CRONOBACTER IN FOOD AND STRATEGIES FOR …civersen.tripod.com/beuchat.pdf · Larry R. Beuchat...

CRONOBACTER IN FOOD AND STRATEGIES FOR CONTROL

Larry R. Beuchat

Center for Food Safety, University of Georgia

An International Meeting on Cronobacter (Enterobacter sakazakii)University College Dublin, Ireland

22 - 23 January 2009

Overview

� Presence of Cronobacter in foods and beverages

� Survival and growth in dry and reconstituted infant formula and cereal

� Biofilm formation and resistance to disinfectants

� Survival and growth on fresh fruits and vegetables, and efficacy of sanitizers

Foods and beverages other than powdered infant

formula, powdered milk and water from which

Cronobacter has been isolated

Animal origin: Beef, pork, poultry, sausage, camel, fish, shellfish, shrimp, cheese, eggs

Plant origin: Cereal, flour, meal, grains, oilseeds, legumes, nuts, bakery products, herbs and spices, tea, lettuce, tomatoes, seed sprouts and other fresh produce, tofu, tempé, fermented beverages (attiéké, sobia, sous)

Survival in Powdered Infant Formula

� Ten-strain inoculum

► Five clinical isolates► Four food isolates► One environmental isolate

� Six powdered infant formulas► Four milk-based formulas

► Two soy-based formulas

� aw range: 0.25 - 0.86► aw 0.25 - 0.50 (10-strain inoculum)► aw 0.43 - 0.86 (1 clinical isolate and 1 food isolate)

� Powdered milk (aw 0.23) was used as a carrier for inoculum

� Two inoculum populations► 0.80 log CFU/g (6.3 CFU/g)► 4.69 - 4.86 log CFU/g

� Three storage temperatures (4, 21, and 30ºC)

� Five storage times (1, 3, 6, 9, and 12 months)

� Three replicate experiments

5.0

0 0.5 1 3 6 9 12

E. sakazakii

(log C

FU

/g)

Storage time (months)

Ι

○ TSAP

□ VRBGP agar

6.0

7.0

5.0

0 0.5 1 3 6 9 12

E. sakazakii

(log C

FU

/g)


Ι

○ TSAP

□ VRBGP agar

6.0

7.0

C.

saka

za

kii

(lo

g C

FU

/g)

5

4

3

2

1

0

4°C

30°C21°C

E. sakazakii (log C

FU

/g)


0 1 3 6 9 12 0 1 3 6 9 12

I

5

4

3

2

1

0

○ aw 0.26

□ aw 0.34

∆ aw 0.49

Formula A5

4

3

2

1

0

4°C

30°C21°C

E. sakazakii (log C

FU

/g)


0 1 3 6 9 12 0 1 3 6 9 12

I

5

4

3

2

1

0

○ aw 0.26

□ aw 0.34

∆ aw 0.49

Formula A

C.

saka

za

kii

(lo

g C

FU

/g)

Milk-based formula

0 1 3 6 9 12 0 1 3 6 9 12

4°C

30°C21°C


I

5

4

3

2

1

0

5

4

3

2

1

0

E. sakazakii

(log C

FU

/g)

○ aw 0.30

□ aw 0.32

∆ aw 0.44

Formula C

0 1 3 6 9 12 0 1 3 6 9 12

4°C

30°C21°C


I

5

4

3

2

1

0

5

4

3

2

1

0

E. sakazakii

(log C

FU

/g)

○ aw 0.30

□ aw 0.32

∆ aw 0.44

Formula CC

. saka

za

kii

(lo

g C

FU

/g)

Soy-based formula

Detection (by enrichment) of C. sakazakii in a milk-based powdered

infant formula initially containing 0.80 log CFU/g (6.3 CFU/g)

a Numbers indicate positive 10-g samples out of two tested (0 month) or three tested (1 - 12 months)

101

100

111

000

010

142130

0.50

110

010

100

000

110

142130

0.33

111

200

000

100

121

142130

0.27

1296310temp.(ºC)aw

Storage time (months)aStorage

8

7

6

5

4

3

2

1

0

0 1 2 4 12 24 0 1 2 4 12 24

4°C

30°C21°C

E.

sa

ka

zakii (

log

CF

U/g

)

Storage time (weeks)

8

7

6

5

4

3

2

1

0

○ aw 0.52

□ aw 0.75

aw 0.81

aw 0.86

Formula A, strain 28558

7

6

5

4

3

2

1

0

0 1 2 4 12 24 0 1 2 4 12 24

4°C

30°C21°C

E.

sa

ka

zakii (

log

CF

U/g

)


8

7

6

5

4

3

2

1

0

○ aw 0.52

□ aw 0.75

aw 0.81

aw 0.86

Formula A, strain 2855C

. saka

za

kii

(lo

g C

FU

/g)Milk-based formula (strain 2855)

○ aw 0.43

□ aw 0.50

aw 0.65

aw 0.81

0 1 2 4 12 24 0 1 2 4 12 24

E. sakazakii (lo

g C

FU

/g)


4°C

21°C 30°C

8

7

6

5

4

3

2

1

0

8

7

6

5

4

3

2

1

0


○ aw 0.43

□ aw 0.50

aw 0.65

aw 0.81

0 1 2 4 12 24 0 1 2 4 12 24

E. sakazakii (lo

g C

FU

/g)


4°C

21°C 30°C

8

7

6

5

4

3

2

1

0

8

7

6

5

4

3

2

1

0


C.

saka

za

kii

(lo

g C

FU

/g)


Milk-based formula (strain 3396)

C.

saka

za

kii

(lo

g C

FU

/g)


Observations

� Rate of death of C. sakazakii in powdered infant formula increases with increased aw and temperature

� Rate of death of specific strains is unaffected by the composition of formulas but rate of death in a given formula may differ among strains

� When initially present in formulas (aw 0.25 - 0.50) at populations as low as 6.3 CFU/g, C. sakazakii can survive for at least 12 months

Survival in Dry Infant Cereal

� Ten-strain inoculum (all cereals)

� Four cereals

► Rice, barley, mixed grain, and oatmeal

� aw range

► 0.30 - 0.69 (rice cereal)► 0.63 - 0.83 (rice, barley, mixed grain, and oatmeal

cereals)

� Rice cereal (aw 0.23) was used as a carrier for the inoculum

� Inoculum populations► Rice cereal at aw 0.30 - 0.69 (0.31 and 5.03 log CFU/g)► Rice, barley, mixed grain, and oatmeal at aw 0.63 - 0.83

(5.39 log CFU/g)

� Three storage temperatures (4, 21, and 30ºC)

� Storage times (1, 3, 6, 9, and 12 months)► Low aw: 1, 3, 6, 9, and 12 months

► High aw: 2, 4, 6, 8, 10, 14, and 24 weeks


Rice cereal

○ aw 0.30

□ aw 0.45

aw 0.68

30°C

4°C

21°C

E.

sa

ka

zakii

(log

CF

U/g

)

0 1 3 6 9 12


0 1 3 6 9 12

6

5

4

3

2

1

0

6

5

4

3

2

1

0

30°C

4°C

21°C

E.

sa

ka

zakii

(log

CF

U/g

)

0 1 3 6 9 12


0 1 3 6 9 12

6

5

4

3

2

1

0

6

5

4

3

2

1

0

C.

saka

za

kii

(lo

g C

FU

/g)


Rice

Barley

Mixed grain

Oatmeal

0 2 4 6 8 14 24


E.

sa

ka

za

kii

(lo

g C

FU

/g)

6

5

4

3

2

1

0

6

5

4

3

2

1

0

0 2 4 6 8 14 24

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83

4°C

Rice

Barley

Mixed grain

Oatmeal

0 2 4 6 8 14 24


E.

sa

ka

za

kii

(lo

g C

FU

/g)

6

5

4

3

2

1

0

6

5

4

3

2

1

0

0 2 4 6 8 14 24

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83

4°C

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83aw 0.82 - 0.83

4°C

C.

saka

za

kii

(lo

g C

FU

/g)

Rice

Barley

Mixed grain

Oatmeal

0 2 4 6 8 14 24


E. sakazakii

(log C

FU

/g)

6

5

4

3

2

1

0

6

5

4

3

2

1

0

0 2 4 6 8 14 24

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83

21°CRice

Barley

Mixed grain

Oatmeal

0 2 4 6 8 14 24


E. sakazakii

(log C

FU

/g)

6

5

4

3

2

1

0

6

5

4

3

2

1

0

0 2 4 6 8 14 24

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83

21°C

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83aw 0.82 - 0.83

21°C

C.

saka

za

kii

(lo

g C

FU

/g)

Rice

Barley

Mixed grain

Oatmeal

0 2 4 6 8 10 14


E. sakazakii

(log C

FU

/g)

6

5

4

3

2

1

0

6

5

4

3

2

1

0

0 2 4 6 8 10 14

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83

30°C

Rice

Barley

Mixed grain

Oatmeal

0 2 4 6 8 10 14


E. sakazakii

(log C

FU

/g)

6

5

4

3

2

1

0

6

5

4

3

2

1

0

0 2 4 6 8 10 14

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83

30°C

○ aw 0.63 - 0.66

□ aw 0.76

aw 0.82 - 0.83aw 0.82 - 0.83

30°C

C.

saka

za

kii

(lo

g C

FU

/g)


Detection (by enrichment) of C. sakazakii in infant rice cereal initially containing 0.31 log CFU/g (2 CFU/g)

aNumbers indicate positive samples out of three tested

210

220

210

221

142130

0.69

000

222

122

022

242130

0.46

202

223

221

112

242130

0.30

96310temp.(ºC)aw

Storage time (months)aStorage

Observations

� At an initial population of 0.31 log CFU/g (2 CFU/g), C.

sakazakii was detected in infant rice cereal (aw 0.30 -

0.69) stored for up to 9 months, depending on storage temperature

� Rate of death increases with increased aw and storage temperature

� Rate of death was more rapid in rice, barley, mixed grain, and oatmeal cereals (aw 0.63 - 0.83) than in rice cereal at aw 0.30 - 0.69)

� Rate of death was unaffected by the composition of the cereals at aw 0.63 - 0.83

Survival and Growth in Reconstituted Infant Formula


� Six powdered infant formulas (four milk-based and two soy-based)

� One reconstitution liquid (water)

� Two inoculum populations► Low: 0.02 CFU/ml (13 CFU/100 g, dry wt)► High: 0.53 CFU/ml (413 CFU/100 g, dry wt)

� Four storage temperatures (4, 12, 21, and 30ºC)

� Six storage times (4, 8, 12, 24, 48, and 72 h)


109876543210-1-2

0 4 8 12 24 48 72 0 4 8 12 24 48 72

Formula A, low inoculum

Formula C, high inoculum

Formula B, high inoculum

Formula A, high inoculum

Formula C, low inoculum

Formula B, low inoculum

E. sakazakii (lo

g C

FU

/ml)

Storage time (h)

109876543210-1-2

109876543210-1-2

○ 4°C□ 12°C∆ 21°C⃟ 30°C

109876543210-1-2

0 4 8 12 24 48 72 0 4 8 12 24 48 72

Formula A, low inoculum

Formula C, high inoculum

Formula B, high inoculum

Formula A, high inoculum

Formula C, low inoculum

Formula B, low inoculum

E. sakazakii (lo

g C

FU

/ml)

Storage time (h)

109876543210-1-2

109876543210-1-2

○ 4°C□ 12°C∆ 21°C⃟ 30°C

C. saka

zakii

(log C

FU

/ml)

Observations

� Powdered infant formula reconstituted with water is an excellent medium for growth of C. sakazakii

� Growth occurs in reconstituted formulas held at 12, 21, and 30ºC, but not at 4ºC

� Growth is unaffected by composition of the formula

Survival and Growth in Reconstituted Infant Cereal


� Three cereals► Rice, oatmeal, and rice with mixed fruit

� Two inoculum populations► Low: 0.005 CFU/ml (3.6 CFU/100 g, dry wt)► High: 0.52 CFU/ml (36 CFU/100 g, dry wt)

� Three reconstitution liquids► Water, milk, and apple juice

� Four storage temperatures (4, 12, 21, and 30ºC)

� Six storage times (4, 8, 12, 24, 48, and 72 h)


0 4 8 12 24 48 72 0 4 8 12 24 48 72

E. sakazakii(log C

FU/m

l)

Incubation time (h)

Rice, low inoculum Rice, high inoculum

Oatmeal, low inoculum Oatmeal, high inoculum

Rice with mixed fruit, low inoculum Rice with mixed fruit, high inoculum9

8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

○ 12°C

30°C

□ 21°C

0 4 8 12 24 48 72 0 4 8 12 24 48 72

E. sakazakii(log C

FU/m

l)

Incubation time (h)




8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

0 4 8 12 24 48 72 0 4 8 12 24 48 72

E. sakazakii(log C

FU/m

l)

Incubation time (h)




8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

○ 12°C

30°C

□ 21°C○ 12°C

30°C

□ 21°C

30°C30°C

□ 21°C

C. saka

zakii

(log C

FU

/ml)

Incubation time (h)

0 4 8 12 24 48 72 0 4 8 12 24 48 72

E. sakazakii

(log C

FU

/ml)

Incubation time (h)




8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

○ 12°C

□ 21°C

30°C

0 4 8 12 24 48 72 0 4 8 12 24 48 72

E. sakazakii

(log C

FU

/ml)

Incubation time (h)




8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

9

8

7

6

5

4

3

2

1

0

-1

-2

-3

○ 12°C

□ 21°C

30°C

○ 12°C

□ 21°C

30°C30°C

C. saka

zakii

(log C

FU

/ml)

Observations

� C. sakazakii grows in infant cereals reconstituted with water or milk and held at 12, 21, and 30ºC, and in cereals

reconstituted with apple juice and held at 21 and 30ºC

� C. sakazakii, at an initial population of 0.005 CFU/ml, survives for at least 72 h but does not grow in cereals reconstituted with water, milk, or apple juice and held at 4ºC

� Higher populations are reached in cereals reconstituted with milk than in cereal reconstituted with water, which in turn are higher than populations in cereals reconstituted with apple juice

Observations

� Rice and oatmeal cereals support better growth than do rice with mixed fruit cereal, regardless of reconstitution liquid

� C. sakazakii, at an initial population of 2.62 log CFU/ml (417 CFU/ml), survives for at least 14 days in rice cereal reconstituted with apple juice and held at 4ºC

� cabbage

� carrot

� cucumber

� lettuce

� tomato

� apple

� cantaloupe

� strawberry

� watermelon

E. saka

za

kii

(log

CF

U/m

l)

Time (h)

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144

4°C

12°C

25°C

C. saka

zakii

(log C

FU

/ml)

� apple

� cantaloupe

� strawberry

� watermelon

� cabbage

� carrot

� cucumber

� lettuce

� tomatoE

. saka

zakii

(lo

gC

FU

/ml)

Time (h)

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144 168

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144 168

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144 168

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144 168

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144 168

0

1

2

3

4

5

6

7

8

9

10

0 24 48 72 96 120 144 168

4°C

12°C

25°C

C. saka

zakii

(log C

FU

/ml)

Efficacy of Sanitizers in Killing C. sakazakii on

Apples, Tomatoes, and Lettuce

� Treatment with chlorine, chlorine dioxide, and peroxyacetic acid reduced populations by 2.4 - 4.3 log CFU/apple, > 3.7 log CFU/tomato, and 4.0 – 5.5 log CFU/30 g of lettuce

0

2

4

6

8

10

0 1 2 3 4 5 6

Time (days)

C. sakazakii

(log C

FU

/coupon)

Populations of C. sakazakii in biofilm formed on stainless steel coupons immersed in M9 medium (■) or RIF (�)

Effectiveness of Disinfectants in Killing C. sakazakii

� Thirteen commercially available disinfectants used in hospitals, day-care centers, and foodservice kitchens were effective in killing planktonic cells (7.4 log CFU/ml of water) within 1 - 5 min

� Reductions in reconstituted infant formula were only 0.02 -3.7 log CFU/ml after 10 min

� Presence of infant formula enhanced the resistance of C.

sakazakii in biofilms or dried on the surface of stainless steel

� Overall order of resistance: Planktonic cells < cells dried on stainless steel < cells in biofilm

Publications from Which Presentation was Drawn

J. Food Prot. 68:2541-2552, 2005

J. Food Prot. 70:1410-1422, 2007

J. Food Prot. 70:1579-1586, 2007

J. Food Prot. 70:2095-2103, 2007

J. Food Prot. 70:2104-2110, 2007

J. Food Prot. 71:940-945, 2008

Appl. Environ. Microbiol. 71:7661-7669, 2005



Food Microbiol. 24:767-777, 2007

Int. J. Food Microbiol. 111:134-143, 2006

E-mail: [email protected]

THANK YOU

CRONOBACTER IN FOOD AND STRATEGIES FOR …civersen.tripod.com/beuchat.pdf · Larry R. Beuchat...

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