932 bytes) Neisseria gonorrhoeae - coccoid prokaryote (dividing); causes gonorrhea (SEM x 40,000)  © Dr Dennis Kunkel, University of Hawaii. Used with permission

neisseria3.jpg (110388 bytes) Positive FA test for Neisseria gonorrhoeae. This strain was penicillin-resistant. CDC


N. gonorrhoeae, 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 may show the presence of Gram negative cocci present in cells. However, culture is essential for definitive diagnosis (go here).

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 ß lactamases are sufficiently common that alternatives are recommended for all gonococcal infections; this includes ceftriaxone (a ß 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.

 

N. meningitidis (the "meningococcus")

neisseria.jpg (72153 bytes) Neisseria meningitidis, group C, in spinal fluid. CDC/Dr. M.S. Mitchell neis2-dk.jpg (12129 bytes)  Neisseria meningitidis - coccoid prokaryote (dividing); causes meningitis and Waterhouse-Friderichson syndrome (a fulminating meningococcal infection occurring mainly in children under ten years old)  © Dr Dennis Kunkel, University of Hawaii. Used with permission

 

This organism 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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