Cytotoxic Activity of Cronobacter spp. Isolated from Indonesian Food
Orga nised By:
In Conjunction With:
Progra
e Boo Sponsored By:
Cytotoxic Activi(v of Cronobacter spp. Isolated from Indonesian Food
Siti Nurjanah fJ, Ratih Dewanti-Hariyadi u ,
Sri Estuningsih 3,Maggy T. Suhartono'
'Department o.l Food Science and Technology, Bogor Agricultural University , IP B Darmaga
Campus Bogor 16680. West Java, Indonesia, Tel/ Fax: +622518626725
2SEAFAST Center. Bogor Agricultural Unive;'sity, IPB Darmaga Campus Bogor 16680, West
Java, Indonesia. Tel/Fax: + 6225 i 88629903
3Department ol Veterinary Clinic Reproduction and Pathology, Bogor AgricuLtural
University , IPB Darmaga Campus Bogor i6680, West Java, Indonesia.Tel/ Fax: +62251
*Corresponding author. email: mthenawidjaja@yahoo. com
Abstract
In previous research. twenty isolates ol Cro nobacter spp. we re obtainedfrom local powdered
infant formula. vlieaning food and other dried products.
Molecular showed that nineteen
isolates were confirmed as Cronobacter :·;akazakii, llihile one isolate was Cronobacter
muytjensii. Cro nobacter virulence factors have bee n reported including toxin production and
its cytotoxic activity. However there is limited report ojits haemoly tic activity. The objective
o.lthis study was to evaluate the cytotoxic activity oltl'l entyfood isolates olCronobacter spp.
obtained/i'om indonesia. In vitro analysis olthe cytotoxic aclivity oj"Cronobacter crude toxin
was conducted by MTT (Methyl Thiazol Tetrazolium) Assay which measures its inhibition to
Vero cell growth. Thirteen out ojtwenty isolates weref(JLmd to have positive cytotoxin activity
which varied between 51-8 0% Vero cell deaths.
Key Words: Cronobacter sakazakii. Cronobacter muytjensii. cytotoxic activity, l1fTT Assay.
Vero cell
Introduction
In Indonesia, the presence of Cronobacter spp. !','om food sources have been reported
(Estuningsih et a!. 2006; Meutia et al .2008 ; Dewanti - Hariyadi et a!. 20 I0; Gitapratiwi et a!.
2012 and Hamdani 2012). All of the isolates were obtained from dried food products such as
powdered infant formula , weaning food , starch , sugar, cocoa powder and dried spices.
2
Molecular ide nti fi cati on showed that most of the iso lates were co nfirm ed as C. sakazakii and
onl y one iso late co nfirm ed as C. muy(jensii (G itaprati wi et al. 20 12 and Hamdani 20 12).
Cronobacter spp. (prev iously known as EnteroiJacter sakazakii) has been reported as
an emergi ng path ogen that causes neonatal menin gi ti s and bacteremia (Bowen and Braden
2006) and necrotizing enterocolitis (NEC) (E mami and Mittal 20 12) . Meningi tis and NEC
di seases caused by (' saku::okii in newborn infa nt ha ve been li nkecl tn con sum ptio n of
powdered in fa nt formu la (Acker et al. 200 1).
Measurement of cytotox ic acti vity of tox in ca n be determ ined quan titati ve ly uSlll g
MTT (Methyl Thiazol Te trazolium) assay meth od Ll slng Vero ce ll s and qualitati ve ly by
observin g the cytotox ic effect to alterati on of the ce ll s morph ology. Cro nobacler spp , have
been reported to have cytotox ic acti vity to Vero ce ll s as in dicated by th e cell s damaged
(Pagotto el al. 2003; Grec ili a 2008), The obj ective of this study was to eva lu ate th e cytotox ic
acti vity of twenty foo d iso lates of Cronobacler spp. obta ined fro m Ind ones ia.
Materials and Meth ods
Bacterial stra ins
The bacteri al strain s used in thi s stud y are li sted in Ta bl e I. C. saka::akii and C. muy tjensii
from SEAFAST Center (Bogor Agri cul tural Uni ve rsity Indones ia) culture co ll ecti on we re
prev iously isol ated fro m dri ed food product. Salmonel/a thypimuriul11 ATCe 14028 was used
as a pos iti ve control for bacteri al cytotox icity.
Culture Preparation and Confirmation
All of Cronobacter spp . from stock culture was rev ived by culturin g ove rni ght at 37°C in
Brain Hearl infusion (B HI , OXO ID).
The overni ght cultu re was grown in Druggan-
Forsythe-Iversen (OFI , OXOID) aga r medium to reconfirm th eir viability. Cronobacter spp.
iso lates we re seen as green co loni es in thi s medium .
Vero Cell Culture Preparation and Propagation
Afri can green monkey kidney ep ithelial ce ll s (Vero Ce ll Lin e, ATCC No. CCL-8 1) we re used
in thi s stud y. Ve ro cell s we re maintained in Eagle' s minimum essenti al medium (Oubbeccos
MEM; In vitrogen) suppl emented with 2% Fetal bov ine serum (FBS ; Gibco), 100 U/ml
fungizo ne and I 00
ｾｬ ｧ Ｏ ｭｬ＠
gentamyc in at 37°C in 5 % CO 2 and subcul tured every 3-4 days. For
ex perim ents, Vero ce ll s suspend ed in OME M suppl emented with 10 % FBS we re seeded in
96-we ll ti ssue culture pl ates and kept at 37°C and 5 % CO 2 for 24 h to fo rm a semi co nfluent
monolaye r (5 x 104 ce ll s/we ll ).
Toxin Extraction
Overni ght cultu res of Cronobacter spp. we re inocul ated in to 5 ml TS B (OX OIO) and
incubated at 37°C fo r 14 h.
Bacteri al ce ll s were separated fro m the supernatant by
centrifugati on (3 ,350 x g at 4°C fo r 10 min). Ce ll -f,"ee fil trates co ntainin g th e crud e tox in
were sterili zed by filtrat ion th ro ugh ＰＮＲ
Ｍｾｬｉｮｰ
ｯ ｲ ･ Ｍ ｳ ｩ ｺ･＠
Ill emb ra ne filters.
Cytotoxic assay
Cytotoxic acti vity was determined by MTT assay. All amount of 20
culture filt rates we re mi xed with 80
ｾｬ ｌ＠
ｾｴＱ＠
of eac h bacteri al
of OM EM . One hundred mi cro liters of tox in aliqu ots
we re add ed into semi co nfluent monolaye r of Vero ce ll s culti vated in 96-we ll ti ssue culture
pl ates. The pl ates we re in cubated in 5% CO 2 atmosph ere at 37°C durin g 48 h. After
incubati on, th e supern atants were eliminated and monolayers we re in cubated with MTT sa lts
(5 mg/ml ) Sigma) as much as I 0
ｾｉ＠
per we ll at 37°C l'or 4 h. Fo ll ow ing the incubati on, the
medium containing MTT was removed. Prec ipitated salts (form aza n) produced by acti ve
4
ce ll s, were so lubil ized with ethanol (Merck). Aliquots of th e prod uct we re transferred to each
we ll of the new 96-we ll ti ssue culture pl ate. The absorbance was determin ed with an enzymelinked immun oso rbent assay (E LI SA) reader at A 595 nm . Ex perim ent was perform ed with
three we ll per bacteri al filtrat e. The ce ll viability was expressed as the mean of percentages of
treated and untreated monolaye rs. Cytotox ic pos iti ve resul t was co nfirm ed if there was >5 1%
of ce ll death and cytotox ic negati ve res ult was confirm ed if thi s va lue was less th an th e
cytotox ic acti vity of th e negati ve co ntro l.
Results and Discuss ion
Cytotox ic acti vity of the bacteri al tox in is one indi cator of th e path ogeni city of these
bacteri a. The cytotox ic acti vity of thi s crude tox in was measured by MTT Assay meth od
using Vero ce ll s. Vero ce ll viability was determined by the MTT redu cti on th at detects
mitochondri al metaboli c alteration s as a resul t of th e effect of cytotox ic age nts on viabl e cell
lines.
The prin cipl e of thi s meth od is measuring dehydrogenase enzyme acti vity in
mitochondria of li vin g Vero ce ll by spectroph otometer. Thi s enzyme will conve rt MTT
reagent into fo rm aza n blue crystals. The abso rbance observed at th e di sso lved fo rmazan
crystals indi cates th e number of surviva l ce ll s.
MTT assay \·vas a comm on method used for meas urin g the cytotox ic acti vity of either
vegetati ve ce ll s or tox in s sampl es. Thi s method was appli ed fo r determin ati on cytotox ic
acti vity of Aeromonas spp. (Krzyminska et al. 20 II ), E. coli (G hadir et al . 20 IOa ),
Staphy lococcus spp . and Pseudomonas spp . (G hadir et al . 20 I Ob ). Appli cati on of thi s
method for measurin g the cytotox ic activ ity of tox in sampl e was reported for Clostridium
difficile tox in (Hurtado et al . 2008), Shiga tox in (Sek ino et al. 2004), E. coli crud e tox in
(Gh adir et al. 20 l Oa ), and Aerol11onas spp . crude toxi n (Co ute 2007, Magda et al . 2009).
Couto et al. (2007) determined pos iti ve cytotox ic results if its acti vity result in more th an 50%
Vero cell death . wh iIe Hurtado et al. (2008 ) confi rm ed pos iti ve results if the absorbance value
is small er th an th e abso rban ce value of negati ve co ntrol.
5
No.
I
2
-.
.)
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
Table I Bacterial isolates used in this stud y
Isolate Codes/
Species
Isolation Sources
(from Indonesia)
GenBank Accession
Number"
Cornstarch
Des c7/JF800180 C. sakazakii
Powdered infant formulae
Des b I0/ J F800 I 81 C. sakazakii
YR c3a/JF800 183 C. sakazakii
Weaning food
Weaning food
YR k2a/JF800 187 C. sakazakii
Powdered infant formulae
YR t2a/JF800 182 C. sakazakii
Powdered infant formulae
YR w3 /JF800 185 C. sakazakii
C. sakazakii
FWH bl5
Sugar
Chili powder
FWH d2u
C. sakazakii
C. muy /jensii
FWH dll
Ca raway powder
FWH b6
C. sakazakii
Flour
Pepper powder
FWHd 16/J X5350 18 C. sakazakii
FWH c3
c. sakazakii
Tapioca
Chili povvder
FWH d IIJX5350 16 C. sakazakii
Weaning food
C. sakazakii
EI
Weaning Food
C. sakazakii
E2
Weaning lood
C. sakazakii
E4
Weaning
food
C sakazakii
E6
Weaning food
E7
C. sakazakii
Weaning lood
C. sakazakii
E9
Weaning food
Ell
C. sakazakii
En/erobacter
sakazakii
References
iso lates
ATCC51329
Salmonella thypimurium References iso lates
ATCC 14028
!S/aphy lococcus aureus References iso lates
ATCC 29252
En/erococcus./aecafis
References iso lates
ATCC 19433
Reference
Dewanti-Hariyadi e/ al.
20 10
Meutia e/ al. 2008
Hamdan i 2012
Estunin gs ih el al. 2006
In this current stud y, twenty iso lates of Crol1obacler sakazakii and Cronobac/er
muy/jensii showed differences in cytotoxic activity that was distributed between 32%-80%
(Fig. I) . The cytotoxicity of Salmonella typhimurium to the human ce ll s have been reported
(Burkholder et al. 2009); therefore its activity was used as a maximum standard (100%). The
growth medium (Tryptic Soy Bro/h) without tox in was used for a negative control. The
positive cytotoxic results \vas confirmed if the cytotoxi c was hi gher than negative control. A
number of 13 out of 20 iso lates (65% sampl es) were confirmed as positive cytotoxic isolates
and 7 were grouped as negative iso lates (Fig. I).
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Isolate
o Salmo ne lla Thypim urium o FW H b6
o FW H b15
• FW H c3
o YRc3a
o E1
o E2
o FW H d 16
. FWHd11
O Des c7
O YRw3
Cil E4
• E11
o E6
• FW H d2 u
o YRk2a
o E9
D E sakaza ki i A TC C 51329
o E7
O Des b1 0
o YRt2a
Fig. I. Distribu tion of the cytotox ic activi ty of Crol1obocl er spp.
Five iso lates with hi ghest cytotox icity we re C sakazakii FWH b6, E2, FW Hb 15,
FW Hc3 and FW Hd 16 (80%, 78%, 76 % , 73 % and 70 % respective ly) . Th ese cytotox icity
we re lower th an th e cytotox icity of Shi ga toxi n (Sekin o el af . 2004) . Iso lates with the lowest
acti vity was YRt2a (32 %) .
When co mpared with sources of the iso lates, there was no corre lati on betwee n th e
cytotox ici ty of tox in and the food so urces. When compared with th e geneti c di ve rsity
dendogram based on 16SrRNA gene sequences (Gi tap rati wi et a!. 20 12). there was no clea r
associ ati on betwee n pos iti ve / negati ve of cytotoxic act ivity and th e gen eti c relati onship .
Conclu sion
Twenty iso lates of C. sakazakii and C. l11uyljensii ori gin from foo d ha ve di ffe rent
cytotox ic acti vity of thei r tox in s. A number of 13 out of 20 iso lates (65% sampl es) were
confirm ed as pos iti ve cytotox ic iso lates and 7 we re grouped as negati ve iso lates Five hi ghest
7
cytotoxic activity isolates were C sakazakii FWH b6 , E2 , FWHb I5, FWHc3 and FWHd 16,
while Cronobacler l11uyljensii FWHd II belonging to moderate group.
Acknowledgements
Acknowledgements submitted to the Directorate General of Higher Education
Indonesia for funding this research through Competenc y Grant at 2012, to SEAFAST Center
and Department of Veterinary Clinic Reproduction and Pathology IPB for isolates and Vero
cell collection.
References
Acke r. .I ., Slllet. F. D.. Muy lclerm ans. G .. BouhgateL A.. Naessen s. A. and Lauwers. S. 2001.
Outbreak s 01' nec rotizing enterocolitis assoc iakd with Ei1lero!Jw;ler saka::okii III
powderedillilk formula . .I Cl in Microbiol. 39: 293 297. doi: 10.1128/jc m.39.1.
Couto C.R.A ,. Oliveira S.S, Queiroz M.L.P . and Freitas-Almeida AC. 2007. Interactions of
clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal
epithelial cells. The Society for Applied Microbiology, Lett Appl Microbiol. 45: 405410. doi:10.IIII /j.1472-765X.2007.02199.
Bowen AB and Braden CR. 2006. Invasive Enterobacter sakazak ii diseases In infants.
Emerg Infect Dis. 12: 1185-1189.
Dewant i- Hmiyadi R. Citaprati wi D. Meutia YR. I-iicla) at SH, NLII:janah S. 20 IO. Iso latio n of
Elllero!Jw;/er sulw::o!iii (Crollo!Jw;ler spp.) frol11 powdered ini'allt lo rmul a and olher
dried foods obw ined I'rom Bogor area. In: inlern atiolla I Sem inar 011 Currellt Issues and
Cha llellges ill Fooe! Safety: science-based app roach for food safety mallagement.
October 3-4. IPI3 Internatiollal Conference Center. Sogor. Indonesia. Southeast Asian
Food and Agricultural Scicnce and Techno logy (S t-: AFAST) Center. Bogor. Illdonesia
Estuningsih S. el al. 2006. Cro nobaclericeae in dehydrated powd ered infant formula
manufactured in Indonesia and Malaysia. J Food Protection. 69:3013-3017.
Ghadir S, Aboulwafa M M. and Hassouna NA. 20 lOa. Cytotoxic Activities of Some
Escherichia coli Isolates: Possible Mechanism s and Approaches for Inhibition . J
American Science. 6( I0): 269-283
8
Ghadir S, Aboulwafa M M. and Hassouna NA. 20 lOb . Adherence, Invasion and Cytotoxicity
of Some Bacterial Pathogens . .I American Science
Gitapratiwi D. Dewa nti-Hari yadi R, H idayat SH. 2012 . Cenet ic related ness or CronoiJacler
spp . (/:;nlel'OhuclC'1' ,\'o!w:;ukii) iso lated frolll dri ed rood products in Indonesia. Int Food
Research.!. 19(4): 1745-1749.
Grecilia G. 2008. Cytotoxic testing of Enlerobacler sakazakii iso lated from weaning food on
Vero cell. Skripsi. Bogor: Institut Pertanian Bogor
Hamdani FW.
2012. Eva luation of genetic divers ity of local isolates Crol1obacler spp.
Bogor Agricultural University,
Bogor. Indonesia.
(En terobacler ,Iakazaki!) frol11 dried foods. Th esis.
S. Mokracka .I , Koczura R, Cw iertnia A,and Kaznowski A. 20 I I.
Aerol11ol1as spp.-mediated cell-contact cytotoxicity is associated with the presence of
Krzymillska
type III secretion system. Antonie Van Leeuwenhoek. 10 I (2): 243- 25 1
Magda el al. 2009. Hi gh frequency of hemolytic and cytotoxic activity in Aerol11onas spp.
isolated from clinical, food and environmental in Rio de Janeiro, Brazil. Antonie van
Leeuwenhoek (2009) 96:53- 61. DOl 10.1 007/s I 0482-009-9335-6
Meutia YR. Dewanti-Hariyadi R, Estuningsih S. 2008. Characteriz,ltion of' 16S rRN!\ gene oj'
Enlero/JacleI' suku:;okii iso lated from pO'vvdered inla nt [()rl11ula. In : Abst racts: An nu al
Semi nar of Indonesian /\ssociati on of Food Tec hno log ists. October 14-1 6. Pa lemba ng.
Indonesia. Indonesian Assoc iation of Food Tec hn o log ists, Jakarta. Indonesia
Pagotto FJ. Maria NW. Sabah B. and Jeffrey MF. 2003 . Enterobacler sakazakii Infectifity and
Enterotoxin Production Invitro and Invivo. J Food Protection. 66(3): 370-375.
Sekino el al. 2004. Characterization of a shiga-toxin I-resistant stock of Vero cell. Microbiol
Immunol. 48(5):377-87 . PMID 15215625
In Conjunction With:
Progra
e Boo Sponsored By:
Cytotoxic Activi(v of Cronobacter spp. Isolated from Indonesian Food
Siti Nurjanah fJ, Ratih Dewanti-Hariyadi u ,
Sri Estuningsih 3,Maggy T. Suhartono'
'Department o.l Food Science and Technology, Bogor Agricultural University , IP B Darmaga
Campus Bogor 16680. West Java, Indonesia, Tel/ Fax: +622518626725
2SEAFAST Center. Bogor Agricultural Unive;'sity, IPB Darmaga Campus Bogor 16680, West
Java, Indonesia. Tel/Fax: + 6225 i 88629903
3Department ol Veterinary Clinic Reproduction and Pathology, Bogor AgricuLtural
University , IPB Darmaga Campus Bogor i6680, West Java, Indonesia.Tel/ Fax: +62251
*Corresponding author. email: mthenawidjaja@yahoo. com
Abstract
In previous research. twenty isolates ol Cro nobacter spp. we re obtainedfrom local powdered
infant formula. vlieaning food and other dried products.
Molecular showed that nineteen
isolates were confirmed as Cronobacter :·;akazakii, llihile one isolate was Cronobacter
muytjensii. Cro nobacter virulence factors have bee n reported including toxin production and
its cytotoxic activity. However there is limited report ojits haemoly tic activity. The objective
o.lthis study was to evaluate the cytotoxic activity oltl'l entyfood isolates olCronobacter spp.
obtained/i'om indonesia. In vitro analysis olthe cytotoxic aclivity oj"Cronobacter crude toxin
was conducted by MTT (Methyl Thiazol Tetrazolium) Assay which measures its inhibition to
Vero cell growth. Thirteen out ojtwenty isolates weref(JLmd to have positive cytotoxin activity
which varied between 51-8 0% Vero cell deaths.
Key Words: Cronobacter sakazakii. Cronobacter muytjensii. cytotoxic activity, l1fTT Assay.
Vero cell
Introduction
In Indonesia, the presence of Cronobacter spp. !','om food sources have been reported
(Estuningsih et a!. 2006; Meutia et al .2008 ; Dewanti - Hariyadi et a!. 20 I0; Gitapratiwi et a!.
2012 and Hamdani 2012). All of the isolates were obtained from dried food products such as
powdered infant formula , weaning food , starch , sugar, cocoa powder and dried spices.
2
Molecular ide nti fi cati on showed that most of the iso lates were co nfirm ed as C. sakazakii and
onl y one iso late co nfirm ed as C. muy(jensii (G itaprati wi et al. 20 12 and Hamdani 20 12).
Cronobacter spp. (prev iously known as EnteroiJacter sakazakii) has been reported as
an emergi ng path ogen that causes neonatal menin gi ti s and bacteremia (Bowen and Braden
2006) and necrotizing enterocolitis (NEC) (E mami and Mittal 20 12) . Meningi tis and NEC
di seases caused by (' saku::okii in newborn infa nt ha ve been li nkecl tn con sum ptio n of
powdered in fa nt formu la (Acker et al. 200 1).
Measurement of cytotox ic acti vity of tox in ca n be determ ined quan titati ve ly uSlll g
MTT (Methyl Thiazol Te trazolium) assay meth od Ll slng Vero ce ll s and qualitati ve ly by
observin g the cytotox ic effect to alterati on of the ce ll s morph ology. Cro nobacler spp , have
been reported to have cytotox ic acti vity to Vero ce ll s as in dicated by th e cell s damaged
(Pagotto el al. 2003; Grec ili a 2008), The obj ective of this study was to eva lu ate th e cytotox ic
acti vity of twenty foo d iso lates of Cronobacler spp. obta ined fro m Ind ones ia.
Materials and Meth ods
Bacterial stra ins
The bacteri al strain s used in thi s stud y are li sted in Ta bl e I. C. saka::akii and C. muy tjensii
from SEAFAST Center (Bogor Agri cul tural Uni ve rsity Indones ia) culture co ll ecti on we re
prev iously isol ated fro m dri ed food product. Salmonel/a thypimuriul11 ATCe 14028 was used
as a pos iti ve control for bacteri al cytotox icity.
Culture Preparation and Confirmation
All of Cronobacter spp . from stock culture was rev ived by culturin g ove rni ght at 37°C in
Brain Hearl infusion (B HI , OXO ID).
The overni ght cultu re was grown in Druggan-
Forsythe-Iversen (OFI , OXOID) aga r medium to reconfirm th eir viability. Cronobacter spp.
iso lates we re seen as green co loni es in thi s medium .
Vero Cell Culture Preparation and Propagation
Afri can green monkey kidney ep ithelial ce ll s (Vero Ce ll Lin e, ATCC No. CCL-8 1) we re used
in thi s stud y. Ve ro cell s we re maintained in Eagle' s minimum essenti al medium (Oubbeccos
MEM; In vitrogen) suppl emented with 2% Fetal bov ine serum (FBS ; Gibco), 100 U/ml
fungizo ne and I 00
ｾｬ ｧ Ｏ ｭｬ＠
gentamyc in at 37°C in 5 % CO 2 and subcul tured every 3-4 days. For
ex perim ents, Vero ce ll s suspend ed in OME M suppl emented with 10 % FBS we re seeded in
96-we ll ti ssue culture pl ates and kept at 37°C and 5 % CO 2 for 24 h to fo rm a semi co nfluent
monolaye r (5 x 104 ce ll s/we ll ).
Toxin Extraction
Overni ght cultu res of Cronobacter spp. we re inocul ated in to 5 ml TS B (OX OIO) and
incubated at 37°C fo r 14 h.
Bacteri al ce ll s were separated fro m the supernatant by
centrifugati on (3 ,350 x g at 4°C fo r 10 min). Ce ll -f,"ee fil trates co ntainin g th e crud e tox in
were sterili zed by filtrat ion th ro ugh ＰＮＲ
Ｍｾｬｉｮｰ
ｯ ｲ ･ Ｍ ｳ ｩ ｺ･＠
Ill emb ra ne filters.
Cytotoxic assay
Cytotoxic acti vity was determined by MTT assay. All amount of 20
culture filt rates we re mi xed with 80
ｾｬ ｌ＠
ｾｴＱ＠
of eac h bacteri al
of OM EM . One hundred mi cro liters of tox in aliqu ots
we re add ed into semi co nfluent monolaye r of Vero ce ll s culti vated in 96-we ll ti ssue culture
pl ates. The pl ates we re in cubated in 5% CO 2 atmosph ere at 37°C durin g 48 h. After
incubati on, th e supern atants were eliminated and monolayers we re in cubated with MTT sa lts
(5 mg/ml ) Sigma) as much as I 0
ｾｉ＠
per we ll at 37°C l'or 4 h. Fo ll ow ing the incubati on, the
medium containing MTT was removed. Prec ipitated salts (form aza n) produced by acti ve
4
ce ll s, were so lubil ized with ethanol (Merck). Aliquots of th e prod uct we re transferred to each
we ll of the new 96-we ll ti ssue culture pl ate. The absorbance was determin ed with an enzymelinked immun oso rbent assay (E LI SA) reader at A 595 nm . Ex perim ent was perform ed with
three we ll per bacteri al filtrat e. The ce ll viability was expressed as the mean of percentages of
treated and untreated monolaye rs. Cytotox ic pos iti ve resul t was co nfirm ed if there was >5 1%
of ce ll death and cytotox ic negati ve res ult was confirm ed if thi s va lue was less th an th e
cytotox ic acti vity of th e negati ve co ntro l.
Results and Discuss ion
Cytotox ic acti vity of the bacteri al tox in is one indi cator of th e path ogeni city of these
bacteri a. The cytotox ic acti vity of thi s crude tox in was measured by MTT Assay meth od
using Vero ce ll s. Vero ce ll viability was determined by the MTT redu cti on th at detects
mitochondri al metaboli c alteration s as a resul t of th e effect of cytotox ic age nts on viabl e cell
lines.
The prin cipl e of thi s meth od is measuring dehydrogenase enzyme acti vity in
mitochondria of li vin g Vero ce ll by spectroph otometer. Thi s enzyme will conve rt MTT
reagent into fo rm aza n blue crystals. The abso rbance observed at th e di sso lved fo rmazan
crystals indi cates th e number of surviva l ce ll s.
MTT assay \·vas a comm on method used for meas urin g the cytotox ic acti vity of either
vegetati ve ce ll s or tox in s sampl es. Thi s method was appli ed fo r determin ati on cytotox ic
acti vity of Aeromonas spp. (Krzyminska et al. 20 II ), E. coli (G hadir et al . 20 IOa ),
Staphy lococcus spp . and Pseudomonas spp . (G hadir et al . 20 I Ob ). Appli cati on of thi s
method for measurin g the cytotox ic activ ity of tox in sampl e was reported for Clostridium
difficile tox in (Hurtado et al . 2008), Shiga tox in (Sek ino et al. 2004), E. coli crud e tox in
(Gh adir et al. 20 l Oa ), and Aerol11onas spp . crude toxi n (Co ute 2007, Magda et al . 2009).
Couto et al. (2007) determined pos iti ve cytotox ic results if its acti vity result in more th an 50%
Vero cell death . wh iIe Hurtado et al. (2008 ) confi rm ed pos iti ve results if the absorbance value
is small er th an th e abso rban ce value of negati ve co ntrol.
5
No.
I
2
-.
.)
4
5
6
7
8
9
10
II
12
13
14
15
16
17
18
19
20
21
22
23
24
Table I Bacterial isolates used in this stud y
Isolate Codes/
Species
Isolation Sources
(from Indonesia)
GenBank Accession
Number"
Cornstarch
Des c7/JF800180 C. sakazakii
Powdered infant formulae
Des b I0/ J F800 I 81 C. sakazakii
YR c3a/JF800 183 C. sakazakii
Weaning food
Weaning food
YR k2a/JF800 187 C. sakazakii
Powdered infant formulae
YR t2a/JF800 182 C. sakazakii
Powdered infant formulae
YR w3 /JF800 185 C. sakazakii
C. sakazakii
FWH bl5
Sugar
Chili powder
FWH d2u
C. sakazakii
C. muy /jensii
FWH dll
Ca raway powder
FWH b6
C. sakazakii
Flour
Pepper powder
FWHd 16/J X5350 18 C. sakazakii
FWH c3
c. sakazakii
Tapioca
Chili povvder
FWH d IIJX5350 16 C. sakazakii
Weaning food
C. sakazakii
EI
Weaning Food
C. sakazakii
E2
Weaning lood
C. sakazakii
E4
Weaning
food
C sakazakii
E6
Weaning food
E7
C. sakazakii
Weaning lood
C. sakazakii
E9
Weaning food
Ell
C. sakazakii
En/erobacter
sakazakii
References
iso lates
ATCC51329
Salmonella thypimurium References iso lates
ATCC 14028
!S/aphy lococcus aureus References iso lates
ATCC 29252
En/erococcus./aecafis
References iso lates
ATCC 19433
Reference
Dewanti-Hariyadi e/ al.
20 10
Meutia e/ al. 2008
Hamdan i 2012
Estunin gs ih el al. 2006
In this current stud y, twenty iso lates of Crol1obacler sakazakii and Cronobac/er
muy/jensii showed differences in cytotoxic activity that was distributed between 32%-80%
(Fig. I) . The cytotoxicity of Salmonella typhimurium to the human ce ll s have been reported
(Burkholder et al. 2009); therefore its activity was used as a maximum standard (100%). The
growth medium (Tryptic Soy Bro/h) without tox in was used for a negative control. The
positive cytotoxic results \vas confirmed if the cytotoxi c was hi gher than negative control. A
number of 13 out of 20 iso lates (65% sampl es) were confirmed as positive cytotoxic isolates
and 7 were grouped as negative iso lates (Fig. I).
r
6
120 .00
100 .00
0
.
+'
C1>
>
.!!l
...
C1>
ｾ＠ｾ＠
ｾ｣Ｚ＠
.-
ｾ＠
80.00
'"
0
ｾ＠ u E
60 .00 I
＼ｴＺｾ＠
.!:!
x
40 .00
u
20 .00
.s
.s;.,
000
Isolate
o Salmo ne lla Thypim urium o FW H b6
o FW H b15
• FW H c3
o YRc3a
o E1
o E2
o FW H d 16
. FWHd11
O Des c7
O YRw3
Cil E4
• E11
o E6
• FW H d2 u
o YRk2a
o E9
D E sakaza ki i A TC C 51329
o E7
O Des b1 0
o YRt2a
Fig. I. Distribu tion of the cytotox ic activi ty of Crol1obocl er spp.
Five iso lates with hi ghest cytotox icity we re C sakazakii FWH b6, E2, FW Hb 15,
FW Hc3 and FW Hd 16 (80%, 78%, 76 % , 73 % and 70 % respective ly) . Th ese cytotox icity
we re lower th an th e cytotox icity of Shi ga toxi n (Sekin o el af . 2004) . Iso lates with the lowest
acti vity was YRt2a (32 %) .
When co mpared with sources of the iso lates, there was no corre lati on betwee n th e
cytotox ici ty of tox in and the food so urces. When compared with th e geneti c di ve rsity
dendogram based on 16SrRNA gene sequences (Gi tap rati wi et a!. 20 12). there was no clea r
associ ati on betwee n pos iti ve / negati ve of cytotoxic act ivity and th e gen eti c relati onship .
Conclu sion
Twenty iso lates of C. sakazakii and C. l11uyljensii ori gin from foo d ha ve di ffe rent
cytotox ic acti vity of thei r tox in s. A number of 13 out of 20 iso lates (65% sampl es) were
confirm ed as pos iti ve cytotox ic iso lates and 7 we re grouped as negati ve iso lates Five hi ghest
7
cytotoxic activity isolates were C sakazakii FWH b6 , E2 , FWHb I5, FWHc3 and FWHd 16,
while Cronobacler l11uyljensii FWHd II belonging to moderate group.
Acknowledgements
Acknowledgements submitted to the Directorate General of Higher Education
Indonesia for funding this research through Competenc y Grant at 2012, to SEAFAST Center
and Department of Veterinary Clinic Reproduction and Pathology IPB for isolates and Vero
cell collection.
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