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Clostridium

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An earlier encounter of botulism will not provide immunity. Sources: Types ... Suspicion of botulism poisoning to initiate therapy. Four objectives in therapy ... – PowerPoint PPT presentation

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Title: Clostridium


1
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

4
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

5
Symptoms and Disease
  • 12-36 hrs post-intake. Can range 6 hrs to 10 days
  • Flaccid paralysis
  • Can be fatal

6
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

7
Host Defenses
  • Immunity specific for each toxin type
  • An earlier encounter of botulism will not provide
    immunity

8
Sources Types of Food Incriminated
  • Home canned foods
  • Fish preserved by salting or smoking
  • Prepared meats eaten uncooked
  • Honey can be problematic for infants

9
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

10
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

11
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

12
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13
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

14
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

15
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

16
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

17
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

18
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

19
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

20
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)

21
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

22
Perfringens Food Poisoning, 2001
23
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.

24
Pancreatic Clostridial Gas Gangrene
25
C. perfringens Analysis and Diagnosis
  • Food
  • Culture and identification of bacteria
  • Molecular techniques
  • Patients
  • Toxin detected in feces

26
Monitoring
  • Detection
  • In patients
  • In food
  • In the environment (soil and water)
  • Prevention
  • Regulation by national agencies

27
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

28
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

29
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?

30
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?

31
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

32
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

33
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

34
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

35
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)

36
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

37
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

38
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

39
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

40
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

41
Summary of Clostridium
  • Detection
  • Combination of phenotypic and molecular
    techniques
  • Prevention
  • Heating and refrigeration of foods
  • Sanitary handling of seafood

42
References
  • .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
    Coliforms, Escherichia coli, and Clostridium
    perfringens Spores. Applied and Environmental
    Microbiology. 71 (1) 65-71.
  • .Centers for Disease Control (2001). US Foodborne
    Disease Outbreaks. Retrieved May 25, 2005 from
    http//www.cdc.gov/foodborneoutbreaks/us_outb.htm.
  • .Centers for Disease Control (1994). Morbidity
    and Mortality Weekly Report 43(8) 137-138.
  • .The Free Dictionary (2005). Retrieved May 11,
    2005 from http//encyclopedia.thefreedictionary.co
    m/Clostridium.
  • .Hughes, K. A. and Thompson, A. (2004).
    Distribution of sewage pollution around a
    maritime Antarctic research station indicated by
    faecal coliforms, Clostridium perfringens and
    faecal sterol markers. Environmental Pollution.
    127 315-321.
  • .Huss, H. H. 1997. Control of indigenous
    pathogenic bacteria in seafood. Food Control. 8
    91-98.
  • .Kato, S., Haruta, S., Cui, Z. J., Ishii, M., and
    Igarashi, Y. (2004). Effective cellulose
    degradation by a mixed-culture system composed of
    a cellulolytic Clostridium and aerobic
    non-cellulolytic bacteria. FEMS Microbial
    Ecology. 51133-142.
  • .Louisiana State University (N.D.) Clostridium
    perfringens Thioglycollate and ANABAP. Retrieved
    May 26, 2005 from http//www.medschool.lsuhsc.edu/
    microbiology/DMIP/cpthio.jpg.
  • .Lukinmaa, S., U.-M. Nakari, M. Eklund, and A.
    Siitonen. 2004. Application of molecular genetic
    methods in diagnostics and epidemiology of
    food-borne bacterial pathogens. APMIS. 112
    908-929.
  • .McGinley, S. (1998). Clostridium perfringens
    New Ways to Type Strains of a Deadly Bacteria.
    Retrieved May 26, 2005 from http//ag.arizona.edu/
    pubs/general/resrpt1998/clostridium.html.
  • .New York University (N.D.) Gram Positive
    Bacilli. Retrieved May 26, 2005 from
    http//education.med.nyu.edu/courses/microbiology/
    courseware/infect-disease/GramPosBacilli3.html.
  • .Purves, D., et al. (2001). Neuroscience, Second
    Edition. Sunderland, MA Sinauer Associates, Inc.
    Retrieved May 18, 2005 from http//www.ncbi.nlm.ni
    h.gov/entrez/query.fcgi?cmdSearchdbbooksdoptcm
    dlGenBookHLtermclostridiumANDneurosci5Bbook
    5DAND2311925Buid5Dridneurosci.box.330.
  • .State of Alaska Health and Social Services
    (1998). Botulism in Alaska. Retrieved May 26,
    2005 from http//www.epi.hss.state.ak.us/pubs/botu
    lism/bot_01.htm.
  • .U.S. Food and Drug Administration (2001).
    Bacteriological Analytical Manual Online.
    Retrieved May 26, 2005 from http//www.cfsan.fda.g
    ov/ebam/bam-toc.html.
  • .U.S. Food and Drug Adminsitration (1992). The
    Bad Bug Book. Retrieved May 24, 2005 from
    http//www.cfsan.fda.gov/mow/intro.html.
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    May 25, 2005 http//www.doh.wa.gov/Notify/guidelin
    es/pdf/ncg20021014.pdf.
  • http//www.ahc-net.at/0001/antibiotika_monitor/4_0
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