Common Foodborne Pathogens - VII

Clostridium botulinum Risk Profile 

Clostridium botulinum is gram-positive, spore-forming rod-shaped, an anaerobic bacterium that produces a protein, which has characteristic neurotoxicity. The spores are heat-resistant and can survive in foods that are incorrectly or minimally processed. C. botulinum may grow in foods under certain conditions while producing toxin(s), where a severe form of food poisoning (botulism) results when the toxin-containing foods are ingested. Although it is rare, its mortality rate is high. 
 
There are seven serotypes of C. botulinum have been identified (A, B, C, D, E, F, and G), based on the antigenic specificity of the toxin produced by each strain. Further, types A, B, E, and F cause human botulism, and types C and D cause botulism in animals including birds and fish. Besides, type G outbreaks have never been reported yet. Although most strains produce only one type of toxin, dual toxin-producing strains also been reported. 
 
Spores produced by the C. botulinum are heat-resistant, and both bacterium and the spores are widely distributed in nature. The spores germinate in the absence of oxygen, grow, and excrete toxins. They are found in both cultivated and forest soils, bottom sediments of streams, lakes, and coastal waters as well as in the intestinal tracts of fish and mammals, and on the gills and viscera of crabs and other shellfish.
 
Growth Factors
Temperature:
            Minimum – 10°C      Maximum – 48°C     (Optimum 35 - 40°C)
pH:
            Minimum – 4.6         Maximum – 9.0         (Optimum -)
Water Activity (aW):
            Minimum – 0.93       Maximum – -            (Optimum -)
Water Phase Salt:
            Maximum – 10%     
 
Sources

The food types that are susceptible to botulism vary according to the specific food preservation and cooking practices. Almost any food that is not very acidic, where pH is above 4.6 can be susceptible to the proliferation, growth, and toxin production by C. botulinum. Hence, 4% to 5% salt concentration is required for inhibition of its spores (especially regarding type E), while a 5% salt concentration can completely inhibiting growth. Salt concentrations slightly lower than those providing inhibition tend to extend spore outgrowth time at low temperatures.
 
Thus, any food that is conducive to outgrowth and toxin production of C. botulinum can be associated with botulism, when inadequate food processing practices that will allow survival of spores and that are not subsequently heated before consumption, to eliminate any live cells. A variety of foods, such as canned corn, peppers, green beans, soups, beets, asparagus, mushrooms, ripe olives, spinach, tuna fish, chicken and chicken livers, liver pate, luncheon meats, ham, sausage, stuffed eggplant, lobster, and smoked and salted fish have been associated with botulinum toxin.
 
Infant botulism can be caused due to various potential environmental sources, such as soil, cistern water, dust, and foods, honey is the one dietary reservoir of C. botulinum spores linked to infant botulism by both laboratory and epidemiologic studies. 
 
Disease

Botulism is a serious and sometimes fatal, rare non-transmittable foodborne infection caused by C. botulinum due to a potent neurotoxin formed during growth. The recommended treatment for foodborne botulism includes early administration of botulinum antitoxin, and intensive supportive care, including mechanical breathing assistance. An antitoxin for infant botulism (Botulism Immune Globulin Intravenous or BIG-IV) also is available, which should be administered as early in the illness as possible. However, antimicrobial therapy is not recommended, due to concerns about increased toxin release as a result of cell lysis. 
 

Mortality: 

The general fatality of botulinum is in-between 5% to 10% of the cases, but the mortality rate is high if treatment is not immediately administered. 

 

Infective dose: 

An extremely small amount such as “a few nanograms” of the toxin can cause infection.

 

Onset:

Adult – Usually 18 to 36 hours after ingesting food containing the toxin, although times have varied from 4 hours to 8 days.

Infant – Generally follows a period of normal development.

Complications: 

The infection results in flaccid paralysis of muscles due to neurotoxin, including those of the respiratory tract. The botulinum causes flaccid paralysis due to toxin by blocking motor nerve terminals at the neuromuscular junction, which progresses symmetrically downward, usually starting with the eyes and face, to the throat, chest, and extremities. Once the diaphragm and chest muscles become fully involved, respiration is inhibited and death can result from asphyxia without intervention. 

 

There are three major types of botulism are to be known and foodborne botulism and infant botulism, which also is foodborne. The third type, wound botulism, is not a foodborne infection. Nonetheless, there is a fourth undecided category, which might be the result of intestinal colonization in adults, with in vivo production of toxin. 

 

Foodborne botulism – A severe type of food poisoning caused by the ingestion of foods contaminated with the toxin produced by C. botulinum, which mostly develops symptoms often after consumption of improperly processed and inadequately cooked home-preserved foods. Home-canned or, occasionally, commercially produced foods have been involved in botulism outbreaks. Foodborne botulism is a rare occurrence in most commercially produced food products, but of considerable concern, because of its high mortality rate if not treated immediately and properly. 

 

Infant botulism – Colonization of C. botulinum in the intestinal tracts of infants due to the ingestion of spores that produce toxin is a serious infection such as intestinal toxemia botulism. 

 

Wound botulism - The rarest form of botulism and it does not involve food, which occurs when C. botulinum colonizes in a wound and produces toxins. The toxins are transported through the bloodstream. Whereas foodborne botulism is limited to the amount of toxin ingested, C. botulinum in wounds produce toxin in situ (gas gangrene) until the pathogen is gone. 

Symptoms:

Adult – Early signs of intoxication consist of marked lassitude, weakness, and vertigo, usually followed by double vision and progressive difficulty in speaking and swallowing. Initial symptoms may include double vision, blurred vision, drooping eyelids, slurred speech, difficulty swallowing, dry mouth, and muscle weakness. If the disease is not treated, symptoms may progress to paralysis of the arms, legs, trunk, and respiratory muscles. Difficulty in breathing, weakness of other muscles, abdominal distention, and constipation may also be common symptoms. 

 

Infant – Constipation is often the first sign of normal development of infant botulism, which is followed by flat facial expression; poor feeding (weak sucking); weak cry; decreased movement; trouble swallowing, with excessive drooling; muscle weakness; and breathing problems.

 

Duration: 

Patients with severe cases that involve paralysis of the respiratory muscles may need mechanical ventilation and intensive care for weeks or months.

 

Route of entry: 

Oral, botulinum toxins are ingested through improperly processed food in which the bacteria or the spores survive, then grow and produce the toxins. Though mainly a foodborne intoxication, human botulism can also be caused by intestinal infection with C. botulinum in infants, wound infections, and inhalation.

 

Pathway: 

Clinical symptoms develop after a person ingests the pre-formed toxin, or if the organisms grow in the intestines or in-wounds, followed by toxin release. The ingested botulinum toxin, which is an endopeptidase enzyme, blocks peripheral cholinergic neurotransmission at the neuromuscular junction and cholinergic autonomic nervous system. The toxin acts by binding presynaptically to high-affinity recognition sites on the cholinergic nerve terminals and decreasing the release of acetylcholine, causing a neuromuscular blocking effect. 

 

C. botulinum produces the toxin as a complex of proteins, among which is the neurotoxic moiety. The toxin is synthesized as a relatively inactive single-chain polypeptide. It becomes an active toxin by selective proteolytic cleavage to yield the heavy and light chains that are linked by a single disulphide bond and non-covalent interactions. The toxin’s light chain is a Zn⁺⁺ containing endopeptidase that blocks acetylcholine-containing vesicles from fusing with the terminal membrane of the neuron, resulting in flaccid muscle paralysis. 

 
Frequency
Although this food illness is rare, its mortality rate is high if the disease is not treated immediately and properly. There are 962 recorded botulism outbreaks in the United States from 1899 to 1990 involved 2320 cases and 1036 deaths. In outbreaks in which the toxin type was determined, 384 were caused by type A, 106 by type B, 105 by type E, and 3 by type F. In two outbreaks, the foods implicated contained both types A and B toxins. Some cases of botulism may go undiagnosed because symptoms are transient or mild or are misdiagnosed as Guillain-Barré syndrome.
 
Target Populations
All people are believed to be susceptible to botulism.
 
Diagnosis

Botulism can be diagnosed by clinical symptoms alone, but differentiation from other diseases may be difficult. Hence, the most direct and effective way in a laboratory to confirm the clinical diagnosis of botulism is to demonstrate the presence of toxin in the serum or feces of the patient or in the food he consumed. The most sensitive and widely used method for detecting toxin is the mouse neutralization test, which takes 48 hours, and the culturing of specimens takes 5 to 7 days.

Food Analysis
Determination of the source of an outbreak is usually based on the detection and identification of toxin in the food since botulism results from the ingestion of preformed C. botulinum toxin. Hence, the most widely accepted method is the mouse neutralization test, where extracts of the food injected into passively immunized mice, and results can be obtained in 48 hours. The mouse neutralization is followed by culturing of all suspected food in an enrichment medium, to detect and isolate the causative organism.

Reference:

FDA Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins. Second Edition. 2013
Preventive Controls for Human Foods. 2016
www.cdc.gov

Common Foodborne Pathogens - VI

Listeria monocytogenes Risk Profile 

Listeria monocytogenes is one of the primary causes of death from foodborne illness, which is a pathogenic, gram-positive, rod-shaped, facultative intracellular bacteria that are motile through its flagella. There are 13 serotypes of L. monocytogenes, including 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7, where serotypes 1/2a, 1/2b, and 4b were associated with the most of foodborne infections. There five other species in the genus Listeria – L. grayi, L. innocua. L. ivanovii, L. seeligeri, and L. welshimeri, but they are not pathogenic to humans, but L. ivanovii is considered pathogenic to ruminants. 

Several L. monocytogenes genes involved in cellular invasion and intracellular parasitism and their functions have been identified, including the pleiotropic regulator of the virulence gene cluster prfA, members of the gene cluster (plcA, hly, mpl, actA, and plcB), and the inl family of invasion genes. Products with roles in phagosomal lysis and escape into the cytoplasm include LLO, a pore-forming toxin encoded by hly, and two C-type phospholipases, a phosphoinositol-specific phospholipase C encoded by plcA and a broad-spectrum phospholipase C encoded by plcB that cleaves phosphatidylcholine (PC-PLC). These enzymes act with LLO to facilitate phagosomal escape and cell-to-cell spread and also may be involved in stimulating intracellular signaling in the eukaryotic target. The mpl gene encodes an enzyme that processes the immature form of PC-PLC into a mature form. Intracellular motility and subsequent cell-to-cell spread are dependent upon the ActA protein, which is essential for polymerization of host F-actin. The recently described inl family of genes encode internalin A and internalin B proteins that are involved in binding and invasion of eukaryotic cells.

L. monocytogenes is a hardy, salt-tolerant bacterium that can survive in temperatures below 1°C and also can grow under psychrotrophic conditions vigorously, unlike many other pathogens. L. monocytogenes is ubiquitous in the moist environments, soil, and decaying vegetation as well as remarkable persistence in food-manufacturing environments.

Sources 
A variety of foods have been implicated with L. monocytogenes, including raw milk, inadequately pasteurized milk, chocolate milk, cheeses (soft cheeses), ice cream, raw vegetables, raw poultry and all kind of meats, fermented raw-meat sausages, hot dogs, and deli meats, and raw and smoked fish and other seafood. Nonetheless, L. monocytogenes can grow in psychotropic conditions, where potential contamination sources include food workers, incoming air, raw materials, and food processing environments. Thus, post-processing contamination at food-contact surfaces poses the greatest threat to product contamination. 

Growth Factors

Temperature:

            Minimum – -0.4°C    Maximum – 45°C     (Optimum 37°C)

pH:

Minimum – 4.4         Maximum – 9.4         (Optimum 7.0)

Water Activity (aW):

Minimum – 0.92       Maximum – -             (Optimum -)

Water Phase Salt:

                        Maximum – 10%      

Disease 

L. monocytogenes is a well-known pathogenic microbe who can infect and replicate within human umbilical vein endothelial cells, and causes central nervous system infections of immunocompromised humans and domesticated animals. 
 

Mortality: 

L. monocytogenes is among the leading causes of death from foodborne infections, and yet it is not a leading cause of foodborne infection. The severe form of the infection can cause a case-fatality rate of 15% to 30%, overall, but if the infection developed into listerial meningitis, the case-fatality rate may increase as high as 70%, and from septicemia, it may go up to 50% overall, further in perinatal/neonatal infections, the mortality rate may be more than 80%.

 

Infective dose: 

The accurate infective dose of L. monocytogenes is not established, where it is believed to be varied with the strain and susceptibility of the host, and the dose-response relationship may affect by the food matrix involved. In cases established against raw or inadequately pasteurized milk has shown that it is likely to be fewer than 1,000 cells for susceptible individuals. However, the infective dose may vary widely and depends on a variety of factors. 

 

Onset: 

Onset can be a relatively short incubation period from a few hours to 2 or 3 days to cause gastroenteritis. However, the severe, invasive form of the infection might take a very long incubation period, before symptoms can be seen which is estimated to vary from 3 days to 3 months. 

 

Complications: 

There are two types of disease caused by L. monocytogenes in humans, which are non-invasive gastrointestinal infections (generally resolves in healthy people) and the much more serious, invasive form of the infection that may cause septicemia and meningitis. Manifestations of L. monocytogenes infection tend to be host-dependent, where people with intact immune systems may cause acute febrile gastroenteritis, which is the less severe form of the disease. However, L. monocytogenes infection tends to be more severe for vulnerable populations. The infection can be grown into a more severe form, where it may result in sepsis and spread to the nervous system, potentially causing meningitis in elderly and immunocompromised people. L. monocytogenes, may cause mild, flu-like symptoms in pregnant women who are disproportionately infected may result in abortion or stillborn of their offspring, and those who born alive may have bacteremia and meningitis. One-third of confirmed cases are usually ending up in abortion or stillbirth due to maternal-fetal L. monocytogenes infections. 

 

Symptoms: 

When infected with L. monocytogenes, healthy people might have mild symptoms or no symptoms, while vulnerable populations may develop fever, muscle aches, nausea and vomiting, and, sometimes, diarrhea. If a more severe form of the infection develops and spreads to the nervous system, the symptoms may further include headache, stiff neck, confusion, loss of balance, and convulsions. 

 

Duration: 

The duration of symptoms generally depends on the health status of the infected person, where symptoms can last from days to several weeks. 

 

Route of entry: 

Oral. 

Pathway: 

L. monocytogenes has unique pathogenesis, where the bacterium can spread directly from one cell to another within the host, instead of traveling interstitially to reach other cells. L. monocytogenes is blood-borne, and once it is attached to the host’s monocytes, macrophages, or polymorphonuclear leukocytes, it starts to reproduce. The phagocytic cell attacks are overcome through a group of proteins on the surface of L. monocytogenes, which further enhance its cell-to-cell spread.

 
Frequency 
Based on a survey conducted in 1997 by the Centers for Disease Control and Prevention (CDC), listeriosis was responsible for approximately 2,500 infections and 500 deaths in the United States annually. L. monocytogenes infections had declined by 36 percent in 2008, compared to the period from 1996 to 1998, but there was a moderate increase in the incidence of L. monocytogenes from 2008 to 2009. More recently, the 2011 CDC report cited above estimated that L. monocytogenes causes 1,591 infections and causes 255 deaths annually, in the United States.  
 
Target Populations 
The main target populations for listeriosis are: 

Pregnant women/fetuses/neonates - perinatal and neonatal infections; 

Persons immunocompromised by i.e., corticosteroids, anticancer drugs, graft suppression therapy, AIDS, 

Cancer patients, particularly leukemic, 

Diabetic, cirrhotic, asthmatic, and ulcerative colitis patients,

The elderly, 

Healthy people.

 
Studies suggest that some of the healthy people are also at risk, because antacids or cimetidine may predispose them to the infection. Furthermore, healthy, uncompromised people could develop the disease, particularly if the food eaten was heavily contaminated with L. monocytogenes. 
 
Diagnosis 

Identification of culture isolated from tissue, blood, cerebrospinal fluid, or another normally sterile site such as placenta or fetus is needed for diagnosis of L. monocytogenes infection. Stool cultures are not informative, since some healthy humans may be intestinal carriers of L. monocytogenes.
 
Food Analysis 
Methods of analyzing foods for purposes of identifying L. monocytogenes are complex and time-consuming. The present FDA method uses a single enrichment broth, buffered Listeria enrichment broth, and requires 24 to 48 hours of enrichment, followed by a variety of agars and, finally, biochemical confirmation. The total time to identification is from 5 to 7 days. Many other enrichment broths, such as UVM broth and Fraser broth, are also included in various protocols. Agars that have been extensively evaluated include Oxford agar, PALCAM, LPM plus esculin, and ferric iron and MOX. New molecular biology techniques have been used to develop various rapid-screening kits for L. monocytogenes. These kits generally rely on ELISA, PCR, and probe-based identification. 
 

 
Reference:
FDA Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins. Second Edition. 2013
Preventive Controls for Human Foods. 2016
www.cdc.gov
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107882/

Common Foodborne Pathogens - V

Bacillus cereus sp. Risk Profile

Bacillus cereus and other bacillus species are gram-positive, facultatively anaerobic, endospore-forming, large rods. The identification and differentiation of serotypes further required biochemical tests to differentiate and confirm the presence of B. cereus, given characteristics are fairly common in B. mycoides, B. pseudomycoides, B. thuringiensis, and B. anthracis. Thus, differentiation depends on several factors specific to each organism such as determination of motility where most B. cereus strains are motile and the presence of toxin crystals distinguishes the B. thuringiensis. B. anthracis is usually non-hemolytic while B. cereus and others are beta-hemolytic. The rhizoid growth is characteristic of B. cereus var. mycoides and B. weihenstephanensis is a psychrotrophic strain that can grow at refrigerated temperatures.

Production of enterotoxin is associated with the vomiting form of B. cereus food poisoning. The same effect can also be caused by B. weihenstephanensis or other bacilli groups, which suggests that the plasmid carrying the emetic toxin can undergo lateral transfer, conferring the same properties to otherwise non-pathogenic strains. B. cereus is widespread in the environment and majorly on soil and vegetation.

 

The maximum temperature recorded is 48°C and the minimum growth temperature is 4°C where the optimal growth temperature is between 28°C to 35°C, and tolerance to pH ranges from 4.9 to 9.3, and the salt tolerates at 7.5%.

 

Sources

A wide range of foods such as meats, milk, vegetables, and fish, have been associated with diarrheal-type food poisoning. Vomiting-type food poisoning has been largely associated with rice products and other starchy foods such as potato, pasta, and cheese products. Besides, food mixtures such as sauces, puddings, soups, casseroles, pastries, and salads, are also been frequently linked with B. cereus food-poisoning outbreaks.

Disease

B. cereus food poisoning is a common term for the infection associated with the B. cereus family of human pathogens. There are two recognized types of infections that are caused by two distinct toxins, where diarrhea is caused by a large-molecular-weight protein, and vomiting is associated with cereulide (an ionophoric low molecular-weight dodecadepsipeptide). The cereulide is one of toughest to beat as its stability against pH and resistance to heat as well as proteases, where this non-antigenic peptide is stable after heating at 121°C for 30 minutes, cooling at 4°C for 60 days, and at a pH range of 2 to 11.

Mortality:

The mortality is a rare occurrence, but the emetic enterotoxin of B. cereus has been implicated in liver failure and death and a newly identified cytotoxin isolated from a B. cereus strain can cause a severe outbreak and deaths.

Infective dose:

The presence of large numbers of B. cereus usually more than 106 organisms/g in a portion of food is an indication of active growth and proliferation of the organism, where most often the number associated with human infection is 105 to 108 organisms/g. The pathogenicity arose from preformed toxin which is a potential human health hazard.

 

Onset:

Diarrheal stains: 6 to 15 hours after consumption of contaminated food.

Vomiting/emetic stains: 0.5 to 6 hours after consumption of contaminated foods.

 

Complications:

B. cereus foodborne infections that are caused by diarrheal or vomiting toxins producing stains generally considered mild and self-limiting, but more severe and fatal forms of the infection have been reported. Further, B. cereus can cause severe systemic and pyogenic infections such as gangrene, septic meningitis, cellulitis, panophthalmitis, lung abscesses, infant death, and endocarditis, and, in cows, bovine mastitis.

 

Symptoms:

Food poisoning of diarrheal stains can cause symptoms such as watery diarrhea, abdominal cramps, and pain where nausea may accompany diarrhea, but vomiting is a rare occurrence. Emetic stains usually cause symptoms of nausea and vomiting.

 

Duration of symptoms:

The symptoms usually subside after 24 hours of onset.

 

Route of entry:

Consumption of food contaminated with enterotoxigenic B. cereus or with the emetic toxin.

 

Pathway:

Cereulide is implicated to be toxic to mitochondria which acts as a potassium ionophore, where a serotonin5-HT3 receptor-mediated mechanism is associated with the emetic syndrome. Dermonecrotic and vascular permeability activities and fluid accumulation are observed in two diarrheal enterotoxins which are composed of multi-component proteins and the third type of enterotoxin is a member of the β-barrel toxin family that is similar to the β-toxin of Clostridium perfringens.

 

Frequency

There are thousands of cases around the world annually, but most of them are underreported or misdiagnosed due to the symptomatic similarities to Staphylococcus aureus intoxication with B. cereus vomiting type and Clostridium perfringens food poisoning with B. cereus diarrheal type. The United States has an annual average of over 60,000 for the B. cereus infections where foods that were associated with outbreaks included beef, turkey, rice, beans, and vegetables.

 

Diagnosis

Confirmation of as the pathogenic agent in a foodborne outbreak requires, either B. cereus diagnosis is carried out through isolation of B. cereus from suspect foods or isolation of large numbers of a B. cereus serotype or isolation of strains of the same serotype from the suspected food and feces or vomitus of the patient known to cause foodborne infection. Then determination of their enterotoxigenicity using serological tests for diarrheal toxin or biological tests for diarrheal and emetic stains. On the other hand, rapid-onset time to symptoms in the emetic form of the disease, coupled with some food evidence, is often sufficient to diagnose this type of food poisoning.

 

Target Populations

All humans are believed to be susceptible to B. cereus food poisoning.

 

Food Analysis

Several methods are used for the recovery, enumeration, and confirmation of B. cereus in foods, where a serological method has been developed for detecting the putative enterotoxin of B. cereus diarrheal type isolates from suspect food sources and the vomiting-type toxin can be detected through animal models or, possibly, by cell culture.

Reference:

FDA Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins. Second Edition. 2013

Preventive Controls for Human Foods. 2016

www.cdc.gov