This document provides information about several gram-positive foodborne bacterial agents: Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Listeria monocytogenes, and Staphylococcus aureus. For each organism, it discusses characteristics such as temperature and pH tolerances, toxins produced, and methods for isolation and inactivation. The document is presented as part of a course on food safety management, with the goal of educating students about important foodborne pathogens.

1. AGM 608 – Food safety Management (1+1)
Presented by
KASTHURI S
2022611003
Dept. of Agrl. Microbiology
TAMIL NADU AGRICULTURAL UNIVERSITY
DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
COIMBATORE-03
Gram positive food borne bacterial agents

2. List of organism
 Bacillus cereus
Clostridium Botulinum
Clostridium Perfringens
Listeria monocytogenes
Staphylococcus aureus

3. 1. Bacillus cereus
Introduction
• A spore forming bacillus was first isolated from the incriminated
meal - named as Bacillus peptonificans -properties resemble as B.
cereus.
• Later, in Norway some people got ill. Found due to Vanilla sauce - It
is now known that B. cereus by sample test.
• Higher incidence of B. cereus occur in pasteurized and other heat-
processed milks compared with raw milk.
• Type of spoilage known as ‘sweet curdling’ or ‘bitty cream’.
• It is a opportunistic pathogen.

4. Organism and its characteristics
• Bacillus are Gram +ve, aerobic, spore-forming rods.
• It is facultative anaerobic with large vegetative cells,
typically 1.0mm by 3.0–5.0 mm in chains.
• Temperature range from 8 to 55 ℃, optimum 30–37 ℃(mesophilic),
and does not have any marked tolerance for low pH and water
activity (0.93 to 0.95).
• Maximum toxin production at 20-25 ℃ and minimum at 10 ℃.
• Oxygen is required for production of emetic toxin

5. • Spores show a variable heat resistance; recorded D values at 95 ℃
in phosphate buffer range between around 1 min up to 36 min.
• Spores are more resistant to dry than moist heat. More resistant in
oily foods. Cooking below 100 ℃ cause spore survival.
• Emetic toxin – stable for 80 min. at 121 ℃ and 60 min at 150 ℃.
Stable between pH 2 and pH 9.
• Spores are resistant to gastric acidity (between pH 1 and pH 5.2).

6. Toxins
• Emetic toxin: Cereulide toxin is formed by growing cells of B. cereus.
Rich in lipophilic potassium ion. Suppress fatty acid oxidation and stop
mitochondrial activity.
• Enterotoxin (Diarrheal): produced during vegetative growth. Not liable at
pH 4-11. get deactivated at 56 ℃ for 5min.
• Haemolysin BL: tripartite enterotoxin. Considered as primary virulence.
• Enterotoxin T: single composite protein. Has lethal effect in mice.
• Enterotoxin FM: involved in biofilm formation
• Cytotoxin K: cause severe food poisoning outbreaks.

7. Isolation and Identification
• B. cereus can be identified after 24 h incubation at 37 ℃
• Colonial morphology - flat or slightly raised, grey-green colonies.
• Polymyxin/pyruvate/eggyolk/mannitol/bromothymol blue agar
(PEMBA) media is one widely used example.
• Polymyxin as a selective agent and where yeasts and moulds are
likely to be problem of acidione.
• Pyruvate in the medium improves the egg-yolk precipitation and a
low level of peptone enhances sporulation.
• Spores appear green in a cell with red vegetative cytoplasm and
containing black lipid globules.
• Biochemical confirmation - produce acid from glucose but not from
mannitol, xylose and arabinose.

8. Inactivation techniques
• Spores (depends on strain and food)
D100℃ - 1.2 to 7.5 min. in rice
D120℃ - 3.4 min. in oily foods.
• Emetic toxin is stable in food.
• Diarrheal toxin inactivated at 56 ℃ for 5min.
• Vegetative cells inactivated in yogurt at pH 4.5; fruit juice pH 3.7, pH 5-6
log10 reduction within a few hours depending on temp.
• Diarrheal toxin unstable at pH 4-11.
• Vegetative cells inhibited at aw < 0.91.

9. 2. Clostridium Botulinum
• In 1793, few people died after eating Blunzen, a type of sausage
made by packing blood and other ingredients into a pig’s stomach.
• It is boiled and then smoked, stable at room temperature for several
weeks and suitable for consumption without reheating.
• Early evidence suggested that botulism was confined to meat
products, it was later found to occur wherever conditions suitable
for survival and growth.
• Low-acid foods are the most common sources like most vegetable,
fruits, milk, all meats, fish and other sea foods.

10. Organism and its Characteristics
• Gram-positive, motile with peritrichous flagella, obligately
anaerobic.
• The most important common feature of the species is the production
of neurotoxins responsible for botulism.
• The rate of growth and toxin production at the lower
temperature limit is slow and will be reduced still further by any
other factors adverse to growth.
• Experimental studies shows that storage periods of 1–3 months are
necessary for toxin production at 3.3℃, reduced at higher
temperatures.
• Vacuum-packed herrings inoculated with 100 spores per pack - toxic
after 15 days storage at 5 ℃.

11. Condition Group I
Toxins A, B, F; proteolytic
& mesophilic
Group II
Toxins B,E,F ; non-
proteolytic & psychrotropic
Vegetative cells
Temperature Min: 10℃
Opt.: 35-40℃
Max.: 48℃
Min: 3.0℃
Opt.:18-25℃
Max.: 45℃
pH 4.6 5.0
Water activity-
aw
0.94 (10% NaCl) 0.97 (5%NaCl)
Spores survival
Temperature Spores and toxins resistant to freezing temp.
pH Spores: <4.6
Toxins: stable at low pH
Spores survive at <5.0
Toxins: stable at low pH

12. Isolation and Identification
• C. botulinum produce a small proportion of the total microflora.
• Enrichment is necessary to improve the chances of isolation.
• Sometimes enrichment cultures are heated prior to incubation to
eliminate non-spore forming anaerobes.
• After enrichment in a medium such as cooked meat broth at 30 ℃
for 7 days, the culture is streaked on egg yolk agar, incubated
anaerobically for 3 days.
• Exhibit smooth colonies with irregular edge
• Showing lipolytic activity on egg-yolk agar - check for toxin
production.
• Incorporating antitoxin into the agar medium, so toxin-producing
colonies are surrounded by a zone of toxin–antitoxin precipitate.

13. Inactivation techniques
Condition Group I
Toxins A, B, F; proteolytic &
mesophilic
Group II
Toxins B,E,F ; non-
proteolytic & psychrotropic
Vegetative cells
Temperature Killed by few min at 60℃
pH <4.6 (sporulation) <5.0(sporulation)
Spores
Temperature D100℃ = 25 min.
D121℃ = 0.1-0.2 min.
D100℃ = <0.1 min
D121℃ = <0.001 min.
pH Low pH (<5.0) or high pH (>9.0) reduce D values
Radiation Mostly resistant. In frozen food, D= 2.0 to 4.5 kGy
Toxin
Temperature D74℃ = <3 min. (type A B E). D74℃ = 25 min. (tomato soup of type
A toxin)
pH Inactivated at pH 11
Radiation Toxins not inactivated by doses used in food preservation

14. 3. Clostridium Perfringens
• Clostridium perfringens, formerly welchii, - gas gangrene.
• Species is classified into five types, designated A–E.
• Type A is responsible for food poisoning and gas gangrene produces
only major toxin - lecithinase (phospholipase C) activity.
• Its ability to hydrolyse lecithin and some other phospholipids plays
an important role in the pathogenesis of gas gangrene.
• Thickened sauce like gravies, pre-cooked foods, soups and sauce,
poultry and meat products.

15. Organism and its Characteristics
• Gram-positive, rod-shaped anaerobe, encapsulated and non-motile, and
occasionally grow in the presence of oxygen.
• Heat shock around 75 ℃ actively germinate spores in food.
• Growth is very slow <20 ℃.
• Optimum 43–47 ℃, growth is extremely rapid with a generation time of
only 7.1 min at 41 ℃.
• Vegetative cells show no marked tolerance to acid (range pH 5.1-9.7), have
a minimum aw for growth of 0.93–0.95, depending on the humectant, and
will not grow in the presence of 6% salt.

16. • Enterotoxin produced only during spore formation.
• Toxin production is at 35-40 ℃.
• Vegetative cells decline at refrigeration temperature.
• Spores survive both 4 ℃ and – 20 ℃ with <1-log reduction in spore
viability after 6 months at both condition.
• Sporulation occur between pH 6 and 8.
• Hardy spores show less than 1.2 log decrease in numbers after 3
months at pH 4 and 10.

17. Isolation and Identification
• Selective plating media to enumerate C. Perfringens; antibiotic as
the selective agent and sulfite reduction - produce black colonies.
• The most popular combinations are tryptose/sulfite/cycloserine
(TSC) and oleandomycin/polymyxin/sulfadiazine/perfringens
(OPSP) medium, incubated anaerobically for 24 h at 37 ℃.
• A better diagnostic reaction is obtained if pour plates are used since
colonies on the agar surface of spread plates can appear white.
• Suspect colonies - confirmed by absence of motility, their ability to
reduce nitrate to nitrite, lactose fermentation, and gelatin
liquefaction.

18. Inactivation techniques
• Temperature:
Cooking for 70 ℃ for 2 minutes achieve 6-log reduction in
vegetative cells but not kill spores.
D120 ℃ - 18 sec.
D120 ℃ - 161 sec. is used.
• D-values vary for different strains.
• Enterotoxin is heat liable protein – inactivated by heating for 5 min.
at 60 ℃. But not generally produced in food.
• Vegetative cells inactivated below pH<5.

19. 4. Listeria monocytogenes
• Dairy products such as raw and pasteurized milk and soft cheeses
• Raw vegetables, in the form of a garnish containing celery, tomatoes
and lettuce
• Soft cheeses are also frequently contaminated with L.
monocytogenes due to the cheese ripening process.
• L. monocytogenes survives poorly in unripened soft cheeses such as
cottage cheese but well in products such a Camembert and Brie.
• During the ripening process, microbial utilization of lactate and
release of amines increase the surface pH allowing Listeria to
multiply to dangerous levels.
• Does not produce toxins in food.

20. Organism and its Characteristics
• It is a Gram-positive, facultative anaerobic, catalase-positive,
oxidase-negative, non –spore former.
• Colonies on tryptose agar viewed as characteristic blue–green sheen.
• L. monocytogenes will grow over a wide range of temperature
between 30 and 37 ℃. Min at -1.5 ℃ and Max. at 45 ℃.
• Below about 5 ℃ growth is extremely slow with lag times of 1 to 33
days and generation times from 13 to more than 130 h being
recorded.
• Water activity 0.97 and pH Min. at 4.3-Max. at 9.6-Opt. at 7.0
• It is also quite salt tolerant being able to grow in 10% sodium
chloride and survive for a year in 16% NaCl at pH 6.0.

21. • Survives and grow slowly at refrigeration temperature.
• Can survive even at low aw of 0.83.
• pH survive but not multiply at 4.3
• Survive in food packaged under vacuum, increased CO2(up to 40%)
and N2 (up to 70%) gas levels.
• Produce biofilms – that helps to survive for long period of time.
• Biofilm – resistant to chlorine, iodine and anionic levels.

22. Isolation and identification
• Low-temperature enrichment at 4 ℃ is the traditional technique for
isolating L. monocytogenes from environmental samples.
• Selective agars with combination of selective agents such as lithium
chloride, phenylethanol and glycine anhydride and antibiotics used.
• Identification of presumptive Listeria colonies was based on
microscopic examination of plates illuminated from below at an
incident angle of 45 degree (Henry illumination), when they appear
blue–grey to blue–green.
• Confirmation of L. monocytogenes: sugar-fermentation tests to
distinguish it from other Listeria species and, in particular, the
CAMP test to differentiate L. Monocytogenes from L. innocua.

23. Inactivation techniques
• Inactivate faster at high temp. (> 50 ℃)
• Organic acid (ex. acetic acid) more effective than mineral acids (ex.
Hydrochloric).
• Low dose gamma radiation emit some , but not all.
• More resistant to UV
• D values: D55 ℃– 95.6 min; D60 ℃– 15.2 min; D70 ℃– 0.4 min.
• Others like High hydrostatic pressure
post packaging pasteurisation used

24. 5. Staphylococcus aureus
• First food poisoning caused by staphylococci is caused by cheese.
• Enterotoxin production is principally asssociated with species Staph.
aureus, although it has also been reported in others including Staph.
intermedius and Staph. hyicus.
• Occur naturally in poultry and other raw meats.
• Milk products such as dried milk and chocolate milk
• Enterotoxin production occurred in the raw milk.
• Toxin production best in oxygen but also grow anaerobically.
• In Japan, rice balls that are moulded by hand (food vehicle) while in
Hungary, it is ice cream.

25. Organism and its Characteristics
• Gram-positive, forming spherical to ovoid, irregular i.e. resembling
bunches of grapes.
• Facultative anaerobes; ability to ferment glucose can be used to
distinguish them from genus
• Staphylococcus aureus is a typical mesophile with a growth
temperature range between 7 and 48 ℃.
• pH: 4.0-9.3. growth inhibited at pH-5.1
• Growth is retarded at 80% CO2
• Particularly important consideration in some foods is its tolerance of
salt (5-7% NaCl) and reduced aw (0.83).

26. Toxins
• Produce enterotoxin in food with low aw as 0.85.
• Temp. 35 – 40 48 ℃
• pH: 5.3-7.0
Survival
• Survives in frozen stage
• Heat resistant is increased in dry, high-fat and high-salt foods.
• Toxins extremely resistant to heat.
– Ex. Dvalue 149 ℃ - 100 min at aw 0.99
– Dvalue 149 ℃ - 225 min at aw 0.90

27. Isolation and Identification
• Most successful and widely used selective plating .
• Lithium chloride and tellurite act as selective agents
• Reduction of the tellurite by Staph. aureus gives shiny, jet-black
colonies - surrounded by clear zone.
• Colonies also often have inner white margin caused by precipitation
of fatty acid.
• Colonial appearance on Baird-Parker (B-P) agar gives presumptive
identification of Staph. aureus - confirmed by tests (production of
coagulase and thermostable nuclease).
• Coagulase is an extracellular substance which coagulates human or
animal blood plasma in the absence of calcium.

28. Inactivation techniques
• Inactive at D60 ℃– 2 min. for salty foods
ex. Cheese, ham.
• Heat resistant reduced at high and low pH
• Rapid destruction at pH-2.3 in lemon juice and lime juice
• Aw – withstand desiccation level.
• High CO2 conc. Substantially reduce growth.

29. References
• Jovanovic, J., Ornelis, V. F., Madder, A., & Rajkovic, A. (2021). Bacillus
cereus food intoxication and toxicoinfection. Comprehensive Reviews in
Food Science and Food Safety, 20(4), 3719-3761.
• Grenda, T., Jarosz, A., Sapała, M., Grenda, A., Patyra, E., & Kwiatek, K.
(2023). Clostridium perfringens—Opportunistic Foodborne Pathogen, Its
Diversity and Epidemiological Significance. Pathogens, 12(6), 768.
• Kanaan, M. H. G., & Tarek, A. M. (2020). Clostridium botulinum, a
foodborne pathogen and its impact on public health. Annals of Tropical
Medicine and Public Health, 23, 49-62.
• Camargo, A. C., Woodward, J. J., & Nero, L. A. (2016). The continuous
challenge of characterizing the foodborne pathogen Listeria
monocytogenes. Foodborne pathogens and disease, 13(8), 405-416.
• Fetsch, A., & Johler, S. (2018). Staphylococcus aureus as a foodborne
pathogen. Current Clinical Microbiology Reports, 5, 88-96.

30. THANK YOU