Professor Dr. Son Radu Centre of Excellence for Food Safety Research Universiti Putra Malaysia
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Transcript of Professor Dr. Son Radu Centre of Excellence for Food Safety Research Universiti Putra Malaysia
Professor Dr. Son Radu
Centre of Excellence for Food Safety Research Universiti Putra Malaysia
How safe is
our food?
Using The Results of a Risk Assessment in Food Safety Risk Management
HACCP is appliedat this level
Operational level
Food safetyassurance
Government level
Food control system
• Determine the policy
• Set public health goals
• Set standards
• Based on risk analysis
• Adjust inspection systems to verify FSO/PO are met
• Design control measures from farm to fork
• Responsible for hazard controlGAPs/GHPs/GMPs
HACCP
PH goalsStandards
Policy
MRA is a used as a tool at this level
FSO, PO
PC
ALOP
•GAP •GMP •GHP •HACCP •Code of practicle
Public health burden??
Safety by design
Performance Objectives
Food Safety Objectives
RISK COMPARISON
Growth pattern of a child with frequent episodes of diarrhoea
Presentation Topics
• Risk assessment of Listeria monocytogenes in Raw Vegetable
• Raw Vegetables Consumption as A Potential Risk Factor for Campylobacteriosis in Malaysia
• Biofilm formation and persistence of Salmonella• Malaysia Fishery Products Export to
the EU & Issues Encountered – An example of a collaborative work between University and the relevant authority
What is the hysteria about Listeria?
• Listeria is a Gram positive, facultative anaerobic, psychrotrophic, rod shaped bacterium
• Listeria is a hardy bacterium- grows across a broad pH (4.3-9.8) and temperature (0.5 -45oC) range, and up to 20% salt tolerance
• Incidence of listeriosis varies between 0.1 to 11.3 per 1,000,000 in different countries
• Listeriosis has an average case-fatality rate of 20-30% despite adequate antimicrobial treatment
• Life-threatening illness in three clinical syndromes: maternofetal listeriosis or neonatal listeriosis, blood stream infection, and meningoencephalitis
• Listeria monocytogenes is widely distributed in soil and water• Can enter the VBNC state and pose problem in routine plating detection methods
What is the hysteria about Listeria?
Incidence of listeriosis per 100,000 of the population in North America, Europe and New Zealand from 1996 to 2005
Country Cases per 100,000 of the population
1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
US CanadaGermanyFranceUnited KingdomEurope AveNew Zealand
0.50.1-0.40.20.1-
0.30.2-0.40.20.10.5
0.30.2-0.40.20.2-
0.20.3-0.40.20.2-
0.30.2-0.40.20.20.5
0.20.20.30.40.30.20.6
0.20.20.30.40.30.2-
0.20.20.30.30.40.2-
0.30.20.40.40.40.2-
0.30.20.60.40.40.30.7
Source: Denny & MaLaughlin, 2008; Fenton et al., 2001; Goulet et al., 2008; Health Canada, 2000; Lake et al., 2002; Vugia et al., 2008
What is the hysteria about Listeria?
Prevalence of Listeria monocytogenes within processing facilities within North America and Europe
Facility
Cheese processing 8%Milk processing 3%Ice cream 6%Beef processing 28-92%Poultry processing 13.3%Fish processing 12.8%Domestic refrigerators 20%
Taken from Kornacki & Gutler, 2007
Key Questions
• To what extent does the consumption of raw vegetables contribute to Listeria monocytogenes infections in humans in Malaysia?
No raw vegetables consumption vs. Raw vegetables consumption
O% 100%
•What should be done to intervene?
The different steps of the infectious process at the cellular level: Left panels. Electron micrographs describing the various steps of entry, lysis of the vacuole, intracellular movement, cell-to-cell spread and lysis of the two-membrane vacuole (Cossart and Lecuit, 1998). Right panel. Schematic
representation of the infectious process with the proteins involved: internalin, InlB, PI-PLC, ActA, and lecithinase (adapted from (Tilney and Portnoy, 1989)). The infectious process by L. monocytogenes at the cell and tissue levels L. monocytogenes is an invasive bacterium which induces its own
entry into cells. Internalization results from the tight apposition of the plasma membrane over the entering bacterium. This process, also called “the zipper mechanism” appears different from the “trigger mechanism” used by bacteria such as Salmonella and Shigella during which dramatic
membrane ruffles rich in filamentous F-actin engulf the bacterium in a process similar to macropinocytosis. Listeria is then present in vacuole that is lysed in less than thirty minutes. When free in the cytosol, Listeria starts to replicate while inducing the recruitment and the polymerisation of
cellular actin. Actin polymerisation only occurs at one pole of the bacterial body and allows the bacterium to propel itself inside the cytosol. From time to time, bacteria reach the plasma membrane where they induce the formation of long protrusions containing a bacterium at their tips. These
protrusions can invaginate in a neighboring cell and give rise to a two-membrane vacuole that the bacterium lyses to get access to a second infected cell and by doing so disseminate into tissues by a direct cell-to-cell process (Tilney and Portnoy, 1989).
The Recipe For Risk
A person must ingest cheese that is contaminated with Listeria monocytogenes in the order of 100-1000 cells
The immunocompromised person that ingests these bacteria must become sick – invade gastro-intestinal epithelium, become bloodborne and associated with monocytes, then subsequently the liver, spleen and lymphatic system and then to the nervous system and placenta barrier
FLOWCHART OF SAMPLING, MPN-PLATING AND MPN-PCR FOR LISTERIA MONOCYTOGENES
MPN 9-TUBES DILUTIONS
Incubate (30oC, 48 h, aerobic condition)
Contaminated sample
Sample & broth (pre-enrichment)1:10 RATIO
Pre-enrichment 4 hours, 30°C
MPN-PLATING of turbid tubes MPN-PCR of turbid tubes
Presumptive Listeria colonies are black centered on PALCAM Agar (selective agar)
VBNC
Detection of Listeria monocytogenes using polymerase chain reaction
Estimated mean intake(g/day/person)
Central Zone 3.27
Urban 3.02
Rural 4.59
Men 3.62
Women 3.78
Malays 4.41
Chinese 1.74
Indian 1.72
Whole Malaysia 3.7
Estimated mean intake (g/day/person) of raw vegetables as ulam in Malaysia according to area, gender and race (Source: MOH, 2008)
Consumption data
Schematic representation of the model framework for retail-to-table risk assessment of L. monocytogenes in raw vegetables.
Dose of L. monocytogenes in a meal
No. of cells
Prevalence
Risk assessment model structure
Exposure Assessment
Prevalence• The distribution for the prevalence of L.
monocytogenes in Japanese parsley, wild parsley, winged bean and Indian pennywort was estimated from the data collected in this study. The prevalence was described by pert distribution assuming a minimum and maximum prevalence accordingly:
Pr = RiskPert( min, max, most likely ) where min and max are minimum and maximum
of the prevalence data, respectively.
Exposure Assessment
Concentration• The distribution of concentration of L.
monocytogenes in contaminated raw vegatables was estimated from the data collected in this study and was assumed to follow a lognormal distribution:
Cr = RiskPert (min, most likely, max)
Exposure Assessment
Log reduction of washing practice• Log reduction of washing practice was estimated from the
data collected in the kitchen simulation study performed in the laboratory.
• The correlation coefficient between initial microbial load on the raw vegetable and log reduction was determined by using StatTools (Palisade Corporation).
• The distributions of microbial load on vegetable before washing and log reduction of washing were fitted to the data collected in the study and the correlation between both inputs was defined with the resulted correlation coefficient with @Risk 5.5.
Risk Characterization
• The output of exposure assessment was combined with the dose response function for hazard characterization to estimate the yearly risk from L. monocytogenes.
• The probability of illness per person per year was described by the equation:
Pill:year = 1 – ( 1 – Pill )365
where Pill is the probability of illness person per day.
Prevalence and MPN count in log10 MPN/g of Listeria monocytogenes in four types of raw vegetables purchased from wet markets and hypermarkets
Sample Location Prevalencelog10 MPN/g
min max mean SD
Indian pennywort (pegaga)
Wet markets 2/16a 0.125 0.97 3.04 2.01 1.46
Hypermarkets 3/16 0.188 0.56 1.97 1.11 0.76
5/32 0.156 0.56 3.04 1.47 1.03
Japanese parsley (selom)
Wet markets 8/16 0.500 0.48 2.46 1.39 0.69
Hypermarkets 1/16 0.063 NAb NA NA NA
9/32 0.281 0.48 2.46 1.29 0.72
Winged bean (kacang botol)
Wet markets 1/16 0.063 NA NA NA NA
Hypermarkets 4/16 0.250 1.04 1.20 1.13 0.08
5/16 0.313 1.04 1.20 1.12 0.08
Wild parsley (ulam raja)
Wet markets 4/16 0.250 0.48 1.18 0.75 0.31
Hypermarkets NDc ND ND ND ND ND
4/16 0.250 0.48 1.18 0.75 0.31
a Numerator: number of positive samples; denominator: total number of samples tested.b NA: Not applicablec ND: Not done
5.0% 90.0% 5.0%
0.1025 0.2036
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prevalence / IP
prevalence / IP
Minimum 0.0762Maximum 0.2364Mean 0.1522Std Dev 0.0308Values 30000
Mea
n =
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522
5.0% 90.0% 5.0%
0.125 0.458
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prevalence / JP
prevalence / JP
Minimum 0.0217Maximum 0.5485Mean 0.2938Std Dev 0.1014Values 30000
Mea
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5.0% 90.0% 5.0%
0.1250 0.2923
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prevalence / WB
prevalence / WB
Minimum 0.0252Maximum 0.3097Mean 0.2205Std Dev 0.0515Values 30000
Mea
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5.0% 90.0% 5.0%
0.1412 0.3587
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prevalence / WP
prevalence / WP
Minimum 0.0772Maximum 0.4216Mean 0.2500Std Dev 0.0661Values 30000
Mea
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(a) (b)
(c) (d)
Figure 2: Distribution of prevalence of L. monocytogenes in (a) Indian pennywort, pegaga; (b) Japanese parsley, selom; (c) winged bean, kacang botol; and (d) wild parsley, ulam raja.
Distribution of prevalence of L. monocytogenes in (a) Indian pennywort, pegaga; (b) Japanese parsley, selom; (c) winged bean, kacang botol; and (d) wild parsley, ulam raja.
Distribution of concentration of L. monocytogenes in retail Indian pennywort, Japanese parsley, winged bean and wild parsley.
-1.0
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0.07After washing / IP
After washing / IP
Minimum -0.6065Maximum 2.9349Mean 1.3062Std Dev 0.4917Values 30000
Concentration / IP
Minimum 0.5674Maximum 2.9776Mean 1.5800Std Dev 0.4612Values 30000
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After washing / JP
Minimum -0.6240Maximum 2.4156Mean 1.0762Std Dev 0.4092Values 30000
Concentration / JP
Minimum 0.4944Maximum 2.4116Mean 1.3500Std Dev 0.3714Values 30000-0
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After washing / WB
Minimum -0.5172Maximum 1.2783Mean 0.8462Std Dev 0.1752Values 30000
Concentration / WB
Minimum 1.0422Maximum 1.1981Mean 1.1200Std Dev 0.0302Values 30000
-1.0
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After washing / WP
Minimum -0.9214Maximum 1.1030Mean 0.5028Std Dev 0.2170Values 30000
Concentration / WP
Minimum 0.4829Maximum 1.0932Mean 0.7750Std Dev 0.1289Values 29850 / 30000Filtered 150
Distribution of microbial load before and after washing of vegetable with tap water. (a) Indian pennywort; (b) Japanese parsley; (c) winged bean; and (d) wild parsley.
(b)
(d)
(a)
(c)
Raw vegetables (ulam-ulaman) consumption in Malaysia based on estimated mean intake (g/day/person) a
Estimated mean intake(g/day/person) F(x)b
Indian 1.72 0.1
Chinese 1.74 0.2
Urban 3.02 0.3
Central Zone 3.27 0.4
Men 3.62 0.5
Whole country 3.7 0.6
Women 3.78 0.7
Malays 4.41 0.8
Rural 4.59 0.9
a The distribution for the mean intake was described by the following cumulative distribution RiskCummul (1.0,5.5,{1.72,1.74,3.02,3.27,3.62,3.7,3.78,4.41,4.59},{0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9})/ F(x) is the cumulative probability(Vose, 1996), i.e. F(x) = i / (n+1), where i is the rank of the observed data point and n is the total number of data points.
The probability of illness per person (high-risk population and low-risk population) per year due to consumption of raw vegetables.
High-risk population Low-risk population
Indian pennywort Japanese parsley winged bean wild parsley Indian pennywort
Japanese parsley winged bean wild parsley
Minimum 8.78E-11 1.15E-11 2.74E-11 3.69E-12 0.00E+00 0.00E+00 0.00E+00 0.00E+00
Maximum 5.55E-04 9.87E-04 3.14E-06 2.39E-06 2.18E-07 5.19E-08 1.65E-10 1.26E-10
Mean 1.38E-05 5.34E-06 2.70E-07 6.93E-08 9.91E-10 2.80E-10 1.42E-11 3.63E-12
Std Dev. 4.59E-05 2.01E-05 2.92E-07 1.04E-07 4.91E-09 1.05E-09 1.53E-11 5.45E-12
Variance 2.11E-09 4.03E-10 8.51E-14 1.08E-14 2.41E-17 1.11E-18 2.34E-22 2.97E-23
Median 9.03E-07 5.78E-07 1.74E-07 3.47E-08 4.83E-11 3.04E-11 9.16E-12 1.82E-12
5% Perc 1.69E-08 1.55E-08 1.61E-08 2.68E-09 8.92E-13 8.10E-13 8.51E-13 1.22E-13
95% Perc 7.01E-05 2.35E-05 8.51E-07 2.55E-07 4.06E-09 1.23E-09 4.47E-11 1.34E-11
Raw Vegetables Consumption as A Potential Risk Factor for
Campylobacteriosis in Malaysia
OUR FINDINGS• We found presence
of campylobacters especially C. jejuni in farm and retail vegetables.
• The prevalence and concentration of C. jejuni in freshly-harvested vegetables are lower compare to retail raw vegetables.
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Field study: vegetable farms
Irrigation waterPrevalence: non detected
Freshly-harvested vegetablesPrevalence:18.8% (3.0-150.0 MPN/g) Aged manure3.0% (9.1 MPN/g) composted manure
SoilPrevalence: 30.4% (3.0-9.1 MPN/g) Aged manure2.7% (6.1 MPN/g) composted manure
Poultry ManurePrevalence:57.1% (3.0-9.3 MPN/g) Aged manureNon detected composted manure
Raw Vegetables
Soil?
poultry manure?
Irrigation water?
workers?
animals? FARM LEVEL
RETAIL LEVEL
vehicles?
containers?
workers?
Raw Vegetables
workers?
containers?
Washing water?
Environmental?Poultry?Other animals?
Cross-contamination from poultry? Meat? Water?
Spraying water?
Contact with meat?
Raw Vegetables
Holding timeHolding temperature
KITCHEN
Cook
NO RISK
Consume raw/ salad
RISK?How to reduce?Washing rate of reductionBlanching rate of reduction
Simulation of cross-contamination and decontamination of C. jejuni during handling of raw vegetables
The simulation was designed to simulate the real preparation of salad in a household kitchen starting from washing of vegetables in tap water; cutting the vegetable on cutting board; followed by slicing cucumber and blanching (heating in hot water) the vegetables in 85oC water.
Vegetables naturally contaminated with C. jejuni were used throughout the simulation to attain realistic quantitative data.
The mean of the percent transfer rate for C. jejuni from vegetable to wash water was 30.1% - 38.2% ; wash water to cucumber was 26.3% - 47.2%; vegetables to cutting board was 1.6% - 10.3%; and cutting board to cucumber was 22.6% - 73.3%.
The data suggest the wash water and plastic cutting board as potential risk factors in C. jejuni transmission to consumers.
Washing of the vegetables with tap water had a 0.4 log10 reduction of C. jejuni attached to the vegetables (MPN/g); while rapid blanching reduced the number of C. jejuni to an undetectable level.
Retail-to-Fork Model: a simple deterministic risk assessment
Retail raw vegetables
* Consumption * Washing
* Blanching
* Consumption
* Consumption
*Washing: wash in a bucket of tap water (30oC) for about 20 min. The log reduction rate is 0.36 log MPN/g.*Blanching: blanching was done by dipping raw vegetables in 85oC hot water for 10 sec. The removal efficiency is 0.95.* Locals consume 3.7g (mean) of raw vegetables per serving (MOH Food Consumption in Malaysia, 2007).* the dose-response model for C. jejuni was adapted from a report by Joint FAO/WHO Expert Consultation on Risk Assessment of Microbiological Hazards in Foods (FAO/WHO 2002).
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WW: Consume without washingW : Consume after washingWB: Consume after washing and blanching
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• The presented risk model is just a preliminary risk assessment aimed to demonstrate the risk of Campylobacteriosis via raw vegetables consumption.
• Washing as proposed in this study has a certain efficiency in reducing the risk. However, washing methods with higher reduction efficiency (1 log reduction) are able to reduce the risk by 80%.
• Other washing practices like wash under running tap water, wash and soak in salt water, etc. might have higher log reduction rate.
Biofilm formation and persistence of Salmonella
• Salmonella clones can persist in many locations for many years
• Persistence not due to resistance against – heat– disinfection– dry conditions
I can’t go with the flow anymore I’m thinking of
joining a biofilm
BIOFILM
Bacteria on surfaces
AdhesionMicrocolonies
Structures
Matrix
Photo from ASM Microbe Library
Two weeks old biofilm in an industrial condencer
Inorganic surfaces
Liquid-air interface (pellicle)
Salmonella biofilm
Organic surface
Close up
Under magnifying glass
SEM
Salmonella biofilm production
20°C 28°C 37°C
LB brothLB broth wo/salt
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Biofilm formation at room temperature on different surfaces
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Agona Biofilm - Biofilm - Biofilm Biofilm
Typhimurium Biofilm - Biofilm - Biofilm Biofilm
Typhimurium Biofilm Biofilm Biofilm - Biofilm Biofilm
Typhimurium Biofilm Biofilm Biofilm - Biofilm Biofilm
Typhimurium Biofilm Biofilm Biofilm - Biofilm Biofilm
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Persistent strains
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Malaysia Fishery Products Export to the EU & Issues Encountered
Malaysia Fishery Export
• Malaysia fishery products export is valued at RM 2260 million per year
• About 50% of Malaysia fishery products are exported to the European Union
(EU) and United States of America (USA)
Department of Fishery Malaysia. Annual Fisheries Statistic 2004 Export / Import (Volume 2). Cawangan Pengutipan Data, Bahagian Pengurusan Maklumat Perikanan, Jabatan Perikanan Malaysia, 2004.
Department of Fishery Malaysia. Annual Fisheries Statistics (Volume 1). Cawangan Pengutipan Data, Bahagian Pengurusan Maklumat Perikanan, Jabatan Perikanan Malaysia, 2006.
Fishery Products Exported to EU
• Shrimp & Prawn (frozen, cooked)• Squid, Cuttlefish & Octopus (frozen, cooked)• Anchovy & Fish (prepared or preserved)
• Anchovy (dried)• Mackerel (dried, salted, in oil)• Catfish fillets (dried, salted, smoked)• Queen fish (salted, dried)• Scad (Selar) (salted)
• Fish (frozen, cooked)• Farmed barramundi • Eviscerated farmed eel• Tuna (steak, strip meat, lion, cube, ground Meat)
• Shell fish (frozen)
Frozen HOSO Prawn
Frozen BarramundiDry Anchovy Mackerel in Oil Smoked Catfish Fillets Dried Salted Queen FishSalted Scad (Selar)
Frozen Squid
Frozen Octopus
EU Import Requirements
• EU import requirements for fishery products:
EUROPA The Food Veterinary Office (FVO) –General guidance on EU import andtransit rules for live animals and animalproducts from third countries. Retrieved 23 January, 2010 from http://ec.europa.eu/food/international/trade/guide_thirdcountries2006_en.pdf
Food and Veterinary Office (FVO) Audits
EUROPA The Food Veterinary Office (FVO) –Third Country Establishments List - Malaysia. Retrieved 25 September, 2010https://sanco.ec.europa.eu/traces/output/FFP_MY_en.pdf
Third Country Approval List
Adobe Acrobat Document
2005
2010
2008
Mission Report 2005 (DG/SANCO/2005 – MR Final)
Mission Report 2008 (DG (SANCO)/2008-7679-MR-FINAL)
Mission Report 2010(DG (SANCO)/2010-8532-MR-FINAL)
EUROPA The Food Veterinary Office (FVO) – Final Report (DG/SANCO/2005 – MR Final). Retrieved 1 October, 2008 from http://ec.europa.eu/food/fvo/ir_search_en.cfmEUROPA The Food Veterinary Office (FVO) – Final Report (DG (SANCO)/2008-7679-MR-FINAL). Retrieved 1 October, 2008 from http://ec.europa.eu/food/fvo/ir_search_en.cfm
EUROPA The Food Veterinary Office (FVO) – Final Report (DG (SANCO)/2009-8319-MR-FINAL). Retrieved 25 September, 2010 from http://ec.europa.eu/food/fvo/ir_search_en.cfm
EUROPA The Food Veterinary Office (FVO) – Final Report (DG (SANCO)/2010-8532-MR-FINAL). Retrieved 25 September, 2010 from http://ec.europa.eu/food/fvo/ir_search_en.cfm
Audit Reports from FVO
2009
Mission Report 2010(DG (SANCO)/2010-8532-MR-FINAL)
Summary
020406080
100
Mar, 05 May, 08 Jun, 08 May, 09 Dec, 09 Sep, 10
No. of Malaysia Fishery Establishments with MOH HACCP & EU Registration Number for the export of fishery products to EU
77
9
EUROPA The Food Veterinary Office (FVO) – Third Country Establishments (Fishery Products).,Retrieved 15 April, 2008 from https://sanco.ec.europa.eu/traces/output/FFP_MY_en.pdfEUROPA The Food Veterinary Office (FVO) – Third Country Establishments (Fishery Products), Retrieved 23 December, 2009 from https://sanco.ec.europa.eu/traces/output/FFP_MY_en.pdfEUROPA The Food Veterinary Office (FVO) – Third Country Establishments (Fishery Products) , Retrieved 25 September, 2010 from https://sanco.ec.europa.eu/traces/output/FFP_MY_en.pdf
52
6
17
Border Inspections
Border Inspections
EUROPA, EU Import Conditions for Seafood & Other Fishery Products, Retrieved 28 August, 2010 from http://ec.europa.eu/food/international/trade/im_cond_fish_en.pdf
What if Non Compliance?
EUROPA, EU Import Conditions for Seafood & Other Fishery Products, Retrieved 28 August, 2010 from http://ec.europa.eu/food/international/trade/im_cond_fish_en.pdf
Rapid Alert for System Food & Feed
EUROPA, Rapid Alert System for Food and Feed, Retrieved 28 August, 2010 from http://ec.europa.eu/food/food/rapidalert/index_en.htm
Notification Types
EUROPA, Rapid Alert System for Food and Feed – annual Report 2008, Retrieved 28 August, 2010 from http://ec.europa.eu/food/food/rapidalert/report2008_en.pdf
RASFF Portal
EUROPA, RASFF Portal, Retrieved 28 August, 2010 from https://webgate.ec.europa.eu/rasff-window/portal/
Breakdown of NCs 2004 - 2010
Breakdown of NCs 2004 - 2010
Breakdown of Microbiological NCs
Fishery Products Rejected 2004 - 2010
Fishery Products Rejected 2004 - 2010
Thank You