Food Microbiology

Food Borne Pathogens

Types of Food Poisoning

• Infective Bacterial Food Poisoning

• Toxic Bacterial Food Poisoning

Infective Bacterial Food Poisoning

• The principal mode of pathogenesis is not thought to be toxin-mediated.

• Pathogenesis is due to the presence of bacteria in large numbers (10^5 – 10^7 organisms/g food).

• Reasons for large bacterial numbers:

  • Food being heavily contaminated initially (receiving of raw materials, storage, or preparation).

  • Food stored in conditions that promoted the growth of large bacterial populations after purchase.

• Examples of bacteria:

  • Salmonella spp.

  • Campylobacter spp.

  • Vibrio parahaemolyticus

  • Escherichia coli (EPEC, EIEC)

    • EnteroPathogenic E. coli (EPEC)

    • EnteroInvasive E. coli (EIEC)

Toxic Bacterial Food Poisoning

• Caused by the action of toxins produced by the organisms.

• Toxins produced either in the food before consumption or during intestinal digestion.

• Examples of bacteria:

  • Clostridium botulinum

  • Bacillus cereus

  • Escherichia coli (ETEC, EHEC)

    • EnteroToxigenic E. coli (ETEC)

    • EnteroHaemorrhagic E. coli (EHEC)

  • Staphylococcus aureus

Infective Bacterial Food Poisoning: Campylobacter jejuni

• Source: Contaminated or raw milk, raw or undercooked poultry, or untreated water.

• Symptoms: Bloody diarrhea, abdominal cramps, fever, nausea, and vomiting.

• Transmission:

  • Consuming food contaminated directly or indirectly by animal feces.

  • Consuming food handled by someone who has not adequately washed hands after using the toilet.

• CDC estimates Campylobacter is the #1 cause of bacterial diarrheal illness in the United States.

• Also the #1 intestinal disease diagnosed in travelers returning to the United States.

• Characteristics:

  • Shape: Helical morphology

  • Size: 0.2–0.9 μm and 0.5–5 μm

  • Gram type: Gram-negative

  • Spore-forming: Non-spore-forming

  • Motility: Motile

Infective Bacterial Food Poisoning: Salmonella spp.

• Not thought to be toxin-mediated, but an infective bacterial food poisoning type.

• Salmonella spp. have to be present in large numbers to cause disease.

• Multiply in the small intestine and colon areas.

• Cause inflammatory response in the intestinal cells.

• The inflammatory response releases prostaglandins which stimulates cAMP and produces active fluid secretion.
  *Prostaglandins are a group of lipid compounds. They are mediators and have a variety of strong physiological effects, such as regulating the contraction and relaxation of smooth muscle tissue.

• Cyclic adenosine monophosphate (cAMP) is a messenger used in signaling. cAMP and its associated kinases function in several biochemical processes, including the regulation of glycogen, sugar, and lipid metabolism as well as liquid release.

• This results in diarrhea.

• Characteristics:

  • Gram-negative

  • Rod-shaped bacteria belong to the family of Enterobacteriaceae

  • Facultative anaerobic

  • Non-spore-forming bacilli

  • Most members of the genus are motile via peritrichous flagella (flagella distributed uniformly over the cell surface)

• Enterobacteriaceae is a large family of bacteria Gram- negative bacteria that includes Escherichia coli, Klebsiella, Salmonella, Shigella and Yersinia.

Aerobic and Anaerobic Bacteria

• Aerobic and anaerobic bacteria can be identified by growing them in test tubes of thioglycollate broth.

  • Obligate aerobes need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest.

  • Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest.

  • Facultative anaerobes can grow with or without oxygen because they can metabolize energy aerobically or anaerobically. They gather mostly at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration.

Salmonella Symptoms

• Loose motions

• Strong thirst

• Progressive weakness

• Sunken eyes

• Sunken fontanelle (in babies under 18 months)

• Hoarse voice

• Dry tongue

• Loose skin

• Reduced urination

• Wrinkled fingers

• Low body temperature

• Feeble pulse, gradually becoming imperceptible

  • Loose skin = Reduced skin turgor (tension). Normally, the skin on the back of the hand flattens immediately after being pinched and released; in severe dehydration, skin loses its fullness, so fold persists for more than 2 seconds. In infants, the test should be performed on the abdominal skin.

Sunken Fontanelle

• The fontanelle is the baby’s “soft spot” where the skull bones have not yet closed. In newborns, the bones of the skull are separate from each other in order to allow the baby to be born. They also remain separate to give the baby’s brain room for rapid growth in the first few months.

• Doctors check the baby’s fontanelle to make sure the skull is not closing up too quickly and to look for a fontanelle that is sunken in, because this can be a sign of trouble.

Types of Salmonella spp. Infection

1. Typhoid fever (Salmonella enterica serotype Typhi or Paratyphi)

   • Appears after an incubation period ranging from 7 to 28 days

   • Gradual onset of high fever (>40°C) over several days. Weakness, abdominal pain, constipation, headache, mild vomiting.

   • Asymptomatic chronic carrier state follows the acute phase.

2. Non-typhoid fever

   • Onset 8 to 72 hours after ingestion of the pathogen

   • Self-limiting, non-bloody diarrhea, may only involve abdominal pain

Infectious Dose of Salmonella spp

• Newborns, infants, and immunocompromised individuals are more susceptible to Salmonella infection than healthy adults.

Infective Bacterial Food Poisoning: Vibrio parahaemolyticus

• V. parahaemolyticus is one of the commonest causes of bacterial food poisoning in Singapore because of the preference of the local population to consume raw or partially cooked seafood.

• 78% of a variety of imported seafood were found to be contaminated with V. parahaemolyticus.

• Morphology

  • Gram negative

  • Curved rod

  • Non-spore forming

  • Halophilic (prefers to stay in a salty environment)

  • Motile

Reservoir for Vibrio parahaemolyticus

• Occurs naturally in coastal water; sediment, suspended particles, plankton, fish, and shellfish.

• Other 30 species. Example, oyster, scallop, clam, lobster, shrimp, crab.

• High percentage of seafood samples tested positive for the species.

• Exclusively associated with seafood that is consumed raw, inadequately cooked, or cooked but contaminated.

• Even though it can survive in seawater, it is neither a psychrophile nor psychrotroph. Optimal growth temperature is around 30-37°C.

V. parahaemolyticus Foodborne Outbreak

• Generation time: the time it takes for a population of bacteria to double in number

  • 8-9 min at 37 °C and 12 to 18 min in seafood

• Mean onset time: 24 hours

• Primary symptoms: abdominal cramps and diarrhoea. Subside in 3 to 5 days

• Human volunteer feeding studies: 10^5 to 10^7 cfu are required to develop the illness

Eating Sashimi at your own Risk

• All raw oysters, sashimi, and sushi sold on the premises should be kept in a refrigerator separated from other food items at a temperature between 0 °C and 4 °C.

• To avoid cross contamination, food eaten raw should be prepared only at specified areas in the food preparation room and should be separated from other food.

• Besides, exclusive cutting boards and knives should be used for food eaten raw. The utensils should be cleaned and disinfected thoroughly before handling different types of food eaten raw.

• Thorough cooking can significantly reduce the food poisoning risk

V. parahaemolyticus and Hemolysin

• V. parahaemolyticus possesses at least three hemolytic gene components known as Kanagawa hemolysin

• Hemolysin – lipid or protein causing lysis (rupture) of red blood cells by destroying their cell membrane

Toxic Bacterial Food Poisoning

• Staphylococcus aureus

• Clostridium botulinum

• Bacillus cereus

• Escherichia coli (certain strains)

Toxic Bacterial Food Poisoning: Clostridium botulinum

• Clostridium botulinum is a bacterium that produces toxins known as botulinum toxins. It will be produced in low-oxygen conditions. Botulinum toxins are one of the most lethal substances known.

• Foodborne botulism is caused by consumption of improperly processed food and is a rare but potentially fatal disease if not diagnosed rapidly and treated with antitoxin. Homemade canned, preserved, or fermented foodstuffs are a common source of foodborne botulism, and their preparation requires extra caution (WHO 2018).

• Characteristics

  • Gram-positive

  • Obligate anaerobic

  • Endospore-forming bacilli

  • Produces botulinum toxins which are neurotoxins. Botulinum toxins block nerve functions and can lead to respiratory and muscular paralysis

Clostridium tetani

• Clostridium tetani produces tetanus toxin.

• Tetanus toxin (or tetanospasmin) is closely related to botulinum toxin (the cause of botulism), the latter produced by Clostridium botulinum.

• Transported by motor neurons to the spinal cord.

• Severe muscular spasms develop

• Stiffness in jaw and neck muscles

• Difficulty swallowing

Toxic Bacterial Food Poisoning: Bacillus cereus

• Characteristics

  • B. cereus produces two toxins

    • Emetic (vomiting) toxin (acts rapidly 0.5 to 6 hours)

    • Diarrheal toxin that causes diarrhoea, cramp, and rectal tenesmus (take longer to act 6 to 14 hours)

  • Both types of illness are mild and underreported

  • Rectal tenesmus is a feeling of incomplete defecation. It is the sensation of inability or difficulty to empty the bowel at defecation, even if the bowel contents have already been evacuated.

  • Endospore-forming bacilli

  • Facultative anaerobes

  • Rod shape

  • Gram positive

• Endospores

  • When B. cereus are killed by cooking, B. cereus endospores survive, grow, and cause illness.

  • Endospores attach to pipelines and other solid surfaces in the dairy, making spores difficult to remove.

  • Endospores contain appendages and/or pili that are involved in adhesion

  • Outbreaks are commonly associated with fried rice, sauces, gravies, meat, dairy products

  • Studies have shown incidence of B. cereus contamination in markets and in street-ended foods

Endospores’ Unique Features

• The endospores are able to attach to pipelines and other solid surfaces present in the dairy food plants. The endospores are very difficult to remove.

• The endospores contain appendages/ bacterial pili that are involved in adhesion and reproduction.

Toxic Bacterial Food Poisoning: Staphylococcus aureus

• Staphylococcus aureus food poisoning is a gastrointestinal illness caused by eating foods contaminated with toxins produced by the bacterium S. aureus bacteria.

• About 25% of people and animals have S. aureus on their skin and in their nose. It usually does not cause illness in healthy people, but it has the ability to make toxins that can cause food poisoning (CDC 2018).

• Characteristics
  *Gram positive
  *Facultative anaerobic (Facultative organisms can survive in the presence, as well in the absence, of oxygen. These organisms generate adenosine triphosphate (ATP) through aerobic respiration when present in the oxygen-enriched environment, however, in an oxygen-deficient environment, these organisms have the capability to derive energy for survival from anaerobic respiration or fermentation process)
  *Non-spore-forming cocci
  *Coagulase and DNase positive (Coagulase positive means the bacteria is able to coagulate and clot the blood plasma. DNase positive means the bacteria is able to hydrolyse DNA and use it as a source of carbon and energy for growth. )

Staphylococcal Enterotoxins

• The Staphylococcal enterotoxins are named by letter in the order of their discovery (A, B, C, D, E)

• The type of enterotoxin most frequently involved in food poisoning is

  • Staphylococcal enterotoxin A (SEA)

  • Found in the food associated with ~79% of outbreaks

S. aureus Colonies Grown on Baird Parker Agar

• Tellurite is present in the Baird Parker Agar (BPA). S. aureus reduces the tellurite to a black compound. Hence you will see a black colony formed.

• Egg york is part of the components in the BPA. S.aureus possesses lecithinase, it can precipitate egg yolk and clear the egg yolk. Hence you see a clear zone surrounding the black colonies.

How do people get S. aureus food poisoning?

• People who carry S. aureus can spread the bacteria to the food if they do not wash their hands before touching the food. The bacteria can then multiply in the food and produce toxins that can sicken people.

• The bacteria can be easily killed by cooking, but its toxins are challenging to be destroyed and will still be able to cause illness.

• Ready-to-eat food such as sandwich, fruits, salad are especially risky if contaminated. Food contaminated with S. aureus toxin may not smell bad or look spoiled (CDC 2018).

What are the Symptoms?

• S. aureus food poisoning is characterized by nausea, vomiting, and stomach cramps. Most people also have diarrhoea.

• Symptoms usually develop within 30 minutes to 8 hours after eating or drinking food containing S. aureus toxin. The symptoms usually last no longer than 1 day. Severe illness is rare (CDC 2018).

How can a food handler prevent S. aureus food poisoning?

• The best way to avoid food poisoning by S. aureus is to prevent food from the danger zone (5 to 60 degree Celcius) for more than 2 hours.

• Bacteria can multiply rapidly if left in the danger zone.

• Remember to always follow these food safety tips:

  • Use a food thermometer and cook foods to their safe minimum internal temperature of 75 degree Celcius.

  • Keep hot foods hot (above 60 degree Celcius) and cold foods cold (below 5 degree Celcius).

  • Frequent hand washing.

  • Do not prepare food if you are ill with diarrhea or vomiting.

  • Wear gloves while preparing food if you have wounds or infections on your hands or wrists.

Toxic Bacterial Food Poisoning as well as infective Food Poisoning: Escherichia coli

• Escherichia coli (E. coli) bacteria normally live in the intestines of healthy people and animals. Most types of E. coli are harmless or cause relatively light diarrhoea. But a few strains, such as E. coli Enterohaemorrhagic O157:H7, can cause severe stomach cramps, bloody diarrhea and vomiting.

• We may be exposed to E. coli from contaminated water or food. The most common food sources with E. coli are especially raw vegetables and undercooked ground beef. Healthy adults usually recover from infection with E. coli O157:H7 within a week. Young children and older adults have a greater risk of developing a life-threatening form of kidney failure (Mayo Clinic 2020).

• Characteristics

  • Gram-negative

  • Facultatively anaerobic

  • Non-sporeforming bacilli (rod-shaped)

Types of Escherichia Coli

The four most important groups are as follows,

1. Pathogenic mechanisms not related to toxins (had to be present in large numbers to cause disease)

   • Enteropathogenic (EPEC)

   • Enteroinvasive (EIEC)

2. Where pathogenicity is related to toxin production

   • Enterotoxigenic (ETEC)

   • Enterohaemorrhagic (EHEC)

EPEC and EIEC

• Non toxin producing

• Some strains can produce urinary tract and wound infections as well as food poisoning

• Occasionally causes septicaemia and meningitis.

ETEC (Enterotoxigenic E. coli)

• ETEC produces 2 types of toxin

  • LT (Heat Labile)

  • ST (Heat Stable)

• Responsible for travelers' diarrhea

EHEC (Enterohemorrhagic E. coli)

• Produces verocytotoxins (VTs) or Shiga Toxin

• Responsible for haemorrhagic colitis

• Hemorrhagic colitis is a type of gastroenteritis in which certain strains of the bacterium Escherichia coli (E. coli) infect the large intestine and produce a toxin that causes bloody diarrhoea

• Hemorrhagic colitis is an illness characterized by crampy abdominal pain, initial watery diarrhoea, and subsequent development of grossly bloody diarrhoea with little or no fever.

Pathogenesis of EHEC (Enterohemorrhagic E. coli)

• Confirmed outbreak of E. coli O157:H7 was associated with eating ground beef, hamburger, at fast food outlets in the States.

• Acid Tolerance: E. coli 0157:H7 are unusually tolerant of acidic environments. Organic acid sprays (acetic acid, citric acid and lactic acid) do not affect the level of E. coli 0157:H7 on beef Antibiotic Resistance:

  • When it was first isolated in 1982, it was susceptible to most antibiotic affecting Gram negative bacteria

  • It is becoming increasingly resistant to antibiotic.

Endotoxins vs Exotoxins

Foodborne Outbreaks Caused by Virus

• Viruses are able to cause enteric disease and thus cause foodborne outbreaks

• e.g. Hepatitis A virus (HAV) and Norwalk viruses have been implicated here

Hepatitis A

• Small and naked

• Polyhedral enteric virus

• About 30 nm in diameter

• Contains RNA which is enclosed in the capsid

Hepatitis A is only Endemic in Developing Countries

1. Hepatitis A is an enterically transmitted viral disease which is highly endemic throughout the developing world but of low endemicity in developed countries such as the United States (U.S.).

• In developing countries, hepatitis A virus (HAV) is usually acquired during childhood, most frequently as an asymptomatic or mild infection.

• Transmission may occur by direct person-to-person contact, from contaminated water, ice, or shellfish harvested from sewage-contaminated water; or from fruits, vegetables or other foods which are eaten uncooked, but which may become contaminated during handling.

Source of Transmission

• Hepatitis A virus is inactivated by boiling or cooking to 85°C (1 minute); cooked foods may serve as vehicles for disease if they are contaminated after cooking.

• Adequate chlorination of water as recommended will inactivate HAV.

Higher Risk

• For travellers to developing countries, risk of infection increases with duration of travel and is highest for those who live in or visit rural areas or frequently eat or drink in settings of poor sanitation.

Symptoms and Incubation Period

• The time between infection and onset of symptoms is usually 4 to 6 weeks.

• Symptoms include fever, feeling unwell, no appetite, nausea, followed after a few days by jaundice (skin and eyes take on a yellowish tinge)

• Usually a fairly mild illness, but rare, very severe cases occur.

• Length of illness from weeks to months depending on severity.

• Severity increases with age

• A bilirubin test measures the amount of bilirubin in a blood sample. Bilirubin is a brownish yellow substance found in bile. It is produced when the liver breaks down old red blood cells. Bilirubin is then removed from the body through the stool (feces) and gives stool its normal color.

How to avoid Hepatitis A?

• Travellers should avoid drinking water (or beverages with ice) of unknown purity and eating uncooked shellfish or uncooked fruits or vegetables that are not peeled or prepared by the traveller.

Recommendations for Vaccination

• Hepatitis A vaccine is recommended for all susceptible travellers to or for persons working in countries with intermediate or high rates of HAV infection.

• Vaccination of children 2 years of age and older, adolescents and adults with the age-appropriate dose of hepatitis A vaccine is preferred for persons who plan to travel repeatedly or reside for long periods in intermediate or high risk areas.

Norwalk virus (Norovirus)

• Described as 27 nm in their shortest diameter and 32 nm in their longest diameter.

• It is the first viruses relevant to gastroenteritis which were subsequently identified.

• They were initially named Norwalk virus, honoring the place of their discovery. Today these viruses are known as noroviruses, and they belong to the genus Norovirus, the family Caliciviridae

Norwalk virus (Norovirus)

• Norwalk virus infection is an intestinal illness that often occurs in outbreaks.

• Norwalk and Norwalk-like viruses are increasingly being recognized as leading causes of foodborne disease in the United States.

• The viruses are passed in the stool of infected persons. People get infected by swallowing stool-contaminated food or water. Outbreaks in the United States are often linked to raw oysters.

Symptoms and Incubation Period

• Illness is characterised by acute onset of diarrhoea, nausea and vomiting, stomach pains, headache and mild fever 24 to 48 hours after infection.

• Symptoms generally last 24 to 48 hours, although a small proportion of cases may feel unwell for several days.

• Usually a mild to moderate self-limited illness

Source of the Norovirus

• Humans faeces

• Shellfish harvested from sewage-contaminated water.

• Method of spread

  • Fecal contaminated food, particularly shellfish and water. Possibly also airborne and via contact with surfaces contaminated with feces and vomit, even if this cannot be seen.

How to Prevent Norovirus?

1. Wash hands with soap and warm water after toilet visits and before preparing or eating food;

2. Cook all shellfish thoroughly before eating;

3. Wash raw vegetables before eating; and

4. Dispose of sewage in a sanitary manner.

What is Mycotoxin?

• Mycotoxins are secondary metabolites produced by fungi that are capable of causing disease and death in humans and other animals.

• Mycotoxins are toxic compounds that are naturally produced by different types of fungi.

• Mycotoxins enter the food chain as a result of infection of crops before or after harvest and are typically found in foods such as cereals, dried fruits, nuts and spices.

Primary and Secondary Metabolites

• Mycotoxins are secondary metabolites

Mycotoxins

• Consequence of mycotoxins are as follows,

  • Mutagenic: Mutagenic means capable of inducing genetic mutation.

  • Carcinogenic: Carcinogenic means having the potential to cause cancer.

  • Some causes specific organ toxicity

  • Most of the mycotoxins (At least 14 mycotoxins) are carcinogens

  • Aflatoxins is the most potent among all mycotoxins.

Aspergillus flavus

• It is a soil-borne opportunistic fungal pathogen that infects important agricultural crops such as maize, peanut, and cotton.

• It produces the most carcinogenic naturally occurring mycotoxin known as aflatoxin, which causes hundreds of millions of dollars in economic losses and serious illnesses in human and domestic animals due to the congestion of aflatoxin-contaminated food or feed, especially in developing countries.

Types of Aflatoxins

• Question: Are we using Aspergillus to make food?

• Answer: Yes, we are using Aspergillus oryzae in soy sauce fermentation. It does not produce aflatoxin (AF).

1. Aspergillus flavus and Aspergillus parasiticus are the most common Aspergillus type to produce aflatoxin.

• Question: What does the alphabet B and G mean?

• Answer: Aflatoxins B1, B2, G1, G2 are produced in nature by the molds. The letters B and G refer to the fluorescent color (Blue and Green observed under ultraviolet (UV) light)

Aflatoxin Toxicity

• Aflatoxins causes acute toxicity in animals and humans: Acute liver damage, liver cirrhosis and tumor induction and immunosuppressive effects *Liver cirrhosis: Involves loss of liver cells and irreversible scarring of the liver.

  • Cirrhosis can cause weakness, loss of appetite, easy bruising, yellowing of the skin (jaundice), itching, and fatigue.

Cases of Aflatoxin Toxicity

• Hepatitis case (liver related disease) in India that affected 400 people, 100 of whom died.

• Traced to corn heavily contaminated with A. flavus and containing up to 15 mg of aflatoxins per kg.

• The affected adults may have consumed 2 – 6 mg on a single day.

Experiments on Rats

• LD_{50} of AFB1 for rats by oral route is 1.2mg/kg and 1.5 - 2mg/kg for AFG1;

• Susceptible animals die within 3 days and show gross liver damage;

• Postmortem examination reveals aflatoxins to be hepatocarcinogens (a substance or agent causing cancer of the liver)

  • Cells grow at a fast rate

  • Swollen

  • Tumour