Jawetz-chapter-11 Spore-Forming Gram-Positive Bacilli PDF

11 Spore-Forming Gram-Positive C H A P...

11 Spore-Forming Gram-Positive C H A P T E R Bacilli: Bacillus and Clostridium Species The Gram-positive spore-forming bacilli are the Bacillus and Morphology and Identification Clostridium species. These bacilli are ubiquitous, and because they form spores, they can survive in the environment for A. Typical Organisms many years. Bacillus species are aerobes, and the Clostridium The typical cells, measuring 1 × 3–4 µm, have square ends species are anaerobes (see also Chapter 21). and are arranged in long chains; spores are located in the cen- Of the many species of Bacillus and related genera, most ter of the bacilli. do not cause disease and are not well characterized in medi- cal microbiology. There are a few species, however, that cause B. Culture important diseases in humans. Anthrax, a classical disease in Colonies of B. anthracis are round and have a “cut glass” the history of microbiology, is caused by Bacillus anthracis. appearance in transmitted light. Hemolysis is uncommon Anthrax remains an important disease of animals and occa- with B. anthracis but common with B. cereus and the sapro- sionally of humans. Because of its potent toxins, B. anthracis phytic bacilli. Gelatin is liquefied, and growth in gelatin stabs is a major potential agent of bioterrorism and biologic war- resembles an inverted fir tree. fare. B. cereus causes food poisoning and occasionally eye or other localized infections. C. Growth Characteristics The genus Clostridium is extremely heterogeneous, The saprophytic bacilli use simple sources of nitrogen and and more than 200 species have been described. The list of carbon for energy and growth. The spores are resistant to pathogenic organisms, as well as novel species isolated from environmental changes, withstand dry heat and certain human feces whose pathogenic potential remains undeter- chemical disinfectants for moderate periods, and persist for mined, continues to grow. Clostridia cause several impor- years in dry earth. Items contaminated with anthrax spores tant toxin-mediated diseases, including tetanus (Clostridium can be sterilized by autoclaving or irradiation. tetani), botulism (C. botulinum), gas gangrene (C. perfringens), and antibiotic-associated diarrhea and pseudomembranous colitis (C. difficile). Other clostridia are also found in mixed BACILLUS ANTHRACIS anaerobic infections in humans (see Chapter 21). Pathogenesis Anthrax is primarily a disease of herbivores—goats, sheep, BACILLUS SPECIES cattle, horses, and so on; other animals (eg, rats) are relatively resistant to the infection. Anthrax is endemic among agrarian The genus Bacillus includes large aerobic, Gram-positive rods societies in developing countries in Africa, the Middle East, occurring in chains. The members of this genus are closely and Central America. A website maintained by the World related but differ both phenotypically and in terms of patho- Health Organization provides current information on disease genesis. Pathogenic species possess virulence plasmids. Most in animals (www.who.int/csr/disease/Anthrax/en/). Humans members of this genus are saprophytic organisms prevalent become infected incidentally by contact with infected animals in soil, water, and air, and on vegetation (eg, B. subtilis). Some or their products. In animals, the portal of entry is the mouth are insect pathogens, such as B. thuringiensis. B. cereus can and the gastrointestinal tract. Spores from contaminated soil grow in foods and cause food poisoning by producing either find easy access when ingested with spiny or irritating veg- an enterotoxin (diarrhea) or an emetic toxin (vomiting). etation. In humans, the infection is usually acquired by the B. cereus may occasionally produce disease in immunocom- entry of spores through injured skin (cutaneous anthrax) or promised humans (eg, meningitis, endocarditis, endophthal- rarely the mucous membranes (gastrointestinal anthrax) or mitis, conjunctivitis, or acute gastroenteritis). B. anthracis, by inhalation of spores into the lung (inhalation anthrax). A which causes anthrax, is the principal pathogen of the genus. fourth category of the disease, injection anthrax, has caused 183 Riedel_CH11_p183-p194.indd 183 04/04/19 4:44 PM  184   SECTION III  Bacteriology outbreaks among persons who inject heroin that has been protein that binds to specific cell receptors, and after proteo- contaminated with anthrax spores. The spores germinate in lytic activation, it forms a membrane channel that mediates the tissue at the site of entry, and growth of the vegetative entry of EF and LF into the cell. EF is an adenylate cyclase; organisms results in formation of a gelatinous edema and with PA, it forms a toxin known as edema toxin. Edema toxin congestion. Bacilli spread via lymphatics to the bloodstream, is responsible for cell and tissue edema. LF plus PA form and they multiply freely in the blood and tissues shortly lethal toxin, which is a major virulence factor and cause of before and after the animal’s death. death in infected animals and humans. When injected into B. anthracis (Figure 11-1) isolates that do not produce a laboratory animals (eg, rats), the lethal toxin can quickly kill capsule are not virulent and do not induce anthrax in test the animals by impairing both innate and adaptive immu- animals. The poly-γ-d-glutamic acid capsule is antiphago- nity, allowing organism proliferation and cell death. The cytic. The capsule gene is present on a plasmid, pXO2. anthrax toxin genes are encoded on another plasmid, pXO1. Anthrax toxins are made up of three proteins: protective In inhalation anthrax (woolsorters’ disease), the spores from antigen (PA), edema factor (EF), and lethal factor (LF). PA is a the dust of wool, hair, or hides are inhaled; phagocytosed in the lungs; and transported by the lymphatic drainage to the mediastinal lymph nodes, where germination occurs. This is followed by toxin production and the development of hem- orrhagic mediastinitis and sepsis, which are usually rapidly fatal. In anthrax sepsis, the number of organisms in the blood exceeds 107/mL just before death. In the Sverdlovsk, inhala- tion anthrax outbreak of 1979 and the U.S. bioterrorism inhalation cases of 2001, the pathogenesis was the same as in inhalation anthrax from animal products. Pathology In susceptible animals and humans, the organisms proliferate at the site of entry. The capsules remain intact, and the organ- isms are surrounded by a large amount of proteinaceous fluid containing few leukocytes from which they rapidly dissemi- nate and reach the bloodstream. In resistant animals, the organisms proliferate for a few A hours, by which time there is massive accumulation of leuko- cytes. The capsules gradually disintegrate and disappear. The organisms remain localized. Clinical Findings In humans, approximately 95% of cases are cutaneous anthrax, and 5% are inhalation. Gastrointestinal anthrax is very rare; it has been reported from Africa, Asia, and the United States when people have eaten meat from infected animals. The bioterrorism events in the fall of 2001 resulted in 22 cases of anthrax—11 inhalation and 11 cutaneous. Five of the patients with inhalation anthrax died. All of the other patients survived. Cutaneous anthrax generally occurs on exposed surfaces of the arms or hands followed in frequency by the face and neck. A pruritic papule develops 1–7 days after entry of the organisms or spores through a scratch. Initially, it resem- bles an insect bite. The papule rapidly changes into a vesicle or small ring of vesicles that coalesce, and a necrotic ulcer B develops. The lesions typically are 1–3 cm in diameter and FIGURE 11-1 A: B. anthracis in broth culture (original have a characteristic central black eschar. Marked edema magnification ×1000). B: In tissue (original magnification ×400). occurs. Lymphangitis, lymphadenopathy, and systemic signs (Courtesy of PS Brachman.) and symptoms of fever, malaise, and headache may occur. Riedel_CH11_p183-p194.indd 184 04/04/19 4:44 PM  CHAPTER 11 Spore-Forming Gram-Positive Bacilli: Bacillus and Clostridium Species   185 After 7–10 days, the eschar is fully developed. Eventually, it detection of the capsule by fluorescent antibody, or identi- dries, loosens, and separates; healing is by granulation and fication of toxin genes by polymerase chain reaction (PCR). leaves a scar. It may take many weeks for the lesion to heal These tests are available in most public health laboratories. and the edema to subside. Antibiotic therapy does not appear A rapid enzyme-linked immunoassay (ELISA) that mea- to change the natural progression of the disease but pre- sures total antibody to PA has been approved by the U.S. Food vents dissemination. In as many as 20% of patients, cutane- and Drug Administration (FDA), but the test result is not ous anthrax can lead to sepsis, the consequences of systemic positive early in disease. infection—including meningitis—and death. The incubation period in inhalation anthrax may be as long as 6 weeks. The early clinical manifestations are asso- Resistance and Immunity ciated with marked hemorrhagic necrosis and edema of the Immunization to prevent anthrax is based on the classic mediastinum. Substernal pain may be prominent, and there experiments of Louis Pasteur. In 1881, he proved that cultures is pronounced mediastinal widening visible on chest radio- grown in broth at 42–52°C for several months lost much of graphs. Hemorrhagic pleural effusions follow involvement of their virulence and could be injected live into sheep and cattle the pleura; cough is secondary to the effects on the trachea. without causing disease; subsequently, such animals proved Sepsis occurs, and there may be hematogenous spread to the to be immune. Active immunity to anthrax can be induced gastrointestinal tract, causing bowel ulceration, or to the in susceptible animals by vaccination with live attenuated meninges, causing hemorrhagic meningitis. The fatality rate bacilli, with spore suspensions, or with PA from culture fil- in inhalation anthrax is high in the setting of known expo- trates. Animals that graze in known anthrax districts should sure; it is higher when the diagnosis is not initially suspected. be immunized for anthrax annually. Animals acquire anthrax through ingestion of spores In the United States, the current FDA-approved vac- and spread of the organisms from the intestinal tract. This cine (AVA BioThrax®, Emergent BioSolutions, Inc, Rockville, is rare in humans, and gastrointestinal anthrax is extremely MD) is made from the supernatant of a cell-free culture of uncommon. Abdominal pain, vomiting, and bloody diarrhea an unencapsulated but toxigenic strain of B. anthracis that are clinical signs. contains PA adsorbed to aluminum hydroxide. The dose Injection anthrax is characterized by extensive, painless, schedule is 0.5 mL administered intramuscularly at 0 and subcutaneous edema and the notable absence of the eschar 4 weeks and then at 6, 12, and 18 months followed by annual characteristic of cutaneous anthrax. Patients may progress to boosters. The vaccine is available only to the U.S. Depart- hemodynamic instability due to septicemia. ment of Defense and to persons at risk for repeated exposure to B. anthracis. Because the current anthrax vaccines provide short-lived immunity and hence require repeated vaccina- Diagnostic Laboratory Tests tions, a number of new recombinant PA vaccines (rPA) have Specimens to be examined are fluid or pus from a local lesion, been developed. These novel vaccines have been shown to be blood, pleural fluid, and cerebrospinal fluid in inhalational very well tolerated and highly immunogenic (see discussion anthrax associated with sepsis and stool or other intesti- under Treatment). nal contents in the case of gastrointestinal anthrax. Stained smears from the local lesion or of blood from dead animals often show chains of large Gram-positive rods. Anthrax can Treatment be identified in dried smears by immunofluorescence stain- Many antibiotics are effective against anthrax in humans, ing techniques. but treatment must be started early. Ciprofloxacin is rec- When grown on blood agar plates, the organisms pro- ommended for treatment; other agents with activity include duce nonhemolytic gray to white, tenacious colonies with penicillin G, doxycycline, erythromycin, and vancomycin. In a rough texture and a ground-glass appearance. Comma- the setting of potential exposure to B. anthracis as an agent of shaped outgrowths (Medusa head, “curled hair”) may project biologic warfare, prophylaxis with ciprofloxacin or doxycy- from the colony. Demonstration of capsule requires growth cline should be given for 60 days, and three doses of vaccine on bicarbonate-containing medium in 5–7% carbon diox- (AVA BioThrax) should be administered. ide. Gram-stain shows large Gram-positive rods. Carbo- Raxibacumab (Abthrax®, GlaxoSmithKline, London, hydrate fermentation is not useful. In semisolid medium, UK), a recombinant human monoclonal antibody, was FDA anthrax bacilli are always nonmotile, but related organisms approved for treatment of and prophylaxis against inhala- (eg, B. cereus) exhibit motility by “swarming.” Clinical labo- tional anthrax in late 2012. The mechanism of action is pre- ratories that recover large Gram-positive rods from blood, vention of binding of PA to its receptors in host cells. The cerebrospinal fluid, or suspicious skin lesions, which pheno- drug is used in combination with appropriate antimicrobial typically match the description of B. anthracis as mentioned, agents. should immediately contact their public health laboratory Anthrax immune globulin intravenous (AIGIV, Can- and send the organism for confirmation. Definitive identi- gene Corp. Winnipeg, Manitoba, CA) is not FDA approved fication requires lysis by a specific anthrax γ-bacteriophage, but could be made available through the Centers for Disease Riedel_CH11_p183-p194.indd 185 04/04/19 4:44 PM  186   SECTION III  Bacteriology Control and Prevention. AIGIV is a human polyclonal anti- including endocarditis, catheter-associated bacteremia, cen- serum that also inhibits binding of PA to its receptors. Like tral nervous system infections, osteomyelitis, and pneumonia; Raxibacumab, it is used as an adjunct to antimicrobial agents the presence of a medical device or intravenous drug use for the treatment of severe forms of anthrax. predisposes to these infections. Outbreaks of bacteremia in neonatal intensive care units and other hospital units dur- ing construction in health care facilities have been reported. Epidemiology, Prevention, and Control B. cereus is resistant to a variety of antimicrobial agents, Soil is contaminated with anthrax spores from the carcasses including penicillins and cephalosporins. Serious non- of dead animals. These spores remain viable for decades. Per- foodborne infections should be treated with vancomycin or haps, spores can germinate in soil at a pH of 6.5 at proper clindamycin with or without an aminoglycoside. Ciprofloxa- temperature. Grazing animals infected through injured cin has been useful for the treatment of wound infections. mucous membranes serve to perpetuate the chain of infec- tion. Contact with infected animals or with their hides, hair, and bristles is the source of infection in humans. Control CHAPTER SUMMARY measures include (1) disposal of animal carcasses by burning or by deep burial in lime pits, (2) decontamination (usually by Bacillus species constitute a large group of mostly sapro- autoclaving) of animal products, (3) protective clothing and phytic, aerobic, spore-forming organisms ubiquitous in soil. gloves for handling potentially infected materials, and (4) The major pathogen in the Bacillus genus is B. anthracis, a active immunization of domestic animals with live attenu- virulent and toxic organism of historical importance. ated vaccines. Persons with high occupational risk should be Humans acquire infection from spores inoculated via con- immunized. tact with animals or animal products, such as hides. B. anthracis causes four categories of disease in humans depending on the point of entry of the spores: cutane- ous (95%), inhalational (5%), gastrointestinal (rare), and BACILLUS CEREUS injection. Food poisoning caused by B. cereus has two distinct forms: PA combines with two factors, EF and LF, to form potent the emetic type, which is associated with fried rice, milk, and toxins, edema toxin and lethal toxin, respectively, both pasta, and the diarrheal type, which is associated with meat of which have cytotoxic and immunomodulating effects. dishes and sauces. B. cereus produces toxins that cause dis- These toxins are responsible for the edema, tissue destruc- ease that is more of intoxication than a food-borne infection. tion, and the hemorrhage characteristic of anthrax. The emetic form is manifested by nausea, vomiting, abdomi- B. cereus causes food poisoning and opportunistic infec- nal cramps, and occasionally diarrhea and is self-limiting, tions in immunocompromised patients. with recovery occurring within 24 hours. It begins 1–5 hours B. cereus can be differentiated from B. anthracis on the after ingestion of a plasmid-encoded preformed cyclic peptide basis of colony morphology, β-hemolysis, motility, and (emetic toxin) in the contaminated food products. B. cereus is antimicrobial susceptibility patterns. a soil organism that commonly contaminates rice. When large amounts of rice are cooked and allowed to cool slowly, the B. cereus spores germinate, and the vegetative cells produce CLOSTRIDIUM SPECIES the toxin during log-phase growth or during sporulation. The diarrheal form has an incubation period of 1–24 hours The clostridia are large anaerobic, Gram-positive, motile and is manifested by profuse diarrhea with abdominal pain rods. Many decompose proteins or form toxins, and some and cramps; fever and vomiting are uncommon. In this syn- do both. Their natural habitat is the soil, marine sediments, drome, ingested spores that develop into vegetative cells of sewage, or the intestinal tract of animals and humans, where B. cereus secrete one of three possible enterotoxins which they live as saprophytes. The clostridia continue to increase induce fluid accumulation and other physiological responses in number as new species are discovered and several species in the small intestine. The presence of B. cereus in a patient’s have been sequenced. There are 19 clusters based on 16SrRNA stool is not sufficient to make a diagnosis of B. cereus disease gene sequence analysis. Most clinically related species are in because the bacteria may be present in normal stool speci- RNA Cluster I. Among the pathogens in this cluster are the mens; a concentration of 105 bacteria or more per gram of organisms causing botulism, tetanus, gas gangrene, and food is considered diagnostic. pseudomembranous colitis. B. cereus is an important cause of eye infections, such as severe keratitis and endophthalmitis. Typically, the organ- isms are introduced into the eye by foreign bodies associ- Morphology and Identification ated with trauma but infections can also occur after surgery. A. Typical Organisms B. cereus has also been associated with localized infections, Spores of clostridia are usually wider than the diameter of such as wound infections, and with systemic infections, the rods in which they are formed. In the various species, the Riedel_CH11_p183-p194.indd 186 04/04/19 4:44 PM  CHAPTER 11 Spore-Forming Gram-Positive Bacilli: Bacillus and Clostridium Species   187 CLOSTRIDIUM BOTULINUM C. botulinum, which causes the disease botulism, is world- wide in distribution; it is found in soil and occasionally in animal feces. Types of C. botulinum are distinguished by the antigenic type of toxin they produce. Spores of the organism are highly resistant to heat, withstanding 100°C for several hours. Heat resistance is diminished at acid pH or high salt concentration. Toxins During the growth of C. botulinum and during autolysis of the bacteria, toxin is liberated into the environment. Seven antigenic varieties of toxin (serotypes A–G) are known. Types A, B, E, and F are the principal causes of human illness. Types A and B have been associated with a variety of foods and type E predominantly with fish products. Type C produces lim- berneck in birds; type D causes botulism in mammals. Type G is not associated with disease. Botulinum toxins have three FIGURE 11-2 Clostridium Gram-stain. Individual Gram-positive domains. Two of the domains facilitate binding to and entry bacilli are present. Many are in chains. Some of the bacilli have of toxin into the nerve cell. The third domain is the toxin spores, which are the unstained or clear ovoid shapes (arrows). which is a 150 kDa protein that is cleaved into a heavy chain (H, 100 kDa) and a light chain (L, 50 kDa) that are linked by a disulfide bond. Botulinum toxin is absorbed from the gut, enters the blood circulation, and binds to receptors of spore is placed centrally, subterminally, or terminally. Most presynaptic membranes of motor neurons of the peripheral species of clostridia are motile and possess peritrichous fla- nervous system and cranial nerves. The toxin does not cross gella. A Gram-stain of a Clostridium species with terminal the blood brain barrier or affect the central nervous system. spores is shown in Figure 11-2. Proteolysis—by the L chain of botulinum toxin—of the target SNARE proteins (soluble-N-ethyl maleimide-sensitive factor B. Culture attachment protein) in the neurons inhibits the release of ace- Clostridia are anaerobes and grow under anaerobic conditions; tylcholine at the synapse, resulting in lack of muscle contrac- a few species are aerotolerant and also grow in ambient air. tion and paralysis. The SNARE proteins are synaptobrevin Anaerobic culture conditions are discussed in Chapter 21. In (also known as vesicle-associated membrane protein or general, the clostridia grow well on the blood-enriched media VAMP), SNAP 25, and syntaxin. The toxins of C. botulinum or other media used to grow anaerobes. types A, C, and E cleave the 25,000 kDa SNAP 25. Type C also cleaves syntaxin. Types B, D, F, and G toxins cleave only C. Colony Forms synaptobrevin. C. botulinum toxins are among the most toxic Some clostridia produce large raised colonies (eg, C. perfringens); substances known: The lethal dose for a human is probably others produce smaller colonies (eg, C. tetani). Some clos- about 1–2 µg/kg. The toxins are destroyed by heating for tridia form colonies that spread or swarm on the agar surface 20 minutes at 100°C. Strains that produce toxins A, B, or F are (C. septicum). Many clostridia produce a zone of β-hemolysis associated with infant botulism. Additional details on toxin on blood agar. C. perfringens characteristically produces a production and function are described in the review by Ros- double zone of β-hemolysis around colonies. setto et al (see References). D. Growth Characteristics Pathogenesis Clostridia can ferment a variety of sugars (saccharolytic), Resurgence of wound botulism caused by types A or B toxin has and many can digest proteins (proteolytic); some species occurred recently in the United States, in the United Kingdom, do both. These metabolic characteristics are used to divide and in Germany in association with skin-popping using con- the clostridia into groups. Milk is turned acid by some and taminated “black tar” heroin. However, most cases of botu- digested by others and undergoes “stormy fermentation” (ie, lism represent an intoxication resulting from the ingestion of clot torn by gas) with a third group (eg, C. perfringens). Vari- food in which C. botulinum has grown and produced toxin. ous enzymes are produced by different species. Clostridium The most common offenders are spiced, smoked, vacuum species produce more toxins than any other group of bacteria packed, or canned alkaline foods that are eaten without (see next). cooking. In such foods, spores of C. botulinum germinate; Riedel_CH11_p183-p194.indd 187 04/04/19 4:44 PM  188   SECTION III  Bacteriology that is, under anaerobic conditions, vegetative forms grow In infant botulism, C. botulinum and toxin can be demon- and produce toxin. strated in bowel contents but not in serum. Other methods In infant botulism, honey is the most frequent vehicle used to detect toxin include ELISAs and PCR, but the latter may of infection. The pathogenesis differs from the way that detect organisms that carry the gene but do not express toxin. adults acquire infection. The infant ingests the spores of C. botulinum, and the spores germinate within the intestinal tract. The vegetative cells produce toxin as they multiply; the Treatment neurotoxin then gets absorbed into the bloodstream. In rare Supportive care, especially intensive care, is key in the man- instances, adults with gastrointestinal anatomical abnormal- agement of patients with botulism. Adequate respiration ities or functional disorders may develop “infant botulism.” must be maintained by mechanical ventilation if necessary Wound botulism is the result of tissue contamination and in severe cases may need to be maintained for up to with spores and is seen primarily in injection drug users. 8 weeks. These measures have reduced the mortality rate Very rarely, inhalational botulism occurs when toxin enters from 65% to below 25%. Potent antitoxins to three types of the respiratory tract. botulinum toxins have been prepared in horses. Because the The toxin acts by blocking release of acetylcholine at type responsible for an individual case is usually not known, synapses and neuromuscular junctions (see earlier discus- trivalent (A, B, E) antitoxin must be promptly administered sion). The result is flaccid paralysis. The electromyogram and intravenously with customary precautions. Antitoxin does edrophonium strength test results are typical. not reverse the paralysis, but if administered early, it can pre- vent its advancement. Although most infants with botulism recover with supportive care alone, treatment with human- Clinical Findings derived botulinum immune globulin (BIG) is recommended. Symptoms begin 18–24 hours after ingestion of the toxic food, with visual disturbances (incoordination of eye muscles, Epidemiology, Prevention, and Control double vision), inability to swallow, and speech difficulty; signs of bulbar paralysis are progressive, and death occurs Because spores of C. botulinum are widely distributed in soil, from respiratory paralysis or cardiac arrest. Gastrointestinal they often contaminate vegetables, fruits, and other materi- symptoms are not prominent. There is no fever. The patient als. A large restaurant-based outbreak was associated with remains fully conscious until shortly before death. The mor- sautéed onions. When such foods are canned or otherwise tality rate is high. Patients who recover do not develop anti- preserved, they either must be sufficiently heated to ensure toxin in the blood. destruction of spores or must be boiled for 20 minutes before In the United States, infant botulism is as common as consumption. Strict regulation of commercial canning has or more common than the classic form of paralytic botulism largely overcome the danger of widespread outbreaks, but associated with the ingestion of toxin-contaminated food. commercially prepared foods have caused deaths. A chief risk The infants in the first months of life develop poor feeding, factor for botulism lies in home-canned foods, particularly weakness, and signs of paralysis (floppy baby). Infant botu- string beans, corn, peppers, olives, peas, and smoked fish or lism may be one of the causes of sudden infant death syn- vacuum-packed fresh fish in plastic bags. Toxic foods may drome. C. botulinum and botulinum toxin are found in feces be spoiled and rancid, and cans may “swell,” or the appear- but not in serum. ance may be innocuous. The risk from home-canned foods can be reduced if the food is boiled for more than 20 minutes before consumption. Diagnostic Laboratory Tests Botulinum toxin is considered to be a major potential Clinicians who suspect a case of botulism should contact agent for bioterrorism and biologic warfare. the appropriate public health authorities before submitting specimens to the laboratory. Detection of toxin and not the organism is required for definitive diagnosis. Toxin can often CLOSTRIDIUM TETANI be demonstrated in serum, gastric secretions, or stool from C. tetani, which causes tetanus, is worldwide in distribution the patient, and toxin may be found in leftover food. Clinical in the soil and in the feces of horses and other animals. Sev- swabs or other specimens obtained from patients should be eral types of C. tetani can be distinguished by specific flagel- transported using anaerobe containers. Suspect foods should lar antigens. All share a common O (somatic) antigen, which be left in their original containers. Mice injected intraperito- may be masked, and all produce the same antigenic type of neally with such specimens from these patients die rapidly. neurotoxin, tetanospasmin. The antigenic type of toxin is identified by neutralization with specific antitoxin in mice. This mouse bioassay is the test of choice for the confirmation of botulism. C. botulinum may Toxin be grown from food remains and tested for toxin production, The vegetative cells of C. tetani produce the plasmid-encoded but this is rarely done and is of questionable significance. toxin tetanospasmin (150 kDa) that is cleaved by a bacterial Riedel_CH11_p183-p194.indd 188 04/04/19 4:44 PM  CHAPTER 11 Spore-Forming Gram-Positive Bacilli: Bacillus and Clostridium Species   189 protease into two peptides (50 and 100 kDa) linked by a Proof of isolation of C. tetani must rest on production of toxin disulfide bond. The larger peptide initially binds to recep- and its neutralization by specific antitoxin. tors on the presynaptic membranes of motor neurons. It then migrates by the retrograde axonal transport system to the cell bodies of these neurons to the spinal cord and brainstem. The Prevention and Treatment toxin diffuses to terminals of inhibitory cells, including both The results of treatment of tetanus are not satisfactory. glycinergic interneurons and γ-aminobutyric acid (GABA)– Therefore, prevention is all important. Prevention of tetanus secreting neurons from the brainstem. The smaller peptide depends on (1) active immunization with toxoids, (2) aggres- degrades synaptobrevin (also called VAMP2, see above under sive wound care, (3) prophylactic use of antitoxin, and (4) C. botulinum toxin), a protein required for docking of neu- administration of penicillin. rotransmitter vesicles on the presynaptic membrane. Release The intramuscular administration of 250–500 units of of the inhibitory glycine and GABA is blocked, and the motor human antitoxin (tetanus immune globulin) gives adequate neurons are not inhibited. Hyperreflexia, muscle spasms, and systemic protection (0.01 unit or more per milliliter of serum) spastic paralysis result. Extremely small amounts of toxin can for 2–4 weeks. It neutralizes the toxin that has not been fixed be lethal for humans. to nervous tissue. Active immunization with tetanus toxoid should accompany antitoxin prophylaxis. Patients who develop symptoms of tetanus should receive Pathogenesis muscle relaxants, sedation, and assisted ventilation. Some- C. tetani is not an invasive organism. The infection remains times, they are given very large doses of antitoxin (3000– strictly localized in the area of devitalized tissue (wound, 10,000 units of tetanus immune globulin) intravenously in burn, injury, umbilical stump, surgical suture) into which the an effort to neutralize toxin that has not yet been bound to spores have been introduced. The volume of infected tissue nervous tissue. However, the efficacy of antitoxin for treat- is small, and the disease is almost entirely a toxemia. Germi- ment is doubtful except in neonatal tetanus, in which it may nation of the spore and development of vegetative organisms be lifesaving. that produce toxin are aided by (1) necrotic tissue, (2) calcium Surgical debridement is vitally important because it salts, and (3) associated pyogenic infections, all of which aid removes the necrotic tissue that is essential for proliferation establishment of low oxidation–reduction potential. of the organisms. Hyperbaric oxygen has no proven effect. The toxin released from vegetative cells reaches the Penicillin strongly inhibits the growth of C. tetani and central nervous system and rapidly becomes fixed to recep- stops further toxin production. Antibiotics may also control tors in the spinal cord and brainstem and exerts the actions associated pyogenic infection. described. When a previously immunized individual sustains a potentially dangerous wound, an additional dose of toxoid should be injected to restimulate antitoxin production. This Clinical Findings “recall” injection of toxoid may be accompanied by a dose of The incubation period may range from 4 to 5 days up to antitoxin if the patient has not had current immunization or 3 weeks. The disease is characterized by tonic contraction of boosters or if the history of immunization is unknown. voluntary muscles. Muscular spasms often involve first the area of injury and infection and then the muscles of the jaw (trismus, lockjaw), which contract so that the mouth cannot be Control opened. Gradually, other voluntary muscles become involved, Tetanus is a totally preventable disease. Universal active resulting in tonic spasms. Any external stimulus may precipi- immunization with tetanus toxoid should be mandatory. tate a tetanic generalized muscle spasm. The patient is fully Tetanus toxoid is produced by detoxifying the toxin with for- conscious, and pain may be intense. Death usually results malin and then concentrating it. Aluminum salt-adsorbed from interference with the mechanics of respiration. The toxoids are used. Three injections comprise the initial course mortality rate in generalized tetanus is very high. of immunization followed by another dose about 1 year later. Initial immunization should be carried out in all children during the first year of life. A “booster” injection of toxoid is Diagnosis given upon entry into school. Thereafter, “boosters” can be The diagnosis rests on the clinical picture and a history of spaced 10 years apart to maintain serum levels of more than injury, although only 50% of patients with tetanus have an 0.01 unit antitoxin per milliliter. In young children, tetanus injury for which they seek medical attention. The primary dif- toxoid is often combined with diphtheria toxoid and acellular ferential diagnosis of tetanus is strychnine poisoning. Anaer- pertussis vaccine. obic culture of tissues from contaminated wounds may yield Environmental control measures are not possible because C. tetani, but neither preventive nor therapeutic use of anti- of the wide dissemination of the organism in the soil and the toxin should ever be withheld pending such demonstration. long survival of its spores. Riedel_CH11_p183-p194.indd 189 04/04/19 4:44 PM  190   SECTION III  Bacteriology CLOSTRIDIA THAT PRODUCE INVASIVE digests collagen of subcutaneous tissue and muscle, are also produced. INFECTIONS Some strains of C. perfringens produce a powerful Many different toxin-producing clostridia (C. perfringens and enterotoxin (C. perfringens enterotoxin, CPE), especially related clostridia) (Figure 11-3) can produce invasive infec- when grown in meat dishes. When more than 108 vegetative tion (including myonecrosis and gas gangrene) if introduced cells are ingested and sporulate in the gut, CPE is formed. into damaged tissue. About 30 species of clostridia may pro- CPE is a protein (35 kDa) that may be a nonessential compo- duce such an effect, but the most common in invasive disease nent of the spore coat; it is distinct from other clostridial tox- is C. perfringens (90%). An enterotoxin of C. perfringens is a ins. It induces intense diarrhea in 7–30 hours. The action of common cause of food poisoning. C. perfringens enterotoxin involves marked hypersecretion in the jejunum and ileum, with loss of fluids and electrolytes in diarrhea. Much less frequent symptoms include nausea, vom- Toxins iting, and fever. This illness is similar to that produced by The invasive clostridia produce a large variety of toxins and B. cereus and tends to be self-limited. Enterotoxin-producing enzymes that result in a spreading infection. Many of these strains of C. perfringens may also play a role in antibiotic- toxins have lethal, necrotizing, and hemolytic properties. associated diarrhea and necrotizing enterocolitis in infants. In some cases, these are different properties of a single sub- stance; in other instances, they are attributable to different chemical entities. The alpha toxin of C. perfringens type A Pathogenesis is a lecithinase, and its lethal action is proportionate to the In invasive clostridial infections, spores reach tissue either rate at which it splits lecithin (an important constituent of cell by contamination of traumatized areas (soil, feces) or from membranes) to phosphorylcholine and diglyceride. Alpha the intestinal tract. The spores germinate at low oxidation– toxin also aggregates platelets, thereby leading to formation reduction potential; vegetative cells multiply, ferment carbo- of thrombi in small blood vessels and adding to poor tissue hydrates present in tissue, and produce gas. The distention profusion and extending the consequences of anaerobiosis, of tissue and interference with blood supply, together with namely, destruction of viable tissue (gas gangrene). The theta the secretion of necrotizing toxins and hyaluronidase, favor toxin has similar hemolytic and necrotizing effects but is not the spread of infection. Tissue necrosis extends, providing an a lecithinase. It is a member of the cholesterol-dependent opportunity for increased bacterial growth, hemolytic ane- cytolysins that act by forming pores in cell membranes. Epsi- mia, and, ultimately, severe toxemia and death. lon toxin is a protein that causes edema, and hemorrhage is In gas gangrene (clostridial myonecrosis), a mixed infec- very potent. DNase and hyaluronidase, a collagenase that tion is the rule. In addition to the toxigenic clostridia, proteo- lytic clostridia and various cocci and Gram-negative organisms are also usually present. C. perfringens occurs in the genital tracts of 5% of women. Before legalization of abortion in the United States, clostridial uterine infections followed instru- mented abortions. C. sordellii has many of the properties of C. perfringens. C. sordellii has been reported to cause a toxic shock syndrome after medical abortion with mifepristone and intravaginal misoprostol. Endometrial infection with C. sordellii is implicated. Clostridial bacteremia, especially that caused by C. septicum, is a frequent occurrence in patients with neoplasms. In New Guinea, C. perfringens type C pro- duces necrotizing enteritis (pigbel) that can be highly fatal in children. Immunization with type C toxoid appears to have preventive value. Clinical Findings From a contaminated wound (eg, a compound fracture, postpartum uterus), the infection spreads in 1–3 days to produce crepitation in the subcutaneous tissue and muscle, foul-smelling discharge, rapidly progressing necrosis, fever, FIGURE 11-3 Gas gangrene bacilli. C. perfringens typically does hemolysis, toxemia, shock, and death. Treatment is with early not form spores when grown on laboratory media. surgery (amputation) and antibiotic administration. Until Riedel_CH11_p183-p194.indd 190 04/04/19 4:44 PM  CHAPTER 11 Spore-Forming Gram-Positive Bacilli: Bacillus and Clostridium Species   191 the advent of specific therapy, early amputation was the only immunization have been prepared, they have not come into treatment. At times, the infection results only in anaerobic practical use. fasciitis or cellulitis. C. perfringens food poisoning usually follows the inges- tion of large numbers of clostridia that have grown in warmed meat dishes. The toxin forms when the organisms sporulate CLOSTRIDIUM DIFFICILE AND in the gut, with the onset of diarrhea—usually without vom- DIARRHEAL DISEASE iting or fever—in 7–30 hours. The illness lasts only 1–2 days. Pseudomembranous Colitis Pseudomembranous colitis is diagnosed by detection of one Diagnostic Laboratory Tests or both C. difficile toxins in stool and by endoscopic observa- Specimens consist of material from wounds, pus, and tissue. tion of pseudomembranes or microabscesses in patients who The presence of large Gram-positive rods in Gram-stained have diarrhea and have been given antibiotics. Plaques and smears suggests gas gangrene clostridia; spores are not regu- microabscesses may be localized to one area of the bowel. larly present. The diarrhea may be watery or bloody, and the patient fre- Material is inoculated into chopped meat–glucose quently has associated abdominal cramps, leukocytosis, and medium and thioglycolate medium and onto blood agar fever. Although many antibiotics have been associated with plates incubated anaerobically. After pure cultures have been pseudomembranous colitis, the most common are ampicillin obtained by selecting colonies from anaerobically incubated and clindamycin and, more recently, the fluoroquinolones. blood plates, they are identified by biochemical reactions The disease is treated by discontinuing administration of the (various sugars in thioglycolate, action on milk), hemolysis, offending antibiotic and orally giving metronidazole, vanco- and colony morphology. Lecithinase activity is evaluated by mycin, or fidaxomicin. Fecal transplantation has become a the precipitate formed around colonies on egg yolk media. successful and routine method for recurrent and refractory Matrix-assisted laser desorption/ionization time-of-flight disease. This usually involves administration of the feces of mass spectrometry (MALDI-TOF MS) is a rapid and sensi- a healthy related donor by way of colonoscopy or less com- tive method for identification of invasive Clostridium species monly via a nasogastric tube into the gastrointestinal tract of recovered in culture. C. perfringens rarely produces spores the patient. when cultured on agar in the laboratory. Administration of antibiotics results in proliferation of drug-resistant C. difficile that produces two toxins. Toxin A, a potent enterotoxin that also has some cytotoxic activity, Treatment binds to the brush border membranes of the gut at receptor The most important aspect of treatment is prompt and exten- sites. Toxin B is a potent cytotoxin. C. difficile toxins have sive surgical debridement of the involved area and excision glycosyltransferase activity and act by modifying signal- of all devitalized tissue, in which the organisms are prone to ing molecules that control various cellular functions. This grow. Administration of antimicrobial drugs, particularly results in apoptosis, capillary leakage, cytokine stimulation, penicillin, is begun at the same time. Hyperbaric oxygen may and other consequences that lead to colitis. Both toxins are be of help in the medical management of clostridial tissue usually found in the stools of patients with pseudomembra- infections. It is said to “detoxify” patients rapidly. nous colitis. However, toxin A–negative, toxin B–positive Antitoxins are available against the toxins of C. perfrin- infections have been described. Not all strains of C. difficile gens, C. novyi, C. histolyticum, and C. septicum, usually in produce the toxins, and the toxin genes are found on a large, the form of concentrated immune globulins. Polyvalent chromosomal pathogenicity island along with three other antitoxin (containing antibodies to several toxins) has been genes that regulate toxin expression. used. Although such antitoxin is sometimes administered Diagnosis is made clinically and supported by demon- to individuals with contaminated wounds containing much stration of toxin in the stool by a variety of methods that devitalized tissue, there is no evidence for its efficacy. Food includes anaerobic toxigenic culture, enzyme immunoassay, poisoning caused by C. perfringens enterotoxin usually and molecular tests that detect the genes that encode toxins requires only symptomatic care. A or B. See the reference by Burnham for a more complete discussion of C. difficile diagnosis. The surge in C. difficile infections since the beginning Prevention and Control of the 21st century is believed to be related to a combina- Early and adequate cleansing of contaminated wounds and tion of host and organism factors. The responsible host fac- surgical debridement, together with the administration of tors include the aging population, the increase in survival antimicrobial drugs directed against clostridia (eg, peni- of immunocompromised susceptible individuals, and the cillin), are the best available preventive measures. Antitox- increase in administration of antibiotics and gastric acid- ins should not be relied on. Although toxoids for active suppressant agents. Organism factors relate primarily to Riedel_CH11_p183-p194.indd 191 04/04/19 4:44 PM  192   SECTION III  Bacteriology emergence of certain strain types that are more virulent due (A) Protective antigen to mutations in the pathogenicity locus. (B) Lipopolysaccharide (C) Pili (D) A toxin that inhibits peptide chain elongation factor EF-2 Antibiotic-Associated Diarrhea (E) Lecithinase The administration of antibiotics frequently leads to a mild 3. A young man sustains major soft tissue injury and open frac- tures of his right leg after a motorcycle accident. One day later, to moderate form of diarrhea, termed antibiotic-associated he has a temperature of 38°C, increased heart rate, sweating, diarrhea. This disease is generally less severe than the classic and restlessness. On examination, the leg is swollen and tense, form of pseudomembranous colitis. As many as 25% of cases with thin, dark serous fluid draining from the wounds. The of antibiotic-associated diarrhea are caused by C. difficile skin of the leg is cool, pale, white, and shining. Crepitus can be infection. Other Clostridium species such as C. perfringens felt in the leg. His hematocrit is 20% (∼50% of normal), and his and C. sordellii have also been implicated. The latter two spe- circulating hemoglobin is normal. His serum shows free hemo- cies are not associated with pseudomembranous colitis. globin. Which of the following microorganisms is the most likely cause of this infection? (A) Clostridium tetani Concept Checks (B) Staphylococcus aureus Clostridium species are large, spore-forming, anaerobic (C) Escherichia coli (D) Bacillus anthracis Gram-positive rods that are found in the environment and (E) Clostridium perfringens in the gastrointestinal tracts of a large number of animals 4. For the patient described in Question 3, which of the following and humans. is likely to be responsible for the hemolysis? The clostridia are categorized by their ability to ferment (A) Elongation factor carbohydrates and to digest proteins as well as by the tox- (B) Tetanospasmin ins they produce. (C) Lecithinase Toxins produced by pathogenic clostridia are responsible (D) Streptolysin O for a variety of serious diseases that include botulism, teta- (E) Toxin B nus, and gas gangrene. 5. The reported incubation period for inhalational anthrax can be C. botulinum produces botulinum toxin, one of the most up to potent neurotoxins on the planet, responsible for botulism, (A) 2 days a disease characterized by flaccid paralysis. (B) 10 days C. tetani also produces a neurotoxin, tetanospasmin, that (C) 3 weeks blocks release of inhibitory neurotransmitters resulting in (D) 6 weeks tetanus, a disease characterized by spastic paralysis. (E) 6 months Other Clostridium species cause invasive wound infections 6. A food commonly associated with Bacillus cereus food poison- (gangrene), septicemia, antibiotic-associated diarrhea, and ing is food poisoning depending on the epidemiologic circum- (A) Fried rice stances and the types of enzymes or toxins elaborated. (B) Baked potato (C) Hot freshly steamed rice (D) Green beans (E) Honey REVIEW QUESTIONS 7. Tetanus toxin (tetanospasmin) diffuses to terminals of inhibi- 1. A housewife who lives on a small farm is brought to the emer- tory cells in the spinal cord and brainstem and blocks which of gency department complaining of double vision and difficulty the following? talking. Within the past 2 hours, she noted a dry mouth and (A) Release of acetylcholine generalized weakness. Last night she served home-canned (B) Cleavage of SNARE proteins green beans as part of the meal. She tasted the beans before (C) Release of inhibitory glycine and γ-aminobutyric acid they were boiled. None of the other family members are ill. On (D) Release of protective antigen examination, there is symmetrical descending paralysis of the (E) Activation of acetylcholine esterase cranial nerves, upper extremities, and trunk. The correct diag- 8. A 45-year-old man who immigrated to the United States 5 years nosis is which one of the following? ago sustained a puncture injury to the lower part of his right (A) Tetanus leg when his rotary lawn mower threw a small stick into his leg. (B) Strychnine poisoning Six days later, he noticed spasms in the muscles of his right leg; (C) Botulism on day 7, the spasms increased. Today—day 8—he had general- (D) Morphine overdose ized muscle spasms, particularly noticeable in the muscles of his (E) Ricin intoxication jaw. He was unable to open his jaw and came to the emergency 2. Which one of the following is an important virulence factor of department (ED). In the ED, you see a man who is alert and Bacillus anthracis? lying quietly in bed. A door slams down the hall, and suddenly Riedel_CH11_p183-p194.indd 192 04/04/19 4:44 PM  CHAPTER 11 Spore-Forming Gram-Positive Bacilli: Bacillus and Clostridium Species   193 he has general muscle spasm with arching of his back. The cor- Answers rect diagnosis is which of the following? 1. C 5. D 9. E (A) Botulism 2. A 6. A 10. C (B) Anthrax 3. E 7. C 11. D (C) Gas gangrene 4. C 8. D 12. B (D) Tetanus (E) Toxic shock syndrome 9. Which of the following statements about tetanus and tetanus toxoid is correct? REFERENCES (A) Tetanus toxin kills neurons. Abbara A, Brooks T, Taylor GP, et al: Lessons for control of heroin- (B) Tetanus toxoid immunization has a 10% failure rate. associated anthrax in Europe from 2009-2010 outbreak case (C) The mortality rate of generalized tetanus is less than 1%. studies, London, UK. Emerg Infect Dis 2014;20:1115–1122. (D) Double vision is commonly the first sign of tetanus. Aronoff DM: Clostridium novyi, sordellii, and tetani: mechanisms (E) Tetanus toxin acts on inhibitor interneuron synapses. of disease. Anaerobe 2013;24:98–101. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Micro- 10. A 67-year-old man had surgery for a ruptured sigmoid colon biol Rev 2010;23:382–398. diverticulum with an abscess. A repair was done, and the Burnham CA, Carroll KC: Diagnosis of Clostridium difficile infec- abscess was drained. He was treated with intravenous gen- tion: an ongoing conundrum for clinicians and for clinical tamicin and ampicillin. Ten days later and 4 days after being laboratories. Clin Microbiol Rev 2013;26:604–630. discharged from the hospital, the patient developed malaise, Campbell JR, Hulten K, Baker CJ: Cluster of Bacillus species bacte- fever, and cramping abdominal pain. He had multiple episodes remia cases in neonates during a hospital construction project. of diarrhea. His stool was positive for occult blood and the Infect Control Hosp Epidemiol 2011;32:1035–1038. presence of polymorphonuclear cells. On sigmoidoscopy, the Hendricks KA, Wright ME, Shadomy SV, et al: Centers for dis- mucosa was erythematous and appeared to be inflamed, and ease control and prevention expert panel meetings on pre- there were many raised white to yellowish plaques 4–8 mm vention and treatment of anthrax in adults. Emerg Infect Dis in diameter. Which of the following is the likely cause of the 2014;20:e130687. patient’s problem? Kalka-Moll WM, Aurbach U, Schaumann R, et al: Wound botu- (A) Staphylococcus aureus enterotoxin lism in injection drug users. Emerg Infect Dis 2007;13:942–943. (B) Bacillus cereus toxin Kummerfeldt CE. Raxibacumab: potential role in the treatment of (C) Clostridium difficile toxins inhalational anthrax. Infect Drug Resist 2014;7:101–109. (D) Clostridium perfringens toxin Liu S, Moayeri M, Leppla SH: Anthrax lethal and edema toxins in (E) Enterohemorrhagic Escherichia coli anthrax pathogenesis. Trends Microbiol 2014;22:317–325. 11. Which of the following food items is most frequently associated Reddy P, Bleck TP: Clostridium tetani (Tetanus). In Mandell GL, with infant botulism? Bennett JE, Dolin R (editors). Mandell, Douglas and Bennett’s (A) Corn syrup Principles and Practice of Infectious Diseases, 8th ed. Churchill (B) Canned infant formula Livingstone, 2015. (C) Liquid multivitamins Rossetto O, Pirazzini M, Montecucco C: Botulinum neurotoxins: (D) Honey genetic, structural and mechanistic insights. Nat Rev Microbiol (E) Jarred baby food 2014;12:535–540. 12. Which of the following statements regarding vaccination for Stevens DL, Bryant AE, Carroll K: Clostridium. In Versalovic J, Bacillus anthracis is correct? Carroll KC, Funke G, et al (editors). Manual of Clinical Micro- (A) It is routinely available for all citizens of the United States. biology, 11th ed. ASM Press, 2015. (B) Recombinant vaccine trials have shown good safety and efficacy. (C) The current vaccine is well tolerated. (D) A single dose is adequate after exposure to spores. (E) Vaccination of animals is not useful. Riedel_CH11_p183-p194.indd 193 04/04/19 4:44 PM