Enterobacter sakazakii

Kang, Dong-Hyun, Ph.D.

Assistant Professor
Food Science and Human Nutrition
Washington State University

History
Name Enterobacter sakazakii proposed in1980

By

DNA-DNA hybridization (Izard et al)

Biochemical characteristics (Muytjens et al)
Yellow- pigment colony (Farmer et al)

General properties

Member of the family Enterobacteriaceae

Motile, gram-negative rod
Oxidase negative
Reservoir and mode of transmission is unknown
Neonatal infection is severe,
case-fatality rates, 40-80%

Thermotolerant microorganism

General properties
difference from other Enterobacter

Yellow pigment production on TSA

Non-fermentation of D-sorbitol
Delayed production of DNAase (7days)
Absence of phosphoamidase
Production of -glucosidase
& Tween 80 esterase

General properties
Identification of enteric bacteria

General properties
Biochemical differentiation of Enterobacter species
Reactionb

Test

E. sakazakii E. cloacae E. aerogenes E. agglomerans E. gergoviae

Lysine decarboxylase

Arginine dihydrolase

Ornithine decarboxylase

KCN, growth in

sucrose

(+)

dulcitol

(-)

(-)

adonitol

(-)

raffinose

D-sorbitol

x-methyl-D-glucoside

(+)

D-arabitol

(-)

(+)

Fermentation
of:

Yellow pigment
a
b

Adapted from Farmer and Kelly, 1992.

Where + : 90-100% positive; (+) : 75-89% positive; v: 25-74% positive; (-): 10-24% positive; -: 0-9% positive

Source from FDA

General properties
Entrobacter sakazakii colony

General properties
Electron micrograph of Entrobactor sakazakii

Reported infection cases

disease involved

Meningitis
infectious disease characterized by inflammation of the meninges
(the tissues that surround the brain or spinal cord)
http://www.nlm.nih.gov/medlineplus/tutorials/meningitis/nr219101.html

Sepsis

the presence of pus-forming bacteria or their toxins in the blood or tissues

http://www.sepsis.com/sepsis_cascade/cascade2.jsp

Seizure

a sudden occurrence (or recurrence) of a disease

Bacteremia
transient presence of bacteria in the blood
Brain cyst
closed sac that develops abnormally in brain

Reported infection cases

by researcher
Author

Course

Outcome

Uremenyi and
Franklin (1961)

Sepsis, meningitis, seizures

Joker et al.

Meningitis, seizures, brain cysts

Death in 48 h
Acute collapse and death
Survived, neurologic deficits

Monroe and Tift

Bacteremia

Survived

Adamson

Meningitis, seizures

Survived, no follow-up

Kleiman et al.

Meningitis, brain cysts

Mental retardation

Muytjens et al.

Meningitis, seizures
Meningitis, seizures

Death in 4 days
Death in 6 days

Meningitis, seizures

severe retardation

Meningitis, ventriculitis

Death in 4 days

Meningitis
Meningitis
Meningitis
Meningitis
Meningitis, seizure

Death in 2 days
Death in 3 days
Death in 4 days
Survived, retarded
Survived, right hemiparesis

Meningitis, seizures, brain cysts

Survived with severe deficits

Meningitis, seizures, brain cysts,

hydroephalus

Survived with severe deficits

Noqvi et al.
Willis and
Robinson

Reported infection cases

by worldwide
Location

Number of cases (death)

I. Sporadic cases
Denmark
Georgia
Oklahoma
Indiana
Greece
Missouri
Maryland
Ohio
England
Netherland
Greece
Massachusetts
Tennessee
Iceland

1(1)
1(0)
1(0)
1(0)
1(0)
(1)
1(0)
1(0)
2(2)
8(6)
11(4)
2(1)
4(0)
3(1)

Ontario

2(?)

Source implicated

Incubator
Birth canal

Dried-infant formula
Dried-infant formula

Thermal resistance

Thermal resistance

Thermotolerant organism
D-value in reconstituted dried-infant formula (min)
Temperature (oC)
Strains

52

54

56

58

60

Clinical

54.76

36.72

10.91

5.45

3.06

Food

54.82

18.57

9.75

3.44

2.15

Thermal resistance
compare with other microorganism
Organism
Aeromonas hydrophilia
Campylobacter jejuni
E. coli
E. sakazakii
Klebsiella pneumoniae
Salmonella muenster
Salmonella senftenberg
Salmonella typhimurium
Shigella dysenteriae
Yersinia enterocolitica

Source from FDA

Heating menstruum

D value (72oC, S)

Raw milk
Skim milk
Whole milk
Human milk
Infant formula
Human milk
Whole milk
Whole milk
Whole milk
Whole milk
Whole milk
Whole milk
Whole milk

0.01476
0.07033
0.15669
0.01443
1.30088
0.00008
0.07214
0.08417
0.22000
0.12125
0.13045
0.46086
0.91208

Thermal resistance

E. sakazakii is one of the most thermotolerant members

(source from 2004 FDA)

Thermal resistance
Comparison of D58C-Values for Different Enterobacteriaceae
E. sakazakii 607

600

D-value (sec)

500
400
300
200
100
0

E. coli O157:H7
E. sakazaki N&Fpooled
K. pneuomoniae
Salmonella Hartford
E. coli
E. aerogenes
E. sakazakii 51329

Thermal resistance
Thermal Death Time Curves for 2 E. sakazakii Heated at 58C

Survivors [Log(CFU/mL)]

9
8

D = 591.9 sec

7
6
5
4

D = 30.5 sec

3
2
0

500

1000

1500

Heating Time (sec)

2000

2500

Thermal resistance
Distribution of D58C-values for 12 E. sakazakii strains

# of Strains

6
5
4
3
2
1
0

0-100 100- 200- 300- 400- 500200 300 400 500 600

D-value (sec)

How they infect human ?

They have relatively short lag time and generation time
Organisms

Medium

E. sakazakii

Infant formula

E. coli
E. aerogens

BHI
Whole milk

Temp
(oC)

Generation
time (h)

Lag time
(h)

23

0.67

2.76

10

4.64

32.8

23

0.74

5.56

10

6.40

47.0

23

0.80

NR

10

4.20

NR

1 CFU/mL in reconstituted milk reach 107/100ml(10h, RT)

35, 37oC is more faster, so, proper storage is important

How they infect human ?

How they infect human ?

Dried-infant formula : 0.36-66.0 CFU/100g, E. sakazakii
In Canada
Company
A
B
C
D
E
Total

Positive samples (%)

3/24 (12)
2/24 (8)
0/24 (0)
1/24 (4)
2/24 (8)
8/120 (6.7)

How they infect human ?

Resistance to Dehydration

S u rv iv in g P o p u la tio n
[L o g (C F U / m L )]

6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
0

Storage Time (mon)

10

How they infect human ?

Temperature Decline During Rehydration of Infant Formula

Temperature (C)

100
90
80
70
60
50
40
30
20
0

10

Sample Time (min)

FDA, 2004

How they infect human ?

90
80

Rehydration of Dried Infant Formula

70
60
50
40
30
20
0

10

Sample Time (min)

6
Survivors
[Log(CFU/ml)]

Temperature (C)

100

Lower Limit of
Detection

4
3
2
1
0

23

50

60

70

80

Water Temperature (C)

90

100

 University of Wales, UK, Joanne. et al (2003)

Stomoxys calcitrans (blood-sucking insects on cattle)
- E. sakazakii is isolated in midgut
insect

cow

milk

House fly (Musca domestica)

Insects are major environmental reservoir ?

Enterotoxin &
Infectivity studies

Enterotoxin & Infectivity studies

4 strains positive for enterotoxin production (18 strains)
(3 clinical and 1 food)

injections lethal at 108 CFU per mouse

(within 3 days of dosing)

per oral route 2 strains caused death

(1 clinical and 1 food)

Enterotoxin & Infectivity studies

Mechanisms of Toxicity of Enterobacter sakazakii

(Un-known !!)

Development of differential medium

Development of differential medium

FDA recommended method (2002)
1 100g, 10g, 1g dissolve in 45oC distilled water (o/n)
2 Remove 10 ml and mix with 90 ml EE broth (o/n, 36oC)
3 Spreading or direct streaking onto VRBG agar (o/n, 36oC)
4 Pick presumptive colonies and streaking onto TSA (48-72 h, 25oC)
5 Select yellow colonies and confirm API 20E kit

6 Oxidase test for positive identification

FDA recommended method need some

improvement.

Development of differential medium

problem 1

Spreading or direct streaking onto VRBG agar (o/n, 36oC)

not sufficiently selective

other microorganisms can grow
and produce characteristic purple
colonies surrounded by a purple
halo of precipitated bile acids
Impossible to differentiate E.
sakazakii from other bacteria

Development of differential medium

problem 2

Pick presumptive colonies and streaking onto TSA (48-72 h, 25oC)

Too long time needed

many yellow-pigmented
Enterobacteriaceae
- E. hermanii,
- E. vulneris,

Development of differential medium

selection marker strategy
(by Dr. Kangs Laboratory)

-glucosidase
(unique E. sakazakii)

4-MU--D-glc.
Fluorescent
colonies

NO
H2S production
Ferric, sulphate

Black colonies

Development of differential medium

Visual Inspection Criteria

Hydrogen sulfide
production

+ (black)
+
-glucosidase
lucosidase

- (colorless)
E. sakazakii

(fluorescent)

Salmonella
Citrobacter
Edwardsiella
Proteus

Klebsiella
E. coli
E. coli O157:H7
E. cloacae

Development of differential medium

(Method)
Basal medium selection
Optimization of component
Optimization of incubation temperature and time
Verification and OK medium

OK medium

Development of differential medium

fluorescent intensity

Pseudomonas aeroginosa

E. sakazakii

4-MU--D-glucoside containing medium, 360 nm

Development of differential medium

Definition
Background noise is a ratio of
Background noise =

Fluorescent colonies
Total colonies

In absence of E. sakazakii strains (Other 16 strain culture cocktails)

Culture cocktails
- E. sakazakii 4 strain cultures
- other 16 strain cultures

Development of differential medium

Trypton Bile
Background
noise

TSA

Inoculation of
Background
Microorganis
ms (16
cultures)
on three
different basal
medium

VRBG

Reducing background noise is most important

Development of differential medium

basal medium selection

Reduction of microbial
flora (%)b
Mediaa

Background
noise (%)c

E. sakazakii
cocktail

16 strain
cocktail

16 strain
cocktail

VRBG agar

69.7

72.2

52.4b

Trytone bile agar

92.2

91.3

1.0a

TSA

100.0

100.0

43.5b

Tryptone bile agar is selected

Development of differential medium

basal medium composition

Formula

gm/liter

Tryptone

20.0

Bile salt No. 3

1.5

Agar

15.0

pH 7.2 0.2

Nitrogen source and concentration can be changed

Development of differential medium

Selection of nitrogen source and optimization

Nitrogen sourcesa

Background
noise (%)b

Tryptone
Concentrations

Background
noise (%)

Bacto peptone

56.352.48c

40 g/l

0.370.65a

Tryptone

0.680.59a

20 g/l

0.620.54a

Proteose peptone I

11.603.38b

10 g/l

10.034.01b

Proteose peptone II

1.380.74a

5 g/l

48.854.28c

Proteose peptone III

72.6610.19d

2.5 g/l

77.3312.20d

Tryptone and 20g/l concentration is selected

Development of differential medium

Selection of nitrogen source and optimization

Nitrogen sourcesa

Background
noise (%)b

Tryptone
Concentrations

Background
noise (%)

Bacto peptone

56.352.48c

40 g/l

0.370.65a

Tryptone

0.680.59a

20 g/l

0.620.54a

Proteose peptone I

11.603.38b

10 g/l

10.034.01b

Proteose peptone II

1.380.74a

5 g/l

48.854.28c

Proteose peptone III

72.6610.19d

2.5 g/l

77.3312.20d

Tryptone and 20g/l concentration is selected

Development of differential medium

The effect of incubation time and temperature

No. of
fluorescent
colonies

No. of total
colonies

Fluorescent
colonies/ total
colonies (%)

Time
(h)

30oC

37oC

30oC

37oC

30oC

37oC

18

0.67

5.33

77.33

87.67

0.87

6.08

24

2.33

7.33

78.67

88.67

2.96

8.27

48

5.33

7.33

84.33

89.33

6.32

8.21

37oC and 24 h incubation time is selected

Development of differential medium

OK medium
Formula

gm/liter

Tryptone

20.0

Bile salt No. 3

1.5

Sodium thiosulphate

1.0

Ferric citrate

1.0

4-MU--D-glc

0.05

Agar

15.0

pH 7.2 0.2

Development of differential medium

Verification of OK medium

No. of fluorescent
colonies
Verified
Temp.(oC) Examined
(%)

No. of non-fluorescent
colonies
Verified
Examined
(%)

30

24

21 (100.00)

22

0 (0.00)

37

24

23 (100.00)

22

0 (0.00)

Verification of OK medium

Development of differential medium

(FDA Recommended VRBG)

Development of differential medium

(OK medium)

Development of differential medium

(OK medium under UV)
E. sakazakii

Summary

E. sakazakii is a pathogenic microorganism

Natural habitat is not known yet
Pathogenic mechanisms is not known yet
Found in infant formula
Thermo-tolerant
FDA recommended method need some improvement
OK media development (by WSU, Dept. FSHN)
There are much more works to be done..

Questions ?