Cytotoxic Activity of Food Isolates ofCronobacter sakazakii and Cronobacter muytjensii

from Indonesia

Supervised by:Prof. Maggy T. Suhartono

Dr. Ratih DewantiDr. Sri Estuningsih

Siti Nurjanah

Department of Food Science and Technology andSEAFAST CenterBogor Agricultural University, Indonesia

• Introduction• Methodology• Result and Discussion• Conclusion

Outline

About C. sakazakii and C. muytjensii:Spesies in Genus Cronobacter spp. (Enterobacter sakazakii)Problem in several countriesAs contaminant in infant formulae, weaning food and dried foodCaused meningitis and necrosis for “unhealthy infant”

Introduction

Vero cells as a model cell line

Pathogenicity

Our previous research: collected 33 isolates from foods

• MTT Assay method using animal culture cell line : •measured the absorbance of the living cell

Using one of indicator :Toxin production and

its cytotoxic activity

Introduction

Vero cell• Cell line from monkey kidney

alteration of morphology caused by toxin

(died cell)Normal morphology

(living cell)

Polygonal shape

Rounded Shape and shrunken

•Cytotoxic activity : calculated by comparing number of living cells and total of cell control (%)

Introduction

The objective of this study were:• to assess the cytotoxic activity of twenty

food isolates of Cronobacter spp. obtained from Indonesia

• to learn the cytotoxic effect to morphology of Vero cell

Objectives

2. Crude toxin (supernatant free cell ) extraction, was sterilized by filtration

Methodology

1. Culture and Vero Cell preparation

20Total

1FWHb15Sugar

4FWHd2u, FWHd11, FWHd16

Spices

8YRc3a, YRsnkn, E1, E2, E4, E6, E7, E9, E11

Weaning Food

3desb10,YRt2a,YRw3Powdered Infant Formulae

Dewanti-Hariyadi et al. 2010 Meutia et al. 2008 Hamdani 2012Estuningsih et al. 2006

4desc13, desc7, FWHb6, FWHc3

Starch/ flour

ReferencesNumber of Isolates

Name of IsolatesSources

3. Cytotoxic evaluation by MTT Assay• Carried out in tissue culture plate 96 wells• Measured the Abs by ELISA reader

% Cytotoxicity = 1- A595 treated cell x 100%A595 cell control

monolayer Vero cell

Methodology

24 h 40 h 48 h

Staining the Vero cell by Haematoxylin-Eosin

4. Cytotoxic effect vs age of cultures

Methodology

Result and Discussion

0.00

20.00

40.00

60.00

80.00

100.00

120.00

Isolates

Cyt

otox

ic a

ctiv

ity (%

rela

tive

to

S. T

hypi

mur

ium

)

Salmonella Thypimurium FWH b6 E2 FWH b15FWH c3 FWH d16 YRc3a E1FWH d11 Des c7 YRw3 Des b10E4 E11 E6 FWH d2uYRk2a E sakazakii ATCC E7 E9YRt2a

NegativeCytotoxic

PositiveCytotoxic

50%

E413Desb1012YRw311Desc710FWHd119E18YRc3a7FWHd16FWHd16    : 70%5FWHc3       : 73%4FWHb15    : 76%3E2               : 78%2FWHb6     : 80%1

Positive Isolates

E1114

YRt2a20E919E718YRk2a17FWHd2u16E615

Negative Isolates

65% of 20 isolates have

cytotoxicactivity

Result and Discussion

Effect of toxin of C. Sakazakii FWHd16

24 h 48 h40 hage of

culture :

Increasing cytotoxic effect by increasing age of bacterial culture

DamagedVero cells

Result and Discussion

24 h 40 h 48 h

Effect of toxin of C. Sakazakii FWHc3

Increasing cytotoxic effect by increasing age of bacterial culture

age of culture:

DamagedVero cells

Result and Discussion

Normal Vero cell :Complete of cytoplasm

and clear nucleus

Damaged Vero cell : Darkening of Nucleus

EOSIN staining Result

Result and Discussion

• 13 out of 20 isolates positive for cytotoxicactivity with varying between 50-80%

• The cytotoxic effect increase by increasing age of bacterial culture.

• Eosin staining of injured Vero cells showed the loss of cytoplasm and condensation of its nuclei

Conclusions

Funding research• This research was supported by funding of

Competency Grant, Directorate General of Higher Education of Indonesia

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