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From: Food Quality & Safety magazine, August/September 2008

Pathogen Detection at the Speed of Light

Rapid methods have revolutionized raw materials testing, but challenges remain

by Linda L. Leake, MS

Many people believe nothing is certain in life but death and taxes. Daniel Y.C. Fung, PhD, professor of food microbiology at Kansas State University, adds another certainty to the list: "Food processors must get accurate results from tests to detect pathogens in raw materials. This holds true regardless of the technology employed, the time involved, or the cost. A rapid test giving bad results is not good whatsoever."

Accuracy may be the top priority of microbial testing, but in light of the fast-paced global food system, speed is critical to every processing plant’s efficiency and profitability. While there are still no magic bullets delivering immediate results, there are many new and exciting rapid methods to test for bacteria, yeast, and molds in raw and processed food samples, Dr. Fung says. “Different microbiological tests measure different attributes of the cell mass, numbers, metabolites, and genetic materials, so it is really difficult to single out the significance of one test over another.”

Scientists currently rely on two major technologies for microbiological testing, molecular and immunological; both are considered highly accurate and fast. Of these, polymerase chain reaction (PCR) and immunoassays are the key methods available, respectively.

“Real time PCR systems and multiplex PCR systems offered by several high-tech companies are making great advances in rapid identification of pathogens,” Dr. Fung says. “A number of eight-hour tests are on the market for a number of pathogens, including E. coli O157H:7, which provide results during one work shift.” Dr. Fung himself developed what’s known as the Fung Double Tube (FDT) system, a six-hour test that has been used to detect Clostridium perfringens in ground beef.

Micro Hits the Big Time

The development of such tests is an indication of the market’s vitality. According to Food Micro—2008 to 2013, a market report published by Strategic Consulting, Inc. (SCI; Woodstock, Vt.), over 738 million food industry microbiological tests, with a market value exceeding $2.06 billion, were completed in the global market in 2008.

“The food sector represents the largest market segment within the industrial microbiology market…almost 50% of the total market,” says Thomas Weschler, MBA, SCI’s president. “The food sector is more than double the size of any other industrial segments, including the pharmaceutical, beverage, environmental, industrial process, and personal care products sectors.”

Since 1998, the market value for food microbiology has grown significantly, experiencing an annual growth rate of 8.7%. “Based on SCI research, the food microbiology testing market is expected to grow to 969.2 million tests in 2013, with a market value approaching $2.4 billion,” Weschler says. “This represents a projected annual growth rate of 5.6% in testing volume.”

Much work is being done to meet that demand. A variety of nanotechnology tests and procedures for image analysis of colonies are being developed, Dr. Fung adds. “These are not yet ready for commercial use but in due time may be available in food microbiology laboratories.”

There are some limitations with current rapid micro tests, Dr. Fung says. Some instruments and systems, like Fourier transform infrared (FTIR) spectroscopy, require only a few minutes to precisely identify a colony on an agar plate but are very expensive. Some rapid micro tests do not measure cell viability, an important factor in food microbiology. And some slow-growing organisms, such as yeast and mold, are difficult to detect using rapid methods.

“At least 50 companies throughout the world are working on a myriad of systems, and we are seeing new breakthroughs relative to rapid micro tests every day,” Dr. Fung says. “In the next three years, new ideas and procedures will emerge on many fronts.”

There will probably be more nanotechnology, microchips, and biosensor systems that can perform tests “faster and faster and cheaper and cheaper,” Dr. Fung says. And robotics will definitely play a big role.

More research will help a new generation of microbiologists develop new and exciting rapid testing tools, Dr. Fung says. “It is important for all players in this field to stay positive and work on the issues with patience, openness, and optimism.”

Demand Drives Growth

Driving this worldwide development and growth is an increase in food consumption, consumer demand, industry food safety priorities, and regulation, Weschler says. The acceleration of the conversion of traditional microbiological testing methods to rapid methods is a function of those phenomena. It’s no surprise that, despite the higher cost per test, these newer methods are being used more frequently; compared with traditional testing, they provide faster results and/or ease of use benefits.

Nonetheless, traditional methods still account for approximately 58% of the microbiology tests performed worldwide in the food market. Their rapid counterparts—including convenience-based, immunoassay-based, and molecular-based methods—account for 42%. In fact, over the last three years, food micro tests utilizing rapid methods have increased by 37% to 307 million tests, up from 224 million in 2005. Some 80% or more of all tests are run to determine non-pathogens or indicator organisms.

“By 2013, much will have changed,” says Weschler. “Traditional methods will still be the predominant ones used, accounting for 491.2 million tests. However, traditional will represent only 50.7% of all tests conducted, which is approximately an 8% decrease based on percentage of tests performed.”

All types of rapid methods will make significant gains in usage during the coming five-year period, Weschler adds. “When combined, the annual test volume of rapid methods will increase by over 55% from current levels and reach 478 million tests in 2013. The gain in market value for rapid methods will be even more pronounced than the testing volume increases, since the rapid methods have much higher average prices per test than traditional methods.”

Roadblocks to Progress

Currently, progress with raw material testing is limited because of the required enrichment step, the time required to grow pathogens to detectable levels. In most enrichment steps, it typically takes 10 to 24 hours or longer to identify pathogens. Ideally, pathogens should be identified in eight hours or less; in a perfect world, the task could be slashed to minutes, even seconds.

Cost is another limiting factor with some rapid test products, according to J. Stan Bailey, PhD, MS, who spent 34 years as a research food microbiologist with the U.S. Departments of Agriculture’s (USDA) Agricultural Research Service. Dr. Bailey joined bioMerieux, Inc. in January as director of scientific affairs for industry. For 25 of the last 28 years, he has served on the faculty of Dr. Fung’s annual two-week rapid testing methods workshop.

“At first glance, some rapid and automated kits may seem expensive, as much as $10 to $15 per test, but you have to be mindful of the total costs of a microbiological test,” Dr. Bailey says. “Total costs of a conventional test include the costs of media and disposable plastics as well as the labor required for media preparation and reading and recording the results.”

Other factors drive the use of commercial test kits, Dr. Bailey says. These include better quality control, consistency of results from one sample batch to the next, time savings, and International Organization for Standardization or Association of Analytical Communities certification and documentation.

Sample handling has a major impact on the success of any test, rapid or conventional, Dr. Bailey says. Much work is being done to improve sample handling, he points out, including advances in concentration techniques, which improve the sensitivity of pathogen assays.

“For the past 20 years we’ve seen a pretty strong movement toward the use of automation to detect pathogens and indicator organisms,” Dr. Bailey says. “Almost all laboratories are being asked to do more with less resources, and the best solution to this pressure is better automation. One of our biggest needs is a rapid test to detect and count yeasts and molds in order to determine shelf life and spoilage potential.”

Automation of data will be a driver in the years ahead, Dr. Bailey adds. “We also need better connectivity so that for both quality indicators and pathogens we can automatically transfer laboratory results into data management systems,” he says. “We need a data management system that will allow companies to monitor the microbiology results from all their locations, even companies in different countries, in real time.”

New Technologies

Arun Bhunia, BVSc, PhD, professor of molecular food microbiology at Purdue University, focuses much of his research on the development and application of optical biosensors—including light scattering, fiber optic, surface plasmon resonance (SPR), and cell-based—all of which are used for pathogen and toxin detection.

A light scattering sensor is a laser-based entity. Shining this sensor on a bacterial colony growing on an agar plate enables researchers to record a scattered image. “That image is a fingerprint unique to a particular organism,” Dr. Bhunia says. “Without needing any DNA, reagents, or antibodies, we can identify a pathogen by comparing the fingerprint to those stored in a huge database. We already have fingerprints for many species and strains of E. coli, Listeria, Salmonella, Vibrio, and Staphylococcus.”

The time to identification depends on the amount of time needed for the colony to grow, as much as 12 to 15 hours for E. coli O157:H7, Salmonella, and Vibrio, and 24 to 30 hours for Listeria. “The benefit of light scattering is that organisms can be identified immediately, once colonies are available,” Dr. Bhunia says. This promises to be an improvement over the two to three hours now needed for PCR results.

Dr. Bhunia and his collaborators, a group that includes Purdue engineers, have built two such prototype instruments, which they call BARDOT (BActerial Rapid Detection using Optical light scattering Technology). One will soon be provided to the USDA for further testing. “We are exploring the possibility of commercializing this instrument,” Dr. Bhunia says. “It holds promise for identifying pathogens in raw and cooked product.”

Measuring the interaction between two molecules, SPR works when antibody is placed on a sensor and bacteria or toxin are added. “The pathogen will react with the antibody and change the angle of reflectance, which can then be measured,” Dr. Bhunia says.

A sensor features optical fibers with different antibodies. Binding of a target pathogen to the antibody-coated fiber can be detected using a fluorescent-labeled second antibody. The resulting fluorescent signal is proportional to the amount of target agents in a sample.

Dr. Bhunia has developed an innovative mammalian cell-based sensor in a 96-well plate format that makes it possible to test a large number of samples at once using a standard plate reader. This functional diagnostic sensor allows quick analysis—one to two hours—of the virulence potential of viable pathogens or active toxins.

“Recent developments in sensor technologies appear to be promising and sound exciting,” Dr. Bhunia says. “However, there are a few challenges we must continue to address to make this technology robust. As we push the boundaries of detection threshold, the sensor device becomes more susceptible to interferences from food matrices, background resident microflora, and inhibitors. In addition, bacterial physiology and genetic regulation of antigens that are essential for immune sensor-based detection should be well understood.”

Dr. Bhunia adds that sensor technology must be made more affordable, portable, and capable of testing multiple organisms. “Currently, light scattering instruments and fiber optic sensors cost $35,000 to $40,000. The challenge to private companies for the next level of commercial development is to make the technology as user friendly as possible.”

Leake is a food safety consultant and writer based in Wilmington, N.C. Reach her at llleake@aol.com or (910) 799-4881.

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