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 Bacterial identification 
  in the diagnostic 
  laboratory versus 
  taxonomy


ANAEROBES AND 
  PSEUDOMONAS - 
  OPPORTUNISTIC 
  INFECTIONS 

 
Anaerobes
 
Anaerobes non-
    spore- formers of 
    clinical importance

 
Anaerobic spore-
    formers Clostridia  

 
Pseudomonas
    aeruginosa  


BACTERIOPHAGE  
EXCHANGE OF  
  GENETIC 
  INFORMATION

Enterobacteriaceae,
  Vibrio, Campylobacter
  and Helicobacter

Streptococci  
Streptococcus  
  pneumoniae
and 
  Staphylococci  

SPIROCHETES AND 
  NEISSERIA  
 

MYCOBACTERIA,  
  CORYNEBACTERIA
  AND LEGIONELLA  

BORDETELLA AND 
  HAEMOPHILUS  

RICKETTSIA, 
  EHRLICHIA, COXIELLA 
  AND BARTONELLA
  

ZOONOSES  
  LISTERIA, 
  FRANCISELLA,   
  BRUCELLA, BACILLUS  
  AND YERSINIA 





















 

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 ANAEROBES AND PSEUDOMONAS - OPPORTUNISTIC INFECTIONS       

 Dr Alvin Fox
 
Medical Microbiology, MBIM 650/720

 

BRIEF OUTLINE OF MAJOR  POINTS
Overview of anaerobic bacteriology
Anaerobic Gram negative and Gram positive non-spore-formers
Anaerobic Gram positive spore-formers (clostridia)
  Pseudomonas (a strict aerobe)

 

  ANAEROBES  

Obligate anaerobes are bacteria that cannot survive in the presence of a high oxidation-reduction potential (redox potential) / high oxygen content. During metabolism bacteria can produce toxic bi-products from oxygen (including superoxide radicals and hydrogen peroxide). Strict anaerobes lack certain enzymes (including superoxide dismutase and catalase) that detoxify these products.

Polymicrobic anaerobic infection

Strict anaerobes cannot grow in healthy tissues due to their oxygen content. When tissue injury occurs with limitation of the blood (and oxygen) supply, conditions are created for opportunistic growth of obligate anaerobes. Often more than one species will infect the same site. Simultaneous infection with a facultative anaerobe (which uses up the already diminished oxygen supply) also encourages growth of obligate anaerobes.

Endogenous versus exogenous infection

Most anaerobes in the normal flora are non-spore formers and anaerobic infections often occur from this source. However, contamination of wounds can also occur with anaerobic spore-formers (e.g. clostridia) which are common in the environment (e.g. soil). Non-spore-formers rarely produce exotoxins in contrast to spore-formers.

Sites of anaerobes in normal flora

Strict anaerobes are present in large numbers in the intestine (95-99% of total bacterial mass), but also in the mouth and genitourinary tract. The most common infections resulting from abdominal surgery or other gut injury are Enterobacteriaceae (facultative anaerobes) and  Bacteroides fragilis (see below). These are minor components of the gut flora and demonstrate the important point that certain organisms more readily produce opportunistic infections than others.

Problems in identification of anaerobic infections

1. They are often derived from the normal flora. One must be confident that one has not isolated a contaminant.

2. If air gets into the sample during sampling or transportation to the clinical laboratory, then the organism may not be isolatable.

3. Slow growth of the organism (due to inefficiency of fermentation) means isolation takes several days or longer.

 

Identification in the clinical laboratory after isolation

Two systems are commonly used: Biochemical systems and/or gas chromatographic identification of volatile fermentation products (short chain fatty acids / alcohols)

 

  ANAEROBIC NON-SPORE-FORMERS OF CLINICAL IMPORTANCE  

1. Gram-negative rods (Bacteroides [e.g. B. fragilis] and Fusobacterium)
2. Gram-positive rods (Actinomyces, Arachnia, Eubacterium, Bifidobacterium, Lactobacillus, Propionibacterium)
3. Gram-positive cocci (Peptostreptococcus and Peptococcus)

4. Gram-negative cocci (Veillonella, Acidominococcus)

 

Bacteroides fragilis

1. The most important strict anaerobic non-spore-former causing clinical disease.
2. Prominent capsule involved in pathogenesis (i) anti-phagocytic (ii) directly involved in abscess formation.
3. Endotoxin which differs in composition from typical endotoxin and is of low toxicity.

 

 

 

  ANAEROBIC SPORE-FORMERS (CLOSTRIDIA))  

Gram-positive rods, reservoir: found in the environment (particularly soil) but also intestine of man.


C. tetani

tet1.jpg (31190 bytes) This baby has neonatal tetanus. It is completely rigid. Tetanus kills most of the babies who get it. Infection usually happens when newly cut umbilical cord is exposed to dirt CDC tet2.jpg (31041 bytes) This baby has tetanus. He cannot breast feed or open his mouth because the muscles in his face have become so tight WHO tet3.jpg (25275 bytes) This adult has a severe case of tetanus. The muscles in his back and legs are very tight. Muscle spasms can  break bones in the person's body CDC

1. Commonly found in the soil; thus contamination of wounds can lead to colonization and tetanus.

2. The exotoxin (tetanospasmin) binds to ganglioside receptors on inhibitory neurones in CNS (where glycine is commonly the neurotransmitter) and stops nerve impulse transmission to muscle leading to spastic paralysis. Continued severe muscle contractions and spasms result which can be fatal.

3. The organism is non-invasive and thus remains in the local wound.

4. Vaccination of infants with tetanus toxoid have almost eliminated this disease in the US.

CDC tetanus manual (requires Acrobat)

 

C. perfringens

1. Causes wound colonization (gas gangrene) after soil, and to a lesser extent intestinal tract, contamination. Primarily seen in time of war. The term gas gangrene refers to swelling of tissues due to release of gas, as fermentation products, of clostridia.

2. The organism produces several tissue degrading enzymes (including lecithinase [alpha toxin], proteolytic and saccharolytic enzymes). Necrosis and destruction of blood vessels and the surrounding tissue result. This creates an anaerobic environment in adjacent tissue and the organism spreads systemically. Death can occur within 2 days. Nowadays, treatment (including anti-toxin, antibiotic therapy, debridement) is extremely effective and amputation and death is rare.

3. The determination of production of lecithinase is important in laboratory identification of the organism.

4. A significant cause of food poisoning by enterotoxin producing strains.

 

C. botulinum

clostridia-dk.jpg (20320 bytes)  Clostridium botulinum - rod prokaryote. Vegetative (yellow arrow) and spore (blue arrow) stages : note the flagella on the vegetative cells. Causes botulism. SEM  x15,400, © Dr Dennis Kunkel, University of Hawaii. Used with permission clostrid-bot-dk.jpg (14135 bytes)  Clostridium botulinum - rod prokaryote. Vegetative (yellow arrow) and spore (blue arrow) stages: note the flagella on the vegetative cells. Causes botulism. SEM x12,800 © Dr Dennis Kunkel, University of Hawaii. Used with permission

 

1. Botulism (a rare but fatal form of food poisoning) is caused by a potent exotoxin (botulinum toxin). This toxin binds to receptors on peripheral nerves, where acetylcholine is the neurotransmitter. The toxin inhibits nerve impulses and flaccid paralysis results and often death (from respiratory and/or cardiac failure). The organism does not grow in the gut, but pre-formed exotoxin from prior germination of spores may be present in inadequately autoclaved canned food (usually at home).

2. Wound botulism can occur but is even rarer.

3. C. botulinum does not readily grow in the adult intestine due to competition with the normal flora. In the neonate, where the flora is not established, colonization with C. botulinum can occur. Infant botulism, although uncommon, is now the predominant form of botulism.

4. Treatment includes administration of anti-toxin and (for neonates) antibiotic therapy.

CDC information on botulism

CDC botulism manual (requires Acrobat)

Seminal facts about botulism (from WHO)

 

C. difficile

When the normal flora of the intestine is altered by antibiotic therapy this organism can colonize. It produces an enterotoxin and pseudomembanous colitis can result. Therapy includes discontinuation of the implicated antibiotic (e.g. ampicillin). Severe cases require specific antibiotic therapy (e.g. with vancomycin).

 

 

  PSEUDOMONAS AERUGINOSA  

pseudo1.jpg (57140 bytes)   Scanning Electron Micrograph of Pseudomonas aeruginosa CDC 

1. Aerobic, gram-negative rods, polar flagella, oxidase positive (in contrast to Enterobacteriaceae).

2. Among the genus Pseudomonas the majority of human infections are caused by P. aeruginosa, although other related organisms also cause disease.

3. Reservoir: soil, water and air. Commonly infects compromised host e.g. a) Burns and wounds - destruction of blood vessels limits access of phagocytes b) After use of cytotoxic drugs in cancer therapy (which destroys the immune system) c) Alteration of the respiratory epithelium in cystic fibrosis commonly allows colonization and development of pneumonia.

4. Identification includes (a) pigments: pyocyanin (blue-green) and fluorescein (green-yellow, fluorescent) and (b) biochemical reactions. Cultures have fruity smell.

5. Slime layer is anti-phagocytic.

6. Toxin A - ADP ribosylates EF2 similar action to diphtheria toxin

Bacterial toxins: Friend or Foe (from CDC)


 

 


 






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