Kang_E.sakazakii.pdf

Enterobacter sakazakiiEnterobacter sakazakii

Kang, Dong-Hyun, Ph.D.Assistant ProfessorFood Science and Human NutritionWashington State University

History

• Name Enterobacter sakazakii proposed in1980

ByDNA-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 propertiesdifference from other Enterobacter

• Yellow pigment production on TSA• Non-fermentation of D-sorbitol• Delayed production of DNAase (7days)• Absence of phosphoamidase • Production of α-glucosidase

& Tween 80 esterase

General properties Identification of enteric bacteria

General propertiesBiochemical differentiation of Enterobacter species

TestReactionb

E. sakazakii E. cloacae E. aerogenes E. agglomerans E. gergoviaeLysine decarboxylase - - + - +Arginine dihydrolase + + - - -Ornithine decarboxylase + + + - +KCN, growth in + + + v -

Fermentationof:

sucrose + + + (+) +dulcitol - (-) - (-) -adonitol - (-) + - -raffinose + + + v +D-sorbitol - + + v -x-methyl-D-glucoside + (+) - - -D-arabitol - (-) + - +

Yellow pigment + - - (+) -

a Adapted from Farmer and Kelly, 1992.b 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 casesdisease involved

• Meningitisinfectious 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

• Sepsisthe presence of pus-forming bacteria or their toxins in the blood or tissueshttp://www.sepsis.com/sepsis_cascade/cascade2.jsp

• Seizurea sudden occurrence (or recurrence) of a disease

• Bacteremiatransient presence of bacteria in the blood

• Brain cystclosed sac that develops abnormally in brain

Reported infection casesby researcher

Author Course OutcomeUremenyi and Franklin (1961)

Sepsis, meningitis, seizures Death in 48 hAcute collapse and death

Joker et al. Meningitis, seizures, brain cysts 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 Death in 4 daysMeningitis, seizures Death in 6 days

Meningitis, seizures severe retardation

Meningitis, ventriculitis Death in 4 days

Meningitis Death in 2 daysMeningitis Death in 3 daysMeningitis Death in 4 daysMeningitis Survived, retarded

Noqvi et al. Meningitis, seizure Survived, right hemiparesis

Willis and Robinson

Meningitis, seizures, brain cysts Survived with severe deficits

Meningitis, seizures, brain cysts, hydroephalus

Survived with severe deficits

Reported infection casesby worldwide

Dried-infant formula3(1)Iceland2(?)Ontario

2(1)MassachusettsDried-infant formula4(0)Tennessee

Birth canal8(6)Netherland11(4)Greece

Incubator2(2)England1(0)Ohio1(0)Maryland(1)Missouri 1(0)Greece 1(0)Indiana1(0)Oklahoma1(0)Georgia1(1)Denmark

I. Sporadic cases

Source implicatedNumber of cases (death)Location

Thermal resistance

Thermal resistance

• Thermotolerant organism• D-value in reconstituted dried-infant formula (min)

2.153.449.7518.5754.82Food3.065.4510.9136.7254.76Clinical6058565452

Temperature (oC)Strains

Thermal resistancecompare with other microorganism

0.91208Whole milk0.46086Whole milkYersinia enterocolitica0.13045Whole milkShigella dysenteriae0.12125Whole milk0.22000Whole milkSalmonella typhimurium0.08417Whole milkSalmonella senftenberg0.07214Whole milkSalmonella muenster0.00008Human milkKlebsiella pneumoniae1.30088Infant formulaE. sakazakii0.01443Human milk0.15669Whole milkE. coli0.07033Skim milkCampylobacter jejuni0.01476Raw milkAeromonas hydrophilia

D value (72oC, S)Heating menstruumOrganism

Source from FDA

Thermal resistance

• E. sakazakii is one of the most thermotolerant members

(source from 2004 FDA)

Thermal resistanceComparison of D58°C-Values for Different Enterobacteriaceae

0

100

200

300

400

500

600

D-v

alue

(se

c)

E. sakazakii 607

E. coli O157:H7

E. sakazaki N&F-pooledK. pneuomoniae

Salmonella Hartford

E. coli

E. aerogenes

E. sakazakii 51329

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

2

3

4

5

6

7

8

9

0 500 1000 1500 2000 2500

Heating Time (sec)

Surv

ivor

s [L

og(C

FU/m

L)]

D = 591.9 secD = 591.9 sec

D = 30.5 secD = 30.5 sec

Thermal resistanceDistribution of D58°C-values for 12 E. sakazakii strains

0

1

2

3

4

5

6

# of

Str

ains

0-100 100-200

200-300

300-400

400-500

500-600

D-value (sec)

How they infect human ?

• They have relatively short lag time and generation time

NR4.2010

NR0.8023Whole milkE. aerogens47.06.4010

5.560.7423BHI E. coli32.84.6410

2.760.6723Infant formulaE. sakazakii

Lag time(h)

Generation time (h)

Temp(oC)

MediumOrganisms

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

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


• Dried-infant formula : 0.36-66.0 CFU/100g, E. sakazakii• In Canada

8/120 (6.7)Total2/24 (8)E1/24 (4)D0/24 (0)C2/24 (8)B3/24 (12)A

Positive samples (%)Company

How they infect human ?Resistance to Dehydration

2.02.53.03.54.04.55.05.56.0

0 2 4 6 8 10

Storage Time (mon)

Surv

ivin

g P

opul

atio

n[L

og(C

FU/m

L)]

How they infect human ? Temperature Decline During Rehydration of Infant Formula

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10Sample Time (min)

Tem

pera

ture

(C

)

FDA, 2004

How they infect human ?Rehydration of Dried Infant Formula

0

1

2

3

4

5

6

Surv

ivor

s[L

og(C

FU/m

l)]

23 50 60 70 80 90 100Water Temperature (C)

Lower Limit of Detection

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10Sample Time (min)

Tem

pera

ture

(C

)

• University of Wales, UK, Joanne. et al (2003)Stomoxys calcitrans (blood-sucking insects on cattle) - E. sakazakii is isolated in midgut

• House fly (Musca domestica)

milkcowinsect

Insects are major environmental reservoir ?

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)


• Mechanisms of Toxicity of Enterobacter sakazakii


(Un-known !!)

Development of differential medium


• FDA recommended method (2002)

65

4321

Oxidase test for positive identification

Select yellow colonies and confirm API 20E kit

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

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

Remove 10 ml and mix with 90 ml EE broth (o/n, 36oC)

100g, 10g, 1g dissolve in 45oC distilled water (o/n)

FDA recommended method need some improvement.

Development of differential mediumproblem 1

• 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

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

Development of differential mediumproblem 2

• Too long time needed• many yellow-pigmented

Enterobacteriaceae- E. hermanii, - E. vulneris,

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

α-glucosidase(unique E. sakazakii)

NOH2S production

Fluorescentcolonies

Black colonies

4-MU-α-D-glc. Ferric, sulphate

Development of differential mediumselection marker strategy

(by Dr. Kang’s Laboratory)

Development of differential mediumVisual Inspection Criteria

KlebsiellaE. coliE. coli O157:H7E. cloacae

SalmonellaCitrobacterEdwardsiellaProteus

-

E. E. sakazakiisakazakii+ (fluorescent)

lucosidase

- (colorless)+ (black)

α-glucosidase

Hydrogen sulfide production

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 mediumfluorescent intensity

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

Pseudomonas aeroginosa E. sakazakii


• DefinitionBackground noise is a ratio of

In absence of E. sakazakii strains

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

(Other 16 strain culture cocktails)

Total coloniesFluorescent colonies

=Background noise


Reducing background noise is most important

Background noise

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

Trypton Bile

TSA VRBG

Development of differential mediumbasal medium selection

• Tryptone bile agar is selected

43.5b100.0100.0TSA

1.0a91.392.2Trytone bile agar

52.4b72.269.7VRBG agar

16 strain cocktail

16 strain cocktail

E. sakazakiicocktail

Background noise (%)c

Reduction of microbialflora (%)b

Mediaa

Development of differential mediumbasal medium composition

Nitrogen source and concentration can be changed

pH 7.2 ± 0.2

15.0Agar

1.5Bile salt No. 3

20.0Tryptone

gm/literFormula

Development of differential mediumSelection of nitrogen source and optimization

• Tryptone and 20g/l concentration is selected

77.33±12.20d2.5 g/l72.66±10.19dProteose peptone III

48.85±4.28c5 g/l1.38±0.74aProteose peptone II

10.03±4.01b10 g/l11.60±3.38bProteose peptone I

0.62±0.54a20 g/l0.68±0.59aTryptone

0.37±0.65a40 g/l56.35±2.48cBacto peptone

Background noise (%)

TryptoneConcentrations

Background noise (%)bNitrogen sourcesa

Development of differential mediumThe effect of incubation time and temperature

37oC and 24 h incubation time is selected

8.216.3289.3384.337.33 5.3348

8.272.9688.6778.677.332.33 24

6.080.8787.6777.335.33 0.67 18

37oC30oC37oC30oC37oC30oCTime (h)

Fluorescent colonies/ total colonies (%)

No. of total colonies

No. of fluorescent

colonies


1.0Ferric citrate

0.054-MU-α-D-glc

1.5Bile salt No. 3

1.0Sodium thiosulphate

pH 7.2 ± 0.2

15.0Agar

20.0Tryptone

gm/literFormula

OK medium

Development of differential mediumVerification of OK medium

Verification of OK medium

0 (0.00)2223 (100.00)2437

0 (0.00)2221 (100.00)2430

Verified (%)ExaminedVerified

(%)ExaminedTemp.(oC)

No. of non-fluorescent colonies

No. of fluorescent colonies

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 ?

Kang_E.sakazakii.pdf

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