Staphylococcus aureus Introduction – California

 

 

Staphylococcus aureus

Introduction

• In 1878, Koch observed

staphylococci.

• Staphylococcus

recognized as a separate
genus in 1880 by Pasteur.

S. aureus Properties

Introduction

• In the Greek language:
(cid:190) staphyle = a bunch of grapes
(cid:190) coccus = round

• 1884 – Rosenback grew

staphylococci on a solid medium.

• 1884 – Sternberg associated

staphylococci with “ptomaine”
formation in cheese that caused
human illness.

Introduction

• 1894 – Denys associated illness with
eating of meat from a cow sick with
pyogenic staphylococci.
• 1907 – Owen recovered

staphylococci from dried beef that
had caused poisoning characteristic
of what now is called
staphylococcal food poisoning.

Introduction

• 1914 – Barber related

staphylococcal food poisoning to a
toxic substance produced in food.

• He isolated staphylococci from

contaminated milk that came from
a sick cow with mastitis.

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Introduction

• 1929 – Dack studied an outbreak of food

poisoning caused from eating X-mas cake.
• Re-discovered the role of staphylococci in

food poisoning.

• He showed with human volunteers that the
isolated staphylococci produced a toxic
substance in culture, this substance caused
typical staphylococcal food poisoning.

Introduction

• 1948–1974 studies demonstrated:
(cid:190)The presence of preformed

enterotoxin in foods that had caused
staphylococcal food poisoning.

(cid:190)Antitoxin in the blood of people that

had suffered from this type of
poisoning.

S. aureus in the US
(estimated)

Agent

Cases

%

Deaths

%

S. aureus

185,060

1.3

2

0.1

Total bacterial

4,175,565

30.2

1,297

71.7

Total foodborne

13,814,924

100

1,809

100

S. aureus

• In 1994, S. aureus was considered to

be the cause of one of the most
common bacterial food intoxications.

• Holt et al. (1994) estimated S. aureus
food intoxication to be the second
most prevalent disease in the US.

Contemporary Problems

• Foods associated with

staphylococcal food poisoning:
(cid:190)In the US

– Meat products (e.g., ham)
– Desserts
(cid:190)In Japan – Rice balls

• Seasonal variations

Illness & Causative Agent

• S. aureus causes foodborne

intoxication.

• The thermostable enterotoxins and
not the bacterium are responsible
for the foodborne illness.

• Staphylococcus / staphylococcal

food poisoning.

2

Illness & Causative Agent

• The pathogen produces the toxins

while growing in the food.

• When the toxins are ingested by a
susceptible person they will cause
the illness.

S. aureus Properties

• Only enterotoxin-producing

staphylococci cause food poisoning.

• The ability to produce enterotoxin(s)

is associated with production of
coagulase and heat resistant DNase.

S. aureus Properties

S. aureus Properties

• It has a coccus shape.

• Occurs in clusters of

irregular arrangement like
the bunch of grapes.

• May occur singly, in pairs,

or in short chains.

• S. aureus is ~0.5-1.5 µm in diameter

• Gram positive, non-sporeforming,
non-motile, facultative anaerobe

• Coagulase and catalase positive

Coagulase Test

• Suspect colonies are incubated in 2 ml
of Brain Heart Infusion (BHI) broth
for 18–24 hr at 35–37°C.

• 0.5 ml coagulase plasma (with 0.5 ml
of EDTA) is added to 0.5 ml of broth
culture and mixed.

• Tubes are incubated and examined

after 4 hr.

3

S. aureus Properties

• S. aureus produces a variety of

extracellular enzymes and metabolites.

• The most important metabolite

produced is a group of heat-stable
toxins called enterotoxins
(staphylococcal enterotoxins).

S. aureus Properties

S. aureus Properties

• Temperature range:

7–47.8°C (Opt. 35–37°C)

• Enterotoxins produced between

10–46°C (Opt. 40–45°C)

• pH range: 4.0 – 9.8 (Optimal 6-7)

• Salt tolerant (10 – 20% NaCl)

S. aureus Properties

Environmental Effects

• Can grow at a sucrose concentration

• >10% NaCl inhibits SEA and SEB

up to 50–60%

• Water activity as low as 0.86 under
aerobic conditions, and 0.90 under
anaerobic conditions.

• Greater toxin production under

aerobic conditions.

production.

• Enterotoxins are not formed:
– Below pH 5.3 at 30°C
– Below pH 5.6 at 10°C

• Minimal water activity — 0.86 for

growth

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Water Activity

• Enterotoxin production occurs at

0.86 – 0.99, Opt. 0.99

• Reducing aw minimizes production

of enterotoxins:
–0.90 aw reduces SEB by 90-99%

(Maradona, 1998)

Microbial Ecology

• S. aureus does not compete well

with the normal flora of most foods.

S. aureus Toxins

S. aureus Toxins

• Staphylococcal enterotoxin A (SEA)

most common in gastroenteritis.

• S. aureus is the common species
associated with food intoxication.

• 12 enterotoxins: A, B, C, D, E, G, H,

I, J, K, L, M.

• Three variants of SEC – C1, C2, C3

(minor antigenic differences)

S. aureus Toxins

• Enterotoxins are simple proteins.
• Easily soluble in water and salt

solutions.

and papain.

• Resistant to trypsin, chymotrypsin,

• Pepsin destroys the toxin at pH 2.
• Toxin is resistant to radiation (200
kGy), and boiling (resists 121.1°C
for 0.5 hr)

S. aureus Enterotoxins

• Low molecular weight (~30 kDa)

simple proteins

• Heat resistant simple
• S. aureus itself is not heat resistant.
• Enterotoxins A and D are the most

heat resistant.

• When active, A and D exhibit
proteolytic enzyme resistance.

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Pathogenesis – target area

Clinical Symptoms

• Enterotoxins expected to act on

the receptors in the gut that
transmit impulse to medullary
centers.

• S. aureus enterotoxins cause:
(cid:190)severe gastroenteritis
(cid:190)nausea, vomiting, retching,

abdominal cramps, sweating,
chills, prostration, weak pulse,
shock, shallow respiration,
subnormal body temperatures.

S. aureus Food Poisoning

S. aureus Food Poisoning

• About $106 cells/gram of S. aureus

• Onset of illness takes <30 min – 8 hr. in food is needed for toxin production. • About 200 ng of toxin can cause illness in humans. following ingestion of the toxin containing food. • Most illness, however, occurs within 2–4 hr. • Recovery is within 24–48 hr. • Illness is rarely fatal. Clinical Symptoms • The enterotoxins acts on the receptors in the gut that transmit impulse to medullary centers. S. aureus Infections • S. aureus is a feared hospital pathogen. • Treatment of patients consists of bed rest and maintenance of body fluids and electrolytes. • Sometimes it can be very virulent, and often resistant to antibiotics. 6 How Is S. aureus Introduced to Food? How Is S. aureus Introduced to Food? • S. aureus is commonly found in: (cid:190) Nose (cid:190) Throat (cid:190) Hands (cid:190) Fingertips (cid:190) Hair and skin • Found in more than 50% of healthy people. • Found on skins or hides of animals. • Found in the environment. • Any food that requires handling and preparation is susceptible for contamination. • S. aureus is also found on the skin or hides of animals. • Cross-contamination may result from these animals during slaughtering. Foods Often Incriminated • Meats and meat products • Poultry and Fish • Cream-filled baked goods • Baked foods • Potato Salad • Salads containing any of the above items • Any nutrient-rich, moist food that is temperature abused. Food Sources for Staphylococcal Outbreaks (1973-1987) 96 120 100 80 60 40 20 0 26 22 20 14 9 Pork Bakery Products Beef Turkey Chicken Eggs (Bean et al., 1990) Contributing Factors Prevention • Improper storage and holding temperatures • Inadequate cooking/processing temperatures • Contaminated Equipment • Unsafe food sources • Poor personal hygiene (cid:190)10 – 50% adults are reservoirs of S. aureus • Adequate storage and refrigeration of foods • Not preparing foods far in advance • Adequate cooking and/or heat processing • Avoiding poor personal hygiene • Not holding foods between 40 – 140°F (4.4– 60°C) for prolonged periods (cid:131) 40-135°F (4.4-57°C); new numbers 7 Pathogen Detection • Laboratory media: (cid:190)Trypticase soy broth with 10% NaCl (cid:190)Mannitol salt agar (cid:190)Baird-Parker agar Indicators for the Presence of S. aureus • Coagulase Test • Thermostable Nuclease Test (TNase) • Polymerase Chain Reaction (PCR) TNase Testing • Culture is boiled for 15 min. • Toluidine blue agar plates are prepared. • 2 mm wells are dug in the plates and filled with the boiled cultures • Plates incubated for 2–4 hr at 37–50°C • Pink halos around wells indicates positive reaction. Polymerase Chain Reaction (PCR) • Thermostable DNA polymerase catalyzes the gene probe amplification. • Amplified DNA is detected by hybridization ring using radio- and non-radiolabeled probes. (Maradona, 1998) • Can amplify a single DNA molecule to 107 molecules. (Maradona, 1998) Detection Methods of Enterotoxins Biological Detection • Each new toxin type had to be detected • Biological • Biological subjects used are cats, kittens, biologically and monkeys. • Kittens--emetic response • Can determine the enterotoxin activity by observing responses. • Monkeys used to simulate human response. (Maradona, 1998) • Immunological (many, including kits) 8