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GOVERNMENT AND SOCIETY Detecting pathogens on produce
Current detection methods An outbreak is declared by the Centers for Disease Control and Prevention after its network of certified laboratories reports multiple cases of food-borne illness caused by the same pathogen. These laboratories test stool samples from affected people by pulsed-field gel electrophoresis (PFGE) to obtain DNA fingerprints of the offending organism. (PFGE facilitates the differential migration of large DNA fragments through gels by constantly changing the direction of the electrical field.) After an outbreak involving products in interstate commerce becomes appar-
ent, the U.S. Food and Drug Administration (FDA) tracks down the source of the contamination. But finding pathogens on produce is much harder than detecting them in stool samples. One of the main challenges, says Keith Lampel of the FDA, is that the infectious dose of some produce-borne pathogens is quite low and the few
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Last September’s outbreak of E. coli O157:H7 sickened 205 people who ate raw spinach. Of those, 31 developed a serious complication called hemolytic uremic syndrome, and 3 victims died. During the same month, 183 people developed salmonellosis after eating tomatoes in restaurants. In 2001, the U.S. Department of Agriculture (USDA) initiated a Microbiological Data Program (MDP) to establish a microbial baseline for the domestic food supply. Each year, an update has been issued (www.ams.usda.gov/science/mpo/download.htm). In 2005, researchers found by PCR that 48 of 11,508 samples of produce tested positive for E. coli, including 12 that were positive for O157:H7. Of 11,512 samples tested for Salmonella, 47 were positive. These results indicated that the two major pathogens of concern weren’t frequently found on produce. Nevertheless, these bacteria have the potential to cause great harm, and contaminated produce can be distributed widely before such organisms are detected. Faster results than those obtained by current protocols would be advantageous for screening lots of produce before distribution and sale or if an outbreak of food-borne illness were suspected. Therefore, many scientists are devising quicker methods.
harmful bacteria may be masked by hordes of benign species found on particular food commodities. “Like looking for a needle in a haystack, our objective is somehow—usually by enrichment—to increase the numbers of the targeted pathogen,” Lampel says. On produce, E. coli O157:H7 and Salmonella are the prime sources of food-borne illness. These pathogens come from animal feces that contaminate runoff water or water used to irrigate, wash, or transport (as ice) produce. Wendy Warren-Serna of Food Safety Net Services says that although a different test is used for each bacterium, the basic principles for isolation and identification are the same. First, a food sample is placed in an enrichment broth that favors the growth of pathogenic bacteria. The enriched culture is then screened for a particular pathogen. If the screen is positive, confirmatory tests can be performed. Detecting one bacterial cell in 25 g of food is often considered the standard of sensitivity. The FDA’s Bacteriological Analytical Manual (www.cfsan.fda.gov/~ebam) outlines one conventional approach to
finding E. coli O157:H7 in produce. A 25-g sample is placed in enrichment broth that has been supplemented with antibiotics to kill competing microorganisms. After a 20–24-h incubation, the bacteria are selected from the broth with anti-O157 immunomagnetic beads. Those organisms are grown into colonies on various types of agar. Then, biochemical tests that look for known features of this pathogen sequentially document the inability to ferment sorbitol, indole positivity, the tendency to produce shiny colonies with dark centers on eosin methylene blue agar, and lack of -glucuronidase activity. Newer, rapid-screening technologies, including ELISA, immunoprecipitation, and PCRbased assays, are often used to decrease the time to results and improve sensitivity, Warren-Serna says. If those screening tests are positive and the biochemical profile is a match, the presence of E. coli O157:H7 can be confirmed by testing for two of its cellsurface proteins, O157 and H7, with commercial antisera. For further confirmation, colonies can be analyzed by PCR for the presence of genes that encode the organism’s toxins, called Shiga toxins. In addition, a DNA probe specific for E. coli O157:H7 can be used. It is directed at base 93 of the -glucuronidase gene, because that base is mutated in O157:H7. It generally takes 9–24 h to enrich and screen for E. coli O157:H7 and 24– 48 h to culture and screen for Salmonella. The confirmatory tests, including biochemical and serological characterization, take a further 3–5 days for E. coli and 4– 8 days for Salmonella, depending on the number of serological tests needed. “By that time, the specific batch of the product could be gone from the grocery store,” notes Arvind Bhagwat of the USDA. Shanker Reddy of the USDA says the researchers associated with the MDP are modifying this protocol to cut detection time. “We’re using better selective
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GOVERNMENT AND SOCIETY media and trying to get into real-time PCR,” he explains. Several rapid-screening methods that provide initial results in 30 min to 3 h after enrichment (which typically takes 6.5–8 h) are now commercially available. Real-time PCR is a popular approach to rapid screening. “But the specificity and sensitivity of some rapid detection methods are not consistent with those of the standard culture procedures that regulatory agencies generally use as a confirmation tool,” Warren-Serna warns. “Therefore, they may yield conflicting data.”
Irudayaraj also has embedded nanoparticles in IR-sensitive films and used spectroscopy and an immunocapture approach to detect pathogens (Anal. Chem. 2006, 78, 2500–2506). “Now [that] we have done biosensor-based approaches and spectroscopy-based approaches, the next step ahead for us is to combine these techniques with a simple sampling protocol to increase sensitivity and specificity,” Irudayaraj says. The infectious dose of pathogens on produce is quite low, and harmful bacteria may be masked by benign species, says Lempel.
Emerging techniques One of the new methods under development is a laser-based device called a “scatterometer” from Arun Bhunia and colleagues at Purdue University (Biosens. Bioelectron. 2006, doi 10.1016/j.bios. 2006.07.028). After enriching the bacteria in broth, the researchers grow colonies overnight on agar plates, which they place in the device unopened. When the laser beam shines through a plate, each colony acts like a lens, scattering the beam forward into a camera. The camera records the scattered light and delivers the images directly to a computer, which analyzes them with an algorithm developed by the team. Some unique features of the images include concentric rings, spokes, and bright central spots, which arise when light is scattered by unique bacterial byproducts, such as extracellular polysaccharides or toxic proteins. Joseph Irudayaraj at Purdue University is using vibrational techniques such as FTIR and Raman spectroscopy to detect wavelength shifts as light is absorbed and scattered from the surfaces of cells. The resulting molecular fingerprints depend on the abundance and types of lipoproteins, lipids, proteins, and polysaccharides on bacterial surfaces. “In the past, we have identified E. coli and Salmonella from apple juice using spectroscopy techniques,” Irudayaraj says. “The future is how we best can enhance the spectroscopy signal to provide the needed specificity.” 8
Evangelyn Alocilja at Michigan State University is also using nanotechnology to create biosensors (Biosens. Bioelectron. 2004, 18, 813–819). In this protocol, a sample is rinsed with buffer, and the buffer is then mixed with nanowires of polyaniline conjugated to antibodies that are specific for the pathogen under investigation. The mixture flows over a bridge coated with another specific antibody, which traps the pathogen in an antibody sandwich. Because the antibody on the bridge is immobile, the trapped nanowires make contact with electrodes on the sides of the bridge, completing an electrical circuit to generate a signal. To increase the likelihood that a 0.1mL sample will contain the pathogen of interest, Alocilja is developing nanowires that can be pulled out of solution with a magnet. Her group has tested the biosensor on lettuce, alfalfa sprouts, and strawberries spiked with E. coli and Salmonella and obtained results in 6 min. “We need a rapid, inexpensive, and user-friendly method that can be used by people in the field or at home,” Alocilja says. A rapid lab-on-a-chip device is the goal of Avraham Rasooly at the FDA. He aims to extract DNA directly from pathogens in food samples with a Flinders Technology Associates (FTA) membrane, which is currently used to collect blood samples from crime scenes.
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Chemicals in the FTA membrane concentrate and lyse bacteria and stabilize the resulting DNA. The DNA would then be eluted from the membrane, amplified by PCR or whole-genome amplification, labeled with a fluorescent dye, and hybridized into a microarray that contains DNA sequences from foodborne pathogens (Biosens. Bioelectron. 2004, 20, 684–698). Thus, results should be possible in a few hours rather than several days. “But this isn’t going to happen next year,” Rasooly cautions. At the University of South Florida, Luis Garcia-Rubio has been developing a rapid pathogen detection system that collects bacteria directly from water and places them in a cuvette for multi-angle, UV spectrophotometric analysis (Biosens. Bioelectron. 2004, 19, 893–903). The spectrophotometric data reflect an organism’s amino acid and nucleic acid composition as well as structural features such as the cell wall. “It’s like a fingerprint that tells us what the organism is and what is its state of development,” Garcia-Rubio says. To detect biowarfare agents in food and the environment, David Gottfried at the Georgia Institute of Technology is developing a planar optical waveguide interferometer. His model organism is Yersinia pestis, which causes bubonic plague. A quartz substrate with special coatings transmits light down two channels, creating an interference pattern. An antibody specific for the pathogen is placed in one channel, and a nonspecific antibody is put in the other. If bacteria bind to the specific antibody, they change the refractive index of the surface layer, and this alters the propagation velocity of the light. When the two beams are combined, the interference pattern is changed in a manner that relates to bacterial concentration. Samples will be liquids, such as water, fruit juice, homogenized produce, or rinse buffer, and they could be enriched through immunomagnetic separation. Mike Doyle at the University of Georgia is developing new Yersinia-specific antibodies for this system, because the current ones tar-
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PEOPLE get an antigen that is produced at body temperature but not at the lower temperatures at which food is maintained. The FDA’s Lampel says that although new detection methods are certainly
range of products, he says, “but along comes bug A in a food matrix not yet tested. Then we’re confronted with a situation in which we may have to tweak an existing method.” a—Linda Sage
needed, it will be just as important to adapt existing methods to the new food products that constantly come on the market. A current technique might be able to detect pathogen A in a broad
New Editorial Advisory Board members
From left to right: Christian Amatore, Harold G. Craighead, Barbara Finlayson-Pitts, Charles (Chuck) Lucy, Richard A. Mathies, Michael J. Sailor.
Six new members have been selected to serve 3-year terms on Analytical Chemistry’s Editorial Advisory Board. Established in the 1940s, the board is a vital link between the journal editors and the analytical chemistry community, providing guidance and advice on editorial content and policy. Christian Amatore, the director of the chemistry department at the École Normale Supérieure (ENS; France), earned his Doctorat d’État ès Sciences there. After serving as a visiting professor at Indiana University, Bloomington, he returned to ENS in 1984. His research focuses on electrochemistry, and he worked on the development of ultramicroelectrodes. He is especially interested in the theoretical aspects of dynamic chemical processes at electrodes and in electrical and mass transport under extreme conditions. He was elected to the French Academy of Sciences in 2002. Harold G. Craighead, a professor of applied physics and the director of the Nanobiotechnology Center at Cornell University, received his Ph.D. in physics at Cornell. His research activity involves the use of fluidic, optical, and mechanical nanostructures for molecular manipulation, chemical analysis, and
studies of biological systems with high spatial resolution. Barbara Finlayson-Pitts, a professor of chemistry at the University of California, Irvine (UCI), received her B.Sc. (Hons) from Trent University in Peterborough (Canada), and her master’s degree and Ph.D. from the University of California, Riverside, where she also conducted postdoctoral research. Her research focuses on understanding atmospheric processes at the molecular level, particularly heterogeneous reactions occurring in and on particles and surfaces. Her studies involve a variety of analytical techniques and are closely coordinated with theory, computer kinetics modeling, and field studies through collaborations with other faculty members and researchers at UCI and elsewhere. Charles (Chuck) Lucy, a professor of chemistry at the University of Alberta (Canada), received his B.Sc. from the University of Victoria (Canada) and his Ph.D. from the University of Alberta. His research seeks an understanding of the physicochemical behavior underlying separation techniques such as HPLC, ion chromatography, and CE. This understanding enables the development of new instrumentation and techniques.
Richard A. Mathies, a professor of chemistry at the University of California, Berkeley, earned his B.S. from the University of Washington and his M.S. and Ph.D. from Cornell University. His work in analytical biotechnology led to the development of high-speed, highthroughput DNA analysis technologies such as capillary array electrophoresis and energy-transfer fluorescent dye labels for DNA sequencing. He also pioneered the development of microfabricated CE devices and microfabricated integrated sample preparation and detection methods for lab-on-a-chip analysis systems applied to genomics, forensics, and space exploration. Michael J. Sailor, a professor of chemistry and biochemistry at the University of California, San Diego, received his B.S. in chemistry from Harvey Mudd College and his M.S. and Ph.D. in chemistry from Northwestern University. His research focuses on the chemistry, electrochemistry, and photophysics of nanophase semiconductors. Particular emphasis is placed on the use of silicon nanostructures in medical diagnostic devices; drug delivery; high-throughput screening; and low-power remote sensing of toxins, pollutants, and chemical or biological warfare agents.
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