A Tool to Manage Vibrio parahaemolyticus growth in Australian

Oysters

Judith Fernandez-Piquer, Tom Ross, John Bowman, Mark Tamplin

Food Safety Centre, Tasmanian Institute of Agricultural Research, University of Tasmania, Hobart, Tasmania 7005

Australia

INTRODUCTION

Actual situation www.usatoday.com (19th October ’09)

Actual situation

• Low numbers of food poisoning cases in Australia

Uncertainties:-Oyster species-Geographical locations

• Some countries already have action levels for Vp • Codex is considering action levels

Microbial Hazards

• Filter-feeders: biological contamination in water accumulation

• Pathogens likely to occur in Australian oysters are virus, Vibrio spp and toxins

• Vibrios are salt tolerant and occur naturally in estuaries

Vibrio spp.

• Vibrio illnesses are mostly caused by V. cholerae, V. vulnificus and V. parahaemolyticus

Temperature (oC) NaCl (% )

Range Optimum Range Optimum

V. cholerae 10-43 37 0.1-4.0 0.5

V. vulnificus 8-43 37 0.5-5.0 2.5

V. parahaemolyticus 5-43 37 0.5-10 3

Vibrio parahaemolyticus (Vp)

• Is a curved, rod-shaped, gram-negative bacterium

• Pathogenic & non-pathogenic strains

60oC 15min

• Raw, undercooked or mishandled seafood

Post-harvest: outgrowth!

Commercial supply chain

• Why cold chain?

Influence of water temperatureSalinity Air temperature after harvest Length of refrigeration until consumption (appropriate)

• What is cold chain?

Primary production Final consumption

Predictive microbiology

• Knowledge of response of microorganisms to different environmental conditions: TEMPERATURE

√SGR= 0.2016x(T-14.3339)x{1-exp[0.0113x(T-55.3482)]}

• Mathematical model is an algorithm describing the effects of different factors on microbial viability

Aim

To develop a predictive model for V. parahaemolyticus growth in live Australian

Oysters that can be used to design commercial supply chains and reduce consumer health risk.

RESULTS

V. parahaemolyticus growth rate in live Australian Pacific Oysters

00.10.20.30.4

0.50.60.70.80.9

10 15 20 25 30 35

time (hours)

squ

are

roo

t g

row

th r

ate

Model development: data

VPOYS_14.9_OCT

0

0.51

1.52

2.5

33.5

4

0 50 100 150 200

time

logc logc

Fit 1

VPOYS_30.4_OCT

0

12

34

5

67

8

0 20 40 60 80

time

logc logc

Fit 1

V. parahaemolyticus growth rate in live Australian Pacific Oysters

00.10.20.30.40.50.60.70.80.9

10 15 20 25 30 35

time (hours)

squ

are

roo

t g

row

th r

ate

Model development: maths

V. parahaemolyticus growth rate in live Australian Pacific Oysters

00.10.20.30.40.50.60.70.80.9

10 15 20 25 30 35

time (hours)

squ

are

roo

t g

row

th r

ate

Observed

Predicted

√SGR= 0.2016x(T-14.3339)x{1-exp[0.0113x(T-55.3482)]}

Model evaluation

Vibrio parahaemolyticus growth rate in live Australian Oysters

00.10.20.30.40.50.60.70.80.9

10 15 20 25 30 35

time (hours)

squ

are

roo

t g

row

th r

ate

Predicted

PO (NSW)

SRO (NSW)

Model comparison

Vibrio parahaemolyticus growth rate in live Australian and American Oysters

00.10.20.30.40.50.60.70.80.9

10 15 20 25 30 35

time (hours)

squ

are

roo

t g

row

th r

ate Predicted

AO (US)

CONCLUSIONS

CONCLUSIONS and BENEFITS

1) This model demonstrates to industry the influence oftemperature on Vp growth

Allows oyster cold chain to be designed less prescriptive and

more flexible

2) Models should be developed for species and possibly a growing region.

Can show a distinction for Australian oyster industry

3) Vp didn’t grow at 15oC (PO), 28oC (SRO)

May provide Australian oyster industry with more cost-effective storage

ACKNOWLEDGEMENTS

• PhD scholarship (Seafood CRC, Oyster Consortium)

• EIPRS scholarship (UTAS)

• Supervisory team (Mark Tamplin, Tom Ross and John Bowman)

• Project team (UTAS, SARDI, NSW DPI, ASQAP in Tas-SA-NSW)

• Oyster growers (Tasmania, NSW, SA)

• Colleagues from UTAS

• My family

Very tasty, very

nutritious!!!

“ This work formed part of a project of the Australian Seafood Cooperative Research Centre, and received funds from the

Australian Government’s CRCs Programme, the Fisheries R&D Corporation and other CRC Participants”.

Oyster Industry in AUS

Sydney Rock Oyster (Saccostrea

glomerata)

Pacific Oyster (Crassostrea gigas)

Safety Management

• Australian Shellfish Quality Assurance Program (ASQAP) Operation Manual, October 2006 (FSANZ standard 4.2.1)

– Storage conditions for consumption as raw product

Species Maximum days T (oC)

Pacific Oyster 6-7 ≤10 (24h)

Sydney Rock Oyster 9-10 Max 25 then ≤15 (72h)

Material & Methods

• Tasmanian Pacific Oysters • Inoculation • Storage conditions (15-30oC)

• Enumeration on TCBS

• Growth curves using DMfit (SGR)

• Secondary model (SGR at different T)

Material & Methods

• NSW Pacific Oysters (PO) and Sydney Rock Oysters (SRO) • Natural Vp

• Enumeration using MPN+PCR

• Storage conditions (15-30oC)

• Growth curves using DMfit (SGR)

Inoculation diagram

1. trh2. tdh3. tlh

1

23

Strains: 39 40 57 58 59 60

1) Drilling 2) Injection

CONCLUSIONS BENEFITS

• Growth model (15-30oC): how fast Vp grows at different temperatures

• Tmin: 15oC (for PO: higher than the 10C recommended at the moment)

• Slower Vp growth in oyster than in broth: necessity of a different model than the one in broth available

• Vp viability is different in PO and SRO: this supports the idea that different oyster species show different behaviour

TCBS

• Counts:1) Selective media for Vibrio spp

CFU/g= [(70+75)/2]*dilution factor

MPN+PCR

MPN/g= MPN result*dilution factor

• Counts:1) Enrichment step 2) Vp confirmation(Most Probable Number) genetically

10-3 10-4 10-5

+ + + + + - - - -

Results: 4 parameters square-root ModelV parahaemolyticus in live CG oysters

0

0.2

0.4

0.6

0.8

1

10 15 20 25 30 35T (oC)

sq

rt(S

GR

)

Observed

Predicted

T (oC)Observed rate (log CFU/ h)

MPD (log)Predicted rate (log CFU/ mL)

(Predicted-Observed)2

15 0.0006 3.78 0.0008 3.3E-0818 0.0302 6.66 0.0341 1.5E-0520 0.0747 6.03 0.0617 1.7E-0426 0.1721 6.09 0.1856 1.8E-0430 0.2823 7.27 0.2762 3.7E-05

RMSE 0.01

Bf 1.05

Af 1.15

Results: Growth Profiles

30C

012345678

0 24 48 72time(h)

log

/mL

20C

0

1

2

3

4

5

6

7

0

24

48

72

96

12

0

14

4

time(h)lo

g/m

L

V. parahaemolyticus in live CG oysters 30oC

0

2

4

6

8

0 12 24 36 48 60time (h)

cou

nts

(lo

g c

fu/m

L)

Injected 1

Injected 2

Natural

V. parahaemolyticus in live CG oysters 20oC

0

2

4

6

8

0 24 48 72 96 120time (h)

cou

nts

(lo

g c

fu/m

L)

Injected 1

Injected 2

Natural

)

FUTURE WORK

• Real performance of the model (oyster shipment with temperature loggers)

• Generating data for understanding of the differences between PO and SRO (bacterial profiles)

Results: Growth Profiles

V. parahaemolyticus in live PO (NSW) 28oC

0

1

2

3

4

5

0 10 20 30 41 51 61 71 81 92time (h)

cou

nts

(lo

g M

PN

/g)

V. parahaemolyticus in live SRO (NSW) 28oC

0

1

2

3

4

5

0 10 20 30 41 51 61 71 81 92time (h)

cou

nts

(lo

gM

PN

/g)

\

28oC

0

1

2

3

4

5

0 50 100time (h)

cou

nts

(lo

g M

PN

/g)

28oC

0

1

2

3

4

5

0 50 100time (h)

cou

nts

(lo

g M

PN

/g)

Vibrio outbreaks in AUSV. cholerae:

–2006: 1 incident (imported whitebait), OzFoodNet

V. vulnificus:–1988-90: 4 incidents in NSW, 2 deaths (raw oysters) Kraa, 1995–1989-90: 3 cases in NSW septicaemia wound infection– 1991: 1 case in Victoria specticaemia Maxwell, 1991

• V. parahaemolyticus; –1977-84: incident in NSW (seafood at a restaurant), Davey 85– 1990: incident in Sydney (prawns from Indonesia), Kraa 95– 1992: 2 incidents (prawns), Kraa 95– 1992: 1 death (raw oyster), kraa 95– 2002: 1 incident (seafood), OzFoodNet– 2005: 1 incident (oysters), Anon 05–4 cases in 1992 nacrozis

•1995-02: no outbreaks related to Vibrio but noroviurs, HepA and Salmonella.• Oysters:

– 2001-07: unknown, norovirus and Salmonella. Data source: OzFoodNet