Staphylococcus aureus food-poisoning outbreak associated

with the consumption of ice-cream

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.

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)

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

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

I. IntroductionIndividual 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

I. Introduction

Foods that favor growth of bacteria:

Milk, Milk productsMeat, Meat products and saladsBakery products; particularly

cream-filled pastries and cakes and sandwich fillings

II. Method

2.1. Outbreak investigating and sampling

- Human samples (stool, vomit), nasal swab samples, food samples from leftovers

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

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

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

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

ciproflaxin clindamycin daptomycin ErythromycinFosfomycinfusidic acid

Gentamicin linezolidMoxifloxacinMupirocinoxacillinPenicillin

rifampicin trimethoprim tetracyclineteicoplaninvancomycin

II. Method2.2.4. Antibiotic Susceptibility Testing

- All isolates were subjected to susceptibility testing by the broth microdilution method

- 18 antimicrobials tested:

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

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.

III. ResultsHuman 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.

III. ResultsEnumeration 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/gPistachio: 1.7 x 103 CFU/gStrawberry: 2.4 x 103 CFU/gYoghurt-lemon: 1.7 x 103

The amount of CPS in all food left-overs was below detection limit which is <102 CFU/g

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

III. ResultsComparison of Strains from Human and Food Origin:

•Ten different isolates five from humans five from food left-overs

almost indistinguishable indicating a common originhuman S. aureus isolates from cases4 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

IV. DiscussionSE 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 unknown1. humans (1 of 3 employees)2. manufacturing process of ice cream3. particular common ingredient 4. contaminated equipment was used

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

IV. Discussion

2. Manufacturing process of the ice-cream harbors several critical points in terms of biological hazards

Insufficient heating of the pre-mixAddition of ingredients after any heat

treatment or application of another risk eliminating processing step

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 first3 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.

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

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

AssessmentQUESTIONS 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.

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.