Clostridia: Sporeforming Anaerobic Bacilli (2025)

Gas Gangrene and Related Clostridial Wound Infections

Tetanus and Clostridium Tetani

Botulism and Clostridium Botulinum

Antibiotic-Associated Diarrhea, Pseudomembranous Colitis, andClostridium Difficile

Other Pathogenic Clostridia

Clostridium perfringens causes food poisoning and necrotizingenteritis. C sordellii causes bacteremia, endometritis andnonbacteremic infections. C septicum is correlated with thepresence of cancer. C tertium is associated withbacteremia.

Clinical Manifestations

Clostridial wound infections may be divided into three categories: gas gangreneor clostridial myonecrosis, anaerobic cellulitis, and superficial contamination.Gas gangrene can have a rapidly fatal outcome and requires prompt, often severe,treatment. The more common clostridial wound infections are much less acute andrequire much less radical treatment; however, they may share somecharacteristics with gas gangrene and must be included in the differentialdiagnosis.

Gas gangrene is an acute disease with a poor prognosis and often fatal outcome(Fig. 18-1). Initial trauma to hosttissue damages muscle and impairs blood supply. This lack of oxygenation causesthe oxidation-reduction potential to decrease and allows the growth of anaerobicclostridia. Initial symptoms are generalized fever and pain in the infectedtissue. As the clostridia multiply, various exotoxins (including hemolysins,collagenases, proteases, and lipases) are liberated into the surrounding tissue,causing more local tissue necrosis and systemic toxemia. Infected muscle isdiscolored (purple mottling) and edematous and produces a foul-smelling exudate;gas bubbles form from the products of anaerobic fermentation. As capillarypermeability increases, the accumulation of fluid increases, and venous returneventually is curtailed. As more tissue becomes involved, the clostridiamultiply within the increasing area of dead tissue, releasing more toxins intothe local tissue and the systemic circulation. Because ischemia plays asignificant role in the pathogenesis of gas gangrene, the muscle groups mostfrequently involved are those in the extremities served by one or two majorblood vessels.

Clostridial septicemia, although rare, may occur in the late stages of thedisease. Severe shock with massive hemolysis and renal failure is usually theultimate cause of death. The incubation period, from the time of wounding untilthe establishing of gas gangrene, varies with the infecting clostridial speciesfrom 1 to 6 days, but it may be as long as 6 weeks. Average incubation times forthe three most prevalent infecting organisms are as follows: Cperfringens, 10–48 hours; C septicum,2–3 days; and C novyi, 5–6 days. Becausethe organisms need time to establish a nidus of infection, the time lag betweenwounding and the appropriate medical treatment is a significant factor in theinitiation of gas gangrene.

Like gas gangrene, clostridial cellulitis is an infection of muscle tissue, buthere the infecting organisms invade only tissue that is already dead; theinfection does not spread to healthy, undamaged tissue. Clostridial cellulitishas a more gradual onset than gas gangrene and does not include the systemictoxemia associated with gas gangrene. Pain is minimal, and although only deadtissue is infected, the disease can spread along the planes between musclegroups, causing the surrounding tissue to appear more affected than it actuallyis. Anaerobic cellulitis may cause formation of many gas bubbles, producinginfected tissue that looks similar to the gaseous tissue of gas gangrene. Sometissue necrosis does occur, but it is caused by decreased blood supply and notinvasion by the infecting organism. With adequate treatment, anaerobiccellulitis has a good prognosis.

Superficial contamination, the least serious of the clostridial wound infections,involves infection of only necrotic tissue. Usually, the patient experienceslittle pain, and the process of wound healing proceeds normally; however,occasionally an exudate may form and the infection may interfere with woundhealing. Superficial wound contamination caused by clostridia usually involvesC perfringens, with staphylococci or streptococci, or both,as frequent co-isolates.

Structure

The clostridia that cause gas gangrene are anaerobic, spore-forming bacilli, butsome species may not readily sporulate, e.g., Cperfringens.

Classification and Antigenic Types

Clostridial wound infections usually are polymicrobic because the source of woundcontamination (feces, soil) is polymicrobic. In gas gangrene and anaerobiccellulitis, the primary pathogen can be any one of various clostridial speciesincluding C perfringens (80%), C novyi (40%),C septicum (20%), and, occasionally, Cbifermentans, C histolyticum, or C fallax. Otherbacterial isolates may be any of a wide number and variety of organisms (forexample, Proteus, Bacillus, Escherichia, Bacteroides,Staphylococcus). The distinctive or unique properties of thecausative agents of gas gangrene are difficult to list; morphologiccharacteristics and biochemical reactions vary among these species, and areliable laboratory manual should be consulted for their proper identification.Isolation of 10⁷ or more clostridia per milliliter of wound exudateis strong evidence for a clostridial wound infection.

The most frequently isolated pathogen, C perfringens, has fivetypes, designated A, B, C, D, and E. Each of these types produces a semi-uniquespectrum of protein toxins. Alpha-toxin (a lecithinase, also calledphospholipase-C) and theta-toxin (oxygen-labile cytolysin) are both consideredimportant in the disease pathology. Alpha-toxin is lethal and necrotizing; itlyses cell membrane lecithins, disrupting cell membranes and causing cell death.Theta-toxin also contributes to rapid tissue destruction by several mechanisms.At the site of infection, theta-toxin acts as a cytolysin, promoting directvascular injury; lower toxin concentrations activate polymorphonuclearleukocytes and endothelial cells, promoting distal vascular injury bystimulating leukocyte adherence to the endothelium. The result is leukostasis,thrombosis, decreased perfusion, and tissue hypoxia. Theta-toxin also mediatesthe production of shock through induction of inflammatory mediators such asplatelet activating factor, tumor necrosis factor, interleukin 1 and interleukin6.

Pathogenesis

All clostridial wound infections occur in an anaerobic tissue environment causedby an impaired blood supply secondary to trauma, surgery, foreign bodies, ormalignancy. Contamination of the wound by clostridia from the externalenvironment or from the host's normal flora produces the infection. The detailedpathogenesis of the disease is intimately associated with the clinicalpresentation as described above (Fig.18-1).

Host Defenses

Host defenses against gas gangrene and other clostridial wound infections aremostly ineffective. Even repeated episodes of clostridial wound infection do notseem to produce effective immunity.

Epidemiology

Clostridial spores are ubiquitous in the soil, on human skin, and in thegastrointestinal tracts of humans and animals. Thus, the causative agents ofclostridial wound infections are not environmentally restricted. Even operatingtheaters can be habitats for infecting clostridial organisms and spores. Theincidence of clostridial wound infections has declined with the advance ofprompt, adequate medical treatment. Historically, war casualties have had thegreatest incidence of gas gangrene; however, the prompt evacuation and medicalattention given United States casualties in the Vietnam war greatly decreasedthe incidence of gas gangrene in these soldiers, emphasizing the importance ofprompt medical treatment.

Diagnosis

Diagnosis of clostridial wound infections is based on clinical symptoms coupledwith Gram stains and bacterial culture of clinical specimens. Gas gangrene, onceinitiated, may spread and cause death within hours. By the time the typicallesions of gas gangrene are evident, the disease usually is firmly establishedand the physician must treat the patient on a clinical basis without waiting forlaboratory confirmation. Characteristic lesions and the presence of largenumbers of Gram-positive bacilli (with or without spores) in a wound exudateprovide strong presumptive evidence. In contrast to tissue infections caused byStaphylococcus aureus, there is typically an absence ofpolymorphonuclear leukocytes at the site of infection, likely due to thepresence of clostridial toxins. Spores are rare in cultures of Cperfringens, the most common etiologic agent of these diseases. Acommonly used laboratory test for presumptive identification of Cperfringens is the Nagler reaction which detects the presence ofalpha-toxin (phospholipase-C), one of the most prominent toxins produced byC perfringens. However, several other species of clostridiaalso have a positive Nagler reaction, and thus this test is not entirelyspecific for C perfringens.

Discussion of the differential diagnosis of clostridial wound infectionsappropriately includes streptococcal myositis, as this disease can becharacterized by an edematous, necrotizing, often gaseous lesion. Like anaerobiccellulitis and superficial contamination with clostridia, streptococcal myositisis a relatively localized disease, but its later stages may include somesystemic toxicity that mimics the toxemia of gas gangrene.

Control

Correction of the anaerobic conditions combined with antibiotic treatment formthe basis for therapy. Penicillin is the drug of choice for all clostridialwound infections; chloramphenicol is a second-choice antibiotic. Successfultreatment of the less severe forms of clostridial wound infections includeslocal debridement and antibiotic therapy; after these measures are taken,patient recovery usually proceeds along a steady, positive course. Treatment ofgas gangrene includes radical surgical debridement coupled with high doses ofantibiotics. Blood transfusions and supportive therapy for shock and renalfailure also may be indicated.

The usefulness of gas gangrene antitoxin is currently a disputed matter. Somephysicians maintain that the efficacy of this polyvalent antitoxin has beenproved in the past, but better medical care now may have eliminated the need forits use. Others believe that because of insufficient data, antitoxin should beadministered systemically as early as possible after diagnosis, and that theantitoxin should be injected locally into tissue that cannot be excised.

Obviously, prevention of wound contamination is the single most important factorin controlling clostridial wound infections. In the past, immunization has beenconsidered a possible preventive measure for gas gangrene; however, severalfactors have discouraged the use of active immunization, including difficulty inpreparing a suitable antigenic toxoid, availability of prompt wound treatment,and accessibility of effective therapeutic agents.

Clinical Manifestations

Tetanus is a severe disease caused by the toxin of C tetani(Fig. 18-2). This organism grows ina wound and secretes a toxin that invades systemically and causes muscle spasms.The initial symptom is cramping and twitching of muscles around a wound. Thepatient usually has no fever but sweats profusely and begins to experience pain,especially in the area of the wound and around the neck and jaw muscles(trismus). Portions of the body may become extremely rigid, and opisthotonos (aspasm in which the head and heels are bent backward and the body bowed forward)is common. Complications include fractures, bowel impaction, intramuscularhematoma, muscle ruptures, and pulmonary, renal, and cardiac problems.

Structure and Classification

C tetani is an anaerobic gram-positive rod that forms terminalspores, giving it a characteristic tennis racquet appearance. Some strains donot sporulate readily, and spores may not appear until the third or fourth dayof culture. Most strains are motile with peritrichous flagella; colonies oftenswarm on agar plates, but some strains are nonflagellated and nonmotile. Thepresence of C tetani should be suspected on isolation of aswarming rod that produces indole and has terminal spherical spores, but doesnot produce acid from glucose. Toxigenic C tetani contains aplasmid that produces a toxin called tetanospasmin, but nontoxigenic strainsalso exist. Tetanospasmin is responsible for the infamous toxemia calledtetanus. The two animal species most susceptible to this toxemia are horses andhumans.

Pathogenesis

As with all clostridial wound infections, the initial event in tetanus is traumato host tissue, followed by accidental contamination of the wound with Ctetani (Fig. 18-2). Tissuedamage is needed to lower the oxidation-reduction potential and provide anenvironment suitable for anaerobic growth. Once growth is initiated, theorganism itself is not invasive and remains confined to the necrotic tissue,where the vegetative cells of C tetani elaborate the lethaltoxin. The incubation period from the time of wounding to the appearance ofsymptoms varies from a few days to several weeks, depending on the infectiousdose and the site of the wound (the more peripheral the wound, the longer theincubation time).

Tetanus can be initiated in two different ways, resulting in either generalizedor local tetanus. In generalized tetanus (also called descending tetanus), allof the toxin cannot be absorbed by local nerve endings; therefore, it passesinto the blood and lymph with subsequent absorption by motor nerves. The mostsusceptible centers are the head and neck; the first symptom is usually trismus(lockjaw), with muscle spasms descending from the neck to the trunk and limbs.As the disease progresses, the spasms increase in severity, becoming verypainful and exhausting. During spasms, the upper airway can become obstructed,resulting in respiratory failure. Spasms often are initiated by environmentalstimuli that may be as insignificant as the flash of a light or the sound of afootstep. In the localized form of tetanus (also called ascending tetanus),toxins travel along the neural route (peripheral nerves), causing a diseaseconfined to the extremities and seen most often in inadequately immunizedpersons. Localized tetanus may last for months but usually resolvesspontaneously. Another unusual form of tetanus is called cephalic tetanus whichresults from head wounds and affects the face, most commonly the musclesinnervated by lower cranial nerves. Curiously, cephalic tetanus can occur infully immunized persons; the outcome is typically poor, but mild cases (oftenassociated with otitis media) have more favorable outcomes. Neonatal tetanus isseen in newborns when the mother lacks immunity and the umbilical stump becomescontaminated with C tetani spores.

C tetani actually produces two toxins: tetanolysin, a hemolysinthat is inactivated by cholesterol and has no role in pathogenesis, andtetanospasmin, a spasmogenic toxin responsible for the classical symptoms of thedisease.

The actions of tetanospasmin are complex and involve three components of thenervous system: central motor control, autonomic function, and the neuromuscularjunction. Toxin enters the nervous system primarily through the neuromuscularjunction of alpha motor neurons. The toxin is then transported to the otherneurons, most importantly presynaptic inhibitory cells, where it is no longeraccessible to be neutralized by antitoxin. (This retrograde transport to thecentral nervous system is similar to that utilized by some viruses, such asherpes virus and rabies.) The toxin also spreads hematogenously, but it stillmust enter the central nervous system via retrograde transport from peripheralneuronal processes. Once the toxin gains access to inhibitory neurons, it blocksthe release of the neurotransmitters glycine and gamma-aminobutyric acid. Theabsence of this inhibition permits the simultaneous spasms of both agonist andantagonist muscles, producing muscle rigidity and convulsions. Tetanospasminalso acts on the autonomic nervous system and is associated with elevated plasmacatacholamine levels; respiratory failure is a frequent complication of thedisease. Peripherally, there is a failure of transmission at the neuromuscularjunction, involving defective release of acetylcholine in a manner similar tothat seen with C botulinum. Tetanospasmin may be as potent asthe toxin of C botulinum; as little as 130 µgconstitutes a lethal dose for humans. In untreated tetanus, the fatality rate is90% for the newborn and 40% for adults. However, with aggressive hospital care,these fatality rates can be substantially reduced. The ultimate cause of deathis usually pulmonary or cardiac failure.

Host Defenses

Although there are scattered reports that tetanus antibodies can be acquired bynatural, presumably enteric, infection with C tetani, innateimmunity to tetanus toxin does not typically exist. In addition, one or moreepisodes of tetanus do not produce immunity to future attacks. The reason forthe lack of immune response may be twofold: the toxin is potent, and the amountreleased may be too small to trigger immune mechanisms but still be enough tocause symptoms and, because the toxin binds firmly to neural tissue, it may notinteract effectively with the immune system.

Epidemiology

C tetani can be isolated from the soil in almost everyenvironment throughout the world. The organism can be found in thegastrointestinal flora of humans, horses, and other animals. Isolation ofC tetani from the intestinal flora of horses, coupled withthe high frequency of equine tetanus, led to the erroneous assumption that thehorse was the animal reservoir of C tetani.

Generalized outbreaks of tetanus do not occur, but certain populations can beconsidered at risk. Historically, wounded soldiers have had a high incidence oftetanus, but this phenomenon declined with widespread use of immunizations.Umbilical tetanus (tetanus neonatorum) usually is a generalized, fulminating,fatal disease that occurs with the neonates of unimmunized mothers who havegiven birth under unsanitary conditions. In the United States, intravenous drugabusers have become another population with an increasing incidence of clinicaltetanus. One million cases of tetanus occur annually in the world. Tetanus israre in most developed countries. The United States has about one case permillion per year, most often seen in the elderly with declining immune statusdue to failure to receive timely tetanus booster vaccinations. In some lessdeveloped countries, tetanus is still one of the ten leading causes of death,and neonatal tetanus accounts for approximately one-half of the cases worldwide.In less developed countries, approximate mortality rates remain 85% for neonataltetanus and 50% for nonneonatal tetanus. This is an unfortunate situationbecause with adequate immunization, tetanus is a completely preventabledisease.

Diagnosis

Diagnosis of tetanus is obvious in advanced cases; however, successful treatmentdepends on early diagnosis before a lethal amount of toxin becomes fixed toneural tissue. The patient should be treated on a clinical basis without waitingfor laboratory data. C tetani can be recovered from the woundin only about one-third of the cases, and a wound is not even evident in10–20% of cases. It is important for the clinician to be aware thattoxigenic strains of C tetani can grow actively in the wound ofan immunized person, but the presence of antitoxin antibodies preventsinitiation of tetanus. Also, because tetanus is commonly found in the soil, themere presence of tetanus in a wound does not imply that the organism is activelyreplicating and secreting toxin.

Numerous syndromes, including rabies and meningitis, have symptoms similar tothose of tetanus and must be considered in the differential diagnosis. Ingestionof strychnine (found in rat poison) can cause symptoms that closely resemblethose of generalized tetanus. Trismus can occur in encephalitis, phenothiazinereactions, and diseases involving the jaw.

Control

Injections of tetanus toxoid are prophylactic. Currently, booster doses arerecommended only every 10 years by the CDC. More frequent boosters areunnecessary and may cause local reactions resembling the Arthus phenomenon or adelayed hypersensitivity reaction. It has been noted that, because of theirimmunodeficiency state, AIDS patients may not respond to prophylactic injectionsof tetanus toxoid. An antibody titer above 0.01 international units (IU) per mlis usually considered protective. Human tetanus immunoglobulin (HTIG) in a doseof 250 IU intramuscularly should be considered for those with questionableimmune status.

Treatment of diagnosed tetanus has a number of aspects. The offending organismmust be removed by local debridement, after the patient's spasms are controlledby benzodiazepines. Penicillin or metronidazole is usually administered to killthe bacteria, but may not be a necessary adjunct in therapy. Although penicillinhas been historically considered to be the drug of choice, it has beenspeculated that penicillin could have an adverse effect by actingsynergistically with tetanospasmin. Metronidazole is currently recommended, andthere is some evidence that it is associated with an improved prognosis. HTIG isinjected intramuscularly: dosage recommendations vary from 500 IU in a singleintramuscular injection to 3000–6000 IU injected intramuscularly inseveral sites. Supportive measures, such as respiratory assistance andintravenous fluids, are often critical to patient survival. Recommendedtreatment includes benzodiazepines, such as diazepam (Valium). Analgesics thatwill not cause respiratory depression should be used, and include codeine,meperidine (Demerol), and morphine. Adequate nutritional support should beprovided and should consider that the patient's nutritional needs areextraordinarily great.

In cases of clean, minor wounds, tetanus toxoid should be administered only ifthe patient has not had a booster dose within the past 10 years. For moreserious wounds, toxoid should be administered if the patient has not had abooster dose within the past 5 years. All patients who have a reasonablepotential for contracting tetanus should receive injections of tetanusimmunoglobulin, including those recovering from diagnosed cases of tetanus.

Clinical Manifestations

Botulism is a disease caused by the toxin of a diverse group of clostridia calledC botulinum. This neurotoxin characteristically causes asymmetrical, descending paralysis (Fig.18-3). The symptoms of botulism can occur in both the nervous systemand the alimentary tract of the patient. Therefore, many diseases enter into thedifferential diagnosis, including pharyngitis, gastroenteritis, sepsis,intestinal obstruction, myasthenia gravis, encephalitis, muscular dystrophy,electrolyte imbalance, meningitis, poliomyelitis, cerebrovascular accident,Guillain-Barré syndrome, chemical food poisoning, tick paralysis,Reye syndrome, hypothyroidism, heavy metal ingestion, carbon monoxide poisoning,and snake bite. For infant botulism, additional syndromes enter into thedifferential diagnosis: failure to thrive, acute infantile polyneuropathy,dehydration, and various hereditary and metabolic disorders. Infant botulismoften is missed by physicians, but it always should be considered if any of thetypical symptoms are present.

Structure, classification and antigenic Types

Unlike most species of bacteria, which comprise strains that have a close geneticrelationship and similar cultural characteristics, the Cbotulinum “species” consists of severaldistinct groups of organisms that have a common name solely because they producesimilar toxins. The name C botulinum is thus only a conveniencethat reflects the medical importance of the species. A strain of Cbotulinum usually produces only one of seven toxin types,designated A, B, C, D, E, F, and G. The toxins produced by Cbotulinum types C and D have been shown to be coded by the geneticmaterial contained in bacteriophages that infect the bacteria. Cbotulinum types C and D can be interconverted by use of specificbacteriophage. Types C and D even can be transformed into Cnovyi by curing bacteria of the botulinum phage, and substituting aphage that codes for a C novyi toxin. To add to the confusion,a few strains of C baratii and C butyricumhave been reported to secrete botulinum toxin. In addition, certain proteolyticstrains of C botulinum are indistinguishable from Csporogenes except by toxin assay. Thus, the production of apharmacologically similar neurotoxin is the single distinctive property of aclostridium that places it in the botulinum “species.” Allthe organisms that produce this toxin are anaerobic rods, Gram-positive, and aremotile by peritrichous flagella. Oval, subterminal spores are produced inextremely variable numbers, depending on the particular isolate and on theculture medium. Cultural reactions vary greatly, and the species includes highlyproteolytic and nonproteolytic strains as well as saccharolytic andnonsaccharolytic strains.

Of the seven serologically distinct neurotoxins produced by Cbotulinum (A, B, C, D, E, F, and G), humans are most susceptible totypes A, B, E, and F. Types C and D are most toxic for animals. Type G is rare,with only a few reported human cases. The toxins often are released from thebacteria as inactive proteins that must be cleaved by a protease to expose theactive site. These proteases may be produced by the cell itself or may be in thebody fluids of the infected host. Type A toxin is the most potent poison known;ingestion of only 10^-8 grams of this toxin can kill a human. Putanother way, the amount of toxin that could be held on the tip of a dissectingprobe could kill 40 medical students.

Pathogenesis

The pathogenicity of C botulinum depends entirely on neurotoxinproduction (Fig. 18-3). In humans, thesetoxins cause disease in three ways: the well-known form of food poisoningresults from ingestion of toxin in improperly preserved food; wound botulism, arare disease, results from C botulinum growing in the necrotictissue of a wound; and infant botulism is caused when the organism grows andproduces toxin in the intestines of infants.

From its site of entry into the body, the toxin travels through the blood andlymphatic systems (and possibly the nervous system). It then becomes fixed tocranial and peripheral nerves, but exerts almost all of its action on theperipheral nervous system. The toxin appears to bind to receptor sites at theneuromuscular junctions of parasympathetic nerves, and inhibits the release ofacetylcholine at peripheral cholinergic synapses. The result is flaccid muscularparalysis.

The cranial nerves are affected first, followed by a descending, symmetricparalysis of motor nerves. The early involvement of cranial nerves causesproblems with eyesight, hearing, and speech. Double or blurred vision, dilatedpupils, and slurred speech are common symptoms. Decreased saliva productioncauses a dryness of the mouth and throat, and swallowing may be painful. Anoverall weakness ensues, followed by descending paralysis with criticalinvolvement of the respiratory tree. Death usually is caused by respiratoryfailure, but cardiac failure also can be the primary cause. Mortality is highestfor type A, followed by type E, and then type B, possibly reflecting theaffinities of the toxins for neural tissue: type A binds most firmly, followedby type E, then type B. Fatality rates are directly proportional to theinfectious dose and inversely proportional to the incubation time of thedisease.

Type A toxin is used therapeutically to treat a variety of conditions involvinginvoluntary muscle spasms, including strabismus and certain focal dystonias.This therapy takes advantage the effect of the toxin as a specific musclerelaxant. The therapeutic toxin is a neurotoxin-hemagglutinin complex isolatedfrom C botulinum cultures. The extreme potency of type Abotulinum toxin requires extreme caution in using this compound as a therapeuticagent.

Host Defenses

Host defenses against C botulinum are undefined. Some people cantolerate ingestion of botulinum toxin better than others. The reason for thisphenomenon is obscure, but could be due to differences in the efficiency ofuptake of the toxin from the intestine or in transporting the toxin to neuraltissue. An attack of botulism does not produce effective immunity. The smallamount of toxin in the circulation and its affinity for neural tissue probablyprevent adequate amounts of toxin from interacting with the immune system.

Epidemiology

C botulinum spores are found worldwide in the soil (including insea sediments) and in low numbers in the gastrointestinal tracts of some birds,fish, and mammals. In the United States, the most frequent isolate is type A,followed by B and E, with an occasional isolate of type F. In Europe, B is themost frequent isolate, whereas A is comparatively rare. Despite the worldwideoccurrence of C botulinum in the environment, wound botulism isa comparatively rare disease.

Originally, botulism food poisoning was thought to be associated only withcontaminated meat, especially sausage; however, it is now known that Cbotulinum can grow equally well in many types of food includingvegetables, fish, fruits, and condiments. Home canning using inadequatesterilization techniques has been responsible for most cases of botulism duringthis century. The spores are heat resistant and can survive 100° C forhours, but the toxin is relatively heat labile. The toxin is usually produced atpH 4.8–8.5. However, even acid foods such as canned tomatoes have beenresponsible for several recent cases of botulism food poisoning. In addition,certain culture conditions have been shown to cause toxin production at pHvalues lower than 4.6. In general, germination of botulinum spores is favored infood kept at warm temperatures under anaerobic conditions for a long period oftime.

C botulinum spores exist throughout the environment; all adultshave likely ingested these spores with no ill effects. Because spores can causepoisoning in infants, obvious sources should be eliminated from the infant'senvironment and especially the infant's diet. Honey is the only dietaryingredient that has been implicated, and honey is no longer recommended forinfants under 1 year of age. Most cases are not caused by ingesting honey,however, so this will not eliminate the disease. The other more commonenvironmental sources of spores, such as soil and dust, are not so easilycontrolled.

Diagnosis

Although all forms of botulism are difficult to diagnose, prompt diagnosis andtreatment are crucial to patient survival. Laboratory tests offer little inestablishing an initial diagnosis of botulism, and accordingly, the finding of anormal cerebrospinal fluid can help to eliminate many of the diseases concernedwith central nervous system disorders. Differential diagnoses are myriad andinclude neurological as well as gastrointestinal disorders. The infant withbotulism is typically afebrile with generalized weakness, a weak cry, pooling oforal secretions, and poor sucking ability. Constipation may precede the illnessby several weeks. An electromyogram pattern of brief, small-amplitudeoverabundant motor reaction potentials often is seen.

Confirmation of the initial diagnosis rests on demonstrating toxin in thepatient's feces, serum, or vomitus. In adult botulism, serum samples rarelyyield type A toxin because of the strong affinity of this toxin for neuraltissue. In infant botulism, circulating toxin can occasionally be found in theserum. Fecal samples are the best specimens for detecting toxin in botulism foodpoisoning or infant botulism because only a small percentage of ingested orin situ formed toxin is absorbed through the intestinalmucosa. Toxin may be excreted for days or even weeks following botulism foodpoisoning. Toxin is usually detected by its lethal effect in mice coupled withneutralization of this effect by specific antisera. In infants, the organism canusually be cultured from the stool.

Control

The best way to control botulism food poisoning is to use adequate foodpreservation methods and to heat all canned food before eating. Becausebotulinum toxin is heat-labile, boiling food for a few minutes will eliminatetoxin contamination; however, the spores themselves are not destroyed byboiling, and proper canning procedures must be followed to kill clostridialspores.

Once a case of wound botulism or food poisoning has been diagnosed, therapy hasfour objectives: to eliminate the source of the toxin, to eliminate anyunabsorbed toxin, to neutralize any unbound toxin with specific antitoxin, andto provide general supportive care.

Clinical Manifestations

Clostridium difficile is a major nosocomial pathogen that causesa spectrum of intestinal disease from uncomplicated antibiotic-associateddiarrhea to severe, possibly fatal, antibiotic-associated colitis. Diarrhea hascome to be accepted as a natural accompaniment of treatment with manyantibiotics. Although this diarrhea usually causes only minor concern, it canevolve into a life-threatening enterocolitis.

Many antibiotics have been associated with diarrhea and with pseudomembranouscolitis, including ampicillin, cephalosporins, clindamycin, and amoxicillin.Patients treated with clindamycin have a higher incidence of Cdifficile disease, but most cases are found in patients treatedwith other antibiotics because of the more widespread use of these agents.Occasionally, antineoplastic agents that alter the normal intestinal flora mayalso induce pseudomembranous colitis, with methotrexate most commonlyimplicated. Chemotherapy-associated C difficile disease may notbe easily recognized due either to an absence of antibiotic therapy or due tothe frequent concomitant use of antibiotics, obscuring true incidence ofchemotherapy-associated C difficile disease.

Clinical symptoms of C difficile disease vary widely from milddiarrhea to severe abdominal pain accompanied by fever (typically>101°F) and severe weakness. Diarrhea is watery and usuallynonbloody (Fig. 18-4), but approximately5 to 10% of patients have bloody diarrhea. Fecal material typically containsexcess mucus, and pus or blood may also be noted. Hypoalbuminemia andleukocytosis are common findings. Pathology involves only the colon where theremay be disruption of brush border membranes followed by extensive damage to themucosa. The disease may progress to a pseudomembranous colitis, possiblyincluding intestinal perforation and toxic megacolon. There is a leukocyticinfiltrate into the lamina propria accompanied by elaboration of a mixture offibrin, mucus, and leukocytes, which can form gray, white, or yellow patches onthe mucosa. These areas are called pseudomembranes; hence the common termpseudomembranous colitis. Pseudomembranes usually develop after 2–10days of antibiotic treatment, but they may appear 1–2 weeks after allantibiotic therapy has stopped. Mortality varies, but may be as high as 10% inpatients with pseudomembranous colitis. The ultimate cause of death often isdifficult to determine, as most patients show a nonspecific deterioration over aperiod of weeks.

The incidence of pseudomembranous colitis has been diminishing in recent years,most likely due to early diagnosis of the disease and prompt antimicrobialtherapy. However, C difficile is now considered a major causeof diarrhea in hospitals and nursing homes. In most instances, once a patientdevelops antibiotic-associated diarrhea and C difficileorganisms and/or toxin is detected in the stool, appropriate antimicrobialtherapy is begun, and the symptoms are not allowed to progress to the formationof colonic pseudomembranes. Thus, in recent years the terms “Cdifficile diarrhea” and “Cdifficile disease” have come to be associated with aspectrum of diseases, including pseudomembranous colitis as well as diarrhea andcolitis in the absence of pseudomembranes. The common factors in all of thesediseases are the presence of diarrhea associated with antibiotic therapy and therecovery of C difficile organisms and/or toxin from thestool.

Etiologic Agent

C difficile is a slender, gram-positive bacillus that produceslarge, oval, subterminal spores. It is an anaerobe, and some strains areextremely sensitive to oxygen. C difficile is nonhemolytic anddoes not produce lecithinase or lipase reactions on egg yolk agar. It producesvarious tissue degradative enzymes, including proteases, collagenases,hyaluronidase, heparinase, and chondroitin-4-sulfatase. The products offermentation are many and complex and include acetic, butyric, isovaleric,valeric, isobutyric, and isocaproic acids; however, only small amounts of eachare produced.

Pathogenesis

C difficile disease is caused by the overgrowth of the organismin the intestinal tract, primarily in the colon (Fig. 18-4). The organism appears unable to competesuccessfully in the normal intestinal ecosystem, but can compete when normalflora are disturbed by antibiotics, allowing overgrowth of Cdifficile. This organism then replicates and secretes two toxins.Toxin A is an enterotoxin that causes fluid accumulation in the bowel, and it isa weak cytotoxin for most mammalian cells; toxin B is a potent cytotoxin. Nearlyall toxigenic strains produce both toxins A and B. Highly toxigenic strainsproduce high levels of both toxins, while weakly toxigenic strains produce lowlevels of both toxins. Results from in vitro studies using cultured intestinalepithelial cells have indicated that toxin A causes necrosis, increasedintestinal permeability, and inhibition of protein synthesis. Toxin A somehowaffects phospholipase A₂, resulting in the production of severalarachidonic acid metabolites including prostaglandins and leukotrienes. Althoughthe exact mechanism of endocytosis is unclear, both toxins A and B areinternalized by host cells, resulting in alterations in the actin-containingcytoskeleton. Toxin A is a chemotactic factor for granulocytes; both toxins Aand B have effects on leukocytes that include alterations in actin cytoskeletalmicrofilaments, and induction of tumor necrosis factor, interleukin 1, andinterleukin 6. These latter effects contribute to the inflammatory responseassociated with C difficile disease.

Both toxins A and B kill experimental animals, and both probably are involved inthe pathology of disease. Toxin production causes diarrhea that may progress topseudomembranous colitis, where the characteristic pseudomembranes are largelylimited to the colon. In the intestinal tract, toxin A damages villous tips andbrush border membranes, and may result complete in erosion of the mucosa. Thistissue damage causes a viscous hemorrhagic fluid response. In contrast, toxin Bdoes not have noticeable enterotoxic activity, but it is lethal when injectedinto experimental animals. Thus, it seems reasonable to speculate that, inhumans, toxin B exerts its pathogenic effect following dissemination through adamaged gut wall to extraintestinal organs. It has been speculated that infantsharboring high levels of intestinal toxins A and B are at risk for the systemictoxicity of toxin B if their intestinal barrier is compromised.

Host Defenses

Host defenses for C difficile disease are not completelyunderstood, but it seems reasonable to assume that the best host defense againstC difficile disease is maintenance of the stability of thenormal intestinal flora. Certain prostaglandins are known to protect the stomachand small intestine from mucosal necrosis caused by harsh chemicals; and, inexperimental animals, prostaglandins have been shown to prevent extensivemucosal damage from C difficile toxin. These prostaglandins arealso produced by toxin A-induced activation of the arachidonic cascade byphospholipase A₂. Production of specific neutralizing antibodies totoxins A and B may participate in host defense, and a specific intestinalsecretory IgA response to toxin A is more evident in the colon than the upperintestinal tract, compatible with the colon as the primary site of intestinaldisease. The intestinal tract responds to C difficile toxins byincreased fluid production, by secretory IgA neutralization of toxin, and bymucus production, which may inhibit the attachment of the toxins to theirputative receptor sites on intestinal epithelial cells.

Epidemiology

C difficile is a member of the normal intestinal flora of<3% of adults. The organism can be acquired as a nosocomial pathogen anda variable incidence of disease is noted in hospitals and nursing homes. Thisseems to be due in part to environmental contamination with Cdifficile spores, and in part to different patient populations invarious institutions. Patients with C difficile diarrheaexcrete large numbers of C difficile spores, andepidemiological studies have shown that the organism can reside on environmentalsurfaces as well as on the hands of health care workers. Healthy adults do notcarry significant numbers of the organism in their intestinal tracts, buthealthy infants may have large numbers of these organisms in their feces. Moststudies report a high carriage rate of approximately 50% in neonates, althoughsome studies report a carriage rate of 0 to 6%, likely due to differences inenvironmental exposure to the organism. The toxins also are present in theseinfants' stools, and the same amounts of toxins are associated with disease inadults. The toxins typically have no adverse effect in infants, but confound thediagnosis of C difficile disease. There is circumstantialevidence supporting the theory that infants do not develop disease because theylack specific intestinal receptors for C difficile toxins. Inrecent years, C difficile as also emerged as one of the causesof chronic diarrhea in AIDS patients.

Diagnosis

It is often difficult to distinguish C difficile disease fromother intestinal diseases, including ulcerative colitis and Crohn's disease.Diagnosis of C difficile disease includes the presence ofdiarrhea associated with antibiotic therapy in the preceding 4 to 6 weeks, andthe recovery of C difficile organisms and/or toxin from thestool. However, the isolation of toxigenic C difficile frompatients is often not a definitive diagnosis because other enteric pathogens areusually not excluded. Many cases of severe diarrhea are caused not by Cdifficile, but are caused by other enteric pathogens such asCampylobacter spp, Salmonella spp,Shigella spp, toxigenic strains of Escherichiacoli, etc. Moreover, antimicrobial therapy increases the likelihoodof isolating C difficile from the fecal flora: Cdifficile can be isolated from the feces of approximately 20 to 40%of asymptomatic hospitalized patients who are receiving antimicrobial therapy.Despite these caveats, C difficile is likely responsible for^≥25% of cases of antibiotic-associated diarrhea andcolitis. Diagnosis of pseudomembranous colitis requires demonstration ofpseudomembranes by colonoscopy, and C difficile can be isolatedfrom the stools of almost all patients with this disease.

A good selective agar medium is used for the isolation of Cdifficile from stool. Toxin detection is also used for diagnosis.Although the most appropriate test for toxin detection remains controversial, acellular cytotoxicity test remains the “gold standard”;here, filter sterilized fecal extract (or filter-sterilized broth containing apure culture of C difficile) is added to a monolayer ofcultured mammalian cells resulting in a cytopathic effect that is neutralizedwith specific antiserum. Unfortunately, this test is time-consuming andcumbersome. A rapid latex agglutination test is widely used, but this test isnot specific, does not detect toxin A or B, and often results in false negativeor false positive reactions. Enzyme-linked immunoassays can be used to detectboth toxin A and toxin B, and these tests are useful for diagnosis of Cdifficile disease.

Control

In many cases, symptoms resolve 1–14 days after the offendingantibiotic is discontinued, and antibiotic treatment is not needed. Vancomycinor metronidazole are the antibiotics of choice to treat active disease. Oralvancomycin is the “gold standard,” and metronidazole is mostoften used to treat milder infections. Some claim that metronidazole should beconsidered the drug of choice in all but the most severe cases, based onrelative cost of the two drugs and based on prevention of development ofvancomycin resistance in enteric bacteria. C difficile issusceptible to both of these antimicrobial agents, but relapses occur in 15 to20% of patients. Some patients have had many repeated relapses. Constipatingagents, such as atropine diphenoxylate (Lomotil) or codeine, should not be used.Supportive therapy is needed to compensate for the often severe fluid andelectrolyte loss. Health care workers caring for patients infected withC. difficile should wear gloves and strictly adhere toproper hand washing procedures.

Necrotizing Enteritis and Clostridium perfringens

Necrotic enteritis in humans has not been well documented. In adults, the diseaseappears to result from ingesting large amounts of food contaminated withC perfringens, usually type C. It generally followsingestion of a large meal, implicating bowel distention and bacterial stasis ascontributing factors. The intestinal pathology varies considerably, and mayinclude sloughing of intestinal mucosa, submucosa, and mesenteric lymph nodes.Intestinal perforations occur frequently. The best-documented cases of thisdisease involve the natives of New Guinea, who develop necrotic enteritis(“pig-bel”) after eating large quantities of improperlycooked pork that has been contaminated with the bowel contents of the animal.The course of the disease is fulminate, and the mortality rate is high.Scattered cases of necrotizing enteritis with C perfringens asthe prominent bacterial isolate have been reported in Western countries. Inthese cases, controversy exists concerning whether Cperfringens is a primary invader, an accidental contaminant, or anopportunistic pathogen.

Some evidence suggests that acute necrotizing enterocolitis of infants may becaused by a clostridium, but definitive evidence is lacking. The theory issupported by the fact that pneumatosis cystoides intestinalis, a syndrome thatcan be caused by C perfringens, often is present in cases ofacute necrotizing enterocolitis of infants. In addition, Cperfringens, C butyricum, C difficile and otherclostridial species are often isolated in cases of neonatal necrotizingenterocolitis, but a clear pathogenic role for clostridia is yet to beelucidated.

Bacteremia, endometritis and nonbacteremic infections with Clostridiumsordellii

C sordellii is part of the normal intestinal flora of humans.The organism produces several exotoxins including toxins serologically relatedto the toxins of C difficile. There are scattered reports inthe literature of C sordellii wound infections, most of whichinvolve significant trauma. C sordellii has been occasionallyimplicated in bone and joint infections, in pulmonary infections, in bacteremia,and in fulminate endometritis. Because many clinical laboratories fail tospeciate clostridial pathogens, the pathogenic potential of Csordellii is likely underestimated.

Malignancy and Clostridium septicum

C septicum is a spindle-shaped rod that is motile in youngcultures. The organism produces toxins designated alpha, beta, gamma, and delta;the alpha toxin is necrotizing and lethal for mice. Whether Csepticum is a member of the host's normal flora or whether it takesadvantage of a compromised host is uncertain. The organism is not stronglyinvasive, but has been associated with gas gangrene. Fewer than 200 cases ofinvasive disease have been reported, but the majority have a malignancysomewhere in the body. The most frequent association is with colorectal cancer,but other types of malignancies have been noted, including leukemia, lymphoma,and sarcoma. In one survey of C septicum bacteremia, 49 of 59(83%) cases had an underlying malignancy and, in 28 of these cases, the portalof entry appeared to be the distal ileum or the colon. Diabetes mellitus is seenin about 20% of cases. In collective review of 162 cases of nontraumaticC septicum infection, 81% of the patients had malignantdisease; in contrast, other clostridial species are associated with malignancyin approximately 10% of cases. Thus, in the absence of an overt infection,isolation of C septicum should alert the physician to thepossible presence of a malignancy, most likely in the ileum or the colon.Immediate antibiotic therapy is indicated because most patients die quickly ofthe infection if not treated. Penicillin is the antibiotic of choice, butchloramphenicol, carbenicillin, and cephalothin also have been usedsuccessfully.

Bacteremia and Clostridium tertium

C tertium is an aerotolerant clostridium that is usuallyconsidered nonpathogenic. However, there are scattered reports of this organismcausing bacteremia. Most cases have involved neutropenic patients, and thegastrointestinal tract appears to be the source of the infection. It is possiblethat this organism causes many more cases of bacteremia than is currentlyappreciated. The aerotolerant nature of C tertium may result inits misidentification as a Bacillus species.

Other clostridia with potential clinical significance

Clostridium butyricum, C clostrdioforme, C innocuum, andC ramosum are isolated with some frequency from clinicalspecimens and may have an unrecognized clinical significance. These species areoften resistant to clindamycin and cephalosporins. C ramosum isusually listed with the ten anaerobic species most frequently isolated fromclinical specimens. C ramosum frequently is misidentified, asthe Gram reaction is lost easily, and spores are difficult to detect. Cclostridioforme stains Gram-negative and forms characteristicfootball-shaped cells, rarely sporulates, and may be easily misidentified asBacteroides sp or Fusobacterium sp.