Bacteria in food safety

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