13 - Bacillus cereus.pdf

Bacillus cereus Bacillus cereus In a daycare center in Virginia, 82 children ate a special catered lunch The children were served fried rice which had been cooked the night before and left to cool at room temperature before being refrigerated The next morning rice and chicken were fried with oil and...

Bacillus cereus Bacillus cereus In a daycare center in Virginia, 82 children ate a special catered lunch The children were served fried rice which had been cooked the night before and left to cool at room temperature before being refrigerated The next morning rice and chicken were fried with oil and delivered to the day care where it was held at room temperature, and served without re-heating Fourteen of the people who ate the lunch became ill with nausea, abdominal cramps, and diarrhea about 2 hours after eating The median time for recovery was about 4 hours after symptom onset Bacillus cereus Members of the genus Bacillus comprise Gram-positive, spore forming, rodshaped, aerobic bacteria Outbreaks of Bc are highly underestimated since they are moderate in severity, and have symptoms that are similar to those caused by other food-borne pathogens Bc is widespread in nature and is frequently isolated from soil and growing plants Bc is readily spread from grass to cows’ udders and into raw milk Bc spores can survive milk pasteurization, after germination they can cause problems in dairy products Most strains are unable to grow at refrigerator temperature (below 10∘C), although psychrotolerant strains can grow at temperatures as low as 4∘C If Bc contaminated milk is on the market it generally only causes one or two cases of illness (and is not identified as an outbreak) Towards the end of milk’s shelf life there is often enough Bc to cause illness The proteases produced by Bc cause off flavors and people consume little if any of the milk Bacillus cereus Toxins Bc has the potential to produce two toxins, an emetic (vomit-inducing) and one of several possible diarrheal enterotoxins Most strains produce one or the other, a few strains are able to produce many Emetic Toxin: Heat stable (like S. aureus toxin) Acts rapidly (0.5-6 hr) after ingestion Produced by cells growing in the food Stimulates the vagus nerve to initiate vomiting Diarrheal Enterotoxin Several different types of heat-liable toxins Acts more slowly (6-14 hr) after ingestion Produced by vegetative cell growth in the small intestine Mode of action not well understood, but it induces diarrhea by stimulating cAMP system (like Vibrio cholerae) Bacillus phylogenetics Three species of the Bacillus (Bacillus thuringiensis, Bacillus anthracis and Bacillus cereus) have huge differences between their impacts on human health B. anthracis is the cause of the acute and often lethal disease anthrax B. thuringiensis is extensively used as a biological control for insects, because of its ability to produce protein crystals with insecticidal activity. B. cereus causes food-borne illness, but some strains have also been developed as biological control agents in the suppression of fungal and crop diseases There is a high level of sequence homology among the strains of B. thuringiensis Phylogenetic studies also indicate that B. thuringiensis and B. cereus are essentially the same Bacillus phylogenetics The definition of these species were based on phenotype as well as clinical and economic significance This is mainly reliant on plasmid content Speciation based on plasmid content is confusing and somewhat subjective B. anthracis harbors two plasmids with toxin proteins and biosynthetic genes for capsule formation Emetic B. cereus strains harbor a plasmid that encodes the nonribosomal biosynthesis of the toxin cereulide B. thuringiensis harbors several plasmids that encode insecticidal toxins Ehling-Schulz et al., 2019. The Bacillus cereus Group: Bacillus Species with Pathogenic Potential. Microbiology Spectrum. 7(3) Bacillus cereus Microbial Ecology B. cereus are endemic in soil and occupy diverse ecological habitats Endospores can persist for years Due to their ubiquitous presence, this organism is hard to control and can enter the food production and processing chain at anytime point Soil associated organism may also serve as major reservoirs for the acquisition of spores Ehling-Schulz et al., 2019. The Bacillus cereus Group: Bacillus Species with Pathogenic Potential. Microbiology Spectrum. 7(3) Bacillus cereus Toxins Characteristic Diarrheal Syndrome Emetic Syndrome Type of Illness Toxico-infection Intoxication Dose causing illness 105-107 cells 105-108 cells/gram Toxin Production In small intestine In foods Type of Toxin Protein; Enterotoxin Cereulide Incubation Period 8-16 hours 0.5-5 hours Duration of Illness 12-24 hours 6-24 hours Symptoms Abdominal pain, watery diarrhea, occasionally nausea Nausea, vomiting, malaise Foods most frequently Implicated Meat products, soups, vegetables, puddings, sauces, milk and milk products Fried and cooked rice, pasta, pastry, noodles Modified From: Montville, T.J., Matthews, K.R., Food Microbiology an Introduction Cereulide Cereulide is a heat-stable toxin that induces vomiting and may cause liver or brain damage at higher dosages Only about 1% of all B. cereus strains encode the genes necessary for cereulide synthsis Emetic syndrome is technically an intoxication since cereulide is formed during the growth of emetic B. cereus Is a cyclic polymer formed of 12 monomers It is produced by nonribosomal protein synthesis The genes responsible for cereulide synthesis are encoded on a large mega-plasmid (pCER270) Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Nonribosomal Peptides Nonribosomal peptides are a class of secondary metabolites They are synthesized by nonribosomal peptide synthases, which unlike ribosomes are independent of messenger RNA These molecules are often cyclic or have branched structures They contain non-proteinogenic amino acids including B-amino acids They are a diverse family of molecules with a broad range of biological activities including toxins, siderophores, pigments, antibiotics, and immunosuppressants. Cereulide Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Cereulide The enzymes responsible for the synthesis of cereulide include cesA and cesB The ces gene locus also contains cesP, cesH, cesT, and cesC/D that encode other proteins necessary for the regulation and secretion of cereulide These genes are all transcribed as a single 23kb polycistronic mRNA Ces transcription is temporarily regulated and starts during the late logarithmic growth phase then switches in the stationary phase Cytotoxic effects of Cereulide Cereulide toxicity is mediated by its ability to bind cations and mediate their transfer through the lipid bilayer Cereulide allows K+ to leak from cells and their mitochondria, changing their conductance, and this eventually leads to cell death Cereulide exposure can also be immunosuppressive since low doses can cause suppression of natural killer T cells Cereulide can also inhibit cell proliferation, RNA synthesis, and ATP synthesis – all leading to cell dysregulation and death Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Can be Fatal! On October 1, 2018 (after school) a 20-year-old male became sick after eating leftover spaghetti with tomato sauce that had been left in the kitchen at room temperature for 5-days!!! 30 minutes after consuming the spaghetti he had a headache, abdominal pain, and nausea. He vomited profusely for several hours and had two episodes of watery diarrhea He did not receive any medication and drank only water He went to sleep after midnight – his parents found him dead in his bed the next morning at 11am (the coroner said he probably died around 4am) On autopsy his liver appeared softened and his pancreas was totally lysed B. cereus was found in his stool as well as in the spaghetti All isolates contained a variety of enterotoxin genes and the genes for cereulide Naranjo, M. et al., 2011. Sudden death of a Young Adult Associated with Bacillus cereus Food Poisoning. Journal of Clinical Microbiology. (49)12: 4379-4381. Cereulide Cereulide can cause many symptoms in addition to emesis including liver failure, respiratory distress, rhabdomyolysis (damaged muscle tissue releases its proteins and electrolytes into the blood. These. damage the heart and kidneys and cause permanent substances can disability or even death), and encephalopathy Cereulide can spread rapidly from the intestine, to blood, to other organs Cereulide has a short half-life in the human body, which explains why emetic disease is shirt Cereulide rapidly excretes in the feces in high amounts Toxico-Infection Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Toxico-Infection B. cereus spores can be ingested through food, germination in the intestinal tract and release of toxins causes illness Spores are considered highly infective If a combination of spores and vegetative cells reach the stomach most vegetative cells are killed by the environment (10-30% may survive) In the small intestine, spores can germinate and produce enterotoxin In the large intestine, B. cereus re-sporulates due to nutrient limitation and is excreted via feces The onset of symptoms in the toxico-infection is 6-24 hours after food consumption – this is a lot longer than the intoxication emetic disease due to the transit time required to reach the small intestine and then germinate Enterotoxins (Nhe) In addition to cereulide, diarrheal B. cereus can contain one or more of several enterotoxins including non-hemolytic enterotoxin (Nhe), hemolysin BL (Hbl), and cytotoxin K (CytK) Nhe is a three-partite protein that forms pores in the cell membrane leading to cellular lysis All three genes are encoded on a single operon Approximately 93% of B. cereus synthesize this protein cluster Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Enterotoxins (Hbl) Hbl is also a three-partite protein that is similar in its lytic ability to Nhe About 42% of B. cereus strains carry the Hbl genes It is likely that based on sequence similarity and common functionality that Hbl and Nhe originate from a common ancestor Enterotoxins (Cytotoxin K) In 1998 a severe food poisoning outbreak in France took place and several elderly people died from necrotic enteritis, B. cereus encoding cytotoxin K was identified as the culprit CytK shows structural similarity to the hemolysin produced by S. aureus CytK forms pores in lipid bilayers, which causes necrosis and hemolysis In the ballpark of 40% of B. cereus strains encode CytK Food Associations Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Food Associations B. cereus is an issue for prepared meals, temperature abused leftovers, and refrigerated processed foods In processed products where a mild preservation treatment has been applied (i.e. prepared rice, custard, herbs, spices, bakery products) the vegetative cells are inactivated but spores survive and have no competition microorganism, therefore, temperature abuse supports outgrowth leading to the intoxication Additional spores will also be present leading to the toxico-infection Although the prevalence of B. cereus in food is high, the cell counts are low and proper preparation techniques and hygiene will prevent infection Food Associations Jovanovic et al., 2021. Bacillus cereus food intoxication and toxicoinfection. Comprehensive Reviews in Food Science and Food Safety. (20) 3719-3716 Food Associations Strains that can produce cereulide are typically found in high-starch foods like potatoes, pasta, and rice – and most strains are only able to produce cereulide when grown in starch rich meida The prevalence of cereulide in food is poorly studied In the few studies that have been preformed about 7.4% of rice dishes obtained from restaurants contained cereulide It was found in 100% of samples from maize-based dishes in Cameroon households Temperature abuse is the culprit for increased cereulide content in foods that cause intoxication Causes and Prevention Factors that contribute to Bc illness are: Improper holding temperature Contaminated equipment Inadequate cooking Poor personal hygiene Cooking does not destroy spores, so the best strategy is to make sure that spores do not germinate and cells do not grow This is achieved by uniform quick chilling food to 4∘C or holding food above 60∘C Since some Bc can grow at 4∘C food should not be refrigerated for long periods of time (well in advance preparation should be avoided) Toxins of Bacillus Species B. anthracis Enterotoxins Anthrax (X) X B. cereus X (X) B. thuringiensis X B. mycoides X B. weihenstephanensis X Insecticidal toxins Cereulide X X Modified From: Fratamico, Liu, and Kathariou., 2011. Genomes of Food and Waterborne Pathogens. Published by American Society for Microbiology. (X) Review of Toxico-Infections C. perfringens C. perfringens is an anaerobic spore-forming bacteria commonly found on meat and meat products The toxicoinfection toxin is released when the vegetative cells release their spores in the gut Improper heating or cooling is the major cause of C. perfringens type A toxicoinfection The cpe gene encodes the enterotoxin B. cereus Is a sporeformer Is a normal inhabitant of soil and is isolated from a variety of foods Two different toxins can cause emetic or diarrheal type of food-associated illness Desserts, meat, and dairy are usually associated with diarrhea, while rice and pasta are associated with emetic illness Some strains of B. cereus will grow at refrigeration temperature. Vibrio cholerae and enterotoxigenic E. coli are also technically toxico-infections