Cronobacter sakazakii
a coliform bacterium of increased
concern to human health.
Milan Marounek, Department of
Microbiology, Nutrition and Dietetics
1
Cronobacter sakazakii

Formerly Enterobacter sakazakii
(till 2007), belongs to
Enterobacteriaceae.

Molite, Gram-negative, nonsporulating straight rod.

Size of cells: 0.5 – 1.5 x 2 – 3 μm

Ferments glucose and other sugars.

Difference from related
Enterobacter species consists in its
inability to ferment D-sorbitol and
ability to produce extracellular
deoxyribonuclease.
2
Scanning electron micrograph of cells
of C. malonaticus DSM 3148. Scale bar
is 5 μm.
3
Genus Cronobacter is
composed of five species:
C. sakazakii
C. malonaticus
C. turicensis
C. muytjensii
C. dublinensis
The first three species are
opportunistic human pathogens.
4
C. sakazakii, C. malonaticus and C.
turicensis have been associated with
neonatal infections:

Necrotizing enterocolitis

Meningitis
Mortality of neonatal C. sakazakii infections
has been reported as high as 50%.
One half of infected infants die within 1
week of diagnosis.
5
Infections among adults

C. sakazakii infections in adults
are rare.

Most adults with C. sakazakii
infections had serious
underlaying diseases (cancers,
alcohol abuse, diabetes mellitus).

Potential victims of C. sakazakii
infections are
immunocompromised patients
6
The most important risk factor for human
health is the incidence of C. sakazakii, in
powdered infant formula.
According to the EU Directive no.
1441/2007 about microbiological criteria
for food the absence of C. sakazakii is
required in infant milk formula and in
dried food for infants.
7
In the Czech Republic:
Directive ČSN 1P ISO TS 22964
„Milk and milk products – Detection of
Enterobacter sakazakii“
has been approved for isolation and
identification of C. sakazakii in powdered
milk, milk products and infant formula.
Standard microbiological techniques for
detection of C. sakazakii are sensitive,
but time-consuming.
There is a need for the elaboration of
rapid, reliable and sensitive methods for
detection of C. sakazakii in foods.
8
Physiological characteristics of
C. sakazakii
 Growth was observed between 25°C
and 45°C. No growth occurred at 4°C
or 50°C.
 Resistance to acid pH.
 Production of extracellular
polysaccharide (formation of biofilm).
 Resistance to dry stress.
 Resistance to high osmolarity.
 Resistance to many antibiotics.
9
Environmental reservoirs of
C. sakazakii ?
 Water, soil, plants.
 Vectors such as flies and rodents.
 Probably not bovine milk,
domesticates animals, grain.
 Environment of food factories and
households.
10

Resistance of Cronobacter sp. to dry
stress.
Cronobacter sp. exhibited a strong
resistance to dry stress.
Viable counts of cells dried in air and
left at 25°C for 46 days decreased
only by 1.0 – 1.5 log CFU/ml.

Resistance of Cronobacter sp. to acid
and alkaline stresses.
The pH optimum was 7.2.
Isolates were resistant to high pH.
The growth was only slightly inhibited
at pH 11.0.
The strains did not survive at pH < 4.0.
11
Survival of Cronobacter sp. in the production
process of infant formula.
 Cronobacter sp. does not survive
pasteurization of milk.
 Efficacy of disinfection in killing
Cronobacter in biofilm is rather low:
Using ethanol or NaOH alone does not
guarantee the inactivation.
 The complete elimination of
Cronobacter from the whole production
line is difficult due to high resistance to
dry stress and disinfectants.
 The presence of Cronobacter in infant
formula is possible due to recontamination in the course of drying
and packaging.
12
Presence of Cronobacter sp. in a range of
samples from Irish farms, abattoir, food
and domestic environments.
a) Farms
Number of
samples
Positive
samples
Bovine faeces
105
0
Soil and grass
105
0
Trough water
84
0
Hay/straw
2
0
Feed/grain
15
10
Cereals
3
1
Sample type
13
Presence of Cronobacter sp. in a range of
samples from Irish farms, abattoir, food and
domestic environments.
b) Beef and pork abattoir
Number of
samples
Positive
samples
Beef hide swabs
15
0
Beef carcass swabs
15
1
Beef cuts
5
1
Beef process equipment
5
0
Air samples
18
2
Pig caecal contents
20
0
Pork cuts
5
2
Pork process equipment
5
0
Sample type
14
Presence of Cronobacter sp. in a range of
samples from Irish farms, abattoir, food and
domestic environments.
c) Retail
Sample type
Number of
samples
Positive
samples
Beef burgers
10
3
Steaks
1
0
Beef mince
1
1
Pork chops
11
0
Cooked meat
2
0
Sausage
1
0
Pork mince
1
0
Salads
14
0
Leafy green
6
2
Cheese
20
0
Dried herbs
5
0
Breakfast cereals
20
9
15
Presence of Cronobacter sp. in a range of
samples from Irish farms, abattoir, food
and domestic environments.
d) Domestic
Number of
samples
Positive
samples
Vacuum cleaner dust
6
1
Kitchen surfaces
19
0
Total
518
33 (6.4%)
Sample type
16
Presence of Cronobacter sp. in samples from
a Czech farm, retail foods and „domestic“
environment.
a) Farms
Number of
samples
Positive
samples
Environment of shed
17
0
Feed
7
0
Trough water
2
0
Hay/straw
2
0
Cows
2
0
Raw milk
1
0
Equipment of employees
5
0
Sample type
17
Presence of Cronobacter sp. in samples from
a Czech farm, retail foods and „domestic“
environment.
b) Retail foods
Number of
samples
Positive
samples
powdered milk formula
10
0
chocolates
7
0
jams
4
0
63
4
herbs
9
0
spices
31
7
tea
6
3
coffee
2
0
cocoa
5
0
Sample type
Infant food
Confections, sweet,
cookies, chips
Herbs & spices
Instant beverages
18
Presence of Cronobacter sp. in samples from a
Czech farm, retail foods and „domestic“
environment.
b) Retail foods
Number of
samples
Positive
samples
Vegetables
45
1
Fruits
12
0
Hams and sausages, salads
17
0
Cheeses
15
0
Cereal products, pastry
7
0
Seeds & nuts, dry fruit
14
0
Powdered porridge, műsli
10
0
Powdered custards
5
0
Instant soups
11
0
Honey
1
0
Jogurt
1
0
Restaurant food
13
0
Others
3
0
Sample type
19
Presence of Cronobacter sp. in samples
from a Czech farm, retail foods and
„domestic“ environment.
c) Domestic environment
Sample type
Number of
samples
Positive
samples
Households
85
27
Restaurants, bars,
hotels
20
13
Dormitory rooms
20
10
Offices
5
2
Nursery schools
7
0
Storehouses
3
0
469
67 (14.3%)
Total
20
Are there effective methods for reduction
of Cronobacter in foods?
 Natural antimicrobial substances
include bacteriocins, essential oils,
antimicrobial lipids.
 Several fatty acids and their
monoacylglycerols inhibit a range of
microorganisms (Escherichia coli,
Salmonella, Campylobacter).
 In our experiment we determined
activity of 4 medium-chain fatty acids,
their monoacylglycerols and sucrose
caprate against C. sakazakii and C.
malonaticus.
21
Inhibitory activities of fatty acids, monoacylglycerols
and sucrose caproate against Cronobacter sp.
expressed as IC50
Compound
C. sakazakii
C. malonaticus
Sorbic acid
2.32 ± 0.69
2.73 ± 0.17
Caprylic acid
1.13 ± 0.41
1.20 ± 0.38
Capric acid
>5
>5
Lauric acid
>5
>5
Monosorbin
>5
>5
1.26 ± 0.09
1.75 ± 0.45
Monocaprin
>5
>5
Monolaurin
>5
>5
Sucrose caprate
>5
>5
Monocaprylin
– concentration (mg/ml) at which 50% of the
initial glucose was depleted within a 16-h incubation
a IC
50
22
Viable cell counts (log10 CFU/ml) of C. sakazakii and C.
malonaticus as determined by bacterial plating following
a 30-min incubation with sorbic acid, caprylic acid and
monocaprylin (2 mg/ml).
Control
Sorbic
acid
Caprylic
acid
Monocaprylin
Cronobacter
sakazakii
8.40 ± 0.66
7.05 ± 0.50
3.00 ± 1.11
3.05 ± 0.96
Cronobacter
malonaticus
8.75 ± 0.83
7.83 ± 0.61
< 2.00
< 2.00
23
Effect of monocaprylin on the ultrastructure of
C. malonaticus (B, C) and C. sakazakii (D) at 0
(A), 2 (B) and 4 (C,D) mg/ml.
A
B
C
Scale bar is 0.5 μm
D
24
Viable cell counts (log10 CFU/ml) of C. sakazakii and
C. malonaticus as determined by bacterial plating
following a 30-min incubation with monocaprylin at
40, 45, 50 and 55 °C.
Temperatur
e (°C)
C. sakazakii
C. malonaticus
Concentration of monocaprylin
(mg/ml)
0
0.5
1
2
40
8.75
8.37
7.90
4.95
45
8.79
8.47
4.90
2.00
50
8.52
6.46
3.85
< 2.00
55
4.79
3.47
< 2.00
< 2.00
40
8.40
8.38
7.67
3.00
45
8.93
8.90
7.95
2.00
50
7.10
5.08
2.11
< 2.00
55
3.21
2.99
2.70
< 2.00
1
25