Diagnostic microbiology lecture 12 Gram Positive EndosporeForming Bacilli

Diagnostic microbiology lecture: 12 Gram Positive, Endospore-Forming Bacilli CLOSTRIDIA Abed El. Kader Elottol MSc. Microbiology 2010 1

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GENERAL CHARACTERISTICS: 1. Anaerobic gram-positive bacilli 2. Spore formers 3. They decompose proteins 4. Exotoxin producers 5. Natural habitat; Soil or the intestinal tract of animals and man. 6. Some motile, others are non-motile. 7. Catalase negative. 4

SPECIES OF MEDICAL IMPORTANCE: 1. Clostridium tetani 2. Clostridium perfringens 3. Clostridium botulinum 4. Clostridium septicum 5. Clostridium dificilli 6. Clostridium novyi 5

Clostridium tetani 6

Disease: Tetanus or "lock jaw" • Cl. tetani is not an invasive organism but rather remains strictly localized in the area of the affected tissue into which the spores has been introduced. • The disease is transmitted to man through infected wounds. • Spores enter into wounds with soil or material contaminated with soil. • The infection is localized in the area of entry. • Growth and toxin production is aided by the followings: 1. Necrotic tissue 2. Calcium salts 3. Associated pyogenic infection. These conditions aids in providing anaerobic conditions. 7

• The toxin "tetanospasmin" is heat-labile protein, inactivated by heating for 5 minutes at 65 o. C. • The toxin may reach the central nervous system. • It blocks the release of inhibitory mediators of the motor neurons. • This results in extreme hyper-reflexia and violent spasm of skeletal muscles in response to any stimuli. • This toxin is inactivated by the proteolytic enzymes of the digestive system. • 1 mg pure toxin has 6 million lethal mice doses. 8

. People have become infected with tetanus following surgery, burns, abrasions, crush wounds, ear infections, dental infections, animal bites, abortion, pregnancy, body piercing and tattooing, and injection drug use. People can also get tetanus from splinters.

The incubation period varies from 3 -21 days, most cases within 14 days with an average of eight days. The further the injury site is from the central nervous system, the longer the incubation period. The shorter the incubation period, the higher the risk of death

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Neonatal tetanus

Neonatal tetanus This baby has neonatal tetanus. It is completely rigid. Tetanus kills most of the babies who get it. Infection usually happens when newly cut umbilical cord is exposed to dirt

This baby has tetanus. He cannot breast feed or open his mouth because the muscles in his face have become so tight

Morphology: 1. Gram-positive anaerobic 2. Motile with peritrichous flagella 3. Terminal rounded spores (Drumstick appearance). Media of Choice: • Any medium with thioglycollate under anaerobic condition most especially Cooked Meat Media. 15

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TREATMENT Tetanus is a life-threatening disease Treatment consists of two main steps : • First, the patient is given antibiotics to kill the bacteria Metronidazole Or penicillin • Second, injections of antitoxin are also given. An antitoxin is a substance that reacts with and destroys the toxin. • Patients may also need medication to control muscle spasms. • In severe cases, the patient may have to be placed on artificial respiration. • Recovery takes six weeks or more. • After a patient is better, he or she should receive the tetanus vaccine to protect against future episodes of the disease.

vaccination : • combination of vaccines that protect against three diseases: , Diphtheria, Tetanus and Pertussis DTP vaccine • The vaccine is given in five doses at the ages of two months, four months, six months, fifteen to eighteen months, and four to six years. • Adults should receive a booster shot against tetanus every years ten • A booster shot is a dose of the vaccine that renews a person's resistance to the disease 19

• Pregnant also should receive vaccine to prevent neonatal tetanus for their babies during cut umbilical cord • Make sure that wounds are thoroughly cleaned. • A wound should also be treated with an antibiotic cream and covered with a bandage. • Wounds that don't heal should be examined by a doctor 20

Clostridium perfringens 21

Disease: Gas gangrene • It causes a variety of infection to man: 1. Wound infection 2. Gas gangrene (Myonecrosis) 3. Necrotizing jejunitis 4. Food poisoning 5. Meningitis 22

Four types of toxins are produced: 1. Alpha = It is a phospholipase C which hydrolysis lecithin. Disrupts cell membranes of RBCs, WBCs & muscle cells. 2. Beta = Is necrotic for the intestinal mucosa and lethal to the CNS. 3. Epsilon = Inactive which is converted to the active form by trypsin in small intestine. pulmonary edema, neurologic symptoms, or gastroenteritis could be seen. 4. Iota = Similar to epsilon but differ in structure 23

Subdivided into 5 types based on the four major lethal toxins they produce. Type A causes most of the human infections. 24

Minor Major

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C. perfringens Pathogenesis Spore Heat Free Spore Vegetative cell Food Developing Spore Toxin inclusion body Free toxin GI tract Cell wall lysis C. perfringens gastroenteritis can include diarrhea, nausea, severe abdominal cramps and bloating for 1 -2 days. Vomiting and fever are not usually seen. 27

Food Poisoning Spore Contamination Cooking Slow cooling and/or storage at moderate temperature Germination Disease cycle Environment GI illness Ingestion Rapid proliferation

Pathology • Not highly invasive; requires damaged and dead tissue and anaerobic conditions. • Conditions stimulate spore germination, vegetative growth and release of exotoxins, and other virulence factors. • Fermentation of muscle carbohydrates results in the formation of gas and further destruction of tissue.

Morphological Characteristics: = Spores are usually oval and central or subterminal. = Not strictly anaerobe and can survive exposure to oxygen for short periods of time and sometimes referred to as aerotolerant anaerobe. = Non-motile = They form capsule in infected patients and animals. – Collagenase, hyaluronidase, and Dnase Enzymes Cultural Characteristics: • On blood agar plates: Colonies are round, domed and grayish white, surrounded by a zone of hemolysis. 31

Micro & Macroscopic C. perfringens NOTE: Large rectangular gram-positive bacilli NOTE: Double zone of hemolysis Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin

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Clostridium botulinum 35

BOTULISM: • It is a disease caused by the ingestion of food containing the neurotoxin produced by C. botulinum. • The earliest symptoms usually are an acute digestive disturbances followed by nausea and vomiting and possibly diarrhea, together with fatigue, dizziness and headache. • Later there is constipation. • Double vision may be evident early and difficulty in swallowing and speaking may be noted. • Patient may complain of dryness of the mouth and constriction of the throat. 36

• Involuntary muscles become paralyzed, paralysis spread to the respiratory system and heart, the death usually results from respiratory failure. INFANT BOTULISM Organisms introduced with dietary supplement Organisms multiply in colon with absorption of small amount of toxin Infant suffers from constipation & feeding problems One of the causes of sudden infant death. Wound botulism • is caused by neurotoxin produced from a wound that is infected with the bacteria Clostridium botulinum. 37

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Toxicity: • Botulinum toxin is heat-labile, if heated to a certain temperature so can be destroyed if heated and held at 80 C for 10 minutes or longer. • Botulinum toxin is the most powerful toxin known to man (1 mg pure toxin has a 20 million lethal mice doses). Toxine Classification: 8 types has been identified (A-H). Botox Only A, B, E, and F affects human. 1. Type A: Most common in western parts of US. More toxic than B. 2. Type B: Less toxic most frequent in occurrence. 3. Type E: Obtained from fish and fish products. 4. Type F: Similar to A and B. Has been isolated in Denmark. 39

Morphological Characteristics: üSpores are oval, and subterminal üMotile with peritrichous flagella 40

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Clostridium septicum 43

• This organism sometimes causes rapidly progressive cases of Clostridial myonecrosis • associated with severe wounds. • Apparently, C. septicum, occasionally escapes from wounds and is carried by the blood stream to other parts of the body. • Generalized C. septicum infection may occur in leukemia patients or by the administration of antimetabolite or other immunosuppressive drugs. 44

Clostridium difficile • Part of the normal flora of the human gut. • It is the major cause of Pseudomembranous colitis that sometimes follows the administration of broad spectrum antibiotics for prolonged periods. • Only when the other anaerobes in the intestine are killed or inhibited by certain antimicrobials, does C. difficile grow in sufficient numbers, and produce sufficiently great amount of toxin to cause colitis. 45

This organism produces two toxins: • Toxin A (an enterotoxin) induces hypersecretion of fluid, and development of hemorrhagic necrosis. • Toxin B (a cytotoxin) causes tissue damage. • Disease occurs if the organism proliferates in the colon and produces toxins: watery or bloody diarrhea, abdominal cramps, leukocytosis and fever. 46

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Clostridium novyi (A & B) • This organism is rarely associated with human infection other than clostridial myonecrosis. • Type A strains are much more common than type B. • Type B strain, almost unknown in human infections. • They are primarily pathogens of sheep. 48

ISOLATION OF CLOSTRIDIA 49

1. From Clinical Sample that are Usually Sterile (e. g. , Blood) • Inject the specimen in Blood Culture bottles with CO 2 and incubate anaerobically in an anaerobic jar. • Subcultures onto freshly prepared Blood Agar are made and incubated anaerobically. 2. From Clinical Specimens that Contain a Mixture of Organisms (e. g. , Abscess): A. Heat Treatment: • Specimen is inoculated to a tube of chopped-meat broth medium. • Heat at 80 o. C for 10 minutes before incubating anaerobically. • Incubate overnight and subculture to Blood Agar and Egg-Yolk Agar. 50

B. Alcohol Treatment: • 1 ml of the specimen is placed in a sterile screw-capped bottle or tube, add an equal amount of 50% ethanol for one hour. • Pick up some of the treated materials, and inoculate egg yolk agar and blood agar and incubate anaerobically. • A good selective media is Neomycin-Egg-Yolk Agar. NB: Some Clostridium species may swarm. To prevent swarming, the amount of gar is increased to 4 -6%. NB: When growth is evident inoculate the following differential media for identification: 51

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IDENTIFICATION OF CLOSTRIDIA: ØTo differentiate between Clostridia and Bacillus especially B. cereus which grow well under anaerobic conditions. Øcatalase test is performed which is negative for Clostridia and positive for Bacillus. ØC. perfringens produces lecithinase (phospholipase C). ØOther species produces lipase that break down fatty acids. 53

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• Identification of toxigenic strains is done in a similar way to that of diphtheria by toxin neutralization in Vivo. • If C. perfringens is to be specifically isolated, incubate the plates at 44 -46 o. C. TREATMENT 1. TETANUS: Since treatment of tetanus are not satisfactory, prevention is all important. Prevention of tetanus depends upon: 1. Active immunization with toxoids 2. Proper care of wounds, contaminated with soil 3. Prophylactic use of antitoxin 4. Administration of penicillin. 55

2. BOTULISIM: • Potent antitoxin to 3 types of botulinus toxins have been prepared. • Trivalent (A, B, E) antitoxin is administered intravenously as early as possible to neutralize the effect of the toxin. 3. GAS GANGRENE: Treatment of the disease is done by : 1. Surgical measures to remove all devitalized tissues. 2. Administration of penicillin 3. Administration of antitoxin. 4. Infection due to C. septicum and C. novyi : Similar to gas gangrene 5. Pseudomembraneous colitis: Administration of oral vancomycin is effective in reducing the number of this organism in the human intestine. 56

Di glycride formation Lactose egg yolk milk agar 57

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END of LECTURE 59