Spore Forming, Gram-Positive Bacilli

Outline: Two types of gram-positive, spore-forming bacilli: 1- Bacillus “aerobic”: a- B. cereus b- B. anthracis 2- Clostridium “anaerobic”: a- C. botulinum b- C. perfringens c- C. difficile d- C. tetanus (not to be taken) Spore Forming, Gram-Positive Bacilli - A spore is a structure produced during unfavorable condition by the process called ‘sporulation’. Under favorable condition spores ‘germinate’ to give vegetative cell which grows and produces toxins. - In this lecture, we will cover 2 gram-positive spore-forming rods (bacilli); Bacillus and Clostridium. - Bacillus and Clostridium both cause disease by the release of potent exotoxins, i.e. they cause toxin mediated diseases. - They differ biochemically, where Bacillus is aerobic, but Clostridium is generally anaerobic. 1- Bacillus Species The genus Bacillus includes large aerobic or facultatively anaerobic, ubiquitous gram-positive, spore forming rods occurring in chains. Some are: - Bacillus cereus and Bacillus subtilis → are saprophytic (gets energy from decaying plants or animals), prevalent in soil, water, and air. - B. thuringiensis → an insect pathogen. - B. anthracis and B. cereus → are the main pathogens of this genus. B anthracis causes the disease anthrax B cereus causes gastroenteritis (food poisoning). “We will only focus on B. cereus” 1|P a ge Bacillus cereus: - Gram-positive aerobic or facultatively anaerobic, motile, spore-forming, rod shaped bacterium that is widely distributed environmentally. - B. cereus is associated mainly with food poisoning. - B. cereus, in immunocompromised patients is fatal, has also been associated with localized and systemic infections, such as: endocarditis, meningitis (in transplant patients), osteomyelitis, and pneumonia. The presence of a medical device or intravenous drug use predisposes to these infections. - Enterotoxins are usually produced by bacteria outside the host (pre-formed toxins) and therefore cause symptoms soon after ingestion of B. cereus. Morphology of B. cereus: - A 3–4 μ m, arranged in long chains; spores are in the center of the motile bacilli. - B. cereus can be differentiated from B. anthracis based on colony morphology, motility and antimicrobial susceptibility patterns: B. Cereus B. Anthracis: Motile and non-encapsulated Non-motile with a glutamic acid capsule Resistant to penicillin and cephalosporins Generally, not resistance to penicillin. Causes food poisoning; nausea, vomiting, and diarrhea. Causes anthrax; a fatal disease of 3 forms: Cutaneous, pulmonary or gastroenteritis. Colonies are β-hemolytic producing Lecithinase, large and more mucoid Colonies are non-hemolytic, flat with a sticky consistency “medusa-head”. Epidemiology: - B. cereus forms heat-resistant spores that contaminate rice mainly or cereals. - B. cereus can be found from the environment or food “rice and cereals”. - The natural environmental reservoir for B. cereus consists of decaying organic matter “saprophytic”, fresh and marine waters, vegetables and fomites, and the intestinal tract of invertebrates, from which soil and food products may become contaminated, leading to the transient colonization of the human intestine if ingested. - Spores germinate when they encounter organic matter, within an insect or animal host. 2|P a ge - Food poisoning occurs when Bacillus cereus deposits its spores in food (rice or cereal), which survives hard conditions (cooking). After that, at room temperature, bacteria germinate in the food and begin releasing their heat-stable enterotoxin. - A heat-labile toxin can also be produced which can survive flash frying; fast frying with oil. B. Cereus Pathogenesis: B. cereus secretes: a- Hemolysins; β-hemolytic. b- Distinct phospholipases; Lecithinase. c- One type of emesis inducing a heat-stable toxin called cereulide, it causes vomiting. d- Three types of pore-forming heat-labile enterotoxins, they cause diarrhea: 1- Hemolysin BL (HBL), 2- Nonhemolytic enterotoxin (NHE) 3- Cytotoxin K. Note: Those 3 enterotoxins work in synergism (1+1< 2), causing diarrhea. B. Cereus Clinical Features: There are two clinical syndromes produced by the previously mentioned toxins: 1- Vomiting type (emetic form) – caused by a heat stable and acid resistant toxin; Cereulide: - Incubation period 0.5–6 hours; a small incubation period because the toxins are pre-formed when ingested, i.e. here the patient ingests the toxin. - Mainly nausea and vomiting are present but rarely diarrhea and cramps may occur. - The illness is usually self-limiting and is over within a day. - Foods involved are: rice and cereals. 2- The diarrheal type – caused by the 3 heat-labile enterotoxins; HBL, NHE, cytotoxin K: - Incubation period 6–15 hours; a longer incubation period because toxins are released inside the gut after ingestion, they are not pre-formed, i.e. here the patient ingests the bacteria. - Mainly diarrhea and abdominal cramps but may be associated with nausea. Vomiting, however, is rare. Symptoms are over within a day. - Foods involved are: contaminated meat, vegetables and sauces. 3|P a ge B. Cereus Diagnosis: - Clinical grounds; the history of food intake would help in the diagnosis. - Isolation of B. cereus from the suspected food, as well as from the vomitus of the patient. - Isolation of the bacteria from stools is not diagnostic since it may be considered as a contaminant only, thus culturing of the suspected food is required to confirm the diagnose. Treatment and prevention of B. Cereus: - Food-poisoning is self-limiting, therefore antimicrobial therapy is not normally required. Fluid and electrolytes replacement are enough. - B. cereus is resistant to a variety of antimicrobial agents, including penicillin and cephalosporins. - To inactivate the spores, the cooked food must be exposed to high temperatures and/or refrigeration. Inactivating the spores for patients in hospitals is very important to prevent the spread of the disease. ----------------------------------------------- End of Bacillus species ----------------------------------------------- 2- Clostridium Species - Clostridium are also gram-positive endospore-forming rods. However, they are anaerobic unlike aerobic Bacillus species. They are ubiquitous and is part of the microbiome (C. difficile). - A few Clostridium species are aerotolerant; meaning that they do not need oxygen but can survive in its presence. - In general, the clostridia grow well on the blood-enriched media or other media used to grow anaerobes. - Spores of clostridia are usually wider than the diameter of the rods and generally subterminal in position. Recall how in Bacillus species spores were central. - Most species of clostridia are motile and possess flagella. - Clostridium harm their human hosts by secreting extremely powerful exotoxins and enzymes. Clostridium Species of Medical Importance: 1- C. Tetani: Disease → Tetanus Characterized by → Rigid paralysis. 2- C. Botulinum: Disease → Botulism Characterized by → Flaccid paralysis. 3- C. Perfringens: Disease → Gas gangrene and rarely gastroenteritis. 4- C. Difficile: Disease → Pseudomembranous colitis (severe form), diarrhea (mild form). 4|P a ge 1- Clostridium Botulinum - It is found in the soil, which may contaminate vegetables cultivated in or on the soil. It also colonizes the gastrointestinal tract of fishes, birds and mammals. - It causes Botulism, which is characterized by symmetrical, descending, flaccid paralysis of motor and autonomic nerves, usually beginning with cranial nerves. C. Botulinum Pathogenesis: - Clostridium botulinum produces an extremely lethal neurotoxin (coded by a prophage) even in low quantities, it causes a rapidly fatal food poisoning. - There are seven serotypes (A-G) based on the antigenicity of the botulinum toxin produced. A, B, E and rarely F are the serotypes that causes the disease in humans. C. Botulinum Mechanism of action: - The most common offenders are spiced, smoked, vacuum packed, or canned alkaline foods that are eaten without proper cooking. In such foods, spores of C. botulinum germinate when placed under anaerobic conditions (jar, can, etc.) where it then matures and produce toxin. - It gets absorbed by gut and carried by blood to peripheral nerve synapses. - The neurotoxins block the release of Ach from presynaptic nerve terminals in the autonomic nervous system and motor endplates, causing a reversible flaccid muscle paralysis. Botulism: There are four clinical categories of botulism: 1- Foodborne botulism (most common); ingesting contaminated food with pre-formed toxins. 2- Wound botulism; traumatic, most commonly amongst drug abusers. 3- Infant botulism; most common offender is honey followed by powdered milk. Bacteria here germinates and produce the toxins inside the gut rather than being pre-formed as in foodborne botulism. 4- Unintentional, following botulinum IM toxin injection; Botox injections. Clinical Findings of Botulism: - Initial symptoms can include nausea, vomiting, abdominal cramps or diarrhea that begin 18–36 hours after ingestion of the toxic food. 5|P a ge - Dry mouth, blurred vision, and diplopia (double vision) are usually the earliest neurologic symptoms. They are followed by the inability to swallow, and speech difficulty. - In severe cases, extensive respiratory muscle paralysis leads to ventilatory failure and eventually death. - In infants, in the first months of life, poor feeding, weakness, and signs of paralysis (floppy baby) develop. - Infant botulism may be one of the causes of sudden infant death syndrome. - There are no prominent gastrointesinal symptoms and no fever in Botulism. C. Botulinum Diagnosis: - Clinical grounds; exploring food intake history. - Bacteria can often be demonstrated in the serum, gastric secretions, or stool from the patient. Toxin may be found in leftover food using ELISAs and PCR. - Mouse bioassay is the test of choice for the confirmation of botulism in vitro. The mouse will be injected with the patient’s serum, if it dies then this confirms botulism. Treatment of C. Botulinum: - Supportive treatment, especially adequate mechanical ventilation, is of prime importance in the management of severe botulism. - Surgical debridement in wound botulism. - Antitoxin administration. A trivalent (A, B, E) antitoxin must be promptly administered intravenously with supportive care. - Although most infants with botulism recover with supportive care alone, antitoxin therapy is recommended. C. Botulinum Prevention: - Canned food must be sufficiently heated to ensure destruction of spores. - The risk from home-canned foods can be reduced if the food is boiled for more than 20 minutes before consumption. - No honey for the first-year infants. 6|P a ge 2- Clostridium Perfringens - Many different toxin-producing clostridia can produce invasive infection if introduced into damaged tissue (e.g. myonecrosis and gas gangrene). - About 30 species of clostridia may produce such an effect, but the most common is C. perfringens (90%). An enterotoxin of C. perfringens is also considered a common cause of food poisoning. C. Perfringens Distinguishing Features: - Large gram-positive, spore-forming, capsulated and non-motile rods. - Anaerobic: Stormy fermentation in milk media where coagulation and bubbles appear. - Double zone of hemolysis. - Reservoir-soil and human colon. - Transmission: Foodborne and traumatic implantation. - Spores are rarely seen in culture media. C. Perfringens Pathogenesis: - In invasive clostridial infections, spores reach tissue either by contamination of traumatized areas (soil, feces) or from the intestinal tract. The spores germinate at anaerobic conditions, vegetative cells then multiply, ferment carbohydrates present in tissue, and produce gas. - Deep wounds create an anaerobic environment that offers an excellent home for C. perfringens. As this anaerobic organism grows, it releases exotoxins inside the tissue causing further destruction. - Toxins have lethal, necrotizing, and hemolytic properties. C. perfringens produces alpha, epsilon and theta toxins. These toxins are associated with soft tissue infections. - Some strains of C. perfringens produce a powerful enterotoxin as well, associated with intestinal infection. Note: There are 2 types of soft tissue infections caused by C. perfringens. One is wound infections, which grows on and damages local tissue. The other is myonecrosis, inoculated with trauma into muscle and secretes exotoxins that destroy adjacent muscle. 7|P a ge C. Perfringens Clinical Findings: 1- From a contaminated wound (e.g. a compound fracture, postpartum uterus), the infection spreads in 1–3 days to produce crepitation in the subcutaneous tissue and muscle, foulsmelling discharge, rapidly progressing necrosis, fever, hemolysis, toxemia, shock, and death. Note: Crepitation is the crackling sensation due to the presence of air (gas) in the subcutaneous tissue. 2- C. perfringens food poisoning usually follows the ingestion of large numbers of clostridia that have grown in warmed meat dishes. The toxin forms when the organisms sporulate in the gut, with the onset of severe diarrhea, usually without vomiting or fever, in 7–30 hours. The illness lasts only 1–2 days. Diagnostic Laboratory Tests of C. Perfringens: - Gram-stained smears of specimens from wounds, pus, and tissue. - Culture material into thioglycolate medium and onto blood agar plates incubated anaerobically. The growth from one of the medias is transferred into milk which should show a stormy fermentation if positive. - C. perfringens rarely produces spores when cultured on agar in the laboratory. - Final identification rests on toxin production and neutralization by specific anti-toxin. e.g. Nagler test, where an egg yolk agar plate is used to detect the Lecithinase activity of the alpha toxin produced by C. perfringens which breaks down lipoproteins in the plate. The addition of an anti-toxin to one half of the plate further confirms. Treatment and prevention of C. Perfringens: - Prompt and extensive surgical debridement of the involved area and excision of all devitalized tissue, in which the organisms are prone to grow. - Administration of antimicrobial drugs, particularly penicillin, is begun at the same time. Hyperbaric oxygen may be of helpful. It is said to “detoxify” patients rapidly. - Antitoxins are available against the toxins of C perfringens, usually in the form of concentrated immune globulins. Antitoxins should not be relied on. - Food poisoning caused by C. perfringens enterotoxin usually is self-limited and requires only symptomatic care of fluid and electrolytes replacement. 8|P a ge 3- Clostridium Difficile - Ubiquitous in the environment and colonizes the intestine of 50% of healthy neonates and 4% of healthy adults. - A major cause of healthcare-associated infection in patients taking antibiotics, where they become at increased risk of developing C. difficile antibiotic associated diarrhea. - Infection may be endogenous (suppression of the normal bowel flora and subsequent overgrowth of C. difficile) or exogenous (through ingestion of environmental spores). C. Difficile Pathogenesis: - Produces two major toxins: 1- Toxin A (enterotoxin): Induces cytokine production with hypersecretion of fluid. 2- Toxin B (cytotoxin): Induces depolymerization of actin with loss of cytoskeleton. - Adhesin factor and hyaluronidase production are also associated virulence factors. - A hypervirulent, hyper-toxin-producing strains are now recognized (e.g. ribotype 027, 078). Disease: - Clostridium difficile is the pathogen responsible for antibiotic-associated: 1- Mild-moderate form: Diarrhea. 2- Severe form: Pseudomembranous colitis (PMC), characterized by severe diarrhea, abdominal cramping, and fever. - It follows the use of broad-spectrum antibiotics (such as ampicillin, clindamycin, and the cephalosporins). These antibiotics can wipe out part of the normal intestinal flora, allowing the pathogenic C. difficile to superinfect the colon. - Once Clostridium difficile grows in abundance, it then releases its exotoxins. Toxin A causes diarrhea, while Toxin B is cytotoxic to the colonic cells. Diagnosis of C. Difficile infections (CDI): - The diagnosis is based on a combination of clinical criteria: 1- Diarrhea (≥3 loose stools per 24 h for ≥2 days) with no other recognized causes. 2- Toxin A or B detected in the stool (through ELISA, latex agglutination, and PCR) or culture of C. difficile on selective agar. 3- Pseudo-membranes seen in the colon. 9|P a ge - PMC is a more advanced, severe form of CDI and is visualized at endoscopy in only ~50% of patients with diarrhea who have a positive stool culture and toxin assay for C. difficile. Note: PMC is seen as red inflamed mucosa and areas of white exudate called pseudo-membranes on the surface of the large intestine with necrosis of the mucosal surface occurs underneath the pseudomembranes. Treatment and prevention of C. Difficile: - Because of Clostridium difficile, it becomes very difficile (difficult) to give patients antibiotics since they are the causative agent. Treatment includes: 1- Discontinuing the initial antibiotic. 2- Oral administration of metronidazole or vancomycin. 3- Fecal transplantation to restore gut microbiome. - Caution in overprescribing broad-spectrum antibiotics (limited-spectrum drugs should be considered first). - In the nursing home setting, patients who are symptomatic should be isolated. - Autoclaving bed pans (to kill spores). Note: The only case where oral vancomycin is given is for CDI. 10 | P a g e Summary Spore Forming, Gram-Positive Bacilli: 1- Bacillus: Aerobic, generally central spores. a- B. anthracis → Anthrax, non-motile, capsulated with sticky ‘medusa-head’ colonies. b- B. cereus → - Food poisoning, motile, non-capsulated. Produce Lecithinase, hemolysin, cereulide and enterotoxins (HBE, NHE, cytotoxin K). Self-limited. Diarrheal form: Long incubation period, from meat, vegetables and sauces. Toxins formed inside the gut. HBE, NHE, cytotoxin K. - Vomiting form: Short incubation period, from rice and cereals. Pre-formed toxins. Cereulide. 2- Clostridium: Anaerobic, generally subterminal spores. a- C. botulinum (Flaccid paralysis) → - Botulinum neurotoxin “most potent toxin” that block Ach, has 7 serotypes. - Dx of choice: mouse bioassay. - Canned food and honey. - Tx: Mechanical ventilation and trivalent antitoxin (A, B, E). b- C. perfringens (gas gangrene and food poisoning) → - Stormy fermentation, spores rarely seen in culture, double zone of hemolysis. – - Forms alpha, epsilon, theta and an enterotoxin. - Dx: Nagler test. Tx: Hyperbaric oxygen and Penicillin are lifesaving. c- C. difficile (PMC) → - AB associated diseases; diarrhea (mild) or PMC (severe). - Toxin A (enterotoxin) and Toxin B (cytotoxin). - Tx: Oral vancomycin and metronidazole. Good Luck 11 | P a g e