Biology Article on Ice Cream Report

1. Staphylococcus aureus food-
poisoning outbreak associated
with the consumption of
ice-cream

2. Terminology
Enterotoxins - A cytotoxin produced
by bacteria that is specific for the
mucous membrane of the intestine
and causes the vomiting and diarrhea
associated with food poisoning.

3. Terminology
Staphylococcal enterotoxins
- members of a family of more than 20
different staphylococcal and streptococcal
exotoxins
- “pyrogenic” bacterial proteins and are
connected to significant human diseases that
include food poisoning and toxic shock
syndrome
- these toxins are for the most part
produced by Staphylococcus aureus (S.
aureus)

4. Terminology
Coagulase - is a protein enzyme produced
by several microorganisms that enables the
conversion of fibrinogen to fibrin
- it is used to distinguish
between different types of Staphylococcus
isolates

5. I. Introduction
Staphylococcus aureus
• an important food-borne pathogen
due to the ability of enterotoxigenic
strains to produce staphylococcal
enterotoxins (SEs) preformed in food
Staphylococcal food poisoning (SFE)
• characterized by a sudden onset of
symptoms, with vomiting, abdominal
pain, and stomach cramps being the
most common

6. I. Introduction
Individual susceptibility to SE and the amount
of SE ingested influence the onset and severity
of the symptoms.
Occurs due to improper handling and
subsequent storage at elevated temperatures.
Diagnosis of SFP is mainly based on:
•The recovery of >10^5 S. aureus/g from
food remnants
•The detection of SEs in food remnants
•The isolation of identical S. aureus clones
from both patients and food remnants

7. I. Introduction
Foods that favor growth of bacteria:
Milk, Milk products
Meat, Meat products and salads
Bakery products; particularly
cream-filled pastries and cakes and
sandwich fillings

8. II. Method
2.1. Outbreak investigating and
sampling
- Human samples (stool, vomit),
nasal swab samples, food samples
from leftovers

9. Isolation and phenotypic
characterization of
Staphylococcus Aureus from
human samples
Enumeration of coagulase-positive
staphylococci, phenotypic
characterization of Staphylococci
Aureus and detection of
Staphylococcal enterotoxins in
food samples
FT-IR: Fourier Transform/
Infrared Spectroscopy
Antibiotic
Susceptibility Testing
Genotypic
characterization of
isolates
2.2. Laboratory methods

10. 2.2.1.Isolation and phenotypic
characterization of Staphylococcus Aureus
from human samples
- Four human samples (three stool
samples, one vomit sample) from affected
people
- Three nasal swab samples from
people who handled/produced the ice cream
- Twelve food samples obtained from
leftovers of the variety of food items served
II. Method

11. 2.2.2. Enumeration of coagulase-positive
staphylococci, phenotypic characterization of
Staphylococci Aureus and detection of
Staphylococcal enterotoxins in food samples
- Food samples analyzed and processed for
detection and enumeration of coagulase-positive
staphylococci
- Isolates were identified as S.Aureus by
detection of staphylococcal thermonuclease and
MALDI-TOF mass spectrometry
- Detection of staphylococcal enterotoxins by
VIDAS SET 2 kit
II. Method

12. 2.2.3. FT-IR: Fourier Transform/ Infrared
Spectroscopy
- FT-IR was used for rapid species
identification and comparison of S. Aureus isolates
- Isolates cultivated on sheep blood agar
plates at 37°C for 24 hours. Cells of each isolate
were harvested with a platinum loop and suspended
in 80μL of deionized water. An aliquot of 35μL was
placed in the sample zone of a zinc selenide optical
plate and dried under reduced pressure for 30
minutes to a homogenous solid which was used
directly for FT-IR spectroscopy
II. Method

13. ciproflaxin
clindamycin
daptomycin
Erythromycin
Fosfomycin
fusidic acid
Gentamicin
linezolid
Moxifloxacin
Mupirocin
oxacillin
Penicillin
rifampicin
trimethoprim
tetracycline
teicoplanin
vancomycin
II. Method
2.2.4. Antibiotic Susceptibility Testing
- All isolates were subjected to
susceptibility testing by the broth
microdilution method
- 18 antimicrobials tested:

14. 2.2.5. Genotypic characterization of
isolates
- DNA extracted using lysostaphin to
obtain bacterial lysis. Isolates were
genotypically confirmed as S. Aureus by
means of multiplex-PCR and simultaneous
detection of 23SrDNA
- Strains were further characterized
by spa-typing and MLST(multi-locus-
sequence-typing)
II. Method

15. III. Results
•On 13th of April 2013, 31 people participated at a
christening party that took place at a hotel in
Freiburg, Baden-Wuerttemberg.
•13 developed typical SFP associated symptoms 3-4
hours after lunch
•7 people were either hospitalized or obtained an
ambulatory treatment.
•All of them recovered fully the next day.
•None of the personnel of the hotel presented
symptoms of SFP.
•Participants in the Christening Party did not meet
before the said Party.

16. III. Results
Human S. aureus Isolates:
•From all of the 4 samples taken from cases;
enterotoxigenic S.aureus of spa-type t127,
displaying phenotypic fusidic acid (FUS) resistance
and harbouring the sea gene together with
enterotoxin genes (seh, sek, seq) could be isolated.
All four isolates produced enterotoxin A.
•Enterotoxigenic, seb and sep (sea N315) gene
positive S. aureus of spa-type160 (CC12) was
identified from one out of three employees of the
hotel restaurant. The isolate produced enterotoxin
B.

17. III. Results
Enumeration of CPS, Detection of S. aureus
and SE from Food:
CPS were detected in varying numbers from
all of the five different types of ice-cream
samples:
Vanilla and chocolate: >3.0 x 106 CFU/g
Pistachio: 1.7 x 103 CFU/g
Strawberry: 2.4 x 103 CFU/g
Yoghurt-lemon: 1.7 x 103
The amount of CPS in all food left-overs was
below detection limit which is <102 CFU/g

18. III. Results
•The enterotoxigenic S. aureus isolates of spa-
t127(CC1) detected in 4 of 5 ice-cream samples
(vanilla, chocolate, pistachio and strawberry)
• Non-enterotoxigenic S.aureus of spa-type t084
was detected in yoghurt-lemon ice-cream sample.
•SE could be detected in three of the five different
types of ice-cream:
vanilla and pistachio displaying the highest
amount of SE
chocolate ice-cream once, though at a lower
level than the previous two
strawberry and yoghurt-lemon: no SE detected

19. III. Results
Comparison of Strains from Human and Food
Origin:
•Ten different isolates
 five from humans
 five from food left-overs
almost indistinguishable indicating a common origin
human S. aureus isolates from cases
4 of the 5 different types of ice-cream
Clearly distinct from each other
from human isolate from the employee of the hotel
the one isolated from the yoghurt-lemon ice-cream
were compared by
means of FT-IR and
DNA microarray
analyses

22. IV. Discussion
SE was detectable in three of the five flavors of
the ice-cream (chocolate, vanilla and pistachio)
SE was detectable neither in strawberry and in
yoghurt-lemon ice-cream
Possible causes of the outbreak: remains unknown
1. humans (1 of 3 employees)
2. manufacturing process of ice cream
3. particular common ingredient
4. contaminated equipment was used

23. IV. Discussion
1. Humans: potential contamination source
~20-30% of humans persistently carry S. aureus as a
commensal of the skin and mucosal membranes
~persistent carriage increases risk of developing
endogenous disease
~ ½ of the human isolated was proved to be
enterotoxigenic
•1 of 3 employees: Enterotoxigenic S. aureus
:Methicilin –sensitive S. aureus clones
: found in the nasal cavity
• S. aureus are not persistently present in the nasal cavity
- none of the employees carried the outbreak strain
during the sampling/ investigation

24. IV. Discussion
2. Manufacturing process of the ice-cream harbors
several critical points in terms of biological hazards
Insufficient heating of the pre-mix
Addition of ingredients after any heat
treatment or application of another risk
eliminating processing step

25. IV. Discussion
3. Common Ingredient
They were produced using the same ice-cream
maker device, but different time of production.
2 fruit-containing ice cream: Produced first
3 milk-ice cream: from pasteurized milk
- Both ice cream types contains milk or milk
products in general
-Pathogen may be introduced via contaminated
milk
It was assumed that the milk or milk products were
the cause, but rejected otherwise.

26. IV. Discussion
4. Equipment used
S. aureus – capable of developing biofilms on food-
processing surfaces : pathway leading to cross contamination
of food
•properly cleaned prior to the inspection by the
food and veterinary office
• so no sampling was done in the course if the
outbreak investigation

27. V. Recommendation
•Suitable personal hygienic measurements
to reduce the possibility of introduction of
a severe hazard in the food chain.
•Investigations should include analysis of
nasal samples taken from the people
handling the food, as well as the food
components.
•Subsequent in-depth analyses of strain are
needed
•Formation of an interdisciplinary team

28. Assessment
QUESTIONS FROM THE INTRODUCTION:
1-2. What are the two factors that influence the onset
and severity of the symptoms?
3. Diagnosis of SPF is based on: (Give only one)
QUESTIONS FROM THE METHODS:
4. Give 1 of the 18 antimicrobials tested in the antibiotic
susceptibility testing of the S.Aureus isolates
5. All isolates were subjected to susceptibility testing by
what type of method.

29. Assessment
QUESTIONS FROM RESULTS:
11.
12.
13.
14.
15.
QUESTIONS FROM DISCUSSION:
6-7. What were the flavors of the ice-cream that gave a
negative result of Staphylococcus enterotoxins?
8-9. Give two possible causes of the outbreak.
10. Give at least one recommendations of the
researchers that should be taken into consideration.