Clostridium Botulinum PDF

Clostridium botulinum Clostridium botulinum Optional reading: Page 441-458 Food microbiology : fundamentals and frontiers Author: Michael P Doyle & Robert Buchanan Publisher: Washington, DC American Society for Microbiology Press, ©2013 Available as an ebook from library. The Four Faces of Clostridi...

Clostridium botulinum Clostridium botulinum Optional reading: Page 441-458 Food microbiology : fundamentals and frontiers Author: Michael P Doyle & Robert Buchanan Publisher: Washington, DC American Society for Microbiology Press, ©2013 Available as an ebook from library. The Four Faces of Clostridium botulinum Intoxication – Ingestion (or injection) of the toxin Infant Botulism – Ingestion and intestinal proliferation of the organism, internal production of the toxin (Infection) Wound Botulism – Deep wound results in anaerobic environment Bioterrorism – Deliberate delivery of toxin to large populations Phylogeny How do you define a bacterial species? By definition all C. botulinum strains are toxic – Non-toxic variant strains, though genetically related, are assigned to different species such as C. sporogenes and C. subterminale Phylogenetic dendrogram of Clostridium species based on 16S rRNA genes. K. K. Hill et al. J. Bacteriol. 2007;189:818-832 Botulinum Toxin Botulinum neurotoxin (BoNT) is an A-B toxin that consists of two subunits (A and B) The toxin is inactive after production There are two types of Cb proteolytic and nonproteolytic – In nonproteolytic Cb the host’s stomach acids cleave the toxin (activating it) – In proteolytic Cb the bacteria produce a protease that activates the neurotoxin Following ingestion, the toxin molecules are absorbed by the gut and are spread via the blood to peripheral nerves Botulinum Toxin Byers, E. B. (2010). "Botulinum Toxins: Bad Bug or Miracle Medicine?" Inquiries Journal/Student Pulse, 2(11). Retrieved from http://www.inquiriesjournal.com/a?id=324 Botulinum Toxin Following ingestion/production the toxin molecules are absorbed by the gut and are spread via the blood to peripheral nerves Normally the axon terminal of the neuron connects with muscle tissues through SNARE proteins and releases acetylcholine into the muscle cells which causes the contraction BoNT acts as a protease cleaving the SNARE proteins This deprives the muscle of the acetylcholine signal and results in flaccid paralysis This becomes a major issue when it comes to lung or heart muscles! Botulinum Toxin There are several structurally similar, but immunologically distinct BoNTs distinguished using the letters A to G Cb strains were grouped into 4 groups based on physiological differences, but these groupings don’t correlate to which groups produce which type of toxin Human illness is generally caused by Group 1 and Group 2 Cb – Group 1: C. botulinum A and proteolytic strains of B and F (Human) – Group 2: C. botulinum E and non-proteolytic strains of B and F (Human) – Group 3: C. botulinum C and D (Animal) – Group 4: C. botulinum G Testing For Cb Toxin in Foods Detection of Toxin 2/2 Mice must have pinched waist, labored breathing or die for test to be considered positive Prepare food, feces, serum or gastric contents Dispense filtrate into 3 tubes No Tube Treatment Boiled Tube For 10 min Trypsin Added to Tube Inject 2 Mice with each tube No Tube Treatment Boiled Tube For 10 min Trypsin Added to Tube Testing For Cb Toxin In Foods Identification of Toxin Inject a mouse with each tube A toxin Antiserum No Treatment Tube B toxin Antiserum F toxin Antiserum A toxin Antiserum B toxin Antiserum F toxin Antiserum Clinical Aspects of Botulism The hallmark of botulism is bilateral descending weakening and paralysis of skeletal muscles The classic triad of symptoms include: 1- symmetric, descending flaccid paralysis with prominent bulbar palsies (difficulty swallowing, lack of gag reflex, lack of speech, drooling) 2- lack of fever 3- a clear sensorium in the patient (ability to think and concentrate) Autonomic disfunction including constipation, urine retention, and mydriasis can also occur Muscles affecting respiration are weakened, and mechanical ventilation to prevent death by suffocation can be required The patients hearing is normal, consciousness is not lost, and the victim is cognizant of the progression of the disease Long-Term Recovery and Effects Recovery from botulism is a long process that usually requires weeks to months depending on the serotype of the toxin and amount ingested There is evidence that complete recovery is rare – In a controlled study of 217 patients who had botulism (median age 37, 49% female) 68% reported having worse health now than 6 years prior (17% of control subjects reported this) Patients were more likely to report fatigue, weakness, dizziness, dry mouth, and difficulty lifting objects than people who did not have botulism Patients with botulism were also more likely to report difficulty breathing caused by moderate exertion Psychological health was also worse in botulism survivors Sami L. Gottlieb, Katrina Kretsinger, Nato Tarkhashvili, Neli Chakvetadze, Maia Chokheli, Marina Chubinidze, R. Michael Hoekstra, Ekaterina Jhorjholiani, Merab Mirtskhulava, Maia Moistsrapishvili, Merab Sikharulidze, Tamar Zardiashvili, Paata Imnadze, Jeremy Sobel, Long-Term Outcomes of 217 Botulism Cases in the Republic of Georgia, Clinical Infectious Diseases, Volume 45, Issue 2, 15 July 2007, Pages 174–180, https://doi.org/10.1086/518890 Main Properties of Cb Group I Group II Toxin Type A; Proteolytic B, F E; non-proteolytic B,F Proteolysis + - Lipase Production + + Main physiological property Highly Thermoresistant spores Moderately Thermoresistant Spores Optimum Growth Temp 37∘C 25∘C Minimum Growth Temp 10-12∘C 2.5-3∘C Causes botulism in… Humans Humans Modified From: Fratamico, Liu, and Kathariou., 2011. Genomes of Food and Waterborne Pathogens. Published by American Society for Microbiology. Cb Genomics Cb genomes consist of one circular chromosome, between 3.6-4.1 Mb, with a G+C content of 27-28% Most strains also contain plasmids that vary in size The BoNT toxin can be carried either on the chromosome or on a plasmid The genomic context in which the BoNT toxin is found matters BoNT Genomics Type of Toxin Strain Group BoNT location BoNT locus A ATCC3502 I Chromosome HA-A1 A Hall I Chromosome HA-A1 A ATCC19397 I Chromosome HA-A1 A/B NCTC2916 I Chromosome OrfX-A1, HA-B A Kyoto-F I Chromosome OrfX-A2 A Loch Maree I Plasmid OrfX-A3 B Orka I Plasmid HA-B1 F Langeland I Chromosome OrfX-F B/F unnamed I Plasmid OrfX-F HA-B B Eklund II Plasmid HA-npB E Beluga II Chromosome OrfX-E1 E Alaska II Chromosome OrfX-E2 E B5262 II Chromosome OrfX-E3 Modified From: Fratamico, Liu, and Kathariou., 2011. Genomes of Food and Waterborne Pathogens. Published by American Society for Microbiology. BoNT Genomics The similarity of the different BoNTs, the ability of different species to produce BoNT, and the fact that some strains contain combinations of bont genes suggests that all bont genes derive from a similar ancestor – and move around Easy horizontal transfer of genes can be achieved by mobilization of the plasmids that carry BoNT toxins The possibility of C. difficile picking up a BoNT toxin is a serious concern Environmental Microbiology Cb is found almost ubiquitously in soils, you probably consume several spores daily Cb survives by secreting numerous hydrolytic enzymes and then transporting the degradation products into the cell (such as amino acids, small peptides, carbohydrates) Over 300 genes for transport systems have been identified Cb lacks several genes for amino acid synthesis Is a strict anaerobe Has a fermentative metabolism Proteolytic groups prefer proteinous compounds for fermentation substrates Many strains can use amino acids as their sole source of carbon and nitrogen Environmental Microbiology C. botulinum causing human botulism is commonly found in the Northern parts of the world Proteolytic group I type A spores are primarily found in temperate regions including soils of the Western United States, southern and eastern European countries, and China – Group I type A spores favor neutral to alkaline soils with low organic content; they are absent from eastern USA and western European soils – Type B spores are more ubiquitous – Type E spores are mainly found in aquatic sediments of colder regions such as Alaska, Canada, Scandinavia, Poland, Russia, and Japan Environmental Microbiology The true purpose of the BoNTs in nature are yet to be fully understood Deletion of the BoNT gene region in the lab does not alter the growth of the bacterium Cb is a saprophytic microorganism that secretes various hydrolytic enzymes to uptake nutrients – It has been isolated from rotting vegetation and from animal carcasses – Cb does not have an obligatory relationship with any animal host The current theory is that Cb may be predatory in some niches and that killing the host is important for the biome of animal decomposition and the recycling of animal biomass Very little work has been done in this area (working on rotting carcasses stinks) Classic Intoxication Case Late fall, a family harvests a variety of vegetables from the family garden (tomatoes, carrots, potatoes) and makes an abundance of vegetable soup Family home cans the soup using standard boiling methods, but does not check the pH of the soup prior to storage (and does not add any type of acid) 6 Months later in the winter they open a can of soup, which has no visible signs of spoilage By the next morning, they are seeing double, have difficulty swallowing and experience paralysis in their arms and legs The canning method had killed all bacterial competitors which were not spores The soup was at a neutral pH allowing the spores to germinate and produce toxin (though not spoil the product) The product was not cooked before consumption (boiling for 10 minutes denatures all of the toxin, and is recommended for home preserved foods) Toxin was consumed resulting in intoxication Canadian Inuit Muktuk is a traditional Inuit food which consists of raw or fermented beluga whale blubber In 2002 residents of a western Alaska village on the Bering Sea shore shared a meal consisting of muktuk harvested from a beached adult beluga whale found near their village The villagers estimated that the whale had been dead for at least several weeks Of 14 persons identified who ate the muktuk, eight (57%) had an illness that met the case definition of botulism intoxication Symptom onset after ingestion of muktuk occurred within 36 hours in all patients Five patients were hospitalized, four received antitoxin, and two required mechanical ventilation The beluga likely had come into contact with Cb spores from the water and water sediment During the weeks the whale had been dead the spores germinated and began producing toxin The whale was consumed with the toxin resulting in intoxication Intoxication Cb is sensitive to low pH and cannot germinate