The three major sources of anaerobic organisms are the dental and mouth area, the lower intestinal tract (ileum and colon), and the female external genital tract (vagina and vulva area). Anaerobes comprise about 95% of the bacterial flora of the colon and outnumber aerobes in the mouth and vagina. From the mouth the organisms can reach the lungs and brain; from the vagina they may involve the remainder of the female genital tract; diseases of the lower intestinal tract may release anaerobes into the abdominal cavity; and all of these locations may be the source of organisms found in the bloodstream. In addition, anaerobic bacteria are frequently associated with chronic infection, such as bronchiectasis, abscess, or chronic osteomyelitis. Infections with anaerobes frequently are mixed infections that also contain aerobes. The types of infection most frequently associated with anaerobes are listed in the box below.

Types of Infection Most Frequently Associated With Anaerobes (Alone or as a Mixed Infection)
Dental or mouth area infection
Chronic sinus infection
Bite wounds
Bronchiectasis and aspiration pneumonia
Gynecologic intraabdominal and extra abdominal infection
Abscess of any area other than skin; including brain, abdominal and thoracic cavities, and any organ
Infections associated with the intestinal tract, especially the colon (diverticulitis, appendicitis, bowel perforation, etc.)
Deep tissue infection or necrosis
Biliary tract infection
Chronic osteomyelitis

Anaerobic infections usually center on three groups of organisms: Clostridia species, Bacteroides species, and the anaerobic streptococci.

Clostridia

These gram-positive anaerobic rods include several important organisms. Clostridium perfringens (Clostridium welchii) is the usual cause of gas gangrene. It is a normal inhabitant of the GI tract and reportedly can be isolated from the skin in about 20% of patients and from the vagina and female genitalia in about 5%. Therefore, just as with S. aureus, a culture report of C. perfringens isolated from an external wound does not necessarily mean that the organism is producing clinical infection. When isolated from abdominal or biliary tract infections, C. perfringens most often is part of a polymicrobial infection and, although serious, is not quite as alarming as isolation from a deep tissue wound. Clostridium perfringens occasionally is a cause of “food poisoning” (discussed later).

Clostridium tetani causes tetanus. The organism can be found in the human GI tract but is more common in animals. Spores are widely distributed in soil. Clinical disease is produced by release of bacterial exotoxin after local infection in a manner analogous to diphtheria. Puncture-type wounds are notorious for high risk of C. tetani infection. The incubation time is 3-14 days in most patients, a few cases being reported as early as 24 hours after exposure. Cultures for C. tetani are said to be positive in less than 50% of patients. Clostridium difficile is the most frequent proven cause of antibiotic-associated diarrhea. In the 1950s and early 1960s, broad-spectrum oral antibiotics were frequently used, and S. aureus was thought to be the major cause of antibiotic-associated enteritis. Beginning in the late 1960s parenteral antibiotics became much more common in hospitals, and C. difficile was eventually proven to be the major etiology. Clostridium difficile enteritis usually occurs during or after therapy with antibiotics, although some patients have not received antibiotics. One report indicated a possible association with diarrhea induced by cancer chemotherapeutic agents. The condition may consist only of varying degrees of diarrhea, may progress to inflammation of portions of the colon mucosa, and in the more severe form (known as pseudomembraneouscolitis) there is inflammation and partial destruction of varying areas of the colon mucosa, with formation of a pseudomembrane of fibrin, necrotic cells, and segmented neutrophils on the surface of the remnants of the affected mucosa. Some patients develop intestinal perforation and sepsis.

Diagnosis of C. difficile colitis is not always easy. Only about 25%-30% (range, 20%-33%) of diarrhea occurring with antibiotic therapy is due to C. difficile, with most of the remainder not associated with currently known infectious agents. Sigmoidoscopy or colonoscopy can be done to document pseudomembrane formation, but this procedure runs the risk of perforation; and even in pseudomembraneous enterocolitis (PMC), pseudomembranes can be demonstrated in only about 50% (range, 40%-80%) of patients. The patient stools in PMC typically contain WBCs and often contain red blood cells, but gross blood is uncommon. However, WBCs on Gram stain actually are present in only about 45%-50% of cases. For several years, culture was the usual means of diagnosis. Today, this is not often done. C. difficile can be cultured, with reliable results, only on special media, so diagnosis usually is made through detection of C. difficile cytotoxin. Stool specimens must be frozen and sent to a reference laboratory with dry ice. The specimen container must be sealed tightly since the carbon dioxide from the dry ice can inactivate the toxin. Another problem is the fact that C. difficile can be found in the stool of some clinically healthy persons, including 30%-40% (range, 11%-63%) of clinically healthy neonates, 10%-15% of children, 3% (range, 0%-10%) of adults, 20% (range, 11%-36%) of hospitalized persons without diarrhea and not taking antibiotics, and about 30% (range, 20%-46%) of patients taking antibiotics but without diarrhea. Even in patients with endoscopy-proven PMC, stool culture is positive for C. difficile in only about 90% (range, 75%-95%) of cases. Most laboratories today rely more on tests to detect C. difficile toxin, which has been shown to correlate much better with C. difficile clinical infection than does stool culture. There are several C. difficile toxins, of which the best characterized are called toxins A and B. Toxin B is a cytotoxin that must be detected by a tissue culture system with an incubation period of 48 hours. In endoscopy-proven PMC, toxin B sensitivity is reported to be about 90% (range, 64%-100%). Results may be positive in some patients with positive C. difficile cultures who are clinically normal, especially in infants. An enzyme immunoassay kit is commercially available that detects both toxin B and toxin A (Cytoclone A+B). Sensitivity reported to date is about 85%-90% (range, 76%-99%). Toxin A is an enterotoxin that originally was assayed with a biologic system, the rabbit ileal loop test. Several EIA tests are commercially available for toxin A, with reported sensitivity of about 85% (range, 65%-97%). There is a simple latex agglutination kit available (CDT or Culturette CDT) that can provide same-day results. This kit was originally thought to detect toxin A but subsequently was found to be detecting an enzyme (glutamate dehydrogenase) from C. difficile. The test detects non-toxin-producing as well as toxin-producing C. difficile and cross-reacts with Clostridium sporogenes and a few strains of other clostridia. Nevertheless, in various studies this test frequently gave results equivalent to those of the cytotoxicity test (about 75%-90%; range, 38%-97% positive in patients with proven PMC, with roughly 90% specificity). Another company has a similar test (Meritec-CD). In one comparison between CDT and Meritec-CD, Meritec-CD showed about 10% less sensitivity.

Clostridium botulinus produces botulism. Botulism is a severe food poisoning due to ingestion of preformed botulinal endotoxin (a powerful neurotoxin) contained in the contaminated food, rather than actual infection of the patient by the organism. C. botulinum spores are widespread, but they can germinate only in anaerobic conditions at a fairly high pH (>4.6). These conditions are met in canned foods that have not been sufficiently heated during the canning process. Therefore, C. botulinum is usually associated with canned food, especially home canned. Vegetables are the most frequently involved home-canned food, but any variety of canned food may become contaminated. Fortunately, the disease is not common. Although the endotoxin is preformed, symptoms most often appear 12-36 hours after ingestion (in adult patients) and commonly include nausea, vomiting, and abdominal cramps (about 50% of patients), constipation (75%), cranial nerve signs of dysphagia, dysarthria, and dry mouth (80%-90%), upper or lower extremity weakness (70%), and diplopia or other eye abnormalities (90%). There is no fever or diarrhea. Differential diagnosis in adults includes Guillain-Barrй syndrome, drug reaction (especially phenothiazines), myasthenia gravis, cerebrovascular accident, chemical poisoning (mercury, arsenic, etc.), diphtheria, and tick bite paralysis (Landry’s ascending paralysis). Botulism may also occur in infants, usually between ages 3 and 26 weeks (median age 10 weeks). The classic syndrome is onset of constipation followed by 4-5 days of progressive feeding difficulty, ptosis, muscle hypotonia, and possibly respiratory distress. Mildly affected infants may have varying degrees of “failure to thrive” with feeding difficulty and mild muscle weakness, whereas severely affected infants may have severe respiratory distress. Some cases of sudden infant death syndrome (SIDS) have been ascribed to infant botulism, whereas some infants and older children (investigated during studies initiated by botulism cases) were found to harbor C. botulinum organisms without symptoms. Honey was incriminated in some cases as the source of infection but was not used in the majority of cases.

Standard laboratory tests, including those on the CSF, are usually normal in botulism. Electromyography (EMG) may be helpful in differentiating adult botulism from certain other neurologic diseases, but the typical EMG findings are neither specific nor always present. The diagnosis is confirmed by stool culture and demonstration of C. botulinum toxin, neither of which can be done in the ordinary laboratory. In addition, food suspected of contamination should always be tested. Patient vomitus and gastric contents have also been tested. The basic specimens in adult botulism are stool (25 gm or more) and serum (at least 5 ml). For diagnosis of infant botulism a stool specimen is required; but infant serum rarely contains C. botulinum toxin and therefore infant serum specimens are not necessary. The specimens should be kept refrigerated, since the toxin is heat labile, and sent to a reference laboratory with a coolant (for short distances) or dry ice (for more than 1-day transit). There are several C. botulinum serotypes, but most cases are caused by types A and B, with type A predominating.

Bacteroides

Bacteroides are the most frequent organisms found in anaerobic infections. They comprise several species of gramegative rods normally found in the mouth, the intestine, and the female genital tract. Isolation of these organisms often raises the question of their significance or pathogenicity. It seems well established that bacteroides occasionally cause serious infection, which frequently results in abscess or gangrene. The most commonly associated clinical situations include septic abortion, aspiration pneumonia, focal lesions of the GI tract (e.g., carcinoma or appendicitis), and pelvic abscess in the female.

Anaerobic streptococci

Anaerobic streptococci are frequently associated with Bacteroides infection but may themselves produce disease. They are normally present in the mouth and GI tract. Septic abortion and superinfection of lesions in the perirectal area seem to be the most commonly associated factors. Anaerobic streptococci are also part of the fusiform bacteria-spirochetal synergistic disease known as Vincent’s angina.

Laboratory diagnosis of anaerobic infections

Anaerobic specimens cannot be processed by the laboratory as easily as those from aerobic infections. Anaerobic bacteria in general do not grow as readily as common aerobic bacteria, and prereduced media or other special media are often needed. Achieving and maintaining anaerobic culture conditions are not a simple matter. However, by far the greatest problem is the specimen received by the laboratory. If the specimen is not properly maintained in an anaerobic environment en route to the laboratory, the best and most elaborate facilities will be of little help. For abscesses or any fluid material, the preferred collection technique is to aspirate the material with a sterile syringe and needle, then expel the residual air from the needle by pushing the syringe plunger slightly, and then immediately cap the needle point with a sterile cork or other solid sterile material. The syringe must be transported immediately to the laboratory. If there is no liquid content, a swab that forms part of one of the special commercially available anaerobic transport systems should be used. Directions for creating the anaerobic environment should be followed exactly. A less satisfactory procedure is to inoculate a tube of thioglycollate medium immediately after the specimen is obtained and immediately recap the tube.