Enterobacter sakazakii in Infant Foods -Should We Be Concerned?

Dr. Jeff FarberDirector

Bureau of Microbial HazardsFood Directorate

Health Products and Food BranchHealth Canada

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Outline of Presentation

1. General Biology2. Ecology3. Methodology4. Epidemiology/Disease 5. Pathogenicity6. Control7. Summary

Enterobacter sakazakii

1. GENERAL BIOLOGY

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Enterobacter sakazakii

• Gram-negative rod; Enterobacteriaceaefamily

• Non-spore forming, Motile• 1980, designated as a unique species

based on differences from E. cloacae in DNA relatedness, pigment production and biochemical reactions (sorbitol, "-glucosidase)

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Enterobacter sakazakii

• 14 or 15 Biogroups• Biochemical

reactions; motility • Produces a capsule • Genetically diverse

(4 clusters; 2 lineages described to-date)

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Resistance to Treatments

Great deal of information available about Enterobacteriaceae in general, not much on E. sakazakii

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Resistance to Dessication

Isolation from dried foods indicates resistance to dryingStationary phase cells of E. sakazakii more

resistant to dessication than other Enterobacteriaceae (E. coli, Salmonella, etc.)Appears to be associated with accumulation

of trehalose

Breeuwer et al., JAM, 2003

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Resistance to Dehydration

1.0

2.0

3.0

4.0

5.0

6.0

7.0

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700

Storage Duration (days)

Pop

ulat

ion

Den

sity

[L

og(c

fu/m

l)]

EdelsonEdelson--Mammel and Buchanan, 2005Mammel and Buchanan, 2005

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Resistance to Heat

Distribution 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)

Edel

son

Edel

son --

Mam

mel

Mam

mel

and

Buc

hana

n, 2

004

and

Buc

hana

n, 2

004

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Summary

Not a particularly thermally-resistant microorganism Substantial diversity in thermal resistance among strainsSpecific information on its resistance to other treatments starting to emerge (e.g., HPP, acid-tolerance) Genetically diverse

Enterobacter sakazakii

2. ECOLOGY

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Environmental Sources of E. sakazakii

• Dust• Fruit flies,

house/stable flies• Rats• Soil, rhizosphere• Sediment, wetlands

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Foods/food ingredients from which the organism has been isolated

• Cured meat, minced beef, sausages

• Lettuce, vegetables, alfalfa sprouts

• Tofu; bread, cheese; rice seed• Herbs & spices• Sous (licorice drink)• Dried products (infant cereal,

vegs., spices, whey, egg yolk/eggnog, flour/meal)

• Mother’s milk

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Incidence of E. sakazakiiin PIF

Samples positive (%) Reference

20/141(14) Muytjens (1988)

8/58 (13.8) Leuschner et al. (2004)35/3,467 (1.0) IFC (2004)

8/120 (6.7) N-White & Farber (1997)8/210 (3.8) Heuvelink et al. (2001)3/141 (2.1) Heuvelink et al. (2003)

1/835 (0.12) WHO (2004)

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Occurrence in food production environments and households

Site Samples positive for E. sakazakii (%)

Milk powder factory 14/68 (21)Chocolate factory 2/8 (25)

Cereal factory 4/9 (44)Potato flour factory 4/15 (27)Pasta factory 6/25 (23)Households 5/16 (31)

Kandhai et al., 2004; Lancet

Growth of E. sakazakii

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E. sakazakii –Growth in PIF

Temp. Lag time Gen. time Reference6oC ND 13.7 h Iversen et al. (2004)10oC 19-47 h 4.2-5.5 h N-White & Farber (1997)21oC ND 1.7 h Iversen et al. (2004)23oC 1.8-3.4 h

3.9-4.7 h37-44 min43 min

N-White & Farber (1997)Lenati (2005)

37oC ND2.2-3.0 h

19-21 min17.4 min

Iversen et al. (2004)Lenati (2005)

ND=Not Determined

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Growth of E. sakazakii in breast milk, breast milk with fortifiers,and infant formula at 23ºC

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 2 4 6 8 10 12 14 16 18 20 22 24

time (h)

log

(CFU

/ml)

breast milk breast milk + fortifier infant formula

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0

1

2

3

4

5

6

7

8

9

8 12 24 48

Time (h)

log

CFU

/ m

l 12oC

21oC

30oC

* Initial population 0.27 CFU/ml

Generation time 43-57 min at 30oC

Growth of E. sakazakii in infant rice cereal rehydrated with liquid infant formula

(Adapted from Richards et al., 2005)

Enterobacter sakazakii

3. METHODOLOGY

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Methodology – E. sakazakii

Distilled water/BPW* Pre-enrichment

Selective enrichmentmLST+van*/EE broth

ESIA*/VRBG Agar Selection

*ISO MethodTSA 25°C; 2-3d

Identification

Yellow colonies, API 20E, Oxidase

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Medium - Druggan-Forsythe-Iversen Agar (DFI)

• Based on the alpha-glucosidase reaction

• Produces blue-green colonies on a pale yellow medium

• Proteus spp.- grey• Deoxycholate for

Gram-positivesIversen & Forsythe 2004

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Composition of R & F® E. sakazakiiChromogenic Medium

Blue-black• Carbohydrates; Sorbitol –

16.1%; D-Arabitol – 8.1%; Adonitol – 12.9%

• Bile Salts – 2.0%• Sodium chloride – 8.1%• Chromogenic substrates (X-

alpha-D-Glucopyranoside & X-β-D-Cellobioside) at 0.25% each

• Phenol red – 0.16%• Cefsulodin and Vancomycin

MelibioseSucrose

Restaino et al., 2006

E. sakazakii Dupont BAX®Detection Method

25g sample

225 ml mLST broth with vancomycin

Incubate 45°C/20-22h

Enrichment

Day 1

Add 10µl to 500 µl BHI broth (RT)

Incubate 37°C/3h

Use 5 µl in PCR ReactionDay 2

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Pathatrix Method

Magnet

Target Bacteria

Antibodies

Magnetic beads

Debris Non Specific bacteria

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Sample Overview for

E. sakazakii in Infant Formula

100g + 900ml BPW

Static Incubation at 42ºC for 6h

Run 250 ml sample on Pathatrix for 30 min

Plate beads onto DFI plates 1 min

Overnight Incubation - ~ 16 h * Current FDA method = 4 days to colony Total test time = ~23h*

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Summary of Benefits of PATHATRIX E. sakazakiiAssay Format

• Analyzes large sample size• Simple to operate • Colonies isolated within

24h• Easy analysis of pooled

samples • Minimal training costs• Ability to rapidly identify

problem in production environment

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Some Methodological Issues

• Yellow non-diffusible pigment production is not a unique trait

• Reports of white E. sakazakii strains• Biochemical kits not all that reliable

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One Possible solution

• An artificial neural network has been applied to identify factors which discriminate E. sakazakii from similar, closely related organisms (Iversen et al., 2006)

• This predicted that tests for the metabolism of glucose-1-phosphate, sucrose and arginine gave the highest discrimination

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Rapid Methods

• Real-time PCR (Malorny & Wagner, 2005; Seo & Brackett, 2005; Liu et al., 2006)

• PCR targeting 16S rRNA(Lehner et al., 2004); ompA(Nair et al., 2006); alpha-glucosidase (Lehner et al., 2006); 16S-23S rDNA spacer region (Liu et al., 2006)

• 16S RNA in-situ Hy test (VIT); uses epiF microscopy

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Molecular typing

• Plasmid typing• RAPD• Ribotyping• PFGE• MLST

Enterobacter sakazakii

4. EPIDEMIOLOGY / DISEASE

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E. sakazakii infections

• About 90 cases worldwide, 80% of them in infants < 1 y old

• Among infants, most cases are 0-2 months of age

• Conclusion: Group at particular risk is infants (<1 year)

• Greatest risk: neonates (<28d) and immunocompromised, especially those of LBW (<2500 g) and also those < 2 months of age

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Symptoms of E. sakazakii infection

• Asymptomatic babies colonized with the organism

• Sepsis/bacteremia with a bleeding tendency

• Devastating CNS involvement, i.e., meningitis, meningoencephalitis

• A whole host of sequelae

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Sequelae –E. sakazakii infection

• Tropism for the CNS– Ventriculitis– Brain cysts and abscesses/lesions– Cerebral infarction– Retarded neural development– Late development of hydrocephalus

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Summary of Infant Characteristics By Syndrome

Characteristics Bacteremia Meningitis

Gestational age Younger Older

Birth weight Extremely low Low

Age at onset > 1 month < 1 week

Severe outcome Rare Usual

Bowen and Braden, 2006

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Hypotheses

• Very premature, extremely low-birth-weight infants are more likely to be fed– IV nutrition early in life– Sterile, liquid formula next– Non-sterile, powdered formula later

• Less premature, heavier infants are more likely to be fed powdered formula from the time they are born

• Greatest risk for meningitis is in the first weeks of life

Bowen and Braden, 2006

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Human sites where the Organism has been isolated

• CSF, Blood• Urine, stool• Stomach aspirate• Respiratory Tract

(sputum, nose, throat)

• Skin, wounds

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Mortality Rates, Minimum Infectious Dose and Incubation Period

• Mortality Rates – reported as high as 50%, but in recent years has declined to <20%

• MID – Unknown at this point; likely depends on age/weight of patient and health status

• IP – appears to be short, but is unknown

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E. sakazakii –Important Chronology

• First reported cases – Urmenyi (1961); pigmented coliforms

• Farmer et al. (1980) – E. sakazakii• Kleiman et al.; Adamson et al. (1981);

previously healthy 5-wk old infants• First case in an adult – Jimenez (1982)• Muytjens (1983) – first to propose possible

link to powdered infant formula

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Outbreaks linked to PIF

Location Cases Comments ReferenceIceland 3 (1 death) 2 normal term infants;

1 Down'sBiering et al., 1989

Tennessee 4; 3 sepsis, 1 bloody diarrhea

Es; 8 cfu/100g Simmons et al., 1989

Belgium 12 6/12 with NEC positive for Es

Van Acker et al., 2001

Tennessee 9 1 confirmed, 2 suspect, 6 colonized

Himelright et al., 2001

Israel 5 3 colonized only Bar-Oz et al., 2001

New Zealand 5 (1 death) 4 colonized 2004France 9 (2 deaths) 5 colonized AFSSA, 2005

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Case study -Van Acker et al. (2001)

• 12 cases – Belgium, 1998• 6/12 neonates had positive cultures for

E. sakazakii vs. 0/38 without NEC (p<0.001)

• 10/12 neonates with NEC drank the same PIF (Alfaré)

• Organism isolated from prepared formula as well as from unopened cans

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Van Acker et al. (2001) –Noteworthy Points

• After use of implicated formula stopped, no more cases were observed

• Coliform results met Codex, but not Belgian standards

• A closely related E. sakazakii strain had been isolated 4 years earlier from the gastrostomy tube of an infant fed the same brand of formula

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Outbreak in France

• Another outbreak due to E. sakazakii occurred in France in 2004 (Coignard and Vaillant, 2006; Coignard et al., 2006)

• A total of 9 cases were reported, with 2 deaths • Syndromes included fatal meningitis (2),

conjunctivitis (1), hemorrhagic colitis (1) and colonization (5)

• All infants were premature and under 2000 g (low-birth-weight), except for the infant with colitis who weighed 3250g and was born at 37 weeks of gestation

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Outbreak in France

• Public health officials saw 2 E. sak meningitis cases a week apart – triggered an investigation

• 4 implicated lots over a 6-month period were contaminated at levels ranging from 1-10 cfu/100 g; the 2 more highly contaminated lots were involved in 8 of the 9 cases

• Hospitals were storing formula for longer than 24h in the refrigerator, with no temperature control

Enterobacter sakazakii

5. PATHOGENICITY

Pathogenesis of E. sakazakii infection

Crossing of gastrointestinal barrier leads to sepsis, bacteremia

Ingestion of contaminated food: most likely route of entry

Blood-brain barrier passage leads to meningitis, ventriculitis, brain abscess, infraction, cyst formation leading in most cases to neurological sequelae

Gastrointestinal symptoms include necrotizing enterocolitis and diarrhoea

How to mimic infectious process seen in LBW neonates?

In-vivo pathogenicity assessment

Non-primate animal models

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Summary – E. sakazakiipathogenicity

• Suckling mouse bioassay: Of 18 E.sakazakii strains evaluated, four were found to test positive for enterotoxin production

• IP Dosing: Death at 108

• Oral Dosing: 2/18 strains caused death in suckling mouse model

Pagotto et al., 2003; JFP

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Method

• Animal models (weight; age):– Pigs (6.3–7.2 kg; 5 weeks)– Chicks (1 day)– Rabbits (2.7–3.0 kg; 2 months)– Guinea pigs (300–400g; 3–4 months)– Gerbils (40–50g; 1–2 months); newborns– Rat pups

• Challenged with three (of 30) isolates from ILSI core list:– SK81 (clinical)– 2001 – 10 – 01 (clinical)– MNW2 (food)

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Method

Oral dose 1x109

cells First phase - 3 isolates

7 and 14 days

Observe signs of Infection / death

Examine organs: spleen, brain, lymph nodes, feces heart, small intestine

Choose best animal model for second

phase (30 isolates)

Collect fecal samples

(Days 1, 3, 7 and 14)

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Results

• None of the young animal models tested to-date (piglets, chicks, rabbits, guinea pigs, gerbils) have mimicked human clinical symptoms of E. sakazakii infection, including no lethality

• E. sakazakii was recovered from fecal samples of all animals tested

• Newborn gerbils appear promising

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Summary - What do we know about the pathogenicity of this organism?

• Differences in virulence between strains • Can attach to intestinal mucus• Produces enterotoxin-like compounds;

acidic metalloprotease• Can survive passage through the stomach• Healthy young animals and children

appear to be resistant to infection• Unique conditions need to exist for

infection to occur

Enterobacter sakazakii

6. CONTROL

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Control Measures –Dry Infant Formula

• PIF are not intended to be sterile

• Levels of microorganisms must be kept as low as possible

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Sites of potential contamination

Raw milk

TransportStorage

Pasteurization

Drying/Blending

Filling Product

StorageTransport

ReconstitutionConsumption

E sakE sak

Factory

FarmHospital/

HomePotential sites forEnvironmentalcontamination

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The presence of E. sakazakiiin packaged PIF is due to recontamination

• Recontamination is related to the following factors, the first two of which are linked:

1. The presence of these micro-organisms in the processing environment, presenting the possibility that they may get into the processing lines

2. The presence of these micro-organisms, originating from the processing environment (1), on internal surfaces of equipment that are in direct contact with the product; and

3. The presence of these micro-organisms in ingredients added and mixed into the dry base powder after the heat-processing step

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Presence of Enterobacteriaceaeand E. sakazakii in PIF ingredients

Ingredients N(10g) Coliform or Enterobacteriaceaepositives

E. sakazakiipositives

Vitamins 793 8 0

Dem. whey powder 23 3 0

Sucrose 1691 28 0

Lactose 2219 70 2Banana powder/flakes

105 3 1

Orange powder/flakes

61 1 1

Lecithin 136 1 1

Starch 1389 155 40

Unpublished Industry Survey; FAO/WHO, 2004

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Control during production

• Reduction of these micro-organisms in the production environment can be achieved through a combination of:

(1)minimizing their entry into high-hygiene zones and

(2)preventing proliferation of those that are already present

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Monitoring for EB

• Monitoring for EB represents an ideal tool to assess the effectiveness of preventive measures and to detect the occurrence of recontamination

• The methods are simple and provide rapid results allowing for rapid corrections when needed

• Such monitoring can be complemented by testing for E. sakazakii in relevant samples, e.g., in finished product

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Monitoring for EB

Increases in the levels of EB and/or E. sakazakiiin processing environments can be due to:

• A massive and sudden entry as a result of, for example, poorly planned construction or maintenance activities or more commonly– The occurrence of conditions which allow the

proliferation of the low number of micro-organisms already present in the environment

– Growth is only possible in the presence of water, therefore this has to be eliminated as much as possible

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Summary - Control of E. sakazakii

Achieving control of EB and/or E. sakazakii requires the implementation of a number of measures modified according to the needs of individual manufacturing facilities. These measures include:

• The effective implementation of preventive measures (GMP/GHP and HACCP) as originally designed to control Salmonella

• The strengthening of these measures to further minimize entry of the micro-organisms and to avoid their multiplication by excluding water from the processing environment, e.g., the implementation of systematic dry-cleaning

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Summary - Control of E. sakazakii

Continued -• Selection of suppliers of dry mix ingredients according to

specified needs (e.g., microbiological requirements for dry-mix ingredients)

• The implementation of monitoring and environmental management programs (environmental samples, product contact surfaces, finished products) based on EB as indicators for process hygiene, and E. sakazakii in relevant samples to demonstrate control or to detect deviations and assess the effect of corrective actions

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Guidelines for safe preparation, storage and handling of PIF

WHO Guidelines

PIF is not a sterile product and may be contaminated with pathogens that can cause serious illness. Correct preparation & handling reduces the risk of illness

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Control measures in Hospitals

• Scenarios that involve periods of holding at room temperatures (both cool and warm) are associated with the greatest risk

• Quick cooling to lower temperatures to minimize growth is essential (i.e., use small containers)

FAO/WHO, 2006

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Reconstitution

• The highest risk scenarios were associated with reconstitution at temperatures of 40 and 50oC when the formula is not consumed immediately

• Reconstitution with liquid of 70oC is an effective risk mitigation strategy for all scenarios investigated

FAO/WHO, 2006

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Control measures in Hospitals

• Use of trained personnel to prepare PIF under as aseptic conditions as possible

• Prepared formula products should be rapidly cooled and then refrigerated right away; discarded if not used within 24h after preparation

• Hang times should not exceed 4h• Preparing only a small amount of formula for

each feeding • Alternatives to powdered form if possible

Enterobacter sakazakii

7. SUMMARY

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Future Work

• ILSI grant to look at animal models

• Risk assessment• Codex – Recommended

International Code of Hygienic Practices for Foods for Infants and Children

• ICMSF –criteria/specifications

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Control measures for Enterobacter sakazakii in PIF

Ho - ΣR + ΣI < FSO

Reducing the concentration/prevalence of intrinsic contamination

Reducing the level of contamination of the reconstituted PIF (e.g., heat) prior to use

Minimise the chance of contamination of reconstituted formula during preparation

Minimize the growth of E. sakazakiifollowing reconstitution prior to consumption

+

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Summary

• E. sakazakii infection is a rare, but very serious disease

• More data needed on true “susceptible”populations

• More growth data needed • Dose-response models needed

to develop FSOs• More global data needed on PIF

handling, preparation and storing practices

Enterobacter sakazakii in Infant Foods -Should We be Concerned?

Yes

Are we moving in the right direction to control this organism?

Yes