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Appropriate interventions to prevent growth of Clostridium Botulinum

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Appropriate interventions to prevent growth of Clostridium Botulinum

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Use approved heat processes for commercially and home-canned foods (i.e., pressure-can low-acid foods such as corn or green beans, meat, or poultry).

Discard all swollen, gassy, or spoiled canned foods. Double bag the cans or jars with plastic bags that are tightly closed. Then place the bags in a trash receptacle for non-recyclable trash outside the home. Keep it out of the reach of humans and pets.

Do not taste or eat foods from containers that are leaking, have bulges or are swollen, look damaged or cracked, or seem abnormal in appearance. Do not use products that spurt liquid or foam when the container is opened.

Boil home-processed, low-acid canned foods for 10 minutes prior to serving. For higher altitudes, add 1 minute for each 1,000 feet of elevation.

Refrigerate all leftovers and cooked foods within 2 hours after cooking (1 hour if the temperature is above 90 °F).

One of the most common causes of foodborne botulism is improperly home-canned food, especially low-acid foods such as vegetables and meats. Only a pressure cooker/canner allows water to reach 240 to 250 °F, a temperature that can kill the spores.

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Interview conducted on 75 / 77 person.
Stated that consumption of any potato salad (homemade/commercial) yield a higher association with propable and confirmed case statis.

Reason:
Attendee who prepare potato salad with home-canned potatoes using a boiling water canner, which do not kill C.botulinum spore.
Other than that, because potato are not heated after removal from the can.

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Outbreak and Food Associated with C. Botulinum

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Outbreak and Food Associated with C. Botulinum

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A outbreak of botulism which associated with Church Potluck Meal.

Location and Date:
- April 19, 2015.
- Farfield Country, Ohio.

Cases : Eat potato salad at church.

- Farfield Medical Centre (FMC) contact Ohio Department about patient suspected having botulism. At afternoon, 1 person died of respiratory failure.

-77 person consumed potluck food
25 (33%) met confirmed case
4 (5%) met propable case
Among 26 (90%) patients stated;
ilness began a median of 2 days after potluck

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If consuming home-canned foods of low acidity, heat to at least 80°C (176°F) for 10 minutes. Canned corn, spinach, and meats should be heated for 20 minutes.

Oils infused with garlic or herbs should be properly refrigerated during storage.

Canned food products, including both those produced at home and commercially, should be inspected before use. Cans with bulging or damaged lids, leakage, or off-odors should not be used, as these are signs indicative of gas-producing bacteria growth within the can. To ensure that the proper time, temperature, and pressure requirements are met to eliminate growth of the bacteria and its spores, a pressure cooker should be used to produce canned food products at home.

Although foodborne botulism is less commonly associated with commercially canned food products, be mindful not to consume the contents of expired or damaged cans. If canning meats at home, consider including food preservatives such as nitrites in the brine solution to reduce the growth of C. botulinum.

Vacuum-packaged meats should be refrigerated or properly stored in the freezer for extended usage.

During holding or storage, maintain proper food temperature. Put simply, keep hot foods hot (above 57°C/135°F) and cold foods cold (below 5°C/41°F) to prevent the formation of spores.

Wash hands, utensils, and kitchen surfaces that come into contact with food using hot soapy water before food preparation, after contact with raw meat or seafood, and after using the bathroom.

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A massive outbreak of type E.botulinum associated with traditional salted fish in Cairo.

*case is about ilness associated with eating faseikh (uneviscerated salted mullet fish)
- 91 hospitalized patients in cairo with botulism intoxication.

-45% was interview and case control investigate among families of 5 hospitalized patients.

- 18 out of 91 was died.

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Appropriate interventions to prevent growth of Clostridium Botulinum

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Appropriate interventions to prevent growth of Clostridium Botulinum

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Primary growth-limiting factors for C. botulinum include :
1)environmental temperature above 50°C or below 10°C
2)high acidity (pH <4.6)
3)low water activity (lack of available moisture)
4)food preservatives such as nitrite, sorbic acid, phenolic antioxidants, polyphosphates, and ascorbates
5)a low redox potential (absence of oxygen)
6)competing microorganisms (CDC 1998).
To be safe, the FDA (2015) recommends food be kept out of the "Danger Zone." Thus for safety against this pathogen and others, store food items below 5°C and hold hot food above 57°C.
Due to their low water activity, dehydrated foods and foods high in salt and/or sugar do not support growth of the bacteria. Some strains of C. botulinum can be mesophilic, with an ideal growth temperature between 20°C–45°C, whereas others are psychotrophic, with ideal growth between 3°C–20°C.
Proper cooking and handling of food is important in the elimination of C. botulinum because growth of these C. botulinum strains is possible at a wide range of environmental temperatures. Although C. botulinum typically will not grow in environments of pH <4.6, food proteins such as those in soy and beef can have a protective effect on the bacteria by providing localized areas or pockets of high pH, thus allowing for growth in high-acid foods (Wong et al. 1988).

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Where is/are the organism(s) found? What kind of food is it associated with?

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Where is/are the organism(s) found? What kind of food is it associated with?

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Clostridium botulinum is ubiquitous, found at almost everywhere (as spore), specifically,

1.    Soil and untreated water/marine sediments worldwide (as spore)

2.    Improperly preserved or canned food (also as spore)

Characteristic of the condition that favor the sporulation process

low-oxygen conditions

Kind of food, clostridium botulinum associated wit
Improper canned food (not canned under pressure or improperly canned using a pressure canner). If boiling, only C. botulimun bacteria will be destroyed, but the spores will not be destroyed.

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Little Billy and Susy May have a bout of Botulism in this 50's public service announcement.

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Examples of food associated with C. Botulinum growth

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Examples of food associated with C. Botulinum growth

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Sources of the spore:
💫Vegetables
💫Fish
💫Meat
💫Poultry

Associated foods:
☄Home canned low acid foods
☄Honey (infant botulism)
☄Baked Potatoes
☄Fried onions
☄Garlic in oil mixtures

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Foods that are exposed to conditions as below are prone to C. Botulinum growth :

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Foods that are exposed to conditions as below are prone to C. Botulinum growth :

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1.   Temperature range: 3-48°C (38-118°F)
2.   Type A and B: 10-50°C (50-122°F)
Type E: 3-45°C (38-113°F)
3.   Optimum Temperature for toxin
development: 35°C (95°F)
4.    pH range: 4.6 - 8.9
5.    Lowest reported Aw for growth: 0.95

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Conditions in food that favors C. botulinum growth 

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Conditions in food that favors C. botulinum growth 

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1. C. botulinum spores are often found on the

• Surfaces of fruits
• Vegetables
• In seafood

2. The organism grows best under low-oxygen conditions and produces spores and toxins. The toxin is most commonly formed when foodis improperly processed (canned) at home.

3. C. botulinum cannot grow below a pH of 4.6. Thus, acidic foods, such as most fruits

• Tomatoes
• Pickles

can be safely processed in a water bath canner. However, foods with a higher pH (most vegetables and meats) must be processed under pressure. Therefore, a pressure cooker should be used. The pressure cooker will reach high-enough temperatures to destroy the C. botulinum spores.

4. For example, if a low-acid food, such as green beans, is canned improperly (not canned under pressure or improperly canned using a pressure canner), C. botulinum bacteria and other bacteria present will be destroyed by the boiling of water and food, but the C. botulinum spores will not be destroyed. The canning process will remove the oxygen from the jar, creating a low-oxygen environment that will allow the spores to grow into active bacteria. When the jars are stored at room temperature, the spores can germinate and produce the toxin. However, the toxin is sensitive to heat and can be destroyed if the food in question is boiled for 10 minutes (longer at high altitudes)

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Polymerase chain reaction:
- The use of the polymerase chain reaction (PCR) to identify the presence of C. botulinum DNA was originally used to detect the presence of bacterial spores.
- The method could detect the presence of as few as 102 spores per reaction mixture for serotypes A, E and F and only 10 spores per reaction mixture for BoNT/B.
- Multiplex polymerase chain reaction:
This approach, known as Multiplex technology, is conceptually simple for PCR based assays.
-  This method was robust and reasonably rapid for use with food samples contaminated with C. botulinum.
- Real-time polymerase chain reaction:
-  Real-time or quantitative PCR is useful in studies of gene expression; specifically differential expression of genes under different environmental conditions or for comparative studies among different organisms.
- For detection of clostridia, real-time PCR methods that examine expression of the NTNH (non-toxic, non-hemagglutinin) genes have been developed, as well as methods to study toxin gene expression in C. botulinum serotypes A, B, E, and F .
- DNA microarrays:
-  Microarray technology for toxin identification of contaminated food has not been widely used.
- This may be due to the challenge in isolating high-quality RNA samples from clostridia in food matrices.
-  Further development of microarray based assay approaches may provide a means to rapidly identify toxin-producing strains.

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Detection methods can exploit the power of sensitive antibodies for enrichment or sample preparation, as well as the signal amplification ability of enzymatic assays.
-ALISSA (assay with a large immunosorbent surface area):  When compared to other established methods for toxin detection in food matrices, the ALISSA assay can detect toxin concentrations as low as 50 fg/mL, more sensitive than the mouse bioassay or either immunoassay or SNAPtide assay alone.
- ENDOPEP-MS: antibodies, activity assays, and mass spectrometry:  The ENDOPEP-MS method uses antibodies to concentrate and extract BoNT serotypes A, B, E, and F from test samples. The concentrated toxins are then subjected to an endopeptidase activity-based assay to generate target cleavage products. Finally, mass spectrometry is used to identify cleavage target products.
- This approach has been successful in identifying BoNT serotypes A, B, E, and F in a variety of food and clinical sample matrices with sub-mouse bioassay sensitivities.

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- Antibodies have been used extensively in traditional ELISA, bead-based, immuno-PCR, microarray assays or in sample preparation before use in a detection assay.

- ELISA is a widely used detection assay format that uses anti-BoNT capture and detector antibodies usually in a sandwich type format.

- Assay sensitivity varies for each botulinum complex serotype.
- Recently, traditional format sandwich ELISA assays using highly sensitive mAbs against BoNT/A and BoNT/B
- The higher sensitivity and less time required for new ELISA assays make them great alternatives or complements for the mouse bioassay.
-  Multiplex antibody-based detection system has been applied to the development of methods to analyze multiple epitopes on a single antigen or multiple targets in a single sample.
- This approach uses multiple mAbs as well as polyclonal antibodies to reduce false-positive and falsenegative results. The Luminex xMAP technology utilizes microsphere beads conjugated with antibodies.
- Affinity immunochromatography column-based methods : Brunt and colleagues (Brunt, Webb, and Peck, 2010) have developed a number of rapid affinity immunochromatography column (AICC) assays for the detection of BoNT serotypes A, B, E, and F in food matrices.
- Lateral flow technology:  captures antibodies are printed on nitrocellulose membranes. Detection antibodies are labeled with materials that can be visualized.
-  Although simple lateral flow tests have poorer sensitivities compared to other methods, they produced rapid results, require no additional reagents or equipment, are easily interpreted, and have many applications.
- Immuno-polymerase chain reaction (I-PCR):  Here, instead of a secondary antibody conjugated to the detection enzyme, template DNA is conjugated to the antibody; and upon binding of antigen by the antibody, an indirect test for the presence of the BoNT is carried out using PCR.

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Other characteristics of Clostridium Botulinum

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Other characteristics of Clostridium Botulinum

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1. Form endospores in undesirable conditions

Normally found in soil and aquatic sediments.

Extremely heat resistant which makes them difficult to kill by just  boiling water.

2. Obligate anaerobe

This bacterium can produce an enzyme called superoxide dismutase which can defend the bacterium from small traces of O2.

3. Gram-positive firmicute
4. Rod-shaped bacterium and can be grown in the laboratory.

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- Neurotoxin botulin works by inhibiting the release of acetylccholine from presynaptic motor neurons
-The toxin acts at four different sites in the body, which are:
i. neuromuscular junction
ii. autonomic ganglia
iii. postgaglionic symphathetic nerve endings
iv. postgnglionic parasymphathetic nerve endings
Long -term effects of botulism
-dizziness
-fatigue
-weakness
-dry mouth
-difficulty performing strenuous tasks

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1. Produce neurotoxin botulin
-This toxin is very deadly and is associated with causing flaccid muscular paralysis which known as botulism
2. Requires a pH between 4.8 - 7
- Typically, C. botulinum will not grow in a pH

below 4.6.
- Some food proteins (such as soy and beef), may have a protective effect on C. botulinum and allow it to grow at a lower pH.

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1. Temperature
- For food that require heating, avoid ones that have cooled
- For food that require refrigeration, avoid ones that have heated up.
2. Know the source of the food if possible
- high risk: home-canned, low acid items, fermented seafood.
-Commercially prepared chili peppers, baked potatoes and garlic oil have also been associated with botulism
3. Irregularities of can
- Presence of bulging ends and leaking
- If glass jar's lid bulges or leaks, do not eat it.
Honey often associated with infant botulism with no sign of contamination. Do not give to children under age 1
4. Air
If air rushes out during opening, food may be spoiled or contaminated
5. Smell
- Presence of spoiled odour but some strains of this bacteria do not cause bad smell of food.
6. Texture
- Observe colour and texture. Liquid should be clear and broth over canned meat or chicken should be gelled.

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- Most sensitive and robust methods to detect BoNT.
- Measures BoNT in minimal lethal dose (MLD) units
-lowest dose at which tested mice
dead
- Mice are usually inject intraperitoneally and then monitored for several days for clinical symptoms of botulism
- ruffled fur
- wasp-waist
- labored breathing
- paralysis
- death
- Duration for the symptoms manifestation range from several hourd to several days after injection.
-depend on injection dose
-depend on types of BoNT
- Has high sensitivity, detect different serotypes and subtypes, measure different aspects of active toxin, amenable to use in different matrices
- Drawbacks : long assay time, specialized animal facilities, trained staffs and use of animals (with death as the endpoint)
- Alternative: mousephrenic nerve hemi-diaphragm assay, in vivo assay using toe spread reflex model.

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Cell-based assay
- It measure Botulinal toxin (BoNT) receptor binding, translocation and enzymatic activity.
- Viable alternative to mouse bioassay.
-Many neuronal and non - neuronal cell has been generated for this method
-rat spinal cord cells
- chick embryo neuronal cells
- neuroblastoma cells
- The most read out for this method is the cleavage of of SNAP-25.
-Antibodies for SNAP-25 allow immunoblot detection of the cleavage products .
-Indication in decrease of size of endogenous SNAP-25 protein

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1. Trypsin treatment
-Non - proteolytic types toxin need trypsin activation    if present.
-The food sample need to be treated with trypsin before testing of toxin.
-The sample can come from food supernatant fluid, liquid food or Tryptone Peptone Glucose Yeast (TPGY) culture except TPGYT culture as the trypsin has been added to the medium
2. Toxicity testing
- Tests conducted with trypsin-treated materials and untreated replicates.
- Serial dilution are made with the samples before injecting it to the mice.
- Heated culture at 100 degree Celsius was also injected to the mice and absence of botulinal toxin should be expected as it was inactivated by heating.
- Death of mice without clinical symptom of botulism is not sufficient to act as proof
3. Typing of toxin
- Rehydrate antitoxins with sterile physiological saline but do not use glycerine water.
- Monovalent antitoxin (A,B,E and F) solution was injected into the mice 30min or 1 hour before challenging them with toxin injection.
- if result indicate death and the toxin was not neutralized. Monovalent antitoxin types C and D with addition polyvalent antitoxin poo types A thro

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1. PATHOGENICITY

Clostridium Botulinum has ability to form endospores in undersirable conditions.

it can produce enzyme called superoxide dismutase which can defend the bacterium from small traces of O₂

Human disease (botulism) is caused by toxins elaborated under anaerobic conditions usually by colonization of food, less often by colonization of wounds, or colonization of the intestinal tract as an infant botulism

Botulism is a rare but serious paralytic illness caused by a nerve poison that is produced by the bacterium

Some toxins, particularly those of the nonproteolytic strains, require typsin activation for effectiveness in laboratory toxin testing

the toxin of Clostridium Botulinum type C is inactivated by bacteria in the rumen of cattle and sheep.

infant botulism occurs when infants eat the spores of the bacteria, which then grow in their intestines and release toxin. Botulism can also be found from an infected wound. All forms of botulism can cause paralysis and be fatal.

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Clostridium Botulinum