1. Anaerobic, Spore-forming, Gram-Positive Rods 미생물학교실 권 형 주

2. Clostridium - anaerobic, gram-positive rods capable of forming endospores (1) presence of endospores, (2) strict anaerobic metabolism, (3) inability to reduce sulfate to sulfite, (4) gram-positive cell wall structure. - Spores are rarely :Clostridium perfringens, Clostridium ramosum, - Aerotolerant : Clostridium tertium, Clostridium histolyticum - Some clostridia gram-negative : C. ramosum, Clostridium clostridioforme BOX 39-1. Important Clostridia OrganismHistorical Derivation Clostridiumcloster, a spindle C. botulinumbotulus, sausage (the first major outbreak was associated with insufficiently smoked sausage) C. difficiledifficile, difficult (difficult to isolate and grow; refers to the extreme oxygen sensitivity of this organism) C. perfringensperfringens, breaking through (associated with highly invasive tissue necrosis) C. septicumsepticum, putrefactive (associated with sepsis and a high mortality) C. tertiumtertium, third (historically, the third most commonly isolated anaerobe from war wounds C. tetanitetani, related to tension (disease caused by this organism characterized by muscle spasms)

3. - The remarkable capacity of clostridia to cause diseases (1)ability to survive adverse environmental conditions through spore formation; (2) rapid growth in a nutritionally enriched, oxygen-deprived environment, (3) production of numerous histolytic toxins, enterotoxins, and neurotoxins. - Ubiquitious – soil, water, sewage - Tetanus - Botulism - Gas gangrene - Diarrhea and colitis

4. SpeciesHuman DiseaseFrequency C. difficileAntibiotic-associated diarrhea, pseudomembranous colitisCommon C. perfringensSoft-tissue infections (e.g., cellulitis, suppurative myositis, myonecrosis, gas gangrene), food poisoning, enteritis necroticans, septicemia Common C. septicumGas gangrene, septicemiaUncommon C. botulinumBotulismUncommon C. tetaniTetanusUncommon C. tertiumOpportunistic infectionsUncommon C. baratiiBotulismRare C. butyricumBotulismRare C. clostridioformeOpportunistic infectionsRare C. histolyticumGas gangreneRare C. innocuumOpportunistic infectionsRare C. novyiGas gangreneRare C. sordelliiGas gangreneRare C. sporogenesOpportunistic infectionsRare Table 39-1. Pathogenic Clostridia and Their Associated Human Diseases*

5. Clostridium Perfringens PHYSIOLOGY AND STRUCTURE - large (0.6 to 2.4 × 1.3 to 19.0 μm), rectangular, gram-positive rod - spores rarely observed either in vivo or after in vitro cultivation. - nonmotile clostridia - rapidly spreading growth on laboratory media : organism grows rapidly in tissues and in culture, is hemolytic, and is metabolically active, Growth of Clostridium perfringens on sheep blood agar. - flat, spreading colonies and the hemolytic activity of the organism. - zone of complete hemolysis (caused by the θ-toxin) - wider zone of partial hemolysis (caused by the α-toxin)

6. TypeLethal Toxins AlphaBetaEpsilonIota A+--- B+++- C++-- D+-+- E+--+ Table Distribution of Lethal Toxins in Clostridium perfringens Types A to E - The production of one or more major lethal toxins by C. perfringens (alpha [α], beta [β], epsilon [ε], and iota [ι] toxins) - subdivide isolates into five types (A through E). PATHOGENESIS AND IMMUNITY

7. Virulence FactorsBiologic Activity α-ToxinLethal toxin; phospholipase C (lecithinase); increases vascular permeability; hemolysin; produces necrotizing activity, as seen in myonecrosis β-ToxinLethal toxin; necrotizing activity ε-ToxinLethal toxin; permease ι-ToxinLethal binary toxin; necrotizing activity; adenosine diphosphate (ADP) ribosylatingadenosine δ-ToxinHemolysin θ-ToxinHeat- and oxygen-labile hemolysin; cytolytic κ-ToxinCollagenase; gelatinase; necrotizing activity λ-ToxinProtease μ-ToxinHyaluronidase ν-ToxinDeoxyribonuclease; hemolysin; necrotizing activity EnterotoxinAlters membrane permeability in ileum (cytotoxic, enterotoxic); superantigen NeuraminidaseAlters cell surface ganglioside receptors; promotes capillary thrombosis Table. Virulence Factors Associated with Clostridium perfringens

8. α-Toxin : Lethal toxin; phospholipase C (lecithinase); increases vascular permeability; hemolysin; produces necrotizing activity, as seen in myonecrosis -Growth of Clostridum perfringens on egg-yolk agar. -The α-toxin (lecithinase) hydrolyzes phospholipids in serum and egg yolk, producing an opaque precipitate (right). -This precipitate is not observed when the organism is grown in the presence of antibodies against the toxin (left). - Nagler's reaction is characteristic of C. perfringens. - Lecithinase (phospholipase C) : lyses erythrocytes, platelets, leukocytes, and endothelial cells. : massive hemolysis : increased vascular permeability and bleeding (augmented by destruction of platelets) : tissue destruction (as found in myonecrosis) : hepatic toxicity, and myocardial dysfunction (bradycardia, hypotension)

9. - responsible for intestinal stasis, loss of mucosa with formation of the necrotic lesions, and progression to necrotizing enteritis (enteritis necroticans 괴사성 소장대장염, pig-bel). ε-Toxin, Lethal toxin; permease, a protoxin, is activated by trypsin and increases the vascular permeability of the gastrointestinal wall. ι-Toxin, the fourth major lethal toxin, Lethal binary toxin; necrotizing activity; adenosine diphosphate (ADP) ribosylating, is produced by type E C. perfringens. This toxin has necrotic activity and increases vascular permeability.adenosine β-Toxin : Lethal toxin; necrotizing activity Enterotoxin - Type A strains. : Alters membrane permeability in ileum (cytotoxic, enterotoxic); superantigen : produced during the phase transition from vegetative cells to spores : released with the formed spores when the cells undergo the terminal stages of spore formation (sporulation). : released enterotoxin binds to receptors on the brush border membrane of the small intestine epithelium in the ileum (primarily) and jejunum but not duodenum. : altered membrane permeability and loss of fluids and ions. : acts as a superantigen simulating T lymphocyte activity.

10. EPIDEMIOLOGY - inhabits the intestinal tract of humans and animals -widely distributed in nature, particularly in soil and water contaminated with feces -Spores are formed under adverse environmental conditions and can survive for prolonged periods. - Type A C. perfringens : responsible for most human infections, including soft- tissue infections, food poisoning, and primary septicemia. - Type C C. perfringens : responsible for one other important infection in humans-enteritis necroticans ( 괴사성장염 )

11. CLINICAL DISEASES Soft-Tissue Infections ( 연조직감염 ) - Clostridia can be introduced into tissue during surgery or by a traumatic injury. - Five days after the injury, the skin became discolored and bullae and necrosis developed. (1)cellulitis ( 무산소성 봉소염 ) : gas formation in the soft tissue (2) suppurative myositis ( 화농성근염 ) : accumulation of pus in the muscle planes, but muscle necrosis and systemic symptoms are absent. (3) myonecrosis ( 근괴사 ) or gas gangrene ( 가스괴저 ). Clostridial myonecrosis : life-threatening disease, histotoxic clostridia - intense pain, - develops within a week after clostridia are introduced into tissue by trauma or surgery - rapidly by extensive muscle necrosis, shock, renal failure, an death, often within 2 days of initial onset. - Gas found in the tissue is caused by the metabolic activity of the rapidly dividing bacteria (hence the name gas gangrene)

12. Food Poisoning (1)a short incubation period (8 to 24 hours), (2)a clinical presentation that includes abdominal cramps and watery diarrhea but no fever, nausea, or vomiting (3)a clinical course lasting 24 to 48 hours. -ingestion of meat products (e.g., beef, chicken, turkey) contaminated with large numbers (10 8 to 10 9 organisms) of enterotoxin-containing type A C. perfringens. -Holding contaminated foods at temperatures below 60°C (46°C is optimal) allows spores that survived the cooking process to germinate and multiply to high numbers. -The refrigeration of food after preparation prevents this bacterial growth. Alternatively, reheating of the food can destroy the heat-labile enterotoxin.

13. Necrotizing Enteritis (enteritis necroticans or pig-bel) - rare, acute necrotizing process in the jejunum characterized by acute abdominal pain, vomiting, bloody diarrhea, ulceration of the small intestine, and perforation of the intestinal wall, leading to peritonitis( 복막염 ) and shock. - mortality in patients : 50%. - β-Toxin produced by C. perfringens Type C - Necrotizing enteritis is most common in Papua New Guinea : eating undercooked, contaminated pork with sweet potatoes : heat-resistant trypsin inhibitor  protects the β-toxin from inactivation by trypsin. Septicemia - When C. perfringens is isolated in the blood from patients with significant infections (e.g., myonecrosis, necrotizing enteritis), the organism is typically associated with massive hemolysis.

14. LABORATORY DIAGNOSIS - Microscopic detection of gram-positive rods in clinical specimens, in the absence of leukocytes, characteristic morphology. - culture anaerobes. - growth on agar media or in blood culture broths - food poisoning : more than 10 5 organisms/g food or more than 10 6 bacteria/g feces - Immunoassays : detection of the enterotoxin in fecal specimens TREATMENT, PREVENTION, AND CONTROL - C. perfringens soft tissue infections (suppurative myositis and myonecrosis) : treated aggressively with surgical débridement and high-dose penicillin therapy : Hyperbaric oxygen treatment - results are inconclusive - Antibiotic therapy for clostridial food poisoning is unnecessary : self-limiting disease (i.e., the diarrhea washes the bacteria out of the intestines and the normal intestinal flora reestablishes itself). - Prevention and control of C. perfringens infections are difficult : organisms are ubiquitous. - wound care and the judicious use of prophylactic antibiotics

15. Clostridium tetani PHYSIOLOGY AND STRUCTURE - large (0.5 to 1.7 × 2.1 to 18.1 μm), motile, - spore-forming rod (round, terminal spores : drumstick) - extremely sensitive to oxygen toxicity, strict anaerobe - when growth is detected on agar media, it typically appears as a film over the surface of the agar rather than discrete colonies - proteolytic but unable to ferment carbohydrates. -Tetani : related to tension (disease caused by this organism characterized by muscle spasms) PATHOGENESIS AND IMMUNITY Two toxins 1) tetanolysin : oxygen-labile hemolysin, clinical significance unknown, inhibited by oxygen and serum cholesterol 2) tetanospasmin : plasmid-encoded, heat-labile neurotoxin nonconjugative.

16. Tetanospasmin (A-B toxin) :150,000Da - Endogenous protease : cleavage - Disulfide bond and noncovalent forces hold the two chains together -B-chain (heavy chain) : carbohydrate-binding domain, carboxyl-terminal portion, binds to specific sialic acid receptors (e.g., olysialogangliosides) and adjacent glycoproteins on the surface of motor neurons. - endosomal vesicles and transported in the neuron axon to motor neuron soma located in the spinal cord  endosome  acidified  conformational change of the heavy chain  passage of the toxin light chain into the cytosol - A-chain (light chain) : zinc endopeptidase  cleaves core proteins involved in the trafficking and release of neurotransmitters  inactivates inhibitory neurotransmitters glycine and gamma-aminobutryic acid (GABA)  spastic (rigid) paralysis ( 경련성 마비 ) : toxin binding is irreversible, so recovery depends on whether new axonal terminals form

17. EPIDEMIOLOGY -ubiquitous, fertile soil, transiently colonizes the gastrointestinal tracts of many animals - relatively rare : high incidence of vaccine-induced immunity - many deaths in developing countries where vaccination is unavailable or medical practices are lax, more than 1 million cases occur worldwide, with a mortality rate ranging from 30% to 50% CLINICAL DISEASES Generalized tetanus ( 전신성 파상풍 ) : most common form - Involvement of the masseter muscles ( 깨물근, trismus or lockjaw, 개구불능, 교경 입이다 물어지지 않음 ), risus sardonicus ( 냉소 ) - early signs : drooling, sweating, irritability, and persistent back spasms ( 경련, 발작 ) (opisthotonos) - autonomic nervous system is involved in patients with more severe disease; cardiac arrhythmias, fluctuations in blood pressure, profound sweating, and dehydration.

18. localized tetanus ( 국소적 파상풍 ) - musculature at the site of primary infection. : cephalic tetanus ( 뇌 파상풍 )- primary site of infection is the head, very poor. Neonatal tetanus (tetanus neonatorum) : initial infection of the umbilical stump  generalized : mortality in infants exceeds 90%, and developmental defects in survivors : almost exclusively a disease in developing countries. LABORATORY DIAGNOSIS - Diagnosis of tetanus : basis of the clinical presentation - Microscopic detection - frequently unsuccessful - Culture results are positive in only approximately 30% of patients with tetanus : disease can be caused by relatively few organisms : slow-growing bacteria are killed rapidly when exposed to air -Neither tetanus toxin nor antibodies to the toxin are detectable in the patient : toxin is rapidly bound to motor neurons and internalized

19. TREATMENT, PREVENTION, AND CONTROL Treatment of tetanus : débridement of the primary wound (which may appear innocuous) : use of metronidazole- eliminate the vegetative bacteria : passive immunization with human tetanus immunoglobulin (TIG-tetanus antitoxin) - binding free tetanospasmin molecules : vaccination with tetanus toxoid (TD) : penicillin should not be used Vaccination : a series of three doses of tetanus toxoid (DPT)  booster doses every 10 years, is highly effective in preventing tetanus.

20. Clostridium botulinum PHYSIOLOGY AND STRUCTURE - heterogeneous group of large (0.6 to 1.4 × 3.0 to 20.2 μm), - fastidious ( 배양하기 까다로운 ), spore-forming, anaerobic rods. - Seven antigenically distinct botulinum toxins (A to G) : human disease is associated with types A, B, E, and F. - Toxin production is associated with specific groups GroupNeurotoxin TypePhenotyptic Properties IA, B, FProteolytic, saccharolytic IIB, E, FNonproteolytic, saccharolytic IIIC, DWeakly proteolytic, saccharolytic IVGWeakly proteolytic, asaccharolytic Table Clostridium botulinum Classification and Toxin Production

21. PATHOGENESIS AND IMMUNITY - Like tetanus toxin - A-B toxin, 150,000-Da : heavy or B chain - large, nontoxic subunit passage through the digestive tract - carboxyl-terminal portion binds specific sialic acid receptors and glycoproteins of motor neurons  stimulates endocytosis of the toxin molecule  Acidification of the endosome  N-terminal, heavy chain mediated release of the light chain : light or A chain - zinc-endopeptidase activity  inactivates the proteins that regulate release of acetylcholine,  blocking neurotransmission at peripheral cholinergic synapses.  flaccid ( 무기력 ) paralysis.

22. - soil and water samples (1)classic or foodborne botulism : consumption of home-canned foods (types A and B toxins) : consumption of preserved fish (type E toxin). (2) infant botulism : consumption of foods (particularly honey) contaminated with botulinum spores. (3) wound botulism - unknown, but the disease is very rare. (4) inhalation botulism. major concern in this era of bioterrorism : Botulinum - concentrated for purposes of aerosolization as a biologic weapon. EPIDEMIOLOGY

23. CLINICAL DISEASES Foodborne Botulism -1 to 2 days after consuming the contaminated food. - initial signs : blurred vision with fixed, dilated pupils( 눈동자 ), dry mouth (indicative of the anticholinergic effects of the toxin), constipation( 변비 ), and abdominal pain. Fever is absent. - flaccid paralysis : Bilateral descending weakness of the peripheral muscles respiratory paralysis - death. Infant Botulism -neurotoxin produced in vivo by C. botulinum colonizing the gastrointestinal tracts of infants. the symptoms are initially nonspecific (e.g., constipation, weak cry, or "failure to thrive"). - flaccid paralysis and respiratory arrest can develop - mortality : very low (1% to 2%) - Sudden infant death syndrome Wound Botulism - toxin production by C. botulinum in contaminated wounds

24. LABORATORY DIAGNOSIS -culture C. botulinum from the feces of patients with foodborne disease and from the implicated food if it is available. - toxin is detected in the serum of more than 90% of infants with botulism. -Isolation of C. botulinum from specimens (feces, food) : heating the specimen for 10 minutes at 80°C to kill all non-clostridial cells  Culture of the heated specimen on nutritionally enriched anaerobic media  the heat-resistant C. botulinum spores to germinate : lipase production  iridescent film on egg-yolk agar, digest milk proteins, hydrolyze gelatin, ferment glucose - Demonstration of toxin production : mouse bioassay (1)C. botulinum is isolated from feces (2) toxin activity is detected in feces or serum. (3) stool cultures in virtually all patients, (4) wound exudate or serum.

25. TREATMENT, PREVENTION, AND CONTROL (1)adequate ventilatory support; Ventilatory support is extremely important in reducing mortality (2)elimination of the organism from the gastrointestinal tract, through the judicious use of gastric lavage and metronidazole or penicillin therapy (3)the use of trivalent botulinum antitoxin versus toxins A, B, and E to bind toxin circulating in the bloodstream.. (1) destroying the spores in food (virtually impossible for practical reasons), (2) preventing spore germination (by maintaining the food in an acid pH or storage at 4°C or colder), (3) destroying the preformed toxin (all botulinum toxins are inactivated by heating at 60°C to 100°C for 10 minutes). (4) Infant botulism has been associated with the consumption of honey contaminated with C. botulinum spores, so children younger than 1 year should not eat honey. Treatment Prevention

26. Clostridium difficile Physiology and Structure - Gram-positive, spore-forming rod, Strict anaerobe Virulence Two toxins 1)enterotoxin (toxin A) - chemotactic for neutrophils, - stimulating the infiltration of polymorphonuclear neutrophils into the ileum with release of cytokines. - cytopathic effect : disruption of the tight cell-cell junction, increased permeability of the intestinal wall, and subsequent diarrhea. 2) cytotoxin (toxin B). - actin to depolymerize, with the resultant destruction of the cellular cytoskeleton both in vivo and in vitro.

27. Table Virulence Factors Associated with Clostridium difficile Virulence FactorBiologic Activity Enterotoxin (toxin A)Produces chemotaxis; induces cytokine production with hypersecretion of fluid; produces hemorrhagic necrosis Cytotoxin (toxin B)Induces depolymerization of actin with loss of cellular cytoskeleton Adhesin factorMediates binding to human colonic cells Hyaluronidase Produces hydrolytic activity Spore formationPermits organism's survival for months in hospital environment EPIDEMIOLOGY - Ubiquitous - normal intestinal flora in a small number of healthy people, hospitalized patients - spores can be detected in hospital rooms of infected patients - disease develops in people taking antibiotics normal enteric flora  overgrowth of resistant organisms or susceptible to the exogenous acquisition of C. difficile -proliferate in the colon and produce their toxins  diseases

28. CLINICAL DISEASES Toxin-producing C. difficile  responsible for antibiotic-associated gastrointestinal diseases : after antibiotic treatment, clindamycin, penicillins, cephalosporins - relatively benign, - self-limited diarrhea to severe, - life-threatening pseudomembranous colitis ( 위막성 대장염 ). Antibiotic-associated colitis: lumen of the colon. white plaques of fibrin, mucus, and inflammatory cells overlying the normal red intestinal mucosa. Antibiotic-associated colitis caused by Clostridium difficile. histologic section of colon shows an intense inflammatory response, with the characteristic "plaque" (black arrow) overlying the intact intestinal mucosa (white arrow)

29. Diagnosis - enterotoxin or cytotoxin in a stool specimen from a patient : immunoassays. : in vivo cytotoxicity assay using tissue culture cells and specific neutralizing antibodies for the cytotoxin Treatment - Discontinuation of the implicated antibiotic (e.g., ampicillin, clindamycin - Severe diarrhea or colitis : specific therapy with metronidazole, vancomycin - Relapses may occur : the spores are resistant  second course of treatment with the same antibiotic is frequently successful. : commonly exists in hospitals, particularly in areas adjacent to infected patients (e.g., beds, bathrooms). : spores of C. difficile are difficult to eliminate