1. Most species are motile and possess peritrichous flagella.
2. Clostidium sp. are known for the production of spores that allow survival of the organism under severe nutrient deprivation and dehydration conditions. Spores are even resistant to the actions of common antimicrobial agents and treatments. Clostridium spores are usually wider than the diameter of the rods in which they are formed. Placement can be central, subterminal, or terminal.

C. CULTURE AND PHYSIOLOGY

1. Anaerobic culture in which the agar plates or tubes are placed in an airtight jar in which a nitrogen/10% CO2 atmosphere is generated.
2. Fluid media are put in deep tubes containing chopped, cooked meat or 0.1% agar and a reducing agent such as thioglycolate.
3. Large raised colonies with entire margins (C. perfringens), or smaller colonies that extend in a meshwork of fine filaments (C. tetani). Many show hemolysis on blood agar (hemolysin production).
4. Possible explanations for the obligate anaerobic requirement include:
• a. Obligate anaerobes lack superoxide dismutase: they have no mechanism to destroy the toxicity due to superoxide anion formation.
• b. In the presence of oxygen, superoxide anions form peroxides, oxidizing agents lethal to obligate anaerobes (lacking catalase and peroxidase).
• c. Obligate anaerobes need a reduced environment. Reduction is evaluated using oxygen and hydrogen electrodes. The hydrogen electrode is the most highly reduced (-480 mV potential: a good anaerobic environment). Oxidation to between +50 and +75 mV results in no growth.
• d. The need for anaerobic culture conditions makes it difficult to isolate anaerobes from patients. A reduced environment can be maintained artificially using reducing agents or biologically by aerobic organisms.

1. BOTULISM
2. TETANUS
3. GAS GANGRENE
4. PSEUDOMEMBRANOUS ULCERATIVE COLITIS (Antibiotic-induced colitis)
5. FOOD POISONING

III. Clostridium botulinum

A. MORBIDITY/MORTALITY

Botulism is fortunately not a serious disease in terms of incidence, but it is serious in terms of its potential mortality.

1. In the mid-1970s there was an increase in botulism due to increased home canning.
2. Infant botulism was first recognized in the 1970s.

B. EXTRACELLULAR PRODUCTS

1. Both C. botulinum and C. tetani involve potent neuroexotoxins (exotoxins that specifically affect neuronal tissue).
• a. C. botulinum toxin acts upon peripheral nerves. There are eight serologica l types of toxin. Types A, B, and E cause most human disease.
• b. C. tetani toxin (tetanospasmin) acts centrally at the level of the anterior horn cells of the spinal cord.
2. In the case of botulism, the toxin is ingested in a preformed state (ie in home-canned vegetables). Soil spores germinate and produce toxin. The botulinum toxin is heat labile. If improperly canned vegetables are boiled for 10 minutes, botulism poisoning is no longer a problem. In contrast, Staphyloenterotoxin is heat stable.
3. The toxin is activated by proteases in the gastric fluid and by gastric acidity. The toxin is absorbed in the intestine and is transported systemically via the bloodstream. Botulinus toxin is quick acting with signs and symptoms of intoxication apparent within 12 hours after ingestion.
4. Once the toxin is fixed to the tissue, its actions are very difficult to reverse with antitoxin treatment. Antitoxin is only effective if it binds to the toxin before the toxin binds the neuromuscular junction (within 12 hours after ingestion). Prognosis is poor for patients diagnosed after this time period.
5. Botulinus toxin is a medium-sized (150 kDa) AB type protein toxin. There is a binding component and a toxic component. The toxin inhibits acetylcholine release at the level of the presynaptic terminals at the neuromuscular junction. This leads to flaccid paralysis and potentially, respiratory failure.

1. Visual disturbances (eye muscles not coordinated, double vision)
2. Inability to swallow and speech difficulties occur within 18-24 h after ingestion.
3. GI involvement is not usually prominent.
4. No fever is apparent.

There is no reason to give antibiotics; normally there are no organisms, except in infant botulism (found in infants under 6 months of age). A potent trivalent (A,B,E) antitoxin is available from the CDC; it must be promptly administered IV. Rapid antitoxin treatment has reduced the mortality rate from 65% to below 25%.

E. INFANT BOTULISM

Between birth and 6 months there is a unique susceptibility to ingested spores that germinate in the intestine and multiply, producing toxin locally. Toxin is taken up by the bloodstream. Symptoms are milder than in adult botulism and mortality is not as high. The amount of toxin produced is less than would typically be ingested by an adult. After the age of 6 months, the permeability of the intestinal mucosa changes dramatically, contributing to the narrow age range for susceptibility to infant botulism.

IV. Clostridium tetani

A. MORBIDITY/MORTALITY

1. Tetanus is a disease of low incidence in the U.S. and is a disease that is entirely preventable with toxoid immunization that gives essentially 100% protection. The toxoid booster confers protection for more than 10 years.
2. Tetanus is a serious clinical disease because it is difficult to reverse, with a mortality rate of approximately 50%. The toxin produces spastic paralysis due to uncontrolled repetitive firing. A sardonic smile is characteristic of early stages of tetanus, because the masseter muscles are very sensitive.
3. Tetanus is different from botulism in that the organism grows in the host. There is a host-parasite interaction. The bacteria remain localized at the site of introduction. Disease is almost entirely a neurotoxemia.

1. Tetanspasmin is a heat labile AB-type toxin, approximately 150 kDa. Subunit "A" binds to the target tissue and subunit "B" has the toxic effect.
2. C. tetani also produce one other virulence factor called tetanolysin. It may or may not be important in local infection. The toxin is a hemolysin.

1. C. tetani grows in traumatized tissue where the blood supply is cut off and ischemia results, producing an anoxic, anaerobic environment, with low redox potential. Acidity increases with inflammation. The more acidic the environment, the more reduced it tends to be. A puncture wound can also cause spores to be injected deeply into the tissue.
2. It takes 2-3 days for the numbers of C. tetani to increase enough so that toxin production will be effective. Tetanus toxin travels intraaxonally to reach the CNS. Gangliosides in spinal cord and brain stem (anterior horn cells) are bound by tetanospasmin; only a small amount of toxin is needed to produce change. Tetanospasmin blocks the inhibitory pathway. Repeated firing at the synapse causes contraction with no relaxation, resulting in generalized muscular spasms, hyperreflexia and seizures result.

1. C. tetani infection can be treated with penicillin to inhibit the organism's growth and stop further toxin production.
2. Surgical debridement of the necrotic tissue is essential.
3. Muscle relaxants, sedation and assisted ventilation are supportive therapy.
4. Antitoxin is administered to neutralize toxin before it has bound to tissue.

V. Clostridium perfringens

Gas gangrene is associate with C. perfringens infection, but in almost every case mixed infections are responsible: Staphylococcus, E. coli, and Bacteroides may also be present (microbial synergism). The counterpart to gas gangrene in muscle is Fusospirocheatel disease in the oral cavity (trench mouth).

A. MORBIDITY/MORTALITY

C. perfringens is the most common organism associated with anaerobic invasive disease. The perfringens enterotoxin is a common cause of food poisoning. Disease is not usually fatal if treated promptly.

B. EXTRACELLULAR PRODUCTS

1. Protease and toxin activity are apparent in gangrenous wounds. One of the most important virulence factors is the alpha-toxin, (a.k.a. phospholipase C).
2. Theta toxin is produced. It has hemolytic and necrotizing effects.
3. DNAse, hyaluronidase and collagenase are spreading factors.
4. Some strains produce a powerful enterotoxin that causes an intense, self-limiting diarrhea in 6-18 h.

C. PATHOGENESIS

1. C. perfringens grows in traumatized tissue with low redox potential. Exotoxins cause necrosis and toxemia.
2. C. perfringens produces proteolytic metabolic enzymes that break down tissue and proteins. Branch chain amino acids are converted to branch chain volatile acids: isobutyric acid, isovalenic acid, and propanoic acid.

D. TREATMENT

1. Passive anti-alpha toxin therapy is fairly effective.
2. Hyperbaric oxygen is another form of wound treatment.
3. Drainage and debridement are used to expose tissues to aerobic conditions.

VI. Clostridium difficile

C. difficile produces pseudomembranous ulcerative colitis as a result of oral clindomycin therapy (up to 25% of antibiotic-associated cases of diarrhea).

DIAGNOSIS OF C. difficile PSEUDOMEMBRANOUS ULCERATIVE COLITIS

PROCTOSCOPY: Multiple, raised, 2-5 cm plaques adherent to (Direct observation) edematous, friable colonic mucosa.

CULTURE: Anaerobic culture of stool specimen

CYTOTOXIN: Demonstration of cytotoxin in stool sample using a specific cytotoxic assay

A. EXTRACELLULAR PRODUCTS

1. Toxin A is a potent enterotoxin and cytotoxin. It binds to the intestinal brush border membranes at specific receptor sites (MW 440-500 kDa). The C. difficile cytotoxin causes cell death and tissue necrosis. Some strains of C. difficile also produce a second enterotoxin.
2. Toxin B is a potent cytotoxin found in the stools of patients with pseudomembranous colitis (MW 360-470 kDa).

1. Both ampicillin and clindamycin can result in drug-resistant C. difficile.
2. Binding of the difficile toxin to the gut brush border cells produces watery or bloody diarrhea associated with abdominal cramps, leukocytosis and fever.

VII. ENDOGENOUS ANAEROBIC INFECTIONS

The majority of anaerobic infections seen clinically are from endogenous microbes. Anaerobes outnumber aerobes on most mucosal surfaces. Aerobes carry out oxidation reactions, leaving the environment in a reduced state that favors anaerobic growth.
Anaerobic infections result from overgrowth by endogenous opportunistic bacteria that occurs with compromised host defense, particularly when tissue pO2 is reduced. Injury is the most common source of entry of anaerobes into normally sterile body sites.

A. CLINICAL SYMPTOMS SUGGESTING ANAEROBIC INFECTION

1. Putrid odor (volatile acidic metabolic byproducts from fermentation, i.e. gangrene)
2. Infection adjacent to a mucosal surface
   
   • anaerobic lung abscess - can originate with oral flora
   • anaerobic peritoneal abscess - can be caused by gut flora
3. Presence of gas within tissue (H2S gas is present with gas gangrene)
4. Infection in necrotic, avascular tissue or at a site suggestive of anaerobic infection.
5. Bowel perforation
6. Gram stain showing mixed, pleomorphic bacterial flora
7. Negative cultures after a positive Gram stain of clinical samples

B. CLINICAL SPECIMEN COLLECTION The following factors are important to keep in mind for anaerobic specimen collection. Most errors in diagnosis occur at the level of collection and transport.

• DO culture aspirated pus.
• DO culture infected tissue.
• DO NOT culture sites normally inhabited by a rich normal flora (such as mucous membranes and GI tract).
• DO NOT culture swabs-most swabs are aerobic and some are treated with substances inhibitory to anaerobes
• RAPID TRANSPORT TO THE LAB: -possibly the most important item on the list

Other factors to bear in mind are temperature, desiccation, oxidation, antimicrobials, and possible overgrowth by facultative anaerobes or aerobes in the specimen.

C. SPECIMEN COLLECTION EQUIPMENT FOR ANAEROBES

1. Sterile applicator sticks and syringes (perhaps the quickest, easiest way to collect)
2. O2-free/CO2-filled tubes
3. Anaerobic culturettes
4. Gas-Pak jars: a packet produces hydrogen and CO2 in the presence of a palladium catalyst and water)
5. Anaerobic chambers (all tests can be done in an anaerobic environment)
6. Cannula systems
7. Gas chromatograph (identification through metabolic by-products from spent medium. Analysis time is reduced to a few days, and is very accurate).

1. Some anaerobic infections form abscesses that are difficult to treat and must be removed surgically or drained (debridement).
2. Some important antimicrobials for anaerobic organisms are clindomycin and metronidazole. (Clindomycin: secondary complications include C. difficile resistance in the colon).
3. The largest concentration of anaerobes in the abdomen is in the large intestine
4. Primary pathogens
   • Bacteroides fragilis
   • Clostridium perfringens
   • Peptostreptococcus-anaerobic genus of streptococcus
5. Clinical signs include intense local pain, chills, fever, tachycardia, sweating, rigid abdominal wall, rebound, tenderness, shock
6. Causes of anaerobic infection
• malignant tumors
• inflammatory changes
• perforations (trauma, surgery, or ulcers)
7. Diagnosis - if the wound is open, the first procedure is Gram staining. A syringe aspirate of exudates is preferred. A mixed flora usually indicates anaerobic infection.

1. Primary pathogens
• Bacteroides fragilis - most significant
• Clostridium perfringens
• Peptostreptococcus
• Bacteroides melaninogenicus and Fusobacteria
2. Clinical signs: foul smelling discharge, lower abdominal pain, fever
3. Causes of anaerobic infection
• anaerobic flora may ascend through fallopian tubes and enter the pelvis
• anaerobic flora may pass through tissue and places of injury and enter the pelvis
4. Diagnosis - malodorous discharge; but lack of an odor does not rule out an anaerobic infection. Vaginal smears are unreliable as commensal anaerobes are abundant. Specimens are aspirated through the vaginal wall.

1. Primary pathogens
• Bacteroides melaninogenicus
• Fusobacterium nucleatum
• Peptostreptococcus
2. Clinical signs - weakness, listlessness, fever, dull chest percussion
3. Causes of anaerobic infection include aspiration of normal flora of mouth and upper respiratory tract (can also cause chronic infections of the sinuses, middle ear, and abscesses of the brain). Anaerobes hide in the crevices of the mucous membranes, especially the gingivi (responsible for the fetid breath of gingivitis).
4. Diagnosis-transtracheal aspirate is needed. This bypasses contamination of specimen by upper respiratory tract (expectorated sputum will not do). X-rays will also show characteristic shadows or cavitations within the consolidated infiltrate.
5. Alcoholics, drug addicts, and anesthesia patients can aspirate upon losing consciousness and develop aspiration pneumonia
6. Anaerobes can also cause empyema (thick, foul-smelling pus)

1. Anaerobic bacteria are found as normal flora in many places. Infections are caused when they gain access to sterile environments.
2. Anaerobic infections are common in the thorax, abdomen and female pelvis.
3. Abscess formation; high mortality is associated with this type of infection.
4. Diagnosis depends on clinical observation, Gram staining and culture.
5. Effective treatment should be started promptly with selected antibiotics.
6. Antibiotics have been developed that are consistently active against anaerobes. Mixed treatment must sometimes be used to cover anaerobes and aerobes at once.

IMPORTANT ANAEROBES TO REMEMBER

E-mail Dr. Johnson for comments or suggestions. johnsomt (at) iupui.edu

Authored by Mary T. Johnson, Ph.D. ©Copyright 1999-2008, Indiana University School of Medicine Last modified April 10, 2008

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