Clostridium

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Clostridium

• Erica Cooper
• Kristi Martinez
• Karen Peterson
• Jen Tran

2
Overview

• Background
• Species and strains of Clostridium
• C. botulinum
• C. perfringens
• Detection
• In patients
• In food

3
Clostridium General Information

• Anaerobic
• Rods
• Form spores
• Produce toxins
• Species of note but not discussed
• C. tetani
• C. cellulolyticum

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Clostridium botulinum

• Gram positive spore forming rods
• Spores are heat resistant
• Widely found in nature
• Toxins produced are most potent
• Seven types of botulism (A, B, C, D, E, F, G)
• Toxin not heat stable

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Symptoms and Disease

• 12-36 hrs post-intake. Can range 6 hrs to 10 days
• Flaccid paralysis
• Can be fatal

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Action of Botulinum Toxin

• 0.1-1 nanograms (ng) needed to cause illness
• Toxin is absorbed in intestine, transported to
  neuromuscular junctions via blood stream
• Cleaves proteins which enable synaptic vesicle
  fusion in neurons
• Neuromuscular neurotransmitter blocked
• Motor dysfunction

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Host Defenses

• Immunity specific for each toxin type
• An earlier encounter of botulism will not provide
  immunity

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Sources Types of Food Incriminated

• Home canned foods
• Fish preserved by salting or smoking
• Prepared meats eaten uncooked
• Honey can be problematic for infants

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Diagnosis

• Clinical
• Gastrointestinal symptoms
• Nervous system
• Laboratory
• Isolation of organism from contaminated food or
  stool sample
• Demonstrate toxin is existent
• CSF (cerebral spinal fluid) rule out

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Treatment and Control

• Suspicion of botulism poisoning to initiate
  therapy
• Four objectives in therapy
• Eliminate unabsorbed toxin
• Eliminate source of toxin
• Neutralize the unbound toxin
• Supportive care
• Antitoxin therapy
• Antibiotics

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Epidemiology

• Generally 10-30 outbreaks/year in U.S.
• Arctic and Alaska
• First reported in 1900s
• Diet
• Traditional preparation vs. modern techniques for
  food preparation

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Clostridium perfringens

• Previously named C. welchii
• Anaerobic, gram-positive, sporulating
• Widely distributed
• Soil and sediments
• Intestinal tracts
• Areas with fecal contamination
• Toxins can cause problems for humans
• At least 12 identified

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C. perfringens Indicator of Water Contamination

• Problem
• Current methods analyzing fecal contamination
  developed in temperate climates
• Not accurate in tropical regions
• Climate
• Increased nutrient levels

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C. perfringens Indicator of Water Contamination

• Uganda study (Byamukama, et al. 2005)
• Fecal coliforms
• Escherichia coli
• C. perfringens
• Advantages to quantifying spores
• Indigenous at low level
• Numbers increase with fecal contamination
• Resistance enables testing of remote areas

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C. perfringens Indicator of Water Contamination

• Disadvantage
• No strong controls
• Sites could have been contaminated without
  researchers knowledge
• Conservative recommendation
• Use two or more indicator organisms, C.
  perfringens being one

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C. perfringens Indicator of Water Contamination

• 2004 study found C. perfringens is a useful
  indicator in Antarctic near-shore marine
  measurements.(Hughes and Thompson)
• Strong environmental control included

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C. perfringens Poisoning Overview

• Perfringens food poisoning
• Necrotic enteritis
• Gas gangrene
• General information
• Infective dose on order of 105-106 cells
• Caused by undercooked and reheated foods

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Perfringens Food Poisoning

• Type A strain
• Symptoms
• Abdominal cramps, diarrhea
• Death rare
• Symptoms appear 8-22 hours after bacteria
  consumed
• Usually over in 24 hours
• Unreported cases

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Perfringens Food Poisoning

• Very common in U.S
• Average of 15 outbreaks each year for the last 2
  decades
• Each outbreak involves 10s to 100s of people
• Second in reported cases (Salmonella poisoning
  most common)

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St. Patricks Day 1993 Outbreak

• March 12-16
• Deli prepared 1,400 pounds of raw beef
• Boiled for 3 hours
• Cooled to room temperature
• Refrigerated
• March 16 and 17
• Corned beef made into sandwiches
• Held at room temperature up to 7 hours
• 171 cases of PFP reported
• Morbidity and Mortality Weekly Report (1994)
  43(8) 137-138

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Perfringens Food Poisoning, 2001
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Gas Gangrene and Necrotic Enteritis

• Wound invasion causes gas gangrene
• Type A strain
• Ingestion causes necrosis of intestines
• Type C strain
• Results in septicemia
• Rare in the united states
• Often fatal
• C. perfringens one of 6 species causing necrotic
  enteritis
• Present in 80-90 of all cases.

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Pancreatic Clostridial Gas Gangrene
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C. perfringens Analysis and Diagnosis

• Food
• Culture and identification of bacteria
• Molecular techniques
• Patients
• Toxin detected in feces

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Monitoring

• Detection
• In patients
• In food
• In the environment (soil and water)
• Prevention
• Regulation by national agencies

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Detection in Patients

• Patient is clinically diagnosed
• Based on history, epidemiology, and symptoms
• This is only a presumptive diagnosis
• To confirm diagnosis, must identify potential
  sources of contamination and test for clostridial
  toxin or cells

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Detection in Food

• Food is often source of clostridial infection
• Test suspect food using a combination of
  techniques
• Phenotypic techniques
• Older, well-established methods
• Often yield results slowly
• Molecular or genotypic techniques
• Newer, more rapid methods
• Efficacy has not yet been established for many

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Phenotypic Techniques to Detect Clostridia

• Culture
• Enrich for and enumerate clostridia from food
  extract
• Disadvantage hard to differentiate between
  pathogenic and non-pathogenic strains of a
  species
• Clostridia were present in the food, but are they
  pathogenic strains?

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Phenotypic Techniques to Detect Clostridia

• Culture
• Mouse bioassay to test for toxin production
• Inject food extract into mice
• If clostridia produce toxin, mouse will get sick
• Disadvantage time- and labor-intensive
• Now we know there is toxin, but what type is it
  and what strain is it from?

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Phenotypic Techniques to Detect Clostridia

• Culture
• Mouse bioassay to test for toxin production
• Antitoxin (antibody) neutralization
• Differentiates between antigenic types
• Measures ability of specific antibodies to
  neutralize toxin
• Disadvantage toxin production requires
  sporulation, which can be hard to induce in lab
  cultures

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Genotypic Techniques to Detect and Type Clostridia

• Polymerase chain reaction (PCR)
• Amplification of DNA sequence
• Fast and easy
• Detects genes for toxinspresumptive only
• Used in conjunction with mouse bioassay to
  confirm toxicity
• Various PCR-based techniques available
• PFGA
• ELISA/EIA

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Genotypic Techniques to Detect and Type Clostridia

• PCR
• Pulsed-field gel electrophoresis (PFGE)
• Comparison of restriction digest pattern of whole
  genomic DNA
• Run on agarose gel with alternating orientation
  of electrical field
• Allows for very large DNA fragments to move
  through agarose gel
• Allows for subtyping of strains
• Accurate, reproducible, and fast
• ELISA/EIA

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Genotypic Techniques to Detect and Type Clostridia

• PCR
• PFGE
• Enzyme-linked immunosorbent assay (ELISA/EIA)
• Serological test for toxin
• Fastonly requires one day
• Disadvantage toxin production
• Requires sporulation, which is hard
• To induce

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Genotypic Techniques to Detect and Type Clostridia

• PCR
• PFGE
• ELISA/EIA
• Other methods Ribotyping (analyzing ribosomal
  genes), plasmid analysis (for plasmid-encoded
  virulence genes), and AFLP (amplified fragment
  length polymorphism)

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Detection in the Environment

• By PCR or culture
• C. perfringens as an indicator of fecal
  contamination
• Normal intestinal microbiota
• Culture from water, sediment, or soil samples and
  enumerate
• Better indicator than traditional fecal coliforms
  in tropical regions

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Prevention in seafood

• Education
• Especially to native peoples who practice
  traditional food preparation
• Heat
• Botulinum cook
• Designed to kill most heat-resistant spores of C.
  botulinum
• Also inactivates toxin
• Refrigerate

• Prepare in high acidity, high salt
  concentration, high preservative concentration,
  or low moisture
• Best is a combination of two or more
• Prevent anaerobic conditions

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Prevention in Seafood

• Education
• Especially to native peoples who practice
  traditional food preparation
• Heat
• Botulinum cook
• Designed to kill most heat-resistant spores of C.
  botulinum
• Also inactivates toxin
• Refrigerate

• Prepare in high acidity, high salt
  concentration, high preservative concentration,
  or low moisture
• Best is a combination of two or more
• Prevent anaerobic conditions

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Examples of Regulation

• The European union regulates the sale of
  shellfish
• US FDA prohibits processing, distribution, and
  sale of kapchunka
• Uneviscerated, dry-salted, air-dried, whole
  whitefish

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Summary of Clostridium

• C. perfringens
• Normal fecal microbiota
• Indicator of fecal contamination
• Common food-borne pathogens
• Gastroenteritis
• Gas gangrene
• Necrotic enteritis
• C. tetani
• Tetanus toxin lethal
• Not associated with aquatic habitats or seafood
• C. cellulolyticum
• Bioengineering

• Clostridium as a genus
• Anaerobes
• Produce endospores
• Widely distributed
• Pathogens and non-pathogens
• C. botulinum
• Botulinum toxin lethal
• Flaccid paralysis
• Foodborne pathogen
• Toxin for therapy and cosmetology

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Summary of Clostridium

• Detection
• Combination of phenotypic and molecular
  techniques
• Prevention
• Heating and refrigeration of foods
• Sanitary handling of seafood

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References

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  Manafi, M., and Farnleitner, A. H. (2005)
  Discrimination Efficacy of Fecal Pollution
  Detection in Different Aquatic Habitats of a
  High-Altitude Tropical Country, Using Presumptive
  Coliforms, Escherichia coli, and Clostridium
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  Microbiology. 71 (1) 65-71.
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  and Mortality Weekly Report 43(8) 137-138.
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  maritime Antarctic research station indicated by
  faecal coliforms, Clostridium perfringens and
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  127 315-321.
• .Huss, H. H. 1997. Control of indigenous
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  91-98.
• .Kato, S., Haruta, S., Cui, Z. J., Ishii, M., and
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