Food Safety and Beyond

Jianrong (Janet) Zhang, Ph.D

Food safety background Safe, nutritious foods are essential to human

health and well-being. However, food-borne diseases pose a significant problem worldwide.

The World Health Organization (WHO) estimates that 1.5 billion cases of food-borne illnesses cause about 3 million deaths each year.

Food safety background (cont.) Although the United States produces the

safest foods in the world, food-borne illnesses continue to threaten this country. The Centers for Disease Control and Prevention estimate that in the United States more than 6 million cases of food-borne illnesses occur annually -- causing 8,000 deaths and costing up to $13 billion in health care and job-related absenteeism.

Food safety background (cont.) Food safety will continue to be important

issue in the future, because: First, the overall U.S. population is increasing

and changing; The U.S. food system has increased in

complexity as our society has become more urbanized;

Food-borne diseases will likely increase in light of global economy.

Federal agencies The Centers for Disease Control and

Prevention (CDC) is recognized as the lead federal agency for protecting the health and safety of people

FDA Center for food safety and applied nutrition(CFSAN)

USDA Food Safety and Inspection Service(FSIS)

Codex Alimentarius Commission Implements the joint FAO/WHO Food

Standards Program To protect the health of consumers and to

ensure fair practices in trade

Codex Alimentarius Commission North American Free Trade Agreement -

NAFTA(Canada, US, Mexico) MERCOSUR (Argentina, Brazil, Paraguay,

Uruguay) APEC (Asia-Pacific Economic

Cooperation)- 18 countries in Asia and the Pacific

European Union

New CDC data CDC published Preliminary FoodNet Data on

Apr.2003, demonstrate a sustained decrease in major bacterial foodborne illnesses caused by Campylobacter and Listeria, indicating progress toward meeting the Agency’s health objectives of reducing the incidence of foodborne infections.

In addition, data from FSIS show a continuing decline in the prevalence of Salmonella in regulatory samples of meat and poultry.

HACCP There was16 percent decline in foodborne

illness over the last 6 years (1996-2002). CDC attributes these results in part to the

implementation of the Hazard Analysis Critical Control Point (HACCP) system in all meat and poultry plants in the United States.

FDA HACCP HACCP for the seafood industry in a final

rule December 18, 1995 For the juice industry, the final rule released

on January 19, 2001. It will take effect on January 22, 2002 for large and medium businesses, January 21, 2003 for small businesses, and January 20, 2004 for very small businesses

USDA HACCP In 1998, the U.S. Department of Agriculture

has established HACCP for meat and poultry processing plants, as well. Most of these establishments were required to start using HACCP by January 1999. Very small plants had until Jan. 25, 2000

HACCP seven principles Analyze hazards Identify critical control points Establish preventive measures with critical limits for each

control point Establish procedures to monitor the critical control points Establish corrective actions to be taken when monitoring

shows that a critical limit has not been met Establish procedures to verify that the system is working

properly Establish effective record keeping to document the

HACCP system

Food Hazard A biological, chemical or physical agent in

a food with the potential to cause an adverse health effect

Current Hazard Biological

Bacteria Viruses Parasites

Chemical Pesticide residues Veterinary drugs

Physical Contaminated raw material Poorly designed or maintained equipment

Microbial growth, survive and death in food

pH Water activity aw

Oxygen absence Temperature Nutrient content Antimicrobial constituents Biological structures

pH Optimum Maximum Minimum Bacteria 6.5-7.5 9.0 4.5 Molds 4.0-6.8 8.0-11 1.5-3.5Yeast 4.5-6.5 8.0-8.5 1.5-3.5 Food can be divided into two major categories:

low acid (pH <4.6) and acid (pH > 4.6). These were established based upon the growth of C.botulinum, whose minimum growth pH requirement is generally accepted as 4.8.

Water Activity Bacteria 0.90-0.91 S.aureus –0.83 Halophilic bacteria – 0.75 Yeasts

0.87-0.94 Osmotolerant yeasts – 0.60 Molds 0.70-0.80 Xeromyces – 0.60

Water activity of foods Fruits/vegetables – 0.97-1.00 Meats – 0.95-1.00 Bread – 0.95-1.00 Cheese – 0.68 – 1.00 Jams/Jellies – 0.75-0.94 Honey – 0.54-0.75

Temperature effect Most pathogenic organisms are mesophilic (Min.,

5-15 ºC, Opt., 35 –37 ºC, Max, 30-45 ºC) A number of foodborne pathogens are

psychrotrophic (Min., -5 to 5 ºC, opt., 12 –15 ºC, Max., 15 –20 ºC)

Thermophiles (Min.40-45 ºC, Opt., 55 –75 ºC, max. 60 – 90 ºC)

Psychorotrops (Min., -5 to 5 C, Opt. 25 –30 ºC, Max., 3—35 ºC)

Examples of food groups and their related spoilage microorganism

Refrigerated foods – psychrotrophs Juice concentrate – osmophilic yeasts Fermented foods – acid tolerant lactic acid

bacteria and yeast Meat products – psychotropic

pseudomonads Hot – filled juices – heat resistance molds

Ten least wanted foodborne pathogens Campylobacter jejuni Clostridium botulinum E.coli O157:H7 Listeria

monocytogenes Salmonella

Staohylococcus aureus Shigella Toxoplasam gondil Vibrio vulnificus Yersinia

eneterocolitica

Microbial detection Traditional methods to detect foodborne bacteria

often rely on time-consuming growth in culture media, followed by isolation, biochemical identification, and sometimes serology

Recent advances in technology make detection and identification faster, more convenient, more sensitive, and more specific than conventional assays

Rapid methods "rapid methods", a subjective term used

loosely to describe a vast array of tests that includes miniaturized biochemical kits, antibody- and DNA-based tests, and assays that are modifications of conventional tests to speed up analysis

Rapid methods First made available in the early 1980s for

several groups of bacteria Alternative approach besides convenient

methods, less time, labor and set up costs Extensively evaluated Now accepted by most microbiologists

Rapid methods Biochemical test kits Antibody assay DNA-based assay

Partial list of miniaturized biochemical kits and automated systems for identifying foodborne bacteria

APIb Cobas IDA Micro-IDb EnterotubeII Spectrum 10 RapID BBL Crystal Minitek Microbact Vitekb

Microlog MISb

Walk/Away Replianalyzer Riboprinter Cobas Micro-ID Malthusb Bactometer

Partial list of commercially-available, antibody-based assay for the detection of foodborne pathogens and toxins

ELISA - Enzyme-Linked Immunosorbent Assay

LA – Latex agglutination IMS – Magnetic beads Major manufacturers: BioMerieux, Foss,

Microgen, Biocontrol, TECRA, Elcatech, etc.

Partial list of commercially-available, nucleic acid-based assays used in detection of foodborne bacterial pathogens

BAX Probelia

Based on PCR assay

AccuProbe GENE-TRACK

Based on Probe assay Bindb

Based on Phage assay

Some other rapid methods To use disposable cardboards containing

dehydrated media, which eliminates the need for agar plates, constituting savings in storage, incubation and disposal procedures

To inncorporate specialized chromogenic and fluorogenic substrates in media to rapidly detect trait enzymatic activity

To measure bacterial adenosine triphosphate (ATP) to rapidly enumerate the presence of total bacteria

VITEK®(BioMerieux)

The VITEK is a completely automated instrument that offers rapid results (with an average of 2-6 hour same-day turnaround).

It is used for bacterial and yeast identification, antimicrobial susceptibility testing and has a complete Data Management System.

VITEK®(BioMerieux) cards ANI............. Anaerobes & Micrococcus BAC............. Bacillus GPI.............. Gram Positives (Staph & Strep) GNI/GNI+... Gram Negatives (Oxidase Neg) NFC............. Gram Negatives (Oxidase Pos) YBC............. Yeast

How does VITEK® work

Colony need to be isolated first Isolates are subcultured to TSA Plates A smear is prepared from original Tryptone

Soy Agar (TSA) plates for Gram Stain Isolates are incubated at 35 oC to be fresh

sample

How does VITEK® work (cont.) Observe subcultured plates Perform preliminary testing (catalase,

oxidase, microdase, coagulase, etc.) Set-up appropriate VITEK Card Insert Card into VITEK Incubator/Reader Come back to read the report

How does ELISA work? Antibody coated wells Sample is added, target antigens, if present,

bind with antibodies Reagent is added, antibodies sandwich the

antigen,, enzyme labeled antibody detectors attach to the sandwich

Substrate is added, color change occurs where the antigen is present

Riboprinter® Microbial Characterization System (Dupont Qualicon)

Fully automated ribotyping system that provides a genetic "fingerprint" of any bacterium in about eight hours.

The system extracts a RiboPrint® pattern from image data, compares it to others in a database for characterization and identification, and prints the results in a report.

The system can process up to eight bacterial isolates at one time, can accept new batches every two hours, allowing up to 32 samples a day.

How does Riboprinter® work Getting a sample

A colony is picked manually from the plate and introduced into the RiboPrinter system, where the colony is suspended in a buffered solution and then heated

Preparing the DNA The sample is treated with a lysing agent, a chemical that

dissolves the bacterial cell walls to release the DNA. This process is completed by adding a restricting enzyme that "cuts" the DNA at specific points and creates identifiable fragments.

How does Riboprinter® work (cont.) Separating and transferring DNA

The DNA fragments are put into eight small wells, and "markers" - synthetic DNA of known weights - are placed in five other wells. The DNA fragments are separated according to molecular size by a process called gel electrophoresis. Through this process the fragments are electrically drawn out of the gel and transferred directly to a moving nylon membrane

Membrane processing At this point, the markers and samples are attached to the

membrane in 13 distinct "lanes." The membrane then goes through a series of biochemical steps, including treatment with a chemiluminescent agent that literally lights up the DNA fragments of interest

How does Riboprinter® work (cont.) Detection and extraction

The glow of the DNA fragments is not visible to the naked eye. However the RiboPrinter system is equipped with a CCD camera that can detect very low light levels. The camera takes a digital picture of the membrane, resulting in an image of the DNA fragments and markers. The system then uses a proprietary algorithm to "understand" and normalize the image. The result is a standard DNA pattern called a RiboPrinter pattern that can be compared with other such patterns from other images.

Riboprinter®

PCR PCR stands for “Polymerase Chain Reaction” First described only 10 years ago, in its short life

PCR has transformed the life sciences utterly. It is far simpler and less expensive than previous

techniques for duplicating DNA, PCR has democratized genetic research, putting it within reach of all biologists, even those with no training in molecular biology.

PCR’s requirement A template molecule - the DNA or RNA

you want to copy two primer molecules (short chains of the

four different chemical components, named as nucleotides or bases, that make up any strand of genetic material - to get the copying process started

PCR’s requirement DNA is double-stranded, consisting of two such

nucleotide chains that wind around each other in the famous shape known as the double helix

Primers are single-stranded Primers must be duplicates of nucleotide

sequences on either side of the piece of DNA of interest, which means that the exact order of the primers' nucleotides must already be known

PCR’s three steps First, the target genetic material must be denatured-that is,

the strands of its helix must be unwound and separated-by heating to 90-96°C.

The second step is hybridization or annealing, in which the primers bind to their complementary bases on the now single-stranded DNA.

The third is DNA synthesis by a polymerase. Starting from the primer, the polymerase can read a template strand and match it with complementary nucleotides very quickly. The result is two new helixes in place of the first, each composed of one of the original strands plus its newly assembled complementary strand.

Commercialized PCR equipment The key to PCR's automation has been Taq

polymerase. Taq is a nickname for Thermus aquaticus, a bacterium that happily survives and reproduces in an environment that is lethal to other organisms: hot springs

So that it can stand rapidly fluctuating temperatures of automated PCR

BAX® (Dupont Qualicon) Process up to 96 unique samples within four

hours after sample preparation. Results are available as soon as the next day

and are clearly displayed on screen with a simple positive or negative report

Provide reagents for screening Salmonella, E. coli O157:H7, L. monocytogenes, etc.  

BAX® (Dupont Qualicon) Samples are enriched according to standard protocols for

the food type. Samples are then heated in a lysis reagent solution to

rupture the bacterial cell wall and release the DNA. PCR tablets, which contain all the reagents necessary for

PCR plus fluorescent dye, are hydrated with lysed sample and processed in the cycler/detector. Within a few hours, the PCR amplifies a DNA fragment that is specific to the target.

The amplified DNA generates a fluorescent signal, which the BAX® system uses to analyze the findings. Results are then displayed as simple positive or negative symbols

Limitation for rapid methods A positive result by a rapid method is only

regarded as presumptive and must be confirmed by standard methods

Most rapid methods lack of sufficient sensitivity and specificity for director testing, foods still need to be culture-enriched before analysis

Rapid methods are food dependent Can detect cell but can’t detect the toxin

occurrence

Future trend Biosensor

A compact analytical device incorporating a biological or biologically-derived sensing element(such as enzyme, antibody, microbe or DNA) either integrated with a physicochemical transducer.

Transducer: Electrochemical Optical Piezoelectric Thermal

Future trend DNA biochip

A miniature silicon surface containing thousands of gene probes in a thumbnail size area