Title: Clostridium
1Clostridium
- Erica Cooper
- Kristi Martinez
- Karen Peterson
- Jen Tran
2Overview
- Background
- Species and strains of Clostridium
- C. botulinum
- C. perfringens
- Detection
- In patients
- In food
3Clostridium General Information
- Anaerobic
- Rods
- Form spores
- Produce toxins
- Species of note but not discussed
- C. tetani
- C. cellulolyticum
4Clostridium 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
5Symptoms and Disease
- 12-36 hrs post-intake. Can range 6 hrs to 10 days
- Flaccid paralysis
- Can be fatal
6Action 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
7Host Defenses
- Immunity specific for each toxin type
- An earlier encounter of botulism will not provide
immunity
8Sources Types of Food Incriminated
- Home canned foods
- Fish preserved by salting or smoking
- Prepared meats eaten uncooked
- Honey can be problematic for infants
9Diagnosis
- 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
10Treatment 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
11Epidemiology
- 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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13Clostridium 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
14C. perfringens Indicator of Water Contamination
- Problem
- Current methods analyzing fecal contamination
developed in temperate climates - Not accurate in tropical regions
- Climate
- Increased nutrient levels
15C. 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
16C. 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
17C. 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
18C. 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
19Perfringens 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
20Perfringens 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)
21St. 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
22Perfringens Food Poisoning, 2001
23Gas 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.
24Pancreatic Clostridial Gas Gangrene
25C. perfringens Analysis and Diagnosis
- Food
- Culture and identification of bacteria
- Molecular techniques
- Patients
- Toxin detected in feces
26Monitoring
- Detection
- In patients
- In food
- In the environment (soil and water)
- Prevention
- Regulation by national agencies
27Detection 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
28Detection 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
29Phenotypic 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?
30Phenotypic 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?
31Phenotypic 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
32Genotypic 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
33Genotypic 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
34Genotypic 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
35Genotypic 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)
36Detection 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
37Prevention 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
38Prevention 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
39Examples 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
40Summary 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
41Summary of Clostridium
- Detection
- Combination of phenotypic and molecular
techniques - Prevention
- Heating and refrigeration of foods
- Sanitary handling of seafood
42References
- .Byamukama, D., Mach, R. L., Kansiime, F.,
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
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