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Countries where outbreaks of C.botulinum has been reported!

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Countries where outbreaks of C.botulinum has been reported!

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Countries in black have recorded incidences of outbreaks of C. botulinum.

North America

Parts of South America

Large parts of Europe

Approximately half of African continent

Australia

Reference: Smith, L. (1977). Botulism. Springfield, Ill.: Thomas.

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Conditions conducive of growth are dependent on various environmental factors. Growth of C. botulinum is a risk in low acid foods as defined by having a pH greater than 4.6 although growth is significantly retarded for pH below 4.9. There have been some cases and specific conditions reported to sustain growth with pH below 4.6.m

C. botulinum is an obligate anaerobethat is widely distributed in nature and is assumed to be present on all food surfaces. Its optimum growth temperature is within the mesophilic range. In spore form, it is the most heat resistant pathogen that can survive in low acid foods and grow to produce toxin. The toxin attacks the nervous system and will kill an adult at concentrations of 10−6.This toxin is detoxified by holding food at 100 °C for 10 minutes.

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	C. botulinum in different geographical locations

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	C. botulinum in different geographical locations

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A number of quantitative surveys for C. botulinum spores in the environment have suggested a prevalence of specific toxin types in given geographic areas, which remain unexplained.

North America

Type A C. botulinum predominates thesoil samples from the western regions, while type B is the major type found in eastern areas. The type-B organisms were of the proteolytic type I. Sedimentsfrom the Great Lakes region were surveyed after outbreaks of botulism among commercially reared fish, and only type E spores were detected. In a survey, type-A strains were isolated from soils that were neutral to alkaline (average pH 7.5), while type-B strains were isolated from slightly acidic soils (average pH 6.25).

Europe

C. botulinum type E is prevalent in aquatic sediments in Norway andSweden, Denmark, theNetherlands, the Baltic coast of Poland, and Russia.[26] The type-E C. botulinumwas suggested to be a true aquaticorganism, which was indicated by the correlation between the level of type-E contamination and flooding of the land with seawater. As the land dried, the level of type E decreased and type B became dominant.

In soil and sediment from the United Kingdom, C. botulinum type B predominates. In general, the incidence is usually lower in soil than in sediment. In Italy, a survey conducted in the vicinity of Rome found a low level of contamination; all strains were proteolytic C. botulinum types A or B.

Australia

C. botulinum type A was found to be present in soil samples from mountain areas of Victoria. Type-B organisms were detected in marine mud fromTasmania
- Type- A botulinum has been found in Sydneysuburbs and types A and B were isolated from urban areas. In a well-defined area of the Darling-Downs region of Queensland, a study showed the prevalence and persistence of C. botulinum type B after many cases of botulism in horses.

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LABORATORY DIAGNOSTICS OF BOTULISM

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INTRODUCTION

Since the first reported cases of food-borne botulism in the late 18th century, botulism has gained attention not only as a threat to food producers and consumers but also as a potential cause of crib death of small babies, as a deadly trip for intravenous drug abusers, and as an inspiration for bioterrorists. Botulism is caused by botulinum toxins, which are highly potent neurotoxins formed during the growth of the spore-forming bacterium Clostridium botulinum.

Based on the serological properties of the toxins they produce, C. botulinum strains are divided into toxinotypes A to G. Noteworthy of the bacterial species C. botulinum is its old-fashioned taxonomy; the only common denominator of all C. botulinum strains is the ability to produce neurotoxins that cause flaccid paralysis. C. botulinum strains form four genotypically and phenotypically distinct groups of organisms, designated I to IV ( 100, 107, 196). Generally, groups I and II cause botulism in humans, while group III is involved with animal botulism, but exceptions occur. Group IV has not generally been associated with illness. Based on its phenotypic and genetic traits, group IV C. botulinum has been proposed to be renamed Clostridium argentinense ( 203). Of the human pathogenic strains, group I cultures produce toxin A, B, or F and group II cultures produce toxin B, E, or F. Dual-toxin-producing strains have also been reported ( 72, 80, 121), as have those producing only one type of toxin but carrying a silent gene for another ( 73, 74, 121). Other clostridia, namely, C. butyricum and C. baratii, are also known to produce type E and F toxins, respectively.

Phenotypically, group I and II C. botulinum strains differ from each other significantly. Group I organisms seem to be more of terrestrial origin and are present in temperate climates, whereas group II strains, particularly type E, are frequently found in aquatic environments in the Northern hemisphere (Table ). Differences in spore heat resistance and growth temperatures are responsible for the safety risks posed by C. botulinum groups I and II in the food industry; group I spores, which have a high heat resistance ( 112, 138, 180, 184, 192, 202), cause problems in canning and home preservation of vegetables and meat, whereas group II spores, with somewhat lower spore heat resistance ( 135, 140, 168- 170), are of great concern in minimally processed packaged foods that have extended shelf lives at refrigerated temperatures ( 134, 167).

TABLE 1.

Botulinum neurotoxins are 150-kDa proteins with zinc-endopeptidase activity ( 155). The toxin molecule is secreted as a progenitor toxin that contains the neurotoxin as well as nontoxic components. The nontoxic components protect the neurotoxin from environmental stress and assist in absorption of the neurotoxin into the body ( 77). The neurotoxin molecule consists of two subunits: a 100-kDa heavy chain, which is responsible for the binding and translocation of the toxin across the synaptic membrane through specific receptors, and a 50-kDa light chain, which cleaves the proteins involved in acetylcholine vesicle docking and fusion to the presynaptic membrane ( 156). Inhibition of the neurotransmitter release causes paralysis of the corresponding muscle. The maximal potency of the botulinum neurotoxins is achieved only after enzymatic activation of the toxin molecule. This has been suggested to be at least partly related to cleavage of the toxin molecule ( 45, 165). Generally, in proteolytic group I C. botulinum this is done by endogenous enzymes of the organism, but neurotoxins produced by nonproteolytic group II strains may require external proteases, such as trypsin ( 45, 55), for activation.

Typical of botulism is a descending flaccid paralysis that, unless treated immediately and appropriately, may lead to death when the respiratory musculature fails. Food-borne botulism, the most well-known form of botulism, is an intoxication that follows when food containing preformed botulinum neurotoxin is ingested. Other significant forms of botulism are infections that result from toxin formation from spores in vivo. The most common of these is infant botulism, where botulinal spores germinate, grow, and subsequently produce toxin in the gastrointestinal lumens of small babies, in whom the normal gut microflora is poorly developed ( 14).

Since botulism is a life-threatening condition, a rapid diagnosis is essential. This challenges not only the awareness of the disease by clinicians but also the laboratory diagnostics. Apart from the ultimate goal of rapid diagnosis and, above all, saving the patient, every attempt to increase our understanding of the epidemiology of botulism should be made. This enables the future development of prevention strategies against the disease. In addition to the detection of botulinum toxin and C. botulinum in the patient, the diagnostics should aim at physiological and genetic typing of the disease isolates. In the following we review the current status of the laboratory diagnostics of human botulism.

HUMAN BOTULISM

Botulism is a potentially lethal condition caused by botulinum neurotoxin. The neurotoxin can enter the body via the gastrointestinal tract or through mucous membranes of, for instance, the eyes or the respiratory tract. Toxin production from otherwise harmless C. botulinum, C. butyricum, or C. baratii spores may also occur in vivo. After entering the body, the neurotoxin is absorbed into the blood and lymphatic circulation, which then mediate the toxin to motor nerve endings where it blocks the neurotransmitter release. Typical clinical symptoms of all forms of botulism include cranial muscle paralysis, such as double vision and dilated pupils, slurred speech, dry mouth, difficulty in swallowing and speaking, and facial paralysis. As the disease progresses, paralysis of the limbs and respiratory dysfunction become apparent. Respiratory muscle paralysis can lead to death. Recovery occurs upon the sprouting of transitory nerve endings that reside when the synaptic activity of the original nerve regenerates ( 148). Recovery time may be several weeks to months and is dependent on the amount of toxin ingested and, to a lesser extent, on the toxin type in question. Type A toxin tends to be more potent than types B and E and causes the longest-lasting disease ( 71).

Food-Borne Botulism

The classical form of botulism is food poisoning, an intoxication that follows the consumption of food containing preformed neurotoxin. Depending on the toxic dose, the incubation period may vary from 12 to 72 h. In addition to the general signs of botulism, the food-borne form may be manifested by gastrointestinal symptoms such as nausea, vomiting, and constipation. The treatment consists mainly of intensive symptomatic care, including respiratory support ( 24). Intravenous administration of specific trivalent antitoxin towards A, B, and E toxins may be used to neutralize the circulating toxin. However, the success of the antitoxin treatment is strongly dependent on the time of administration; if the toxin has entered the nerve endings and disappeared from blood circulation, the treatment will be irrelevant. Furthermore, due to the risk of severe allergic reactions to the antiserum, it is no longer used in some countries ( 25).

Typical differential diagnoses include Guillain-Barré syndrome, Miller-Fisher syndrome, chemical intoxication, stroke, and staphylococcal food poisoning ( 36, 105). Suspected drug and alcohol abuse may occasionally prolong the time for a diagnosis to be made.

Compared with food-borne illness or death due to more common pathogens, such as Campylobacter, Salmonella, and Clostridium perfringens, food-borne botulism outbreaks are relatively rare. However, regions with a high incidence of botulism that causes a considerable public health hazard include the Republic of Georgia, Poland, China, Russia, Kyrgyzstan, and certain ethnic populations in the North, e.g., Alaskan Inuits. Japan, Italy, Portugal, Germany, France, and the former Yugoslavia have also reported high numbers of cases. The foods most frequently involved, if traced, are home-prepared items such as cured meats, canned vegetables, and fermented fish products. These outbreaks are usually sporadic and restricted to a family. Commercial foods have less frequently been involved in food-borne botulism, but such outbreaks may be large, and they cause significant economic losses to the food industry ( 134).

The case-fatality rate of food-borne botulism in developed countries is 5 to 10% ( 199). Worldwide, more than half of the cases (52%) were due to type B toxin, with types A and E accounting for 34% and 12%, respectively, in 1995 ( 91). On rare occasions, type F toxin has been associated with food-borne botulism ( 90). Apart from the type of toxin causing the outbreaks, no information on the physiologic group of C. botulinum type B or F strains involved is usually provided.

Infant Botulism

Unlike food-borne botulism, the other forms of human botulism are actually infections where the toxigenesis occurs in vivo. Infectious botulism is related mainly to group I C. botulinum, as its temperature optimum for growth and subsequent toxin production is close to the body temperature, while the growth of group II organisms at the same temperature is limited ( 197). For infant botulism ( 150, 172), in addition to group I C. botulinum types A and B ( 92, 93), Bf ( 21), and F ( 99), cases due to type E and F toxins produced by Clostridium butyricum ( 18, 92, 144) and Clostridium baratii ( 22, 88, 119), respectively, have been reported. Infant botulism typically affects babies under 1 year of age, with the youngest reported patient being only 54 h old ( 22, 119). The condition develops as a consequence of ingesting spores of toxin-producing clostridia ( 9). As the gut microflora of small babies is poorly developed, C. botulinum spores may germinate and form a toxin-producing culture in the intestine. The clinical manifestation of infant botulism varies from a subclinical condition to sudden death ( 12). It often starts with constipation that can last for several days, followed by the distinctive flaccid paralysis manifested by impaired feeding due to muscle weakness in the mouth and throat, facial muscle paralysis, ptosis, and general weakness ( 10). Infant botulism has been suggested to be a causative agent of crib death ( 6, 14).

The main treatment for infant botulism is high-quality supportive care, with special attention focused on the patient's nutrition and respiratory functions ( 13, 116). The use of antitoxin is often not required ( 13), and the case-fatality rate is less than 2% ( 39). However, since 2003 a human-derived immune globulin ( 75) has been available in the United States and has been shown to significantly shorten the hospitalization period and decrease the treatment costs ( 209). The only foodstuffs that have been associated with infant botulism are honey ( 19), which may carry high numbers of C. botulinum spores ( 11, 52, 158, 159), and infant milk powder ( 29). Dust and other materials in the environment also seem to be important sources of spores ( 11, 52a, 161).

Wound Botulism

Wound botulism is a rare form of botulism, although it is increasingly diagnosed among injecting drug abusers due to the use of contaminated needles or impure heroin ( 3, 16, 17, 157, 166, 213). Wound botulism follows when C. botulinum spores germinate and grow in profound wounds or abscesses that provide anaerobic conditions. The clinical outcome is similar to the food-borne form, albeit with an absence of gastrointestinal signs. Wound botulism is often accompanied by a mixed infection and may be due to more than one botulinum toxin-producing strain ( 3). The median incubation period is 7 days. In addition to respiratory support, the treatment of wound botulism includes thorough surgical debridement, antibiotics, and the administration of antitoxin. The estimated case-fatality rate is 15% ( 91).

Infectious Botulism in Adults

The adult form of infectious botulism is rare and resembles infant botulism in its pathogenesis and clinical status. It results from colonization of the intestinal tract by toxin-producing clostridia ( 40, 64, 143, 145). People with altered intestinal flora due to abdominal surgery ( 76, 111), prolonged antimicrobial treatment, or gastrointestinal wounds and abscesses are particularly vulnerable ( 40). Infectious botulism in adults is distinguished from the food-borne form by a different history of food consumption, with a missing association to foods with high risk of botulism ( 143).

Other Forms of Botulism

Inhalation botulism may result from aerosolization of the neurotoxin. A few human cases have been reported ( 101). Iatrogenic botulism with local or generalized weakness is rare but can develop as a consequence of therapeutic injection of the neurotoxin ( 20, 149). A distinctive "outbreak" of iatrogenic botulism due to maladroit botulinum toxin treatment caused botulism in four people in Florida in November 2004 ( 7).

LABORATORY DIAGNOSTICS OF BOTULISM

As botulism is a life-threatening condition, a rapid diagnosis is required for successful therapy. Apart from the clinical manifestation and patient history, the diagnosis is based on positive laboratory findings. Of these, detection of toxin in the patient's serum and/or feces remains the standard method ( 118) (Fig. ). The detection of C. botulinum in patient samples, such as feces, gastric and intestinal contents, and wound swabs and tissues, supports the diagnosis ( 3, 39, 118, 162), but should not exclusively be considered pathognomonic of the disease. Sometimes the presence of several toxin-producing strains may complicate the diagnostics ( 3, 29, 131). In some cases of infant botulism, the presence of C. botulinum alone in the feces or intestinal content of the patient may be sufficient for diagnosis even if the neurotoxin could not be detected in samples from the patient ( 38).

Whereas a rapid diagnosis of botulism is a prerequisite for the patient to survive and recover, a more thorough investigation of botulism outbreaks is needed to provide epidemiological information about the disease (Fig. ). This includes the isolation of C. botulinum from the patient and from vehicle foods or other sources of spores or toxin and genotypic analysis (see below) of the disease isolates. Furthermore, characterization of the physiological group of these isolates is fundamental to improve our knowledge of the risk factors for and prevention strategies against different forms of botulism.

In the following we discuss the problems and developments in the laboratory diagnostic tools and procedures established for investigation of human botulism.

Laboratory Safety

The extreme potency of botulinum neurotoxin necessitates rigid requirements to ensure the safety of laboratory workers. Despite the potency of its neurotoxin, the noninvasive and noncontagious C. botulinum has been graded as a class II pathogen. Appropriate biosafety level 2 containment facilities and trained personnel are therefore a minimum requirement for work with C. botulinum. Additional contingencies should be considered whenever aerosol or droplet formation from toxic materials is expected. As the neurotoxins form a potential threat for bioterrorism, restricted entrance to the laboratory facilities containing toxic cultures is a necessity. The definition of additional contingency measures should be based on a thorough risk assessment of activities in each laboratory handling C. botulinum and its toxin. The efficacy of the pentavalent toxoid formerly used to immunize laboratory staff worldwide has been shown to be lower than expected (Centers for Disease Control and Prevention, unpublished data). Therefore, careful attention needs to be paid to the everyday safety of the laboratory staff. Attempts to develop new vaccines against botulism are being made both in the United States and in Europe ( 24).

Detection of Botulinum Neurotoxin

While the mouse lethality assay has remained the standard test for the detection of botulinum neurotoxins, there has been tremendous progress in the development of alternative tests during the past decade (Table ) ( 188). The latest advances in assay development have successfully aimed at rapidity and sensitivity; the fastest tests are now performed in 20 min, and the test sensitivities have surpassed that of the mouse bioassay. However, further research is warranted to gain speed and sensitivity with a single test that would optimally detect all seven neurotoxin types simultaneously. Furthermore, many of the recently developed test systems have been targeted to the medical industry for testing the potencies of therapeutic preparations of purified botulinum toxins, and validations on foods or clinical materials have not been done. Components of complex matrices, such as feces, blood, pus, and foods, may interfere with test reactions, and high concentrations of competitive microbes in many sample materials can retard the growth of and toxin production by C. botulinum. Fecal proteinases may degrade botulinum toxin, causing false-negative results. Therefore, there is a need for systematic validation of botulinum toxin detection tests in clinical sample materials and foods.

Culture Methods for Clostridium botulinum

C. botulinum requires strict anaerobic conditions for growth. This creates challenges for laboratory work with the organism. All culture media must be deoxygenated by heating in a boiling water bath or by a continuous flow of an anaerobic gas mixture. Reducing agents, such as thioglycolate, may be added in media to maintain anaerobiosis during culture and incubation. The use of appropriate oxygen indicators in culture media as well as in anaerobic jars and anaerobic workstations is part of the daily routine in a C. botulinum laboratory. All glassware and plastic supplies, such as containers, tubes, and pipette tips, should be deoxygenated before contact with C. botulinum cultures. Continuous work in an anaerobic workstation is a necessity for successful diagnostics.

The species C. botulinum consists of physiologically distinct organisms (Table ), which hampers the development of diagnostics. Conventional detection and isolation of C. botulinum are based on culturing in liquid medium and subsequent detection of botulinum toxin in the culture supernatant by the mouse bioassay ( 118). Positive samples are streaked on solid media, and the toxin formation by colonies is traditionally further confirmed by the mouse test. Clinical samples, such as serum and feces, can be cultivated directly or pretreated with ethanol to eliminate vegetative bacteria while recovering bacterial spores ( 163). Heating may alternatively be used to eliminate nonsporeformers, but care should be taken in selecting the temperature; 80°C for 10 min has been recommended for group I spores, but this temperature may injure group II spores ( 197). Heating at 60°C for 10 to 20 min is thus a safer choice for group II spores. The addition of a heat-resistant lytic enzyme, such as lysozyme (5 μg/ml), to the culture medium may enhance germination of heat-stressed spores ( 169, 170).

Routine liquid media include chopped-meat-glucose-starch medium ( 39); cooked-meat medium ( 175, 176); broths containing various combinations of tryptone, peptone, glucose, yeast extract, and trypsin (e.g., tryptone-peptone-glucose-yeast extract medium) ( 129); reinforced clostridial medium ( 79); and fastidious anaerobe broth ( 179). All of these media are nonselective and thus allow the growth of a range of other bacteria.

Blood agar and egg yolk agar (EYA) ( 94) serve as the most common unselective plating media, with EYA enabling the lipase reaction typical of C. botulinum (Table ). However, many other clostridia produce lipase and may therefore confuse the identification ( 35). EYA medium alone does not contain any inhibitory compounds, but when supplemented with cycloserine, sulfamethoxazole, and trimethoprim, EYA-based selective media (e.g., C. botulinum isolation medium) have been reported to select group I C. botulinum ( 50, 153, 190). These media have been successfully used in the investigation of fecal samples in cases of infant botulism ( 48, 50, 82). However, as these media may suppress the growth of group II C. botulinum ( 214), their value in the diagnostics of food-borne botulism is limited.

The identification of botulinum toxin in and around C. botulinum colonies grown on agar plates would facilitate the identification and isolation of C. botulinum and neurotoxin-producing C. butyricum and C. baratii among competitive microflora. Procedures based on immunodiffusion ( 69, 152, 190, 210) or immunoblotting ( 83) have been published. As a single C. botulinum colony may produce as much as 10 ⁵ minimal lethal doses (MLD) of toxin within 24 h ( 69), the colony immunoblot assay, which detects 10 to 25 50% MLD (MLD ₅₀) of toxin per spot ( 83), would be sufficiently sensitive for the identification of C. botulinum on an agar plate. No reports on the performance of these tests with naturally contaminated clinical or food samples or mixed bacterial populations have been published. Fluorescent antibodies against the cell walls of vegetative C. botulinum have also been used to identify C. botulinum in a culture ( 81), but this method suffers from cross-reactivity between C. botulinum and its nontoxingenic counterparts.

A dilemma arising from the different physiologies of group I and II strains is the selection of the correct incubation temperature (Table ). Group I strains grow optimally at 35 to 37°C, whereas group II strains favor lower temperatures of 25 to 30°C ( 197). If both groups are being sought in a single sample, a compromise of 30°C has been proposed ( 197). However, investigating replicate samples at 26 to 30°C and at 35 to 37°C is preferable to ensure optimal growth for both groups. The optimal incubation time varies with the sample material and the detection method selected to identify the presence of C. botulinum in the culture tube. When the mouse bioassay is used to identify toxin formation in culture tubes, an incubation period of 5 to 7 days has been recommended ( 197). Shorter incubations may become relevant if molecular biological techniques, such as PCR (see below), are employed ( 133).

As the prevalence of C. botulinum in naturally contaminated samples is generally relatively low (10 to 1,000 spores/kg) and as there are no proper selective media available, quantification of C. botulinum in samples containing other bacteria by use of plate count procedures is difficult, if not impossible. Therefore procedures for enrichment in liquid media are often needed. In such cases the quantification of the organism is obtained with a most-probable-number technique. The most-probable-number estimate is based on the amount of sample material containing the target organism and the total amount of sample material investigated. In general, the greater the number of culture tubes that are used, the more accurate is the estimate obtained. The presence of C. botulinum in the culture tubes can be determined, e.g., with the mouse bioassay or by PCR ( 98).

As the group I and II strains differ in their epidemiologies, discrimination between the two groups should be done whenever an outbreak strain is isolated (Fig. ). To be able to distinguish between the two groups, the proteolytic activity of group I strains can be determined on a casein-containing agar, e.g., reinforced clostridial medium supplemented with milk ( 169). Alternatively, molecular biological techniques such as probe hybridization or ribotyping (see below) can be employed. Although group I and II C. botulinum strains are dissimilar in many respects, both are able to grow at an incubation temperature range of 30 to 37°C, which is often used. Therefore, the ability to grow at a certain temperature should not be used as a feature determining the physiological group of a C. botulinum isolate.

Commercial test systems based on series of biochemical reactions have been developed for the identification of anaerobic bacteria. Contradictory reports on the ability of these tests to identify Clostridium spp. are available; different tests have been able to correctly identify 54% to 96% of the clostridial strains studied to the species level ( 31, 85, 95, 141). Due to the diverse physiology of C. botulinum, not all strains can be identified to the species level ( 28, 132). The biochemical test systems are also susceptible to technical bias, and factors such as incubation environment ( 171), incubation time ( 85), and concentration of the cell suspension ( 28) have been reported to drastically affect the success of identification.

For the reasons discussed above, the identification and isolation of C. botulinum from samples with high levels of competitive bacterial flora, such as fecal and environmental samples, are laborious and time-consuming. Several subsequent broth cultures and platings are required to isolate a pure culture, and often the isolation simply fails. The presence of nontoxigenic C. botulinum-like strains ( 30, 127, 132) disturbs the culture of C. botulinum. Whether these strains are completely different bacterial species from C. botulinum or whether they derive from toxigenic C. botulinum ( 221) by complete or partial deletion of the toxin gene cluster during the exhaustive subculture procedures warrants further investigation. In botulism cases due to type E and F toxins, the possibility of toxin-producing C. butyricum or C. baratii causing botulism should not be ignored unless C. botulinum can be identified. These bacteria differ physiologically from C. botulinum strains ( 35), and the laboratory routines established for C. botulinum are not directly applicable for the isolation and identification of these species.

Molecular Detection of Clostridium botulinum

DNA-based detection methods have overtaken conventional techniques, with numerous molecular detection protocols published for C. botulinum (Table ). Ascendant techniques include PCR and Southern hybridization, both of which are sensitive and specific and are rapid compared to culture techniques and the mouse bioassay. Many laboratories have already invested in PCR thermocyclers and electrophoresis equipment, and their staff are experienced with molecular techniques. In the case of C. botulinum, molecular detection techniques are based merely on the detection of the botulinum neurotoxin gene, bot, in the sample, and thus they do not detect the activity of the gene nor the toxin. Naturally this is a disadvantage, but, apart from the advantages mentioned above, molecular techniques do not require the use of laboratory animals. This makes them a suitable tool for screening bacterial colonies, pure liquid cultures, and sample enrichments for the presence of the neurotoxin genes, indicating the presence of C. botulinum and other toxin-producing clostridia.

Most reports on PCR detection of C. botulinum are based on a single detection of one of the seven types of toxin genes in a reaction (Table ). An improvement has been the development of a multiplex PCR assay that enables the simultaneous detection of the botA, botB, botE, and botF genes in a single reaction ( 133). This significantly decreases the assay time and labor and naturally the reagent costs. The multiplex PCR products are detected using conventional gel electrophoresis ( 133) or by further hybridization onto a membrane coated with cDNA probes to the PCR products ( 78). The two methods provide similar sensitivities (Table ).

As both the botB and botF genes of group I and II C. botulinum are highly similar, the molecular detection methods based on these neurotoxin genes do not reveal the physiological group of C. botulinum. An oligonucleotide probe specific for the 23S rRNA gene specifically detects group II strains ( 33), but as this probe also recognizes the nontoxigenic C. botulinum-like strains within group II, its value as the only diagnostic tool is limited. A HindIII fragment-based DNA probe specific for group I C. botulinum would better suit the purpose, as it does not hybridize to C. sporogenes ( 147). Some molecular typing methods such as ribotyping and amplified fragment length polymorphism (AFLP) (see below) are also suitable for differentiation between C. botulinum groups I and II ( 97, 120).

The sensitivities of the PCR and probe hybridization assays reported for the detection of C. botulinum in feces, serum, and food samples vary markedly (Table ). Greater sensitivity is generally obtained when extracted DNA, as opposed to crude cell lysates, is used as a template. The DNA extraction procedures applied for C. botulinum, however, may be laborious and time-consuming ( 109, 120, 142), while a cell lysate is easily prepared in an hour ( 73).

The sensitivity of PCR can also be increased by a nested design, where two primer sets targeted to the same gene are used in subsequent reactions. Nested PCR protocols have been published for the botB, botE, and botF genes ( 43, 117). The nested PCR format may allow the shortening ( 43) or complete exclusion of enrichment steps that are often required with normal PCR. C. botulinum type B was successfully detected directly from feces of an infant botulism case when using a nested PCR format ( 117). However, the detection limit of the assay was not determined, and this should ideally be done before the method can be reliably applied in diagnostics.

Some components in clinical and food samples, such as bile salts or competitive microflora in feces, immunoglobulins, and other components in blood and high protein and fat contents in foods, may inhibit PCR or at least drastically decrease its sensitivity ( 4, 5, 124). For example, the PCR detection limit for C. botulinum type E in feces was shown to be 4 log units higher than that in a fish or meat matrix ( 133). This should be borne in mind when setting up PCR protocols for clinical specimens. Each type of sample material requires thorough optimization of PCR as well as prior sample preparation. Sample pretreatment should focus on concentrating target cells and eliminating competitive flora and PCR-inhibitory substances. Internal amplification controls as well as other appropriate control setups should be routinely used in diagnostic PCR to control inhibition by the sample material or contamination ( 102, 103). Reports on their use in the molecular diagnostics of C. botulinum are so far scarce ( 63; G. M. Wyatt, J. Plowman, C. F. Aldus, M. W. Peck, and W. Penaloza, Abstr. Rapid Methods 2005, Noordwijk, The Netherlands, p. 8, 2005).

As the number of C. botulinum organisms in naturally contaminated samples is often very low and the target organism forms resistant spores, PCR detection directly from sample materials may often fail due to insufficient sensitivity of the assay. Enrichment is thus required to germinate spores and increase the target cell concentration in the sample (Table ). Again, when investigating samples where both group I and group II C. botulinum may be present, careful consideration of enrichment conditions is urged, and optimally replicate samples will be incubated both at 35 to 37°C (for group I) and at 26 to 30°C (for group II). The duration of the enrichment step for optimal performance of PCR is also an important issue and should always be determined for each sample material ( 133) and enrichment medium. Too short an enrichment may result in competitive bacteria overtaking C. botulinum, whereas extensive incubation may result in lysis or sporulation of C. botulinum cells. Ideally PCR is done from a culture on several subsequent days to determine the optimal length of enrichment for each type of sample ( 133).

Another problem of direct PCR analysis from sample material is the possible detection of dead cells due to intact DNA after cell lysis ( 218). Enrichment procedures partly solve the problem by increasing the number of live cells in proportion to dead cells ( 98). Another approach to ensure that only live cells are detected is reverse-transcription PCR (RT-PCR), where gene expression rather than the gene itself is detected. Reverse transcription-PCR protocols for the botB and botE genes have been described ( 136, 137, 146). In a case of a human botulism outbreak, however, laborious procedures to obtain high-quality RNA may prove to be too time-consuming.

The different chemistry (intercalating fluorescent dyes or probes with a quencher dye) of real-time PCR from that of conventional PCR allows real-time monitoring of the amplification of the target gene after each PCR cycle, enabling rapid interpretation of results. In some experimental settings the melting temperature of the amplicons also can be calculated, which is essential to differentiate the specific PCR product from unspecific amplicons. Real-time PCR has been applied in the qualitative detection of C. botulinum types A, B, and E in foods and feces from food-borne and infant botulism cases ( 2). The same PCR protocol was recently employed in investigation of tissue, wound swab, pus, and aspirate samples from cases of wound botulism ( 3). When subjected directly to DNA extraction and PCR, only a small number of tissue samples from two patients were positive in the real-time PCR analysis. PCR inhibition was suggested to explain some of the negative results; however, after a 1- to 5-day enrichment in liquid medium, most samples from patients with toxin detected in their sera were shown to carry one or more neurotoxin genes ( 3).

Apart from rapidity, another advantage of real-time PCR is its ability to quantify the target sequence, reflecting the level of the organism. To obtain reliable quantification, the copy number of the target gene in the genome of the organism in question must be known. Furthermore, the requirements for selection of reagents, reaction standardization, and sample pretreatment are higher than those for conventional PCR. Quantitative detection of the botB gene has been employed for research purposes ( 136, 137), whereas a recent application allowed the quantitative detection of C. botulinum type A spores spiked in canned corn and sausage ( 223) (Table ). In this method, DNA is extracted directly from spores and no enrichment steps are employed. This allows the whole analysis to be finished within a working day. However, the speed is achieved at the cost of sensitivity; no fewer than 100 spores are detected ( 223). As the natural contamination levels of C. botulinum in food, clinical, and environmental samples may be 10 to 1,000 spores/kg, it is obvious that enrichment steps are needed for successful detection.

As discussed above, C. botulinum strains carrying silent toxin genes may confuse PCR detection by causing false-positive results ( 73). Conversely, point mutations or natural genetic variation in the template at the site of one or both PCR primers may impair primer annealing, thus causing false-negative results even if the gene is present and fully functional (Wyatt et al., Abstr. Rapid Methods 2005). Hence, although PCR can be a powerful screening tool in the laboratory diagnostics of botulism outbreaks, the results should ideally be confirmed by other methods. The specificity of PCR should be confirmed by combining PCR with bot gene-specific probe hybridization or by DNA sequencing of the PCR product instead of or in addition to plain fragment size-based gel electrophoresis analysis. Relying merely on PCR results is somewhat risky and should ideally be avoided if possible. It should also be borne in mind that PCR does not detect botulinum neurotoxin.

Genetic Characterization of Clostridium botulinum

A number of molecular typing tools have been used for the genetic characterization of C. botulinum. These include DNA sequencing, pulsed-field gel electrophoresis (PFGE), and ribotyping, as well as PCR-based techniques such as AFLP, randomly amplified polymorphic DNA analysis (RAPD), and repetitive element sequence-based PCR (Rep-PCR) (Table ). International molecular typing libraries with genetic fingerprints, preferably obtained by use of more than one technique, of C. botulinum strains isolated in botulism epidemics would serve as a valuable source of information when investigating new outbreaks. As the modern food industry focuses on long shelf lives and refrigerated storage, product delivery routes may be long and the same product may be transported to many different countries. It is therefore likely that botulism outbreaks, instead of being sporadic and restricted to a limited geographic area, will be distributed over a wide area and thus be difficult to solve ( 122).

FUTURE PERSPECTIVES

While there has been tremendous progress in the development of in vitro tests to potentially replace the mouse assay in the detection of botulinum neurotoxins and molecular biological tools for the detection and characterization of C. botulinum, far more needs to be done. The in vitro toxin tests need further rapidity and sensitivity in a single test, and all seven toxin types should ideally be identified simultaneously. Furthermore, the tests should be validated with a range of complex matrices, such as feces, foods, and environmental samples, in addition to testing with purified toxins.

Although the molecular biological methods for the detection and identification of C. botulinum are promising, the conventional culture techniques should not be overlooked. Selective media suitable for both group I and II C. botulinum and other toxin-producing clostridia are needed to harvest all possible toxin-producing strains from sample materials containing competitive bacteria. As the distinct physiologies of the different groups of C. botulinum apparently complicate the detection and isolation procedures, the taxonomy of the species should be reconsidered to correspond to the true genetic variation among the botulinum neurotoxin-producing strains.

Whereas the main goal of the laboratory diagnostics of botulism is to achieve a rapid diagnosis and to save the patient, appropriate laboratory procedures should aim at a more thorough understanding of the epidemiology of, risk factors for, and prevention of botulism due to either of the two groups of C. botulinum. The isolation and genetic characterization of C. botulinum originally from the patient and the source should be done whenever possible (Fig. ).

Bacterial genome sequencing has created new possibilities in genetic typing as well as in researching the genetic mechanisms behind different metabolic traits such as neurotoxin formation and its regulation. DNA microarrays enable genome-wide analysis of microorganisms and will probably overtake the more conventional genetic typing tools within a few years. While bacterial genomes are being sequenced at an increasing speed, more genomic information about closely related species and bacterial strains is needed. The genome of a group I C. botulinum strain (type A, ATCC 3502) has been elucidated (Sanger Institute, Cambridge, United Kingdom), enabling the development of a C. botulinum microarray ( http://pfgrc.tigr.org/organisms.shtml). However, due to differences between group I and II organisms, the applicability of the type A array in genomic analysis of group II organisms may prove to be limited. Attempts to sequence a group II C. botulinum genome are therefore necessary. Naturally, genomic information on a single strain will not provide a good overall understanding of the genetic diversity within the species. Thus, additional research funding allocated to C. botulinum genome sequencing projects is needed to shed light on the genetic diversity and the mechanisms underlying different metabolic phenomena among botulinum neurotoxin-producing clostridia.

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Since the first reported cases of food-borne botulism in the late 18th century, botulism has gained attention not only as a threat to food producers and consumers but also as a potential cause of crib death of small babies, as a deadly trip for intravenous drug abusers, and as an inspiration for bioterrorists.

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The symptoms and treatment of Botulism the disease caused by Botulinum

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Botulism (Latin, botulus, a sausage[1]) is a rare and potentially fatal illness caused by a toxin produced by the bacterium Clostridium botulinum. The disease begins with weakness, trouble seeing, feeling tired, and trouble speaking. This may then be followed by weakness of the arms, chest muscles, and legs.

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mechanism of Botulinum Toxin

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Protein Lounge animations @ http://www.proteinlounge.com/Animation/Animation.aspx Botulinum is a bacterial toxin produced by a bacteria Clostridium botulinum that causes the most severe form of food poisoning. The disease is called Botulism, the potentially deadly food poisoning characterized by muscle paralysis.

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treatment and prevention   to the diseases 

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treatment and prevention   to the diseases 

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1.Treatment
-If found early, botulism can be treated with an antitoxin that blocks circulation of the toxin in the bloodstream. This prevents the patient’s case from worsening, but recovery still takes several weeks

2.prevention to the diseases

-Since botulism poisoning most commonly comes from foods improperly canned at home, the most important step in preventing botulism is to follow proper canning procedure.
Further botulism prevention techniques include:

Not eating canned food if the container is bulging or if it smells bad, although not all strains on Clostridium Botulinum smell

Storing garlic or herb-infused oil in the refrigerator

Not storing baked potatoes at room temperature.

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Four groups of neurotoxins produced by Clostridium botulinum that differs physiologically and genetically:

Group 1
Toxin type: A, B, F
Toxin gene:  chromosome/plasmid
Characteristics:

Proteolytic

Grows in temperature between 12.8 to 48 °C (optimum at 37°C) (Peck et al. 2010)

Effects:

Infant botulism

Food contamination

Also found in:
C. sporogenes, C. putrificum

Group 2
Toxin type: B,E, F
Toxin gene:  chromosome/plasmid
Characteristics:

Non-proteolytic

Saccharolytic

Grows in temperature below 25 °C

Effects:

Food contamination

Also found in:
C. butyricum, C. beijerinickii

Group 3
Toxin type: C,D
Toxin gene:  bacteriophage
Characteristics:

Weak proteolytic effect

Effects:

Animal botulism

Also found in:
C. haemolyticum, C. novyi type A

Group 4
Toxin type: G
Toxin gene:  plasmid

Effects:

Unknown

Also found in:
C. subterminale, C. haemolyticum

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We cover an overview of Botulism as well as look at the method of transmission, symptoms (signs), diagnosis and treatment.

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Protect Yourself from Botulism

Botulinum toxin is produced by the germ Clostridium botulinum. This toxin can affect your nerves, paralyze you, and even cause death. Foodborne botulism is a rare but serious illness caused by eating foods that are contaminated with a nerve toxin called botulinum toxin. Home-canned vegetables are the most common cause of foodborne botulism outbreaks in the United States. Taking even a small taste of food containing botulinum toxin can be deadly.

When in doubt, throw it out!

Any food that may be contaminated with botulinum toxin should be thrown out. Find out how to safely dispose of food and cans that may be contaminated.

Never taste the product to determine if it is safe. 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.

When you open a jar of commercially or home-canned food, thoroughly inspect the product. Do not taste or eat foods that are discolored, moldy, or smell bad. Do not use products that spurt liquid or foam when the container is opened.

Inspect your commercial and home-canned foods

Don't open or puncture any unopened cans, commercial or home-canned, if you suspect contamination.

Suspect contamination if

The container is leaking, bulging or swollen, looks damaged or cracked or seems abnormal in appearance,

The container spurts liquid or foam when opened, or

The food is discolored, moldy, or smells bad.

Use a bleach solution to wipe up spills of food that may be contaminated

Add ¼ cup bleach for each 2 cups of water.

Completely cover the spill with the bleach solution.

Place a layer of paper towels, 5 to 10 towels thick, on top of the bleach.

Let the towels sit for at least 15 minutes.

Wipe up any remaining liquid with new paper towels.

Clean the area with liquid soap and water to remove the bleach.

Wash your hands with soap and running water for at least 2 minutes.

Discard sponges, cloths, rags, paper towels, and gloves that may have come into contact with contaminated food or containers with the food.

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This tutorial explains the general properties of clostridium species. It also describes the infection, pathogenesis caused by Clostridium bacteria. It also explains the method of treatment against clostridium infection using antibiotics. For more information, log on to- http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html

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In August and September 2001, several cases of botulism, a life-threatening illness caused by the bacteria Clostridium botulinum, were reported in the United States. Frozen, fully-cooked products were suspected of causing these illnesses.

In the Store

Before buying frozen, fully-cooked products, carefully inspect the container or package. If the package is punctured, torn, partially opened, or damaged in any other way that might expose the contents to the outside environment, do NOT purchase the product.

Do not purchase frozen products that appear to have thawed and refrozen.

Reject all swollen or gassy containers or spoiled foods.

Buy food from reputable dealers, with a known record of safe handling. Buy frozen products only if they are frozen solid and only if stored in the freezer case.

Observe any use-by or sell-by dates on the package.

At Home

When you open the container, inspect the product. Do not use products that are discolored, moldy, or have an off odor. Do not use products that spurt liquid or foam when the container is opened. Do not taste the product to determine if it is safe.

Follow the preparation instructions on the product label.

Reference: https://www.fsis.usda.gov/wps/portal/fsis/topics/food-safety-education/get-answers/food-safety-fact-sheets/foodborne-illness-and-disease/frozen-fully-cooked-products-and-botulism/CT_Index

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The incidence of the disease is low, but the mortality rate is high if not treated immediately and properly. There are generally between 10 to 30 outbreaks a year in the United States. Some cases of botulism may go undiagnosed because symptoms are transient or mild, or misdiagnosed as Guillain-Barre syndrome.
Reference: https://www.fda.gov/food/foodborneillnesscontaminants/causesofillnessbadbugbook/ucm070000.htm

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What is the Best Way to Prevent Botulism?

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What is the Best Way to Prevent Botulism?

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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.

reference: https://www.fsis.usda.gov/wps/portal/fsis/topics/food-safety-education/get-answers/food-safety-fact-sheets/foodborne-illness-and-disease/clostridium-botulinum/ct_index/!ut/p/a1/jZDRbsIwDEW_ZR8QxaUMjUdUaaLdaIUQW5YXFIhbLKUJatxt7OvptL1sGgz7yfa5utaVWiqpvXmlxjAFb9znrCcbWMIkmWZQVNPkHvLyaVk9ZBncrW4H4OUCUKZX6s_UDP7TF1cYjLpFtmikPhjeC_J1kKpBFsbHN-yiVHUIVkRTIx9FbXYs4h6Rvw_b0HkU5JzHGAeNFZYimohS7VyI3JGlvhXbwL0j37fDmjfkLb7LZ6l_fgfJ0HmZrsbzokyhGv8G_ojvCzifz6Fdq4_H2RwovzkBuNfPlA!!/#10

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Domain- Bacteria Kingdom-Bacteria

Kingdom-Bacteria

Phylum – Firmicutes

Class — Clostridia

Order — Clostridiales

Family — Clostridiaceae

Genus — Clostridium

Species – Clostridium botulinum

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A 14-year-old with botulism. Note the weakness of his eye muscles and the drooping eyelids in the adjacent image, and the large and non moving pupils in the right image. This youth was fully conscious

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A 14-year-old with botulism. Note the weakness of his eye muscles and the drooping eyelids in the adjacent image, and the large and non moving pupils in the right image. This youth was fully conscious

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a)food-borne b)wound c) infant botulism d) Adult infectious e)Inadvertent botulism

a) Food born is acquired by consuming contaminated home canned food.

b) Wound botulism is cause by the Clostridium bacteria infecting the wound and releasing bacteria.

c) Infant botulism occurs when the infant consumes spores which grow in the intestines, and release neurotoxin.

d) Adult infectious is very similar to that of Infant botulism, however it occurs in older children and adults with bowel abnormalities.

c) Inadvertent botulism has been seen in patients with intramuscular injections of the botulism toxin

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Humans get botulism from eating or drinking contaminated products.

Within 12-36 hours symptoms will begin to show and if left untreated will lead to dead.

Symptoms include  Nausea, vomiting, double vision, fatigue, dry mouth, etc.

But there is an antitoxin treatment followed by intensive respiratory care, however even after that, there is a chance of being hospitalized for a year.

Unfortunately even with treatment severe case of botulism can result in paralysis, respiratory failure and death.

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Clostridium botulinum reproduce asexually, so basically cloning of one cell, so that the offspring inherits the genes of it parent. In suitable conditions, Clostridium botulinum reproduces by binary fission, a process by which a cell copies it’s DNA and splits itself in half, so that each half has a copy of the DNA.

However in unfavorable conditions, the bacterium then remains dormant in endospore form.

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When processing canned foods, heat is the main objective in killing bacteria, however botulism tends to cultivate in contaminated canned foods, and different strains have arrived. The spores can survive in in temps 49 degrees celceus and up, so the heat actually aids in the growing of these cultures.

For Radiation adaptation, too much radiation would spoil the food, however eliminate dangerous bacteria, and yet with not enough or too little radiation, the weaker bacteria are eliminated while the stronger ones(botulism) remain.

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It is the wonderful surgical treatment aging woman with lots of money use.

Botox! Or in more technical terms, it is the chemical used in making botox.

It is used medically to treat certain muscular conditions and cosmetically remove wrinkles by temporarily paralyzing muscles.

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The bacterium is an obligate anaerobe and ferments carbohydrates and other nitrogenous compounds such as amino acids.

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C. botulinum can have multiple habitats, mainly in the soil and possibly food products. So,basically in moist oxygen poor environments. The ideal environment for Botulism would be in canned foods, and honey. If the canned food has been contaminated during preservation, fermentation etc. there is a possibility the spoilage bacteria will become corrupted and die off, leavings the perfect environment for the nasty botulism bacteria to grow and cultivate before unknowingly you ingest these little suckers and possible die or become paralyzed, either way it sucks.

However, as sneaky as these nasty bacterium are, they’re also the most stubborn. Being able to survive in environments not suited for us mere humans (eg. in temp of 120 degrees Celsius). As well surviving through radiations, and powerful chemicals but we’ll talk more about that in the Adaptations post.

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Stain dye : Crystal violet
Gram stain : Gram-positive
Shape : Rod-shaped
Size : Around 4,039 kbp
Other characteristics
1. Anaerobic
2. Spore forming
3. Motile bacterium
4. pathogenic bacterium which produce toxin called botulin

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Factors that can limit the growth and toxin production

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Factors that can limit the growth and toxin production

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1. Temperature
-->The optimal growth temperature for most strains of C. botulinum is around 40°C (104°F). Some strains, can grow even at 3°C (37°F), which is lower than ordinary refrigeration.
2.  pH
--> 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.
3. Water activity
-->C. botulinum requires a water activity of at least 0.94 to grow and produce toxin.
4. Food preservatives
--> example: nitrite, sorbic acid, parabens, phenolic antioxidants, and ascorbates,
--> effect: inhibit the growth of C. botulinum and limit toxin production.

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1. Life-threatening disease caused by the ingestion of a potent neurotoxin produced during growth of the C. botulinum bacteria.
2. Neurotoxin is among the most toxic substances known; even microscopic amounts can cause illness or death.

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What is important about this bacterium ?

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What is important about this bacterium ?

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1. Clostridium botulinum are rod-shaped bacteria also called C. botulinum.
2. They are anaerobic, meaning they live and grow in low oxygen conditions.
3. The bacteria form protective spores when conditions for survival are poor.
-->The spore has a hard protective coating and has layers of protective membranes. Within these membranes and the hard coating, the dormant bacterium is able to survive for years.
4.  C. botulinum is responsible for a disease called botulism.

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A video to introduce Clostridium botulinum

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Clostridium botulinum - animated quick review

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Fmgsindia.com provides free, useful resources for medical students preparing for postgraduate entrance Examinations. Check our website for more information.Short, comprehensive video lectures designed to integrate knowledge of the basic sciences to clinical applications and context. ENGAGE Subscribe to our channel: http://www.youtube.com/user/fmgsindia?sub_confirmation=1 Leave a comment, thumbs up the video (please!)

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http://www.sciencedirect.com/science/article/pii/S0923250814001910

http://www.sciencedirect.com/science/article/pii/S2214799316301035

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Clostridium botulinum

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https://www.youtube.com/watch?v=4spmAQVlka8&t=46s

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http://popplet.com/app/#/3929564

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UPM-UMP-UiTM

UPM
1. Nur Afiqah Shalihin
2. Zulaika
3. Ayne Talib

UMT
1. Dayana
2. Munirah
3. a'fifah
4. afiqah
5. Ezzati
6. Intan

UiTM
1. Ayunni
2. Faizah