Lecture 3 The Endospore Formers (Bacillus and Clostridium) 2023 PDF

Gram positive bacteria – Topic 4A The endospore formers Bacillus The Firmicutes- Actinobacteria – Low GC Gram-positive bacteria the high GC Gram positive bacteria Non Spore forming bacteria The lactic acid bacteria Streptomyces Streptococcus Lactococc...

Gram positive bacteria – Topic 4A The endospore formers Bacillus The Firmicutes- Actinobacteria – Low GC Gram-positive bacteria the high GC Gram positive bacteria Non Spore forming bacteria The lactic acid bacteria Streptomyces Streptococcus Lactococcus Nocardia Lactobacillus Corynebacteria Staphylococcus Mycobacteria Listeria Mycoplasma The spore forming bacteria Clostridium Bacillus  The endospore forming bacteria Main genera: Bacillus - aerobic or facultative anaerobes Clostridium - anaerobic Form a distinctive type of dormant cell - the endospore. Usually formed when a population reaches conditions of nutrient limitation. Highly resistant to heat, uv, toxic chemicals, ionising radiation. Heat resistance is used for selection. Typical G + cell wall, but stain variably. Typical habitat is soil.  The sporulation cycle of Bacillus From Jeff Errington Nature Reviews Microbiology volume 1, pages117–126 (2003)  Spore Structure Exosporium – thin delicate layer of mostly protein Spore coat(s) – multiple layers of spore specific proteins Cortex – loosely packed peptidoglycan Spore protoplast or core – normal cell wall plasma membrane cytoplasm and nucleoid Properties of the Core Dipicolinate – protects DNA Dehydrated – only 10-30% of the water content of the vegetative cell –gives heat and stress resistance pH is more acidic than vegetative cell Abundant small acid-soluble spore proteins, SASPs – give resistance to dry heat, dessication, UV – protecting DNA Carbon source during outgrowth Properties of the Core Low metabolic activity (low O2 uptake) No macromolecular synthesis Low enzymatic activity Low or absent mRNA synthesis  Germination Spores will remain dormant for decades, even if placed in optimal conditions. State of dormancy can be broken by a variety of treatments - activation. heat shock. eg. several hours at 65C storage at low temperature (4C) When activated spores are placed in favourable conditions - germination. 1. Activation 2. Germination 3. Outgrowth How old are the oldest living cells in Nature?  Longevity of spores. Spores are extremely stable and can survive for long periods. Previously the best documented is 70 years however recently (1995!) spores have supposedly been revived from the gut of a bee preserved in fossil amber 25-40 million years old! Sequenced rRNA genes - related to extanct Bacillus sphaericus. Cano and Borucki Science 268, 1060-64. New record is (maybe!!!) the halophilic spore forming bacterium isolated from salt crystals over 250 million years old. Vreeland Rosenzweig and Powers, Nature 407, 897-900  Bacillus Aerobic or facultative anaerobes. Variable Gram stain. Habitat: mainly soil organisms, although some parasites and pathogens.  Bacillus anthracis Agent of anthrax. Disease of sheep, goats & cattle, that is also transmissable to humans. Isolated by Koch in 1877 - first bacterial pathogen. Survive in soil for 30 years or more. Bacillus cereus Bacillus anthracis From CDC  Cutaneous Anthrax Most common form (2000ish cases per year) Animal workers mostly Spores germinate in skin abrasions Skin ulcer – black eschar – can become systemic Untreated 20% mortality, responds well to antibiotic Gastrointestinal Anthrax ingestion of undercooked contaminated meat 2 types - oral-pharyngeal (most rare) and abdominal (rare) Abdominal Spores germinate in lower gastrointestinal tract primary intestinal lesion forms Symptoms - nausea, severe abdominal pain, vomiting, bloody diarrhea Intestinal perforation or anthrax toxemia are the usual causes of death Mortality very high Three dead and 93 infected after reported Anthrax outbreak in Indonesia Anthrax is a serious infectious disease that occurs naturally in soil and commonly affects domestic and wild animals ByNicola Smith, ASIA CORRESPONDENT 6 July 2023 10:09am Three people have died after an anthrax outbreak in Indonesia that reportedly infected more than 90 people who consumed contaminated meat. Health officials in Yogyakarta province, in the south of the country’s most populous island, Java, confirmed this week that three people had died from the bacterial infection.  Pulmonary anthrax Inhalation of spores Some spores are mopped up by macrophages Others are trafficked to the draining lymph nodes Germination in the lymph nodes may take up to 60 days Anthrax bacilli replicate in the lymph nodes and disease immediately follows. Capsule inhibits phagocytosis. Hemorrhage, edema, and necrosis are the results of bacterial exotoxins released during replication Symptoms- flu-like with fever, myalgia, cough, headache, vomiting, chills, abdominal pain, and chest pain. Cyanosis and hypotension result in death 80% mortality in 2 to 4 days whether antibiotics given or not. Inhalational anthrax is 99% lethal in unvaccinated individuals. Biological warfare Virulence of Bacillus anthracis Capsule made of poly-D-glutamate polypeptide. Smooth mucoid colonies. pX02 plasmid. Resists phagocytosis and complement. Toxin encoded on plasmid pX01. 3 parts - PA (binding domain) - EF (Edema factor) - LF (Lethal factor) Tested as biological warfare agent on remote Scottish island of Gruinard. Island was eventually decontaminated.  Bacillus anthracis Diagnosis G+ rod. Central (non-staining spore) occurs in soil and in culture, but not in clinical samples. Sometimes occur in chains. Identified in blood, skin lesions, or respiratory secretions or by measuring specific antibodies in the blood. Extreme caution is required when handling samples. Treatment - penicillin, doxycycline, and fluoroquinolones (such as ciprofloxacin). To be effective, treatment should be initiated early. Prevention Pasteur demonstrated efficacy of vaccine. Attenuated vaccine possesses some toxicity. Used for occupational exposure. Control also relies on sterilisation of wool etc. from areas where anthrax is endemic. There are presently three vaccines: the Georgian/Russian, the UK, and the US vaccines. All derived from an avirulent strain of B. anthracis - lacks the plasmid pX02 which encodes the protecive coating of the bacteria. The Russian vaccine is inoculation with live spores - high of side effects. UK and US is dead cell free preparation of bacterial filtrates. Bacillus cereus One of the most abundant aerobic sporeformers found in soil. Distinctive loosely spreading (Medusa) colonies, superficially resembling fungi. Bacillus cereus – food poisoning First recognised as a cause of food poisoning in 1955 Two types of poisoning short-incubation or emetic , occurs between 1 and 6 hours after eating Symptoms – nausea, vomiting and abdominal pain Caused by heat stable emetic toxin which cannot be destroyed by cooking – cooked rice kept warm Bacilli cultured from the food long-incubation or diarrheal , occurrs 6-18 hours after eating Symptoms – diarrhea Caused by an enterotoxin that can be destroyed by cooking. Toxin activates intestinal adenylate cyclase and causes intestinal fluid secretion. Bacilli found in stools Also causes disease in immunocompromised. Gram positive bacteria Topic 4B The endospore formers Clostridium Anaerobic sporeformers. Fermentative mode of metabolism. Mostly obligate anaerobes, though some are aerotolerant. Habitat: Widely distributed in nature, soil, water, and intestinal tract of animals. They are principle agents of protein decomposition. Most are saprophytes. Some pathogenic species. Diseases are a consequence of production of highly toxic proteins (exotoxins). Diverse group - over 60 species described. From Batzing, Microbiology and Introduction Clostridia - Fermentation Clostridia lack a cytochrome system and a mechanism for electron transport phosphorylation – therefore they obtain ATP only via substrate-level phosphorylation. The wide variety of fermentable substrates used and mechanisms to derive energy from these can be used to group the clostridia. Some ferment sugars producing end products which may include butyric acid, butanol and acetone (once industrially important). From Brock  Pathogenic Clostridia Natural habitat is soil and the intestinal tract of man and animals. Invasive capability of pathogenic clostridia varies considerably. At one end the organisms remains at the site of inoculation (tetanus) or may not even enter tissue at all (botulism) whereas at the other extreme the organisms elaborates destructive enzymes that allow progressive invasion of necrotic tissue. Toxins are amongst the most powerful poisons known to man - 1 million times more toxic than rattlesnake poison. C. Botulinum - Botulism C. botulinum is found in soil, water and decaying vegetation. Fatal food poisoning that follows on ingestion of preformed toxin from growth of organism in food. Intoxification. Animals are frequently affected from feeds. Endospores are very resistant to heat and may withstand boiling for several minutes Anaerobic conditions (incompletely sterilised canning) may germinate spores - grow into vegetative cells and eventually produce the deadly toxin. Botulinum toxin C. botulinum - May occasionally infect wounds. Infant botulism can result from ingestion of spores in soil and dust - germinate in intestine (anaerobic). Honey added to formula feed found to contain spores. Diagnosis Clinical. ELISA for toxin in food or suspect food is injected into mice. Culture is attempted. Prevention Antitoxin is given to suspected cases. In contrast to spores, toxin is heat labile therefore cooking inactivates.  BOTOX Botulinum toxin type A injected into muscle, lasts for several months. Used to treat patients with overactive muscles Eg. cross eyes (1960s) dystonia  Clostridium tetani Causative agent of tetanus. First described by Hippocrates Etiology discovered in 1884 by Carle and Rattone Commonly found in soil and intestines of animals (humans transiently). >more than 300,000 cases per year Charles Bell, Royal College of Surgeons Terminal spore produces a drumstick shape Tetanus - Clostridium tetani Spores are introduced into the body through wound. Wounding producing necrotic (dead) cells allows anaerobic conditions to develop and spores to germinate and formation of toxin. 2 types of tetanus Generalised tetanus Neonatal tetanus occurs if umbilical stump becomes infected - from soil or bindings containing spores. Normally due to lack of passive immunity from the mother High fatality rate >200,000 deaths each year Tetanus - Pathogenesis Toxin migrates along peripheral nerve axons to the central nervous system (site of action) Symptoms take 3-21 days to develop spastic paralysis - convulsive contractions of voluntary muscles. Since spasms often involve neck and jaws disease is referred to as lockjaw (teeth knocked out!). Body may be severely contorted. Death results from muscular spasms affecting respiration. 50% mortality. Symptoms last 3-4 weeks, complete recovery not for months (if at all!) Tetanus toxin Tetanus - Clostridium tetani Diagnosis Diagnosis is usually by clinical picture, although organism can be isolated - G+, drumstick shaped due to terminal spore. Haemolytic toxin production can be demonstrated by growth on agar plates half spread with antitoxin - prevents haemolysis. Prevention and treatment Prevention is by immunity produced by vaccination with formalin-inactivated toxin (in DPT triple vaccine). Tetanus toxoid vaccine has 100% efficacy. Following injury, non-immunised persons are given human tetanus immunoglobulins. Even in treated people, mortality may be as high as 20 - 30%.  C. perfringens - Gas Gangrene Normal flora in GI and vagina Caused by C. perfringens usually although several other species of clostridia may also cause gangrene. Results from contamination of wounds with spores of clostridia. Able to grow in anaerobic conditions in deep necrotic wounds with reduced blood supply. After germination organisms secrete exotoxins causing more tissue damage, resulting in rapid spread of the organisms. Carbohydrate may be fermented resulting in the production of large quantities of gas (unpleasant odour) in tissue - constrict blood vessels. surgery battlefield casualties. motor vehicle crashes farm injuries. Post-abortal sepsis  C. perfringens - Gas Gangrene Diagnosis and Identification: Usually on clinical picture (smell from protein digestion is characteristic). X-ray for gas. Since other organisms (eg. bacteroides) can produce similar infections, definitive diagnosis requires isolation and identification. Organism may be cultivated on Robertsons meat media and on blood agar incubated anaerobically. G+ large rods. Rancid smell on meat media. hemolytic  C. perfringens – more diagnosis Organisms producing alpha toxin (phospholipase or lecithinase) will hydrolyse lecithin in egg yolk medium -> opaque area around colony. - Neutralised by presence of antitoxin antibody in media - Nagler reaction - organism streaked across plate half smeared with antitoxin. C. perfringens - Gas Gangrene Prevention and Treatment Cleansing of wounds. Antibiotics have little effect in necrotic areas. Surgery may be necessary to remove affected parts. Additionally, C. perfringens is an important cause of food poisoning In man and animals by production of enterotoxins C.difficile - Pseudomembranous Colitis Severe colitis following antibiotic therapy. Overgrowth of Clostridium difficile. Normal flora of many people. Produces toxins. Diarrhea is a common complication of antibiotic therapy 20% of cases of antibiotic-associated diarrhea are caused by Clostridium difficile C difficile is recognized as the most common nosocomial gastrointestinal infection Lesions 2-10 mm. Two major toxins have been identified: toxin A is a 308kD enterotoxin, and toxin B is a 269kDa cytotoxin. Both toxins are capable of stimulating production of proinflammatory cytokines that have been implicated in the pathogenesis of pseudomembranous colitis Strains resistant to certain antibiotics common. C.difficile - Colitis Diagnosis. Most common test is an ELISA test for toxins A and B in the stools Treatment. Stop the causative antibiotic therapy. Anti diarrhoea drugs. Metronidazole, vancomycin.