October 5, 2004
New biosensor rapidly detects deadly foodborne pathogen
WEST LAFAYETTE, Ind. - The pathogen responsible for a precooked chicken recall last summer will become easier to detect in ready-to-eat meats, thanks to a new biosensor developed by scientists at Purdue University.
A team of food scientists has developed a sensor that can detect the potentially deadly bacteria Listeria monocytogenes in less than 24 hours at concentrations as low as 1,000 cells per milliliter of fluid - an amount about the size of a pencil eraser. The sensor also is selective enough to recognize only the species monocytogenes.
"The selectivity, sensitivity and rapidity of this sensor represent a vast improvement over the types of test kits that are currently available commercially," said Arun Bhunia, professor of food microbiology and one of the sensor's developers. "Taken together, those qualities make this research an important contribution in the field of food safety."
Listeriosis, the illness caused by consuming Listeria-contaminated foods like deli meats or cheese, leads to higher rates of hospitalization and mortality than any other foodborne illness, said Tao Geng, research associate in the department of food science and the sensor's co-developer.
"The mortality rate for people with listeriosis is very high, and for this reason, the FDA has a zero-tolerance rule for Listeria. There should be none at all in any ready-to-eat products," he said.
According to the Centers for Disease Control and Prevention, approximately 2,500 people develop listeriosis every year, and approximately one in every five cases is fatal. The elderly, pregnant women, newborn infants and individuals with compromised immune systems are most at risk of contracting the disease.
The bacteria classified as Listeria include six different species, but only L. monocytogenes can infect humans. This makes it especially important to develop highly selective sensors that can detect only L. monocytogenes, Bhunia said.
"The ability to distinguish this one species from all others makes this a very powerful sensor. No other sensor today can do that," he said.
The sensor also is selective enough to recognize cells of L. monocytogenes when other types of foodborne contaminants, such as salmonella or E. coli, are present.
Known as an "optical biosensor," the device uses light to detect the presence of a target organism or molecule. Bhunia and his colleagues have been developing this sensor for three years and demonstrate its function in the current issue of the journal Applied and Environmental Microbiology.
The sensor is made of a small piece of optical fiber - a clear, solid, plastic material that transmits light through its core. The fiber is coated with a type of molecule called an antibody, which specifically recognizes L. monocytogenes and captures it, binding it to the fiber. When the fiber is placed in a liquid food solution, any L. monocytogenes in the sample will stick to the fiber.
The presence of L. monocytogenes is verified by the addition of a second antibody, which not only recognizes L. monocytogenes but also carries a molecule that produces a fluorescent glow when exposed to laser light. This antibody attaches to the L. monocytogenes bound to the fiber and acts as a flag, signaling the pathogen's presence when laser light is passed through the liquid.
Bhunia expects the sensor to be ready for industrial use in another year.
Many tests currently in use require a high concentration of pathogen cells - typically from 1 million to 10 million cells per milliliter of fluid, Geng said. The tests also rely on a process known as "enrichment," which occurs when a sample believed to be contaminated grows for a period of time in a nutrient broth to allow any pathogen cells present to multiply.
The enrichment process increases the concentration of cells present, making it possible for today's sensors to detect their presence, but it can take as long as seven days to complete a test using conventional methods, Geng said.
Other tests rely on DNA markers, but these also can take days to process, he said. That's a problem because by the time test results come back, products may already be in food suppliers' warehouses or on store shelves, he said.
Last summer, for example, a Georgia company recalled nearly 37,000 pounds of precooked chicken products that may have been contaminated by Listeria. The chicken products had been distributed to warehouses in Georgia and Arkansas, as well as to grocery stores in Maryland and New York, when the recall was issued.
"To overcome the time delay and allow for rapid detection before foods are shipped, you need to be able to detect a lower number of the pathogen cells at the processing plant," Geng said.
The ability to detect L. monocytogenes at low levels is essential because most of the foods susceptible to Listeria contamination are ready-to-eat products, which are cooked or otherwise processed for human consumption before they make it to a grocer's shelves.
"Since precooked meats have already been processed, the bulk of microorganisms that were present in the raw product have been eliminated," Bhunia said.
"We don't expect high numbers of microorganisms in processed products, so we need to be able to detect extremely low levels of contamination."
Detection at low levels also is important for another reason, Bhunia said.
"Listeria can grow at refrigeration temperatures, so if a product has a level of Listeria low enough to evade detection when it's tested at the processor, that Listeria still can grow in the home refrigerator to a level that makes it infective to people at risk."
While Bhunia said there's no known precise number of cells it takes to infect someone, most food safety experts suggest from 100 to 1,000 cells can cause illness.
Cooking would kill many of the L. monocytogenes cells that can grow at refrigeration temperature, but many ready-to-eat products, such as deli meats, smoked fish, cheeses and hot dogs, aren't always cooked by consumers before consumption, Bhunia said.
Bhunia said his next goal is to optimize the test conditions of the biosensor so a sample can be processed in one working day and be monitored remotely via computer.
"Our end goal is to use this technology to keep Listeria monocytogenes-tainted foods out of the food supply," Bhunia said. "To do this, we will continue to develop ways to make this device more user-friendly."
Bhunia's research was supported through a cooperative agreement with the Agricultural Research Service of the U.S. Department of Agriculture and the Center for Food Safety Engineering at Purdue. Mark Morgan, associate professor with the Purdue Department of Food Science Sensors and Controls Laboratory, also participated in the research.
Writer: Jennifer Cutraro, (765) 496-2050, firstname.lastname@example.org
Sources: Arun Bhunia, (765)494-5443, email@example.com
Tao Geng, (765) 496-3824, firstname.lastname@example.org
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A publication-quality photo is available at http://ftp.purdue.edu/pub/uns/+2004/bhunia-sensor.jpg
Detection of low levels of Listeria monocytogenes cells using a fiber optic immunosensor
Biosensor technology has great potential to meet the need for sensitive and near real-time microbial detection from foods. An antibody-based fiberoptic biosensor to detect low levels of Listeria monocytogenes following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody C11E9 was used to generate a specific fluorescent signal, which was acquired by launching a 635 nm laserlight from an Analyte-2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed significantly higher signal than other Listeria species or other microorganisms including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcesces in pure or mixed culture setup. Fiberoptic results could be obtained within 2.5 hours of sampling. Sensitivity threshold was about 4.3 x 103 CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1x104 CFU/ml or 2.8x107 CFU/ml, respectively. In less than 24 hours, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 100 CFU/g after enrichment in buffered Listeria enrichment broth.
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