Chapter 29: Clostridium, Bacteroides, and Other Anaerobes

The bacteria discussed in this chapter are united by a common requirement for anaerobic conditions for growth. Organisms from multiple genera and all Gram stain categories are included. Most of them produce endogenous infections adjacent to the mucosal surfaces, where they are members of the indigenous microbiota. The clostridia form spores that allow them to produce diseases such as tetanus and botulism after environmental contamination of tissues or foods. Another anaerobic genus of bacteria, Actinomyces, is discussed in Chapter 28.

BACTERIOLOGY

THE NATURE OF ANAEROBIOSIS

Anaerobes not only survive under anaerobic conditions, they require them to initiate and sustain growth. By definition, anaerobes fail to grow in the presence of 10% oxygen, but some are sensitive to oxygen concentrations as low as 0.5% and are killed by even brief exposures to air. However, oxygen tolerance is variable, and many organisms can survive briefly in the presence of 2% to 8% oxygen, including most of the species pathogenic for humans. The mechanisms involved are incompletely understood, but clearly represent a continuum from species described as aerotolerant to those so susceptible to oxidation that growing them in culture requires the use of media prepared and stored under anaerobic conditions.

Anaerobes lack the cytochromes required to use oxygen as a terminal electron acceptor in energy-yielding reactions and thus to generate energy solely by fermentation (see Chapter 21). Some anaerobes do not grow unless the oxidation–reduction potential is extremely low (–300 mV); because critical enzymes must be in the reduced state to be active, aerobic conditions create a metabolic block.

Another element of anaerobiosis is the direct susceptibility of anaerobic bacteria to molecular oxygen. For most aerobic and facultative bacteria, catalase and/or superoxide dismutase neutralize the toxicity of the oxygen products hydrogen peroxide and superoxide. Most anaerobes lack these enzymes and are injured when these oxygen products are formed in their microenvironment. As discussed in the following text, many of the more virulent anaerobic pathogens are able to produce antioxidant enzymes like catalase or superoxide dismutase.

CLASSIFICATION

The anaerobes indigenous to humans include almost every morphotype and hundreds of species. Typical biochemical and cultural tests are used for classification, although this is difficult because the growth requirements of each anaerobic species must be satisfied. Characterization of cellular fatty acids and metabolic products by chromatography has been useful for many anaerobic groups. Nucleic acid base composition and homology have been used extensively to rename older taxonomy. The genera most commonly associated with disease are shown in Table 29–1 and discussed later.

Anaerobic Cocci

The medically important species of anaerobic gram-positive cocci include species of the genus, Peptoniphilis, Aerococcus, and others. With Gram staining, these bacteria are most often seen as long chains of tiny cocci. On the gram-negative side Veillonella deserves mention because of its potential for confusion with Neisseria although there are others (Megasphera, Anaeroglobus).

Clostridia

The clostridia are large, spore-forming, gram-positive bacilli. Like their aerobic counterpart, Bacillus, clostridia have spores that are resistant to heat, desiccation, and disinfectants. They are able to survive for years in the environment and return to the vegetative form when placed in a favorable milieu. The shape of the cell and location of the spore vary with the species, but the spores themselves (Figure 29–1) are rarely seen in clinical specimens.

The medically important clostridia are potent producers of one or more protein exotoxins. The histotoxic group including Clostridium perfringens and five other species (Table 29–2) produces hemolysins at the site of acute infections that have lytic effects on a wide variety of cells. The neurotoxic group including Clostridium tetani and Clostridium botulinum produces neurotoxins that exert their effect at neural sites remote from the bacteria. Clostridium difficile produces enterotoxins and disease in the intestinal tract. Many of the more than 80 other nontoxigenic clostridial species are also associated with disease.

Nonsporulating Gram-positive Bacilli

Propionibacterium is a genus of small pleomorphic bacilli sometimes called anaerobic diphtheroids because of their morphologic resemblance to corynebacteria. They are among the most common bacteria in the resident microbiota of the skin. Eubacterium is a genus that includes long slender bacilli commonly found in the colonic flora. These organisms are occasionally isolated from infections in combination with other anaerobes, but they rarely produce disease on their own. Other anaerobic gram-positive bacilli play roles in dental caries (see Chapter 41).

Gram-negative Bacilli

Gram-negative, non–spore-forming bacilli are the most common bacteria isolated from anaerobic infections. In the past, most species were lumped into the genus Bacteroides, which still exists along with five other genera. Of these, Fusobacterium, Porphyromonas, and Prevotella are medically the most important. The Bacteroides fragilis group contains B fragilis and 10 similar species noted for their virulence and production of β-lactamases. (Species outside this group generally lack these features and are more similar to the other anaerobic gram-negative bacilli.) Bacteroides fragilis is a relatively short gram-negative bacillus with rounded ends sometimes giving a coccobacillary appearance. Almost all B fragilis strains have a polysaccharide capsule and are particularly oxygen tolerant. Prevotella, Porphyromonas, and Fusobacterium are distinguished by biochemical and other taxonomic features. Prevotella melaninogenica forms a black pigment in culture, and Fusobacterium, as its name suggests, is typically elongated and has tapered ends.

ANAEROBIC INFECTIONS

EPIDEMIOLOGY

Despite our constant immersion in air, anaerobes are able to colonize the many oxygen-deficient or oxygen-free microenvironments of the body. These conditions are created by the presence of resident microbiota whose growth reduces oxygen and decreases the local oxidation–reduction potential. Such sites include the sebaceous glands of the skin, the gingival crevices of the gums, the lymphoid tissue of the throat, and the lumina of the intestinal and urogenital tracts. Except for infections with some environmental clostridia, anaerobic infections are almost always endogenous with the infective agent(s) derived from the patient’s own microbiota. The specific anaerobes involved are linked to their prevalence in the flora of the relevant sites as shown in Table 29–1. In addition to the presence of clostridia in the lower intestinal tract of humans and animals, their spores are widely distributed in the environment, particularly in soil exposed to animal excreta. The spores may contaminate any wound caused by a nonsterile object (eg, splinter, nail) or exposed directly to soil.

PATHOGENESIS

The anaerobic microbiota normally lives in a harmless commensal relationship with the host. However, when displaced from their niche on the mucosal surface into normally sterile tissues, these organisms may cause life-threatening infections. This can occur as the result of trauma (gunshot, surgery), disease (diverticulosis, cancer), or isolated events (aspiration). Host factors such as malignancy or impaired blood supply increase the probability that the dislodged flora will eventually produce an infection. The anaerobes most often causing infection are those both present in the microbiota at the adjacent mucosal site and which possess other features enhancing their virulence. For example, B fragilis represents a small percent of the normal colonic flora but is the bacterial species most frequently isolated from intraabdominal abscesses.

The relation between the microbiota and site of infection may be indirect. For example, aspiration pneumonia, lung abscess, and empyema typically involve anaerobes found in the oropharyngeal flora. The brain is not a particularly anaerobic environment, but brain abscess is most often caused by these same oropharyngeal anaerobes. This presumably occurs by extension across the cribriform plate to the temporal lobe, the typical location of brain abscess. In contaminated open wounds, clostridia can come from the intestinal flora or from spores surviving in the environment.

Although gaining access to tissue sites provides the opportunity, additional virulence factors are needed for anaerobes to produce infection. Some anaerobic pathogens produce disease even when present as a minor part of the displaced resident flora, and other common members of the microbiota rarely cause disease. Classic virulence factors such as toxins and capsules are known only for the toxigenic clostridia and B fragilis, but a feature such as the ability to survive brief exposures to oxygenated environments can also be viewed as a virulence factor. Anaerobes found in human infections are far more likely to produce catalase and superoxide dismutase than their more docile counterparts of the microbiota. Exquisitely oxygen-sensitive anaerobes are seldom involved, probably because they are injured by even the small amounts of oxygen dissolved in tissue fluids.

A related feature is the ability of the bacteria to create and control a reduced microenvironment, often with the apparent help of other bacteria. Most anaerobic infections are mixed; that is, two or more anaerobes are present, often in combination with facultative bacteria such as Escherichia coli. In some cases, the components of these mixtures are believed to synergize each other’s growth either by providing growth factors or by lowering the local oxidation–reduction potential. These conditions may have other advantages such as the inhibition of oxygen-dependent leukocyte bactericidal functions under the anaerobic conditions in the lesion. Anaerobes that produce specific toxins have a pathogenesis on their own, which are discussed in the sections devoted to individual species.

ANAEROBIC INFECTIONS: CLINICAL ASPECTS

MANIFESTATIONS

Bacteroides, Fusobacterium, and anaerobic cocci, alone or together with other facultative or obligate anaerobes, are responsible for the overwhelming majority of localized abscesses within the cranium, thorax, peritoneum, liver, and female genital tract. As indicated earlier, the species involved relate to the pathogens present in the microbiota of the adjacent mucosal surface. Those derived from the oral flora also include dental infections and infections of human bites.

In addition, anaerobes play causal roles in chronic sinusitis, chronic otitis media, aspiration pneumonia, bronchiectasis, cholecystitis, septic arthritis, chronic osteomyelitis, decubitus ulcers, and soft tissue infections of patients with diabetes mellitus. Dissection of infection along fascial planes (necrotizing fasciitis) and thrombophlebitis are common complications. Foul-smelling pus and crepitation (gas in tissues) are signs associated with, but by no means exclusive to, anaerobic infections. As with other bacterial infections, they may spread beyond the local site and enter the bloodstream. The mortality rate of anaerobic bacteremias arising from nongenital sources is equivalent to the rates with bacteremias due to staphylococci or Enterobacteriaceae.

DIAGNOSIS

The key to detection of anaerobes is a high-quality specimen, preferably pus or fluid taken directly from the infected site. The specimen needs to be taken quickly to the microbiology laboratory and protected from oxygen exposure while on the way. Special anaerobic transport tubes may be used, or by expression of any air from the syringe in which the specimen was collected. A generous collection of pus serves as its own best transport medium unless transport is delayed for hours.

A direct Gram-stained smear of clinical material demonstrating gram-negative and/or gram-positive bacteria of various morphologies is highly suggestive, often even diagnostic of anaerobic infection. Because of the typically slow and complicated nature of anaerobic culture, the Gram stain often provides the most useful information for clinical decision-making. Isolation of the bacteria requires the use of an anaerobic incubation atmosphere and special media protected from oxygen exposure. Although elaborate systems are available for this purpose, the simple anaerobic jar is sufficient for isolation of the clinically significant anaerobes. The use of media that contain reducing agents (cysteine, thioglycollate) and growth factors needed by some species further facilitates isolation of anaerobes. The polymicrobial nature of most anaerobic infections requires the use of selective media to protect the slow-growing anaerobes from being overgrown by hardier facultative bacteria, particularly members of the Enterobacteriaceae. Antibiotics, particularly aminoglycosides to which all anaerobes are resistant, are frequently incorporated in culture media. Once the bacteria are isolated, identification procedures include morphology, biochemical characterization, and metabolic end-product detection by gas chromatography.

TREATMENT

As with most abscesses, drainage of the purulent material is the primary treatment, in association with appropriate chemotherapy. Antimicrobial agents alone may be ineffective because of failure to penetrate the site of infection. Their selection is empiric to a large degree because such infections typically involve mixed species. Cultural diagnosis is delayed by the slow growth and the time required to distinguish multiple species. In addition, antimicrobial susceptibility testing methods are slow and not generally available for anaerobic bacteria. The usual approach involves selection of antimicrobials based on the expected susceptibility of the anaerobes known to produce infection at the site in question. For example, anaerobic organisms derived from the oral flora are often susceptible to penicillin, but infections below the diaphragm are caused by fecal anaerobes including B fragilis which is resistant to many β-lactams. These latter infections are most likely to respond to metronidazole, imipenem, or cefotaxime, a cephalosporin not inactivated by the β-lactamases produced by anaerobes.

BACTERIOLOGY

Clostridium perfringens is a large, gram-positive, nonmotile rod with square ends. It grows overnight under anaerobic conditions, producing hemolytic colonies on blood agar. In the broth containing fermentable carbohydrate, growth of C perfringens is accompanied by the production of large amounts of hydrogen and carbon dioxide gas, which can also be produced in necrotic tissues; hence the term gas gangrene.

Clostridium perfringens produces multiple exotoxins that have different pathogenic significance in different animal species and serve as the basis for classification of the five types (A-E). Type A is by far the most important in humans and is found consistently in the colon and often in soil. The most important exotoxin is the α-toxin, a phospholipase that hydrolyzes lecithin and sphingomyelin, thus disrupting the cell membranes of various host cells, including erythrocytes, leukocytes, and muscle cells. The θ-toxin alters capillary permeability and is toxic to heart muscle. This toxin also has pore-forming activity similar to streptolysin O. A minority of strains (less than 5%) produce an enterotoxin, which inserts into enterocyte membranes to form pores leading to alterations in intracellular calcium, membrane permeability, and the integrity of cell-to-cell tight junctions. This leads to loss of cellular fluid and macromolecules.

CLOSTRIDIUM PERFRINGENS DISEASE

EPIDEMIOLOGY

Gas Gangrene

Gas gangrene (clostridial myonecrosis) develops in traumatic wounds with significant avascular muscle necrosis when they are contaminated with dirt, clothing, or other foreign material containing C perfringens or another species of histotoxic clostridia (see Table 29–2). The clostridia can come from the patient’s own intestinal flora or spores in the environment. Compound fractures, bullet wounds, or the kind of trauma seen in wartime are prototypes for this infection. A significant delay (many hours) between the injury and definitive surgical management is required for bacterial multiplication and toxin production to develop. In peacetime these conditions are more likely to be satisfied in a remote hiking accident than in an automobile collision. The difference is the time between injury and medical intervention.

Clostridial Food Poisoning

Clostridium perfringens can cause food poisoning if spores of an enterotoxin-producing strain contaminate food. Outbreaks usually involve rich meat dishes such as stews, soups, or gravies that have been kept warm for a number of hours before consumption. This allows time for the infecting dose to be reached by conversion of spores to vegetative bacteria, which then multiply in the food. Clostridial food poisoning is common in developed countries and is second among foodborne illnesses in the United States with over a million cases per year.

PATHOGENESIS

Gas Gangrene

If the oxidation–reduction potential in a wound is sufficiently low, C perfringens spores can germinate and then multiply, elaborating α-toxin. The process passes along the muscle bundles, producing rapidly spreading edema and necrosis as well as conditions that are favorable for growth of the anaerobes. Very few leukocytes are present in the myonecrotic tissue (Figure 29–2). As the disease progresses increased vascular permeability and systemic absorption of the toxin leads to shock. α-Toxin is the major cause of both local destruction and shock. θ-Toxin and oxygen deprivation due to the metabolic activities of C perfringens are probable contributors. The basis for the profound systemic effects is not known, but α-toxin absorption and circulation seems probable because fatal cases occur without bacteremia.

Clostridial Food Poisoning

The spores of some C perfringens strains are often particularly heat-resistant and can withstand temperatures of 100°C for an hour or more. Thus, spores that survive initial cooking can convert to the vegetative form and multiply when food is not refrigerated or is rewarmed. After ingestion, the enterotoxin is released into the upper gastrointestinal tract, causing a fluid outpouring in which the ileum is most severely involved.

CLOSTRIDIUM PERFRINGENS: CLINICAL ASPECTS

MANIFESTATIONS

Gas Gangrene

Gas gangrene usually begins 1 to 4 days after the injury but may start within 10 hours. The earliest reported finding is severe pain at the site of the wound accompanied by a sense of heaviness or pressure. The disease then progresses rapidly with edema, tenderness, and pallor, followed by discoloration and hemorrhagic bullae. The gas is apparent as crepitance in the tissue, but this is a late sign. Systemic findings are those of shock with intravascular hemolysis, hypotension, and renal failure leading to coma and death. Patients are often remarkably alert until the terminal stages.

Anaerobic Cellulitis

Anaerobic cellulitis is a clostridial infection of wounds and surrounding subcutaneous tissue in which there is marked gas formation (more than in gas gangrene), but in which the pain, swelling, and toxicity of gas gangrene are absent. This condition is much less serious and can be controlled with antimicrobial therapy.

Endometritis

If C perfringens gains access to necrotic products of conception retained in the uterus, it may multiply and infect the endometrium. Necrosis of uterine tissue and bacteremia with massive intravascular hemolysis due to α-toxin may then follow. Clostridial uterine infection is particularly common after an incomplete abortion with inadequately sterilized instruments.

Food Poisoning

The particularly short incubation period of 8 to 24 hours is followed by nausea, abdominal pain, and diarrhea. There is no fever, and vomiting is rare. Spontaneous recovery usually occurs within 24 hours.

DIAGNOSIS

Diagnosis is based substantially on clinical observations. Bacteriologic studies are adjunctive. Clostridium perfringens is readily isolated in anaerobic cultures, which are routine for all wound cultures. It is common, for example, to isolate C perfringens from contaminated wounds of patients who have no evidence of clostridial disease. The organism can also be isolated from the postpartum uterine cervix of healthy women or from those with only mild fever. In clostridial food poisoning, isolation of high numbers of C perfringens in the ingested food in the absence of any other cause is usually sufficient to confirm an etiology of a characteristic food poisoning outbreak.

TREATMENT AND PREVENTION

Treatment of gas gangrene and endometritis must be initiated immediately because these conditions are almost always fatal if untreated. Excision of all devitalized tissue is of paramount importance because it denies the organism the anaerobic conditions required for further multiplication and toxin production. This often entails wide resection of muscle groups, hysterectomy, and even amputation of limbs. Administration of massive doses of penicillin is an important adjunctive procedure. Because nonclostridial anaerobes and members of Enterobacteriaceae frequently contaminate injury sites, broad-spectrum cephalosporins are often added to the antibiotic regimen. Placement of patients in a hyperbaric oxygen chamber, which increases the tissue level of dissolved oxygen, has been shown to slow the spread of disease, probably by inhibiting bacterial growth and toxin production and by neutralizing the activity of θ-toxin.

The most effective method of prevention of gas gangrene is the surgical debridement of traumatic injuries as soon as possible. Wound cleansing, removal of dead tissue and foreign bodies, and drainage of hematomas limit organism multiplication and toxin production.

Antimicrobial prophylaxis is indicated but cannot replace surgical debridement, because the antimicrobial agents may fail to reach the organism in devascularized tissues.

Prevention of food poisoning involves good cooking hygiene and adequate refrigeration. There is growing evidence that enterotoxin-producing strains of C perfringens may also be responsible for some cases of antimicrobial agent-induced diarrhea in a setting similar to that from C difficile (see following discussion).

BACTERIOLOGY

Clostridium botulinum is a large gram-positive rod much like the rest of the clostridia. Its spores resist boiling for long periods, and moist heat at 121°C is required for certain destruction. Germination of spores and growth of C botulinum can occur in a variety of alkaline or neutral foodstuffs when conditions are sufficiently anaerobic.

The major characteristic of medical importance is that when C botulinum grows under these anaerobic conditions, it elaborates a family of neurotoxins of extraordinary toxicity. Botulinum toxin is among the most potent toxins known in nature, with an estimated lethal dose of less than 1 μg for humans. Botulinum toxin is an enzyme (metalloproteinase) that acts at neuromuscular junctions (Figure 29–3). Once bound, it cleaves attachment protein receptors, which effectively block the release of the neurotransmitter acetylcholine from vesicles at the presynaptic membrane of the synapse. Because acetylcholine mediates activation of motor neurons the blockage of its release causes flaccid paralysis of the motor system.

Clostridium botulinum is classified into multiple types (A-G) based on the antigenic specificity of the neurotoxins. All the toxins are heat-labile and destroyed rapidly at 100°C, but are resistant to the enzymes of the gastrointestinal tract. If unheated toxin is ingested, it is readily absorbed and distributed in the bloodstream.

BOTULISM

EPIDEMIOLOGY

Spores of C botulinum are found in soil, pond, and lake sediments in all parts of the world. If spores contaminate food, they may convert to the vegetative state, multiply, and produce toxin in storage under certain conditions. This may occur with no change in food taste, color, or odor. The alkaline conditions provided by vegetables, such as green beans, and mushrooms and fish particularly support the growth of C botulinum. Botulism most often occurs after ingestion of home-canned products that have not been heated at temperatures sufficient to kill C botulinum spores, although inadequately sterilized commercial fish products have also been implicated. Because the toxin is heat-labile, in order to produce disease the food must be ingested uncooked or after insufficient cooking. Botulism often occurs in small family outbreaks in the case of home-prepared foods or less often as isolated cases connected to commercial products. Infant and wound botulism result when the toxin is produced endogenously, beginning with spores that are either ingested in difficult to sterilize foods (honey) or contaminate wounds.

PATHOGENESIS

Foodborne botulism is an intoxication not an infection. The ingested preformed toxin is absorbed in the intestinal tract and reaches its neuromuscular junction target via the bloodstream. Once bound there, its inhibition of acetylcholine release causes paralysis due to lack of neuromuscular transmission. The specific disease manifestations depend on the specific nerves to which the circulating toxin binds. Cardiac arrhythmias and blood pressure instability are believed to be due to effects of the toxin on the autonomic nervous system. The damage to the synapse once the toxin has bound is permanent, and recovery requires growth of presynaptic axons and formation of new synapses.

BOTULISM: CLINICAL ASPECTS

MANIFESTATIONS

Foodborne botulism usually starts 12 to 36 hours after ingestion of the toxin. The first signs are nausea, dry mouth, and, in some cases, diarrhea. Cranial nerve signs, including blurred vision, pupillary dilatation, and nystagmus, occur later. Symmetric paralysis begins with the ocular, laryngeal, and respiratory muscles and spreads to the trunk and extremities. The most serious finding is complete respiratory paralysis. Mortality is 10% to 20%.

Infant Botulism

A syndrome associated with C botulinum that occurs in infants between the ages of 3 weeks and 8 months is now the most commonly diagnosed form of botulism. The organism is apparently introduced on weaning or with dietary supplements, especially honey, which is virtually impossible to sterilize. Ingested spores yield vegetative bacteria, which multiply and produce small amounts of toxin in the infant’s colon. The infant shows constipation, poor muscle tone, lethargy, and feeding problems and may have ophthalmic and other paralyses similar to those in foodborne botulism. Infant botulism may mimic sudden infant death syndrome. The benefits of antitoxin and antimicrobial agents have not been clearly established.

Wound Botulism

Very rarely, wounds infected with other organisms may allow C botulinum to grow. Wound botulism in parenteral users of cocaine and maxillary sinus botulism in intranasal users of cocaine has been reported. Disease similar to that from food poisoning may develop, or it may begin with weakness localized to the injured extremity. Botulism without an obvious food or wound source is occasionally reported in individuals beyond infancy. It is possible that some such cases result from ingestion of spores of C botulinum with subsequent in vivo production of toxin in a manner similar to that in infant botulism.

DIAGNOSIS

The toxin can be demonstrated in blood, intestinal contents, or remaining food by immunoassay or nucleic acid amplification (NAA) methods but these tests are available only in reference laboratories. Clostridium botulinum may also be isolated from stool or from foodstuffs suspected of responsibility for botulism.

TREATMENT AND PREVENTION

The availability of intensive supportive measures, particularly mechanical ventilation, is the single most important determinant of clinical outcome. With proper ventilatory support, mortality rate should be less than 10%. The administration of large doses of horse C botulinum antitoxin is thought to be useful in neutralizing free toxin. Frequent hypersensitivity reactions related to the equine origin of this preparation make it unsuitable for use in infants. Antimicrobial agents are given only to patients with wound botulism.

Adequate pressure cooking or autoclaving in the canning process kills spores, and heating food at 100°C for 10 minutes before eating destroys the toxin. Food from damaged cans or those that present evidence of positive inside pressure should not even be tasted because of the extreme toxicity of the C botulinum toxin.

In an interesting twist, botulinum toxin as Botox has itself become a therapeutic agent. Originally licensed as a treatment of spasmotic neuromuscular conditions by direct injection into muscle, it has found a far larger use for cosmetic applications. For those that can afford it, a temporary respite from the wrinkles of aging can be gained from Botox injections administered by dermatologists and plastic surgeons.

BACTERIOLOGY

Clostridium tetani is a slim, gram-positive rod, which forms spores readily in nature and in culture, yielding a round terminal spore that gives the organism a drumstick-like appearance (Figure 29–1). C tetani requires strict anaerobic conditions. Its identity is suggested by cultural and biochemical characteristics, but definite identification depends on demonstrating the neurotoxic exotoxin. C tetani spores remain viable in soil for many years and are resistant to most disinfectants and to boiling for several minutes.

The most important product of C tetani is its neurotoxic exotoxin, tetanospasmin or tetanus toxin, a metalloproteinase that has structural and pharmacologic features similar to those of botulinum toxin. Tetanus toxin degrades a protein required for neurotransmitter release from vesicles at the appropriate site on presynaptic membranes (Figure 29–3). The most important difference from botulinum toxin is that the neurotransmitters in this case (glycine and γ-aminobutyric acid [GABA]) are the ones that affect inhibitory neurons. The result is unopposed firing of the active motor neurons, generating spasms and spastic paralysis, which are the opposite of the botulinum flaccid paralysis. The toxin is heat-labile, antigenic, readily neutralized by antitoxin, and rapidly destroyed by intestinal proteases. Treatment with formaldehyde yields a nontoxic product or toxoid that retains the antigenicity of toxin and thus stimulates the production of antitoxin.

TETANUS

EPIDEMIOLOGY

The spores of C tetani exist in many soils, especially those that have been treated with manure, and the organism is sometimes found in the lower intestinal tract of humans and animals. The spores are introduced into wounds contaminated with soil or foreign bodies. The wounds are often small (eg, a puncture wound with a splinter). In many developing countries, the majority of tetanus cases occur in recently delivered infants when the umbilical cord is severed or bandaged in a nonsterile manner. Similarly, tetanus may follow an unskilled abortion, scarification rituals, female circumcision, and even surgery performed with nonsterile instruments or dressings.

PATHOGENESIS

The usual predisposing factor for tetanus is an area of very low oxidation–reduction potential in which tetanus spores can germinate, such as a large splinter, an area of necrosis from introduction of soil, or necrosis after injection of contaminated illicit drugs. Infection with facultative or other anaerobic organisms can contribute to the development of an appropriate anaerobic nidus for spore germination. Tetanus bacilli multiply locally and neither damage nor invade adjacent tissues. Tetanospasmin is elaborated at the site of infection and enters the presynaptic terminals of lower motor neurons, reaching the central nervous system (CNS) mainly by exploiting the retrograde axonal transport system in the nerves. In the spinal cord, it acts at the level of the anterior horn cells, where its blockage of postsynaptic inhibition of spinal motor reflexes produces spasmodic contractions of both protagonist and antagonist muscles. This process takes place initially in the area of the causative lesion, but may extend up and down the spinal cord. Minor stimuli, such as a sound or a draft, can provoke generalized spasms.

TETANUS: CLINICAL ASPECTS

MANIFESTATIONS

The incubation period of tetanus is from 4 days to several weeks. The shorter incubation period is usually associated with wounds in areas supplied by the cranial motor nerves, probably because of a shorter transmission route for the toxin to the CNS. In general, shorter incubation periods are associated with more severe disease.

The diagnosis is clinical; neither culture nor toxin testing is useful. Although tetanus may be localized to muscles innervated by nerves in the region of the infection, it is usually more generalized. The masseter muscles are often the first to be affected, resulting in inability to open the mouth properly (trismus); this effect accounts for the term lock-jaw. As other muscles become affected, intermittent spasms can become generalized to include muscles of respiration and swallowing. In extreme cases, massive contractions of the back muscles (opisthotonos) develop (Figure 29–4).

Untreated patients with tetanus retain consciousness and are aware of their plight, in which small stimuli can trigger massive contractions. In fatal cases, death results from exhaustion and respiratory failure. Untreated, the mortality rate caused by the generalized disease varies from 15% to more than 60%, according to the lesion, incubation period, and age of the patient. Mortality is highest in neonates and in elderly patients.

TREATMENT

Specific treatment of tetanus involves neutralization of any unbound toxin with large doses of human tetanus immune globulin (HTIG), which is derived from the blood of volunteers hyperimmunized with toxoid. Most important in treatment are nonspecific supportive measures, including maintenance of a quiet dark environment, sedation, and provision of an adequate airway. Benzodiazepines are also used to indirectly antagonize the effects of the toxin. The value of antimicrobials is not clear. Because toxin binding is irreversible, recovery requires the generation of new axonal terminals.

PREVENTION

Routine active immunization with tetanus toxoid, combined with diphtheria toxoid and pertussis vaccine (DTaP) for primary immunization in childhood and DT for adults, can completely prevent tetanus. It has reduced the incidence of tetanus in the United States to less than 50 reported cases per year. Five doses of DT are recommended, to be given at the ages of 2, 4, 6, and 18 months, and once again between the ages of 4 and 6 years. Thereafter, a booster of adult-type tetanus diphtheria toxoid should be given every 10 years. Unfortunately, routine childhood immunization is not administratively and economically feasible in many less well-developed countries, where as many as 1 million cases of tetanus occur annually. In such settings, immunization efforts have been focused on pregnant women, because transplacental transfer of antibodies to the fetus also prevents the highly lethal neonatal tetanus.

Unimmunized subjects with tetanus-prone wounds should be given passive immunity with a prophylactic dose of HTIG as soon as possible. This immunization provides immediate protection. Those who have had a full primary series of immunizations and appropriate boosters are given toxoid for tetanus-prone wounds if they have not been immunized within the previous 10 years in the case of clean minor wounds or 5 years for more contaminated wounds. If immunization is incomplete or the wound has been neglected and poses a serious risk of disease, HTIG is also appropriate. Penicillin therapy is a prophylactic adjunct in serious or neglected wounds, but in no way alters the need for specific prophylaxis.

BACTERIOLOGY

Clostridium difficile is a gram-positive rod that readily forms spores both in the environment and in vivo. Under circumstances described as follows, spores present in the intestinal microbiota may germinate to the metabolically active vegetative form. The C difficile germination mechanism differs from that of most other spore-forming bacteria in that it is triggered by bile salts. In the vegetative form C difficile has a most important medical feature: its ability to produce toxins. In this species, two distinct large polypeptide toxins, toxin A (TcdA) and toxin B (TcdB), with similar structure (45% homology) are released during late growth phases, perhaps at the time of cell lysis. Both toxins act in the cytoplasm by disrupting signal transduction proteins (G proteins), particularly those involving the actin cytoskeleton. This results in the disruption of intercellular tight junctions followed by altered membrane permeability and fluid secretion. Within hours of contact with enterocytes cell rounding and neutrophilic infiltration also appear. In recent years a third toxin, C difficile binary toxin (CDT), has been discovered which exerts an ADP-ribosylating action which inhibits actin cytoskeleton polymerization within the enterocyte.

CLOSTRIDIUM DIFFICILE INFECTION (CDI)

EPIDEMIOLOGY

Clostridium difficile is present in the stool of 2% to 15% of the general population, sometimes at higher rates among hospitalized persons and infants. Infants remain asymptomatic possibly due to lack of toxin-binding receptors. More than two decades of the antibiotic era had elapsed before the medical importance of C difficile was recognized through its association with antibiotic-associated diarrhea (AAD). Although CDI is endogenous in most cases, hospital outbreaks have clearly established that the environment can be the source as well. CDI is clearly on the rise worldwide and is now the leading cause of death due to an acute diarrheal illness. New strains combining more potent A and B toxin delivery with the CDT toxin have been particularly virulent.

Clostridium difficile is not the only cause of AAD, but it is the most common identifiable cause. In simple diarrhea following antimicrobial administration, this organism is responsible for approximately 30% of cases. As the disease is colitis, the association is stronger, rising to 90% if PMC is present.

Although CDI is primarily an endogenous infection, the generation of spores from excretions provides the prospect for person-to-person spread. This is the basis of the hospital outbreaks but can occur in any situation where C difficile spores lurk in a closed space.

PATHOGENESIS

When C difficile becomes established in the colon of individuals with normal gut microbiota, few, if any, direct consequences result, probably because its numbers are dwarfed by the other flora. Alteration of the colonic flora with antimicrobials (particularly ampicillin, cephalosporins, and clindamycin) favors C difficile in two ways. First, strains resistant to the antimicrobial agent can grow in its presence and assume a larger if not dominant position in the flora. Second, in an antimicrobial milieu, the readiness with which C difficile forms spores may favor its survival over non–spore-forming bacteria. A distinctive feature of C difficile spores is that their germination is triggered by taurocholate, a bile salt through a receptor in the spore itself. Thus, in the situation of general suppression of intestinal flora by antimicrobial agents C difficile has a double advantage. Its spores are specifically triggered to germinate by normal intestinal secretions, and the resultant vegetative cells have less competition for nutrients. Eventually, the minor niche of the species is improved to the point where the effect of its toxins on the colonic mucosa becomes significant.

Although most strains produce both toxins, the relative contribution of TcdA and TcdB has been much debated. The toxins have similar actions and it seems both are important. The recent hypervirulent clones produce higher levels of both toxins and also secrete the new CDT. This deadly combination has been responsible for more cases and more deaths. In PMC, the colonic mucosa is studded with inflammatory plaques, which may coalesce into an overlying “pseudomembrane” composed of fibrin, leukocytes, and necrotic colonic cells (Figure 29–5).

IMMUNITY

Antibody against the TcdA and TcdB have been associated with resolution of disease in experimental animals. This is a long way from concluding that humoral antitoxin immunity is protective when we know serial relapses with toxin production are common.

CLOSTRIDIUM DIFFICILE DIARRHEA: CLINICAL ASPECTS

MANIFESTATIONS

Diarrhea is a common side effect of antimicrobial treatment. In C difficile-caused diarrhea, the onset is usually 5 to 10 days into the antibiotic treatment, but the range is from the first day to weeks after cessation. The diarrhea may be mild and watery or bloody and accompanied by abdominal cramping, leukocytosis, and fever. In PMC, it progresses to a severe, occasionally lethal inflammation of the colon that can be demonstrated by endoscopic examination. Systemic signs of inflammation are common and WBC counts more than 15 000 per cubic millimeter considered ominous. Toxic megacolon is the most serious complication leading to colectomy or death.

DIAGNOSIS

Although selective media have been developed for isolation of C difficile, direct detection of toxins in the stool has largely replaced culture for diagnostic purposes. Clostridium difficile is the only pathogen for which detection of its toxin has become routine. The original cell culture toxin assays were replaced by immunoassays, which demonstrate toxin TcdA and/or TcdB in stool. These tests are now being superseded by NAA methods with improved sensitivity and specificity.

TREATMENT

In AAD discontinuing the implicated antimicrobial often results in the resolution of clinical symptoms. Once C difficile toxins are detected in the stools, treatment with metronidazole, vancomycin, or fidaxomicin is indicated. Vancomycin is not absorbed orally which is an advantage in this situation because the toxin production is taking place in the bowel lumen. Metronidazole is only used for mild to moderate CDI. Clostridium difficile is susceptible to the penicillins and cephalosporins in vitro, but these drugs are ineffective because of access in the intestinal lumen and the hazard of destruction by β-lactamases produced by other bacteria. With all antimicrobial regimens relapses are common and often multiple (more than 20) presumably due to the survival of the inert spores following a treatment course. The relapse rate appears to be less with fidaxomicin, a newer drug. Other treatment strategies under investigation include probiotics, toxin-specific antibodies, and molecules that bind the toxin(s) or its intestinal toxin receptor. These approaches are usually combined with antimicrobial therapy. The emergence of PMC with toxic megacolon requires a high-risk colectomy.

PREVENTION

Strategies to prevent recurrences of CDI have generated some highly creative approaches. In pulsed-treatment, a single dose of vancomycin is given once every few days rather than multiple times a day as in standard treatment. The idea is to allow time for the vegetative bacteria to emerge from the inert spore and then block their cell wall synthesis as they start to multiply. After multiple “hits,” this approach has ended long sequences of relapses. The most recent and sensational approach has been the infusion of donor feces into the intestine in an effort to reestablish an effective competitive flora. This “fecal transplant” has now moved from anecdotal relapse cures to greater than 90% success in controlled trials including the use of standardized preparations in capsules. The elements of the microbiota included in these capsules is a matter of great debate. Finally, another strategy is aimed at preventing germination of C difficile spores by administration of competitive inhibitors of the bile salts known to trigger germination. If successful, this could be applied to any situation where CDI was a risk.

BACTERIOLOGY

The B fragilis group constitutes the most common opportunistic pathogens of the genus Bacteroides. These slim, pale-staining, capsulate, gram-negative rods form colonies overnight on blood agar medium incubated anaerobically. The implication of fragility in the name is misleading, because they are actually among the hardier and more easily grown anaerobes. Most strains produce superoxide dismutase and are relatively tolerant to atmospheric oxygen. Bacteroides fragilis has adhesive surface pili and a capsule composed of a polymer of two polysaccharides. The LPS endotoxin in the B fragilis outer membrane is less toxic than that of most other gram-negative bacteria, possibly owing to modification or absence of the lipid A portion.

BACTEROIDES FRAGILIS DISEASE

EPIDEMIOLOGY

Like the other gram-negative anaerobes, B fragilis infections are endogenous, originating in the patient’s own intestinal microbiota. Given the mass and diversity of intestinal anaerobes, the frequent presence of B fragilis in clinically significant infections is striking. It is typically mixed with other anaerobes and facultative bacteria. Human-to-human transmission is not known and seems unlikely.

PATHOGENESIS

The relative oxygen tolerance of B fragilis probably plays a role in its virulence by aiding its survival in oxygenated tissues in the period between its displacement from the intestinal flora and the establishment of a reduced local microenvironment. Bacteroides fragilis cells can withstand up to 3 days exposure to atmospheric levels of oxygen due to activation of an oxidative stress response which deploys detoxifying enzymes like catalase and superoxide dismutase.

The polysaccharide capsule confers resistance to phagocytosis, inhibits macrophage migration, and mediates binding to the peritoneum. The capsule is also involved in the most distinguishing pathogenic feature of B fragilis, its ability to cause abscess formation. Experimentally, the B fragilis capsular polysaccharide stimulates abscess formation, even in the absence of live cells, a property is not found in the capsules of bacteria like Streptococcus pneumonia, or Neisseria meningitidis. Within the bowel B fragilis polysaccharides have immunomodulatory effects which may influence the presence and course of inflammatory bowel disease. That the same polysaccharides cause abscesses outside their usual habitat may involve their triggering of Toll-like receptors. Bacteroides fragilis and other Bacteroides species produce a number of extracellular enzymes (collagenase, fibrinolysin, heparinase, hyaluronidase) that may also contribute to the formation of the abscess.

Some strains of B fragilis produce an enterotoxin that causes enteric disease in animals, and in some studies they have been associated with a self-limited, watery diarrhea in children. Because these enterotoxin-producing strains are found in up to 10% of healthy individuals, their pathogenic importance is still undetermined.

IMMUNITY

Although it has been demonstrated that antibody to capsular polysaccharide facilitates classical complement pathway killing, there is no evidence that this confers immunity to reinfection. In contrast, there is some evidence that cell-mediated immunity may be protective.

BACTEROIDES FRAGILIS: CLINICAL ASPECTS

MANIFESTATIONS

Some event that displaces B fragilis along with other members of the intestinal flora is required to initiate infection; there is no evidence the organism is invasive on its own. This mucosal break may be the result of trauma or other disease states such as diverticulitis.

The local effects of the developing abscess include abdominal pain and tenderness, often with a low-grade fever. The subsequent course depends on whether the abscess remains localized or ruptures through to other sites such as the peritoneal cavity. This may cause several other abscesses or peritonitis. The course of illness is strongly influenced by the other bacteria in the abscess, particularly members of the Enterobacteriaceae. Spread to the bloodstream is more common with B fragilis than any other anaerobe.

TREATMENT

Drainage of abscesses and debridement of necrotic tissue are the mainstays of the treatment of B fragilis infections, as with anaerobic infections in general. The accompanying antimicrobial therapy is complicated by the fact that abdominal B fragilis isolates almost always produce a β-lactamase, which not only inactivates penicillin but other β-lactams, including many cephalosporins. Resistance to tetracycline is also common, but most strains are susceptible to clindamycin, and metronidazole. Among the β-lactams, aztreonam, imipenem, and cefotaxime have been used effectively, as have combinations of a β-lactamase inhibitor (cilastatin, tazobactam) and a β-lactam (imipenem, piperacillin).