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  1. 1. 06.12.2013 ClOSTRIDIA ASHUTOSH KUMAR MAHAFIZUR RAHMAN ASHIK TILMILSINA Outlines of presentation 1.What is clostridium 2.Taxonomy 3.Transmission 4.Clinical Presentation 5.Treatment and Prevention 6.Diagnostic Method 1
  2. 2. 06.12.2013 What is clostridium ? Genus of 1.Gram positive 3. Endospores 2.Obligate anaerobes 4.Rod shaped Scientific Classification Domain :- Bacteria Kingdom :- Eubacteria Phylum :-Firmicutes Class :-Clostridium Order :-Clostridiales Family :- Clostridiaceae Genus :-Clostridium Species :- >100 2
  3. 3. 06.12.2013 Important Pathogenic Species of Clostridium Clostridium botulinum Clostridium perifringers Clostridium Clostridium difficile Clostridium tetani Clostridium botulinum First recognized and isolated in 1896 by Van Ermengem 1. Gram positive rods 2. Obligate anaerobes :- oxygen is poisonous to it but it can tolerate a little amount of it because of presence of superoxidase dismutase(SOD) enzymes which is a antioxidant defense present in some cells 3. Can produce endospores only in absence of oxygen 4. Able to produces neurotoxins during the formation of spores 3
  4. 4. 06.12.2013 Transmission Clostridium botulinum is a soil bacterium and its spores are found in soil.I It can easily transmitted in humans by soil contaminated foods. most common human diseases: 1. Food poisoning 2. Wound botulinum 3. Infant botulism The spores of C.botulinum are heat resistant, they can not be killed by simple boiling Clostridium perfringens Clostridium perfringens are: 1. Strongly Gram positive rods 2. Non motile 3. Short, thick, straight parallel sides with round edges 4. Spore forming Sporulation favored by alkaline environment and absence of fermentable CHO If spores are produced, they are large, oval and central Capsulated especially at the site of infection Capsule disappears when organism is grown on culture 4
  5. 5. 06.12.2013 Transmission C. perfringens is present in the environment and can be found in - decaying vegetation - marine sediments - intestinal tract of humans - meats and poultry - soil The most common diseases are 1.Food poisoning 2.Gas gangrene Despite its potential danger C. perfringens is used as the leavening agent in salt rising bread. The baking process is thought to reduce the bacterial contamination, preventing negative effects Clostridium difficile It was first described in 1935 by Hall and O’ Toole and then later in year 1970 recognized as cause of antimicrobial associated diarrhea Clostridium difficile is a Gram positive, rod shaped, spore forming, anaerobic bacterium which is ubiquitous in nature and mainly prevalent in soil and human intestine It best grows in blood agar at human body temperature The spores are able to tolerate high heat and resistance to normal cleaning (except diluted bleach) 5
  6. 6. 06.12.2013 Transmission The transmission of C. difficile is mainly due to nosocomially (hospital acquired infection) at hospitals, nursing homes and other medical institution. The rate of C. difficile acquisition is estimated to be 13% in patients with hospital stays up to 2 weeks and 50 % in those with hospital stays longer than 4 weeks Clostridium tetani 1884 - Arthur Nicolaier isolated the strychnine like toxins of tetanus from free living anaerobic soil bacteria 1890 - Antonie Carl and Giorgio Rahtone demonstrated the transmission of tetanus for the first time 1897 - Edmond Nocard showed tetanus antitoxin induced passive immunity in humans and use of prophylaxis treatment. 1924 - Pdescombey developed the first tetanus toxoid vaccine to prevent the induced tetanus Rod shaped anaerobic but able to tolerate little amount of oxygen Usually gram positive Motile by flagella Heat resistant spore survive up to 40+years 6
  7. 7. 06.12.2013 Transmission Pathophysiology 7
  8. 8. 06.12.2013 Clostridium tetani Transmission: Contact with non intact skin, usually injuries from contaminated objects It is also seen in the umbilical stump of infants in underdeveloped countries Incubation time: It may take from three to 21 days to develop any symptoms. In infants, symptoms may take from three days to two weeks to develop Symptoms: The following are the most common symptoms of tetanus Stiffness of the jaw Stiffness of the abdominal and back muscles Contraction of the facial muscles Fast pulse Fever Sweating Painful muscle spasms near the wound area 8
  9. 9. 06.12.2013 C. tetani infection/intoxication Spores of C. tetani in wound Germination and toxin production under anaerobic conditions Transport of toxins via lymphatic- bloodstream through motor neurons to spinal cord Wound contaminated with soil Symptoms [Madigan et al. 2009] 17 Mechanism of action Inhibitory interneurons Excitation signals from the central nervous system Release of acetylcholine inhibited by glycine  Muscle relaxation Tetanus toxin Uncontrolled release of acetylcholine  Spastic paralysis 18 Modified according to [Madigan et al. 2009] 9
  10. 10. 06.12.2013 Mechanism of action  Inhibitory neurotransmitter glycine usually stops the release of acetylcholine from the motor neurons   muscle relaxation  AB toxin (= Tetanospasmin, 50 kDa light chain + 100 kDa heavy chain) inhibits release of glycine from inhibitory  interneurons  No glycine  constant release of acetylcholine  spastic paralysis / uncontrolled contraction of the muscle [Madigan et al. 2009] 19 Treatment:  Immediate treatment with human tetanus immune globulin (TIG)  Medication to control muscle spasm  Aggressive wound care  Antibiotics: Metronidazole, Clindamycin, Erythromycin, Penicilin Prevention:  Being fully immunized is the best tool to prevent tetanus  Immediate and proper wound care can also help prevent infection 10
  11. 11. 06.12.2013 Clostridium perfringens Transmission:  C. perfringens is one of the most common causes of foodborne illness  eating food contaminated with large numbers of C. perfringens bacteria that produce enough toxin in the intestines to cause illness  Beef, poultry, gravies, and dried or pre-cooked foods are common sources of C. perfringens infections  C. perfringens infection often occurs when foods are prepared in large quantities and kept warm for a long time before serving Incubation Time: Persons infected with C. perfringens develop diarrhea and abdominal cramps within 6 to 24 hours (typically 8-12) Symptoms: Diarrhea, usually without vomiting or fever Abdominal cramps Nausea 11
  12. 12. 06.12.2013 Mechanism of action Treatment: Oral rehydration in severe cases, intravenous fluids and electrolyte replacement can be used to prevent or treat dehydration Prevention: foods commonly associated with C. perfringens infections should be cooked thoroughly in temperatures Sufficient cooling of pasteurized and cooked food (especially meat products) No long storage periods Good cleaning of food 12
  13. 13. 06.12.2013 Clostridium botulinum Transmission: Botulism is caused by exposure to botulinum toxin, humans can become infected in a number of ways:  Inhalation of toxin  Consumption of toxin (foodborne)  Consumption of C. botulinum spores (infant; adult intestinal toxemia)  Contamination of a tissue with C. botulinum spores (wound) Incubation Period:  12-80 hours (range 2 hours to 8 days) Botulism Foodborne botulism C. botulinum in food Toxin produced Toxin ingested Toxin in bloodstream Attacks neurons (flaccid paralysis) Infant botulism C. botulinum spores ingested Wound botulism C. botulinum spores in wound C. botulinum grows in intestinal tract C. botulinum grows in wound Toxin produced Toxin produced Modified according to [Salyers et al. 1994] 13
  14. 14. 06.12.2013 Botulinum toxin 7 related AB toxins (A‐G) most potent biological toxins known [Madigan et al. 2009] Relevant toxins for human diseases: A, B, E [Lindstöm et al. 2006] Light chain (50 kD) A Heavy chain (100 kD) B AB toxin (150 kD) [Zhang et al. 2012] Mechanism of C. botulinum intoxication  Absorption of toxin by mucosal surfaces in the gastrointestinal system, the eye or non intact skin.  Botulinum toxin blocks acetylcholine release at the neuromuscular junction of skeletal muscle neurons and peripheral muscarinic cholinergic autonomic synapses  It binds irreversibly to presynaptic receptors to inhibit the release of acetylcholine and cause neuromuscular weakness and autonomic dysfunction 14
  15. 15. 06.12.2013 Symtomps: Nausea Constipation Paralysis Double vision , blurred vision Dry mucous membranes Dysphagia 15
  16. 16. 06.12.2013 Treatment: Foodborne botulism • Within 1 hour of ingestion of suspected food, the recommended course of action is a gastric lavage, or enemas and the administration of a cathartic (sorbitol) • Administration of antitoxin: only useful, if toxin has not yet bound to neural cell Wound botulism • Antitoxin, wound debridement, and antibiotic treatmen Infant botulism • Requires meticulous supportive care • An investigational human-derived botulinum immunoglobulin (BIG) is available for the treatment Prevention: Foodborne botulism can be controlled by safe canning and food manufacturing processes The risk factors for infant botulism are poorly described. Honey should not be fed to infants because it has been identified as a food source Wound botulism cases have occurred in persons who used illicit drugs 16
  17. 17. 06.12.2013 Clostridium difficile Transmission:  C. difficile bacteria and their spores are found in feces. People can get infected if they touch surfaces contaminated with feces, and then touch their mouth  Healthcare workers can spread the bacteria to their patients if their hands are contaminated Incubation period:  The exact incubation time for CDI is unknown, the time from acquisition to disease is relatively short, perhaps no longer than 7 days Symptoms: Mild C. difficile colitis:  a low-grade fever  mild diarrhea (5-10 watery stools a day)  mild abdominal cramps and tenderness Severe C. difficile infection:  Watery diarrhea, up to 15 times each day  High fever (temperature of 38°C to 40°C)  Severe abdominal pain  Loss of appetite  Blood or pus in the stool  Weight loss 17
  18. 18. 06.12.2013 Mechanism of infections Mechanism of action Toxin A: Enterotoxin that disturbs the transport of  electrolytes  loss of fluid, diarrhea Toxin B: Cytotoxin that damages cells of the colon 36 [Jones et al. 2012] 18
  19. 19. 06.12.2013 Treatment:  Antibiotics: Metronidazole, Vancomycin  Probiotics  Rehydration Prevention:  To prevent the spread of C. difficile hospitals and other health care facilities follow strict infection control guideline • Hand washing • Contact precautions • Thorough cleaning DIAGNOSTIC METHODS ASHIK TIMILSINA 19
  20. 20. 06.12.2013 CONTENTS Culture Methods Test for Clostridia Identification Gram Staining Equipment for Anaerobic culturing Diagnostic Methods – Mouse Lethality Assay – Endopeptidase Assay – Immunological Methods – Molecular Detection Culture Media Clostridia are obligate anaerobes Media used for isolation are Non-selective media: AEA Sporulation Broth (Base), modified, Reinforced Clostridial Agar……… Selective Media: Clostridium difficile Agar (Base), Anaerobic Blood Agar with Neomycin…….. Differential Media: Egg-Yolk Agar Selective Differential Media: HiCrome™ M-CP Agar Base, SPS Agar modified….. 20
  21. 21. 06.12.2013 Egg Yolk Agar Special kind of culture media  Contains suspension of egg yolk for identification of lecithinase and lipase activity of different clostridium species  Lecithinase degradation in the yolk results in opaque colonies  Lipase enzyme hydrolizes the fat and results in iridescent sheen of the colony LEC/LIP Activity of different clostridia species Species LEC LIP C. botulinum + + C. prefringens + _ C. tetani _ _ C. difficile _ _ 21
  22. 22. 06.12.2013 Egg Yolk Agar Lecithinase activity Lipase activity Anaerobic Equipment • • • • Anaerobe Atmosphere Generation Bags Anaerobe Indicator Test Anaerobe Jar Insert for Petri Disks Anaerobic Jar 22
  23. 23. 06.12.2013 Gram Staining C. botulinum C. difficile C.tetani C. perfringens 23
  24. 24. 06.12.2013 Mouse lethality assay (C. botulinum) • standard procedure to detect C. botulinum • standard procedure to detect botulinum neurotoxins • Mice are injected with the cultivated microorganisms (fluid extract) or with fluid extract of sample (fecal, serum, gastric, wound, food samples) • The mice get different antitoxins. Fluid extract of cultivated microorganisms or sample fluid Antitoxin Observed for 48h Mouse with Antitoxin A Mouse with Antitoxin B Mouse with Antitoxin E Mouse with Antitoxin F Mouse With No Antitoxin 24
  25. 25. 06.12.2013 After 48 h: Signs of botulism:  Fuzzy hair  Muscle weakness  Respiratory failure ‐> wasp‐like narrow waist Mouse with Antitoxin A Mouse with Antitoxin B Mouse with Antitoxin E Mouse with Antitoxin F Mouse With NO Antitoxin Comments • Generally group I and II clostridium causes Botulism in humans • Group III is involved with animal botulism • Group I culture produces A, B or F toxin • Group II culture produces B, E or F toxin 25
  26. 26. 06.12.2013 Pro’s and Con’s Pro’s : Very Sensitive Con’s: Laborious, expensive, ethical problem, detects only C. botulinum Endopeptidase Assay (C. botulinum) • These assay method is applied for detection of type A and type B botulinum neurotoxins • Type B neurotoxin is based on cleavage of synthetic peptide substrate • Type A neurotoxin is based on cleavage of peptide substrate derived from protein SNAP-25 • These peptide cleave shows the type of neurotoxin 26
  27. 27. 06.12.2013 Pro’s and Con’s Pro’s: Highly specific, no cross reactivity between different botulinum toxins Con’s: Needs more research, only C. botulinum detectable, only biologically active neurotoxin Enzyme Linked Immunosorbent Assay (ELISA) • Combines specificity of antibodies with sensitivity of simple enzyme essay • ELISA measures antigen or antibody concentration • Are used to detect antigens that are recognized by antibody or vice-versa. 27
  28. 28. 06.12.2013 Pro’s and Con’s Pro’s: Technical simple, fast to perform and interpret, C. botulinum, C. perfringens, C.tetani, C. difficile can be detected by this technique Con’s: Inactivated toxins  false positive result Genetic variation in serotypes  false negative results 28
  29. 29. 06.12.2013 Molecular Detection DNA-based detection methods have overtaken all the conventional techniques Technique is based on the detection of the clostridia toxin genes in the sample Detects clostrida toxin genes:  botA , botB , botE , botF (C. botulinum)  tcdA, tcdB (C. difficile)  cpe, plc, cpa, cpb (C. perfringens)  tetR (C. tetani) Sample DNA Exraction Polymera se Chain Reaction PCR Multiplex PCR qPCR 29
  30. 30. 06.12.2013 Principle Polymerase Chain Reaction Primers of PCR for bolinum toxin genes Toxin type Primer Product size (bp) A IOAf/IOAr 101 B CBMLBf/CBMLBr 205 E CBMLEf/CBMLEFr 389 F CBMLFf/CBMLFr 543 IAC IACf/IACr 698 30
  31. 31. 06.12.2013 Multiplex PCR Multiplex PCR • Multiple primer pairs are used  more than one gene can be amplified at the same time • Specific amplified gene = defined amplicon size 31
  32. 32. 06.12.2013 Quantitative real-time PCR • One method used is Taq-Man real-time PCR • Taq-Man probe is used in addition to primers in PCR • Probe is an oligonucleotide consisting of a sequence matching to the middle of the template sequence (DNA) • Probe has attached: Flurophore and Quencher 32
  33. 33. 06.12.2013 Pro’s and Con’s Pro’s: Clinic sample can be used directly, fast procedure, sensitive Con’s: Expensive (rt‐qPCR), detects only the toxin gene and not the toxin itself ASSAY Time to perfrom Application Pro’s Con’s Mouse Lethality 1‐4 days Bacterial culture  serum, gastric  content, food and  environment  sample etc.. Very Sensitive Laborious, ethical  problem,  expensive, detects  only       C.  botulinum Endopeptidase 1‐2 days Medical industry,  infant faecal sample, human  serum etc… High Specific, no  cross reaction  between  botulinum toxin Only  C. botulinum detectable ELISA 8 hrs Bacterial culture,  innoculate meat,  cheese food etc.. Technically  simple, fast to  perform and  interpret,  different toxins  detectable Inactivated toxins  false result PCR Fast, few hours Medical field,  Clinic sample can  laboratory analysis be used directly,  fast procedure,  sensitive, different  clostridia genes  detectable Expensive  (rt‐qPCR), detects  only the gene, not  the toxin itself 33
  34. 34. 06.12.2013 References • Clinical Microbiology Reviews – Laboratory Diagnostics of Botulism [Lindström,Korkeala] • Manual of Clinical Microbiology [Patrick R. Murray, Ellen Jo Barron et al.] • Medizinische Mikrobiologie [Herbert Hof, Rüdiger Dörries] • Dario et al, Multiplex PCR for Detection of Botulinum Neurotoxin – Producing Clostridia in clinical, food, and environment samples; (20):6457-6461; Appl. Environmental Microbiology, 2009 References • • • • • • Lindström M, Korkeala H: Laboratory Diagnostics of Botulism. Clin. Microbiol. Rev. 2006, 19(2):298 Madigan MT, Martinko JM, Dunlap P V, Clark DP: Biology of  Microorganisms. 12th edition, Pearson Education Inc.,  San Francisco 2009 Salyers AA, Whitt DD: Bacterial Pathogenesis: A molecular  approach. ASM Press, Washington, D.C. 1994 Zhang Y, Lou J, Jenko KL, Marks JD, Varnum SM:  Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme‐linked immunosorbent assay‐based  protein antibody microarrays. Analytical Biochemistry 430 (2012) 185–192 “Development of real‐time PCR tests for detecting botulinum neurotoxins A,B,E,F producing Clostridium botulinum, […].” [P.Fach et al.], Journal of AppliedMicrobiology Taq‐Man‐Picture [] 34
  35. 35. 06.12.2013 35