Spore Forming Gram Positive Bacilli PDF

Spore Forming Gram Positive Bacilli Spore-forming Bacilli Genus Bacillus Genus Clostridium 2  Bacillaceae Bacillus Aeribacillus Allobacillus Geobacillus Halobacillus Lentibacillus Halobacillus...

Spore Forming Gram Positive Bacilli Spore-forming Bacilli Genus Bacillus Genus Clostridium 2  Bacillaceae Bacillus Aeribacillus Allobacillus Geobacillus Halobacillus Lentibacillus Halobacillus Gracilibacillus Salinibacillus Clostridiaceae Clostridium Anaerobacter Caloramator Oxobacter Sarcina Thermobrachium  Bacillus anthracis  Gram + rod  Facultative anaerobe  1 - 1.2µm in width x 3 - 5µm in length  Belongs to the B. cereus family  Thiamin growth requirement  Glutamyl-polypeptide capsule  Nonmotile  Non haemolitic  Forms oval, centrally located endospores  Genetics  1 chromosome  2 plasmids  px01 184 kbp Pathogenicity island  pX02 95.3 kbp Capsule  Anthrax receptor  Occurs > than ten thousendfold on macrophage cell  ATR/TEM8 gene Chromosome 4  Anthrax  From the Greek word anthrakos for coal  Caused by spores  Primarily a disease of domesticated & wild animals  Herbivores such as sheep, cows, horses, goats  Natural reservoir is soil  Does not depend on an animal reservoir making it hard to eradicate  Cannot be regularly cultivated from soils where there is an absence of endemic anthrax  Anthrax zones  Soil rich in organic matter (pH < 6.0)  Dramatic changes in climate  Anthrax Infection & Spread  May be spread by streams, insects, wild animals, birds, contaminated wastes  Animals infected by soilborne spores in food & water or bites from certain insects  Humans can be infected when in contact with flesh, bones, hides, hair, & excrement  nonindustrial or industrial  cutaneous & inhalational most common  Risk of natural infection 1/100,000  Outbreaks occur in endemic areas after outbreaks in livestock  Pathogenesis  The infectious dose of B. anthracis in humans by any route is not precisely known.  Rely on primate data  ID50 : Minimum Infection dose of ~ 1,000-8,000 spores  LD50 : Minimum Lethal dose of 8,000-10,000 spores for inhalation  Virulence depends on 2 factors  Capsule  3 toxins  Capsule  Glycocalyx  Sticky, gelatinous polymer external to cell wall  pX02 plasmid  Made up of D-glutamic acid  Non-toxic on its own  Only encapsulated B. anthracis virulent  Most important role during establishment of disease  Protects against phagocytosis & lysis during vegetative state  Toxins  pX01 plasmid  AB model  Binding  Activating  Protective antigen (PA), edema factor (EF) & lethal factor (LF)  Make up 50% of proteins in the organism  Individually non-toxic  PA+LF  lethal activity  EF+PA  edema  EF+LF  inactive  PA+LF+EF  edema & necrosis; lethal  Toxins (2)  Protective antigen (PA, 83kDa)  Pag gene  Binds to receptor & helps internalize other 2 proteins  Edema factor (EF, 89 kDa)  Cya gene  Adenylate cyclase  Affects all cells  Lethal factor (LF, 87 kDa)  Lef gene  More important virulence factor  Metalloprotease  Cleaves mitogen activated protein kinase(MAPK)  Affects only macrophages  PA, EF & LF  PA is an 83-kDa protein that binds to one of two receptors on host cell surfaces that are present on many cells and tissues (e.g., brain, heart, intestine, lung, skeletal muscle, pancreas, macrophages).  After PA binds to its receptor, host proteases cleave PA, releasing a small fragment and retaining the 63-kDa fragment (PA63) on the cell surface.  The PA63 fragments self-associate on the cell surface, forming a ring- shaped complex of seven fragments (pore precursor or “prepore”). This heptameric complex can then bind up to three molecules of LF and/or EF.  Both factors recognize the same binding site of PA63, so the binding is competitive. Formation of the complex stimulates endocytosis and movement to an acidic intracellular compartment. In this environment, the heptameric complex forms a transmembrane pore and releases LF and EF into the cell interior.  LF is a zinc-dependent protease capable of cleaving mitogen-activated protein (MAP) kinase, leading to cell death. EF is a calmodulin-dependent adenylate cyclase that increases the intracellular cyclic adenosine monophosphate (cAMP) levels and results in edema.  EF is related to the adenylate cyclases produced by Bordetella pertussis and Pseudomonas aeruginosa.  Mechanism of Infection  Anthrax spores enter body  Germinate & multiple in entry location & lymph nodes  PA, EF, LF excreted  PA binds to TEM8.  PA nicked by protease furin  leaving 63-kDa peptide  Heptamer forms  EF and/or LF binds  Complex internalized by endocytosis  Acidification of endosome  LF or EF crosses into cytosol via PA mediated ion- conductive channels  LF cleaves MAPK 1 & 2  EF stimulates cAMP  Outcome  EF converts ATP to cAMP  Increases cAMP levels over 1,000 fold  Impairs neutrophil function  Alters water homeostasis  Edema  LF cleaves MAPK at its N terminus  Disrupts pathways involved in cell growth & maturation  Increased synthesis of tumor necrosis factor-α & interleukin-1β  Macrophage lysis  More cells infected with bacteria & toxin  Septic shock & death  Death probably results from high levels of bacteria secreting LF toxins in blood  At death, blood contains as many as 10 10 bacilli/ml 7- 9 (depending on the strains)  Regulators  Bicarbonate or CO2 stimulates capsule and PA formation  LF requires zinc ions  EF requires calmodulin, a major intracellular calcium receptor  Transcriptional regulator AcpA on pX02 controls expression of capsule  atxA on pX01 is a positive regulator necessary for transcription of all 3 toxin genes  Infection of Anthrax  The estimated number of naturally occurring human cases of anthrax in the world is 20,000 to 100,000 per year.  Humans are infected through contact with infected animals and their products because of human intervention.  Anthrax spores contaminate the ground when an affected animal dies and can live in the soil for many years.  Anthrax can also be spread by eating undercooked meat from infected animals.  Anthrax is NOT transmitted from person to person.  Humans can be exposed but not be infected. What are the symptoms for anthrax?  There are two phases of symptom.  1) Early phase - Many symptoms can occur within 7 days of infection  2) 2nd phase - Will hit hard, and usually occurs within 2 or 3 days after the early phase.  - Early Phase Symptoms -  Fever (temperature > 100 degrees F)  Chills or night sweats  Headache, cough, chest discomfort, sore throat  Joint stiffness, joint pain, muscle aches  Shortness of breath  Enlarged lymph nodes, nausea, loss of appetite, abdominal distress, vomiting, diarrhea  Meningitis  - 2nd Phase Symptoms -  Breathing problems, pneumonia  Shock  Swollen lymph glands  Profuse sweating  Cyanosis (skin turns blue)  Death  Three forms of Anthrax in Human  Cutaneous(inoculation) anthrax (95% of all cases)  Skin  Most common  Spores enter to skin through small lesions  Inhalation anthrax(5% of all cases)  Spores are inhaled  Gastrointestinal (GI) anthrax( very rare)  Spores are ingested  Oral-pharyngeal and abdominal  Cutaneous Anthrax  95% human cases are cutaneous infections  1 to 5 days after contact  Small, pruritic, non-painful papule at inoculation site  Papule develops into hemorrhagic vesicle & ruptures  Slow-healing painless ulcer covered with black eschar surrounded by edema  Infection may spread to lymphatics w/ local adenopathy  Septicemia may develop  20% mortality in untreated cutaneous anthrax REVIEW  Inhalation Anthrax Wool sorter Disease  Virtually 100% fatal (pneumonic)  Meningitis may complicate cutaneous and inhalation forms of disease  Pharyngeal anthrax Fever Pharyngitis Neck swelling REVIEW  Inhalation Anthrax  The infection begins with the inhalation of the anthrax spore.  Spores need to be less than 5 microns to reach the alveolus.  Macrophages lyse and destroy some of the spores.  Survived spores are transported to lymph nodes by macrophages.  At least 2,500 spores have to be inhaled to cause an infection.  Inhalation Anthrax  Disease immediately follows germination.  Spores replicate in the lymph nodes.  The two lungs are separated by a structure called the mediastinum, which contains the heart, trachea, esophagus, and blood vessels.  Bacterialtoxins released during replication result in mediastinal widening and pleural effusions (accumulation of fluid in the pleural space).  Inhalation Anthrax  Death usually results 2-3 days after the onset of symptoms.  Now today natural infection is extremely rare  Inhalation Anthrax is the most lethal type of Anthrax.  Incubation period:  1–7 days  Possibly ranging up to 42 days (depending on how many spores were inhaled).  Case fatality after 2 days of infection:  Untreated (97%)  With antimicrobial therapy (75%) Gastrointestinal (Ingestion) Anthrax Virtually 100% fatal Abdominal pain Hemorrhagic ascites Paracentesis fluid may reveal gram- positive rods REVIEW Gastrointestinal Anthrax  GI anthrax may follow after the consumption of contaminated, poorly cooked meat.  There are 2 different forms of GI anthrax: 1) Oral-pharyngeal 2) Abdominal  Abdominal anthrax is more common than the oral- pharyngeal form.  GI Anthrax  Oral-pharyngeal form: results from the deposition and germination of spores in the upper gastrointestinal tract.  Local lumphadenopathy (an infection of the lymph glands and lymph channels), edema, sepsis develop after an oral or esophageal ulcer.  Abdominal form: develops from the deposition and germination of spores in the lower gastrointestinal tract, which results in a primary intestinal lesion.  Symptoms such as abdominal pain and vomiting appear within a few days after ingestion.  GI Infection  GI anthrax cases are uncommon.  There have been reported outbreaks in Zimbabwe, Africa and northern Thailand in the world.  GI anthrax has not been reported in the US.  Incubation period:  1-7 days  Case fatality at 2 days of infection:  Untreated (25-60%)  With antimicrobial therapy (undefined) due to the rarity  Treatment & Prophylaxis  Treatment Penicillin was drug of choice but now ciprofloxacin is accapted Erythromycin, chloramphenicol acceptable alternatives Doxycycline and ciprofloxacin now commonly recognized as prophylactic  Vaccine (controversial)  Four countries produce vaccines for anthrax. Russia and China use attenuated spore-based vaccine administered by scarification. The United States and Great Britain use a bacteria-free filtrate of cultures adsorbed to aluminum hydroxide.  Laboratory workers  Employees of mills handling goat hair  Active duty military members  Potentially entire populace of U.S. for herd immunity Key Characteristics to Distinguish between B. anthracis & Other Species of Bacillus Characteristic Bacillus anthracis Other Bacillus spp. Hemolysis Neg Pos Motility Neg Pos (usually) Gelatin hydrolysis Pos Neg Salicin fermentation Neg Pos Growth on PEA blood agar Neg Pos  Bacillus cereus  Bacillus cereus cells are Gram positive and similar in size to those of B. anthracis but are usually motile, and single organisms, pairs and short chains are more common than in B. anthracis cultures.  Capsules are not formed, but spore and sporangial morphology are similar to those of B. anthracis.  Bacillus cereus is chemoorganotrophic and grows above 10–20 °C and below 35–45 °C with an optimum temperature of about 37 °C.  Bacillus cereus toxins  Bacillus cereus is known to produce six toxins: five enterotoxins and the emetic toxin. 1. Hemolysin BL (Hbl), a 3-component proteinaceous toxin which also has dermonecrotic and vascular permeability activities and causes fluid accumulation in ligated rabbit ileal loops; Hbl is produced by about 60% of strains tested and it has been suggested that it is a primary virulence factor in B. cereus diarrhea, but the mechanism of its enterotoxic activity is unclear 2. Nonhemolytic enterotoxin (Nhe) is another 3-component proteinaceous toxin which is produced by most strains tested. 3. Enterotoxin T (BceT) 4. Enterotoxin FM (EntFM) , BceT and EntFM are single- component proteinaceous toxins whose roles and characteristics are not known.  Bacillus cereus toxins 5. Enterotoxin K (EntK) is similar to the β-toxin of Clostridium perfringens and was associated with necrotic enteritis. 6. The emetic toxin, cereulide, is a dodecadepsipeptide comprising a ring of four amino- and oxy-acids: [D-O-Leu-D- Ala-L-O-Val-LVal] thrice repeated; it is closely related to the potassium ionophore valinomycin. It is resistant to heat, pH and proteolysis, but it is not antigenic. Cereulide is probably an enzymatically synthesized peptide rather than a direct genetic product; it is produced in larger amounts at lower incubation temperatures. Its production does not appear to be connected with sporulation and it is produced in aerobic and microaerobic but not anaerobic conditions. Its mechanism of action is unknown, but it has been shown to stimulate the vagus afferent through binding to the 5-HT3 receptor. The earliest detection system for emetic toxin involved monkey-feeding tests. Foodborne Diseases of B. cereus (Intoxication) (Foodborne Infection) Bacillus cereus clinical presentation Gastroenteritis EMETIC FORM DIARRHOEAL FORM Incubation period > 6 hours Incubation period < 6 hours Diarrhoea Severe vomiting Lasts 20-36 hours Lasts 8-10 hours  Other complications B cereus is an important cause of eye infections, severe keratitis, endophthalmitis, and panophthalmitis. Typically, the organisms are introduced into the eye by foreign bodies associated with trauma.  B cereus has also been associated with localized infections and with systemic infections, including endocarditis, meningitis, osteomyelitis, and pneumonia; the presence of a medical device or intravenous drug use predisposes to these infections.  Diagnosis  The enterotoxin may be preformed in the food or produced in the intestine. The presence of B cereus in a patient's stool is not sufficient to make a diagnosis of B cereus disease, since the bacteria may be present in normal stool specimens; a concentration of 105 bacteria or more per gram of food is considered diagnostic.  Clostridium Anaerobic gram positive spore forming bacilli  Characteristics  Obligate anaerobes  Gram positive  Capable of producing endospores  Rod-shaped, named after Greek word for spindle, kloster  Club-shaped, as well: endospores form club end  Most Clinically Relevant Clostridium Species  Clostridium tetani  Clostridium botulinum  Clostridium perfringens  Clostridium difficile  Most Clostridium spp., including C. perfringens and C. botulinum, have ovoid subterminal (OST) spores  C. tetani have round terminal (RT) spores  Clostridium spp. Anaerobic Gram-Positive Spore-Forming Bacilli Four broad types of pathogenesis: 1. Histotoxic group — tissue infections (C. perfringens type A, exogenously acquired more commonly than endogenously) (C. septicum; endogenously-acquired) a. cellulilitis b. myonecrosis c. gas gangrene d. fasciitis 2. Enterotoxigenic group — gastrointestinal disease a. clostridial foodbome disease (8-24h after ingestion of large numbers of organisms on con-taminated meat products, spores germinate, enterotoxin produced (C. perfringens type A) b. necrotizing enteritis (beta toxin-producing C.perfringens type C) (C. difficile endogenously-acquired or exogenously-acquired person-to-person in hospital) c. antibiotic-associated diarrhea d. antibiotic-associated pseudomembrane colitis 3. Tetanus (exogenously acquired) — C. tetani neurotoxin a. generalized (most common) b. cephalic(primary infection in head, comnnonly ear) c. localized e. neonatal (contaminated umbilical stump) 4. Botulism (exogenously acquired) — C. botulinum neurotoxin a. foodborne (intoxication,1-2days incubation period) b. infant (ingestion of spores in honey) c. wound (symptoms similar to foodborne, but 4 or more days incubation) Clostridium perfringens Clostridium perfringens — histotoxic or enterotoxigenic infections Morphology and Physiology large, rectangular bacilli (rod) staining gram-positive spores rarely seen in vitro or in clinical specimens (ovoid, subterminal) non-motile, but rapid spreading growth on blood agar mimics growth of motile organisms aerotolerant, especially on media supplemented with blood grow at temperature of 20-50°C (optimum 45°C) and pH of 5.5-8.0 Pathogenicity Determinants (note that toxins include both cytolytic enzymes and bipartite exotoxins) four major lethal toxins (alpha (α), beta (β), epsilon (ε), and iota (ι) toxins) and an enterotoxin six minor toxins (delta(δ), theta(θ), kappa(κ), lambda(λ), mu(µ), nu(η)toxins) & neuraminadase C. perfringens subdivided into five types (A-E) on basis of production of major lethal toxins C. perfringens Type A (only major lethal toxin is alpha toxin) responsible for histotoxic and enterotoxigenic infections in humans; Type C causes necrotizing enteritis (not in U.S.) Lab Identification direct smear and Gram stain, capsules upon direct examination of wound smears culture takes advantage of rapid growth in chopped meat media at 45° C to enrich and then isolate onto blood agar streak plate after four to six hours gas from glucose fermentation in vivo toxicity testing and identification of the specific toxin types involved double zone of hemolysis on blood agar (p-hemolytic theta(e) toxin, a-hemolytic alpha(oc) toxin) Nagler rxn; precipitation in serum or egg yolk media; oc -toxin (phospholipase C) is a lecithinase "stormy" fermentation (coagulaltion) of milk due to large amounts of acid and gas from lactose Diagnosis/Treatment of systemic infection — Early diagnosis and aggressive treatment essential removal of necrotic tissue (surgical debridement) Penicillin G in high doses if more serious infection Of poorly defined clinical value are: administration of antitoxin hyperbaric oxygen (dive chamber) adjunct therapy (??inhibit growth of anaerobe??)  C. perfringens Virulence Factors Major Minor Micro & Macroscopic C. perfringens NOTE: Large rectangular NOTE: Double zone of hemolysis gram-positive bacilli Inner beta-hemolysis = θ toxin Outer alpha-hemolysis = α toxin Clostridial Cellulitis  C. perfringens Nagler Reaction NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes phospholipids in egg-yolk agar around streak on right. Antibody against α-toxin inhibits activity around left streak.  Food intoxication and enterotoxin  Bacterium can persist and multiply in animal intestinal tracts  Temperature-abuse in cooked or raw food causes food contamination  When more than 108 vegetative cells are ingested and sporulate in the gut, enterotoxin is formed.  The enterotoxin is a protein (MW 35,000) that may be a nonessential component of the spore coat; it is distinct from other clostridial toxins. It induces intense diarrhea in 6–18 hours Clostridium tetani Summary of C. tetani Infections Clostridium tetani — agent of tetanus Morphology and Physiology- long thin gram-positive organism that stains gram negative in old cultures round terminal spore gives drumstick appearance motile by peritrichous flagella grow on blood agar or cooked meat medium with swarming beta-hemolysis exhibited by isolated colonies spores resist boiling for 20 minutes Antigenic Structure- flagella (H), somatic (0), and spore antigens. Single antigenic toxin characterizes all strains. Pathogenicity Determinants" play a role in local infection only in conjunction with other bacteria that create suitable environment for their invasion systemic-acting, plasmid-mediated A-B neurotoxin (tetanospasmin) produced intracellularly  Mode of Action — one of most poisonous substances binds gangliosides in synaptic membranes (synapses of neuronal cells) and blocks release of inhibitory neurotransmitters; continuous stimulation by excitatory transmitters muscle spasms (spastic paralysis) (trismus (lockjaw), risus sardonicus, opisthotonos), cardiac arrhythmias, fluctuations in blood pressure Lab Identification" use characteristics of resistance to heat, motility, and toxin production to help identify Diagnosis/Treatment/Prevention empirical diagnosis on basis of clinical manifestations treat to prevent elaboration and absorption of toxin  clean wound (debridement), control spasms  metronidazole administered to eliminate vegetative bacteria that produce neurotoxin  passive immunity (human tetanus immunoglobulin); vaccination (active) as preventative  antitoxin administered to bind free tetanospasmin Clostridium tetani Gram Stain NOTE: Round terminal spores give cells a “drumstick” or “tennis racket” appearance.  Mechanism of Pathogenicity  Production of two exotoxins  Tetanolysin: function not determined  Tetanospasim  Neurotoxin  One of most potent toxins known: minimum human lethal dose: 2.5 ng/kg body weight 175 nanograms for 154 lb. person  Zinc-dependent metalloproteinase  Tetanospasim  Absorption into CNS  Absorbed into axon and transported across synaptic junctions until reaches CNS  Circulates through circulatory and lymphatic systems  Rapidly fixed to gangliosides at the presynaptic junction of inhibitory motor nerve endings  Mode of Action  Blocks inhibitory impulses via interference with neurotransmitter release, including that of glycine and gamma-amino butyric acid (GABA)  Prevents neurotransmitters by cleaving synaptobrevin II, component of synaptic vesicles  Causes unopposed muscle contraction and spasm and seizures Mechanism of Action of Tetanus Toxin Clinical Forms of Tetanus  Common Classifications of Tetanus  Common types 1) Local tetanus: persistent muscle contraction in region of injury 2) Cephalic tetanus: concurrent with otitis media, associated with head injuries and cranial nerves 3) Generalized tentanus:(80% prevalence) lockjaw other symptoms include elevated blood pressure, sweating, elevated temperature, rapid episodic heart rate, spasms continue for 3-4 weeks 4) Neonatal tetanus: born without passive immunity, usually through infection of unhealed umbilical stump Trismus or lockjaw Opisthotonos in Tetanus Patient Risus Sardonicus in Tetanus Patient  Treatment  3 Foci 1) Control of muscle spasms 2) Halting of toxin production 1) Metronidazole 2) Intramuscular penicillin G 3) Neutralization of toxin effects  TIG(Teta Bolin): binds to and eliminates unbound toxins from body but cannot affect already bound toxins, as this is an irreversible event  Prevention 1) Rigorous hygienic response to injury 2) Vaccination First 4 immunization shots (DTP: diphtheria- tetanus-pertussis) given within 2 years Every 10 years: booster shot Clostridium botulinum Summary of C. botulinum Infections Summary of C. botulinum Infections C. botulinum — agent of botulism, a rare, but severe (lethal) neuroparalytic disease Morphology and Physiology heterogeneous group of fastidious, strictly anaerobic bacilli motile by peritrichous flagella heat-resistant spores (ovoid, subterminal) proteolytic and non-proteolytic Antigenic Structure species divided into four groups (I-IV) based on type of toxin produced and proteolytic activity seven antigenically distinct botulinum toxins (types A to G) somatic antigens - heat stable and heat labile; spore antigens - more specific Pathogenicity Determinants lethal foodbome intoxication with toxin types A,B,E,or F; shorter incubation period, poor prognosis phage-mediated, systemic-acting A-B neurotoxin (botulinum toxin = botulin) released at cell lysis  Mode of Action - one of most extremely potent neurotoxins known (1 ng of purified toxin contains about 200,000 minimal lethal doses (MLDs) for a 20g mouse) A-B toxin ingested, binds specific receptors on peripheral cholinergic nerve endings (neuromuscular junctions) where it blocks release of presynaptic acetylcholine (excitatory neurotransmitter) blocking muscle stimulation & resulting in flaccid paralysis Early: nausea, vomiting, weakness, lassitude (lack of energy), dizziness, constipation Later: double vision, difficulty in swallowing and speaking Final: death due to respiratory paralysis Lab Identification microscopic detection or Cx (culture) are often unsuccessful (few organisms and slow growing) toxin detected and typed in lab via toxicity and antitoxin neutralization tests in mice or by ELISA Diagnosis/Treatment/Prevention crucial to rapidly diagnose (symptoms often confusing); note the type of botulinum toxin involved Tx (treatment) should be administered as quickly as possible on basis of clinical Dx (diagnosis)  ventilatory support & trivalent (A, B, E) antitoxin (polyvalent) binds free toxin in bloodstream  administer gastric lavage & metronidazole or penicillin eliminates organisms from Gl tract  care in home canning and in heating of home-canned food; toxoid is available  Clinical Syndromes  Food-bourne, Wound, Infant and Unidentified 1) Food-borne: ingested from foods that spores have germinated and grown in, considered an intoxication – most common form 2) Wound: infects a wound and then produces toxins that spread through the bloodstream – very rare 3) Infant: infection, establishes itself in the bowels of infants, colonizes and produces the toxin – common source is honey 4) Unidentified: source is unknown, usually from intestinal colonization with in vivo production of toxin – usually from surgeries  Action of Toxin  Structure: Synthesized as a polypeptide chain that cleaves into two chains, a light and heavy linked by disulfide bonds  Binding occurs at the carboxy terminal  Enters receptors via endocytosis  Blocks release of Ach = failure to release neurotransmitter  Zinc-dependent endopeptidase that cleaves synaptobrevins  Flaccid Paralysis  Permanent damage Mechanism of Action of Botulinum Toxin  Symptoms  Begin 8-36 hours after ingestion  Length: 2 hours to 14 days after entering circulation  Preliminary symptoms: weakness, dizziness, dryness mouth, nausea, vomiting  After Neurological disturbance: blurred vision, inability to swallow, difficulty in speech, descending weakness of skeletal muscles(Drunk walking) and respiratory paralysis  Rates of Isolation of C. botulinum and Botulinum Toxin  Alternative botulism Uses  Botox  A-Type botulism is an active ingredient  Biological Warfare  Poisonous to Humans  World War II – Stanley Lovell Gelatin capsules with a lethal dose Slipped into food or drink Tested on donkeys  1gram crystalline toxin dispersed evenly and inhaled = 1 million deaths 70μg orally = lethal (70kg person) 0.09μg – 0.15μg intravenously = lethal (70kg person) Clostridium difficile Summary of C. difficile Infections Summary of C. difficile Infections C. difficile Virulence Factors  Disease: Pseudomembranous Colitis  Those most susceptible to disease:  Antibiotic therapy such as cephalosporins and clindamycin, which are frequently used in hospital settings.  Advanced age over 65. (80% reported cases)  Multiple, severe underlying diseases  Faulty immune response to toxins produced by C. difficile toxins.  The rate of Clostridium difficile acquisition is estimated to be 13 percent in patients with hospital stays of up to two weeks and 50 percent in those with hospital stays longer than four weeks.  Those taking medications to suppress gastric acid production: H2-receptor antagonists increased the risk twofold, Proton pump inhibitors increased the risk threefold, mainly in the elderly. It is presumed that increased gastric pH leads to decreased destruction of spores C. difficile Mode Of Infection: C. difficile: Patient Susceptibility  Treatment  Fecal Bacteriotherapy:  Procedure related to probiotic research, has been suggested as a potential cure for the disease. Involves infusion of bacterial flora acquired from the feces of a healthy donor in an attempt to reverse bacterial imbalance responsible for the recurring nature of the infection. Has a success rate of nearly 95% according to some sources.  Two Specific Antibiotics can be used as treatment  Metronidazole  Vancomycin  20-30% of patients treated with this method experience a relapse. Other Clostridium Clostridium Associated Human Disease  Virulence Factors Associated with Other Clostridium