In the world of virulent foodborne pathogens, Campylobacter and Salmonella have become the chief culprits, responsible for nearly 70 percent of all such illnesses in the U.S. last year. Detecting these and other bacteria in the food supply is a matter of growing urgency for both government regulators and the food industry. As the food chain expands globally, manufacturers are being held increasingly responsible for preventing outbreaks under the Food Safety Modernization Act (FSMA).
“We are making progress in detecting and responding more quickly to foodborne illness, but our priority remains preventing illnesses from happening in the first place,” says Susan Mayne, PhD, director of FDA’s Center for Food Safety and Applied Nutrition (CFSAN).
Campylobacter is commonly associated with consumption of raw or undercooked poultry and meat, while Salmonella is an issue in many types of food, including eggs, meat, poultry, fruits, vegetables, spices, and nuts. Both bacteria can cause mild to severe illness, from uncomplicated diarrhea to severe systemic infections, such as Guillain-Barré syndrome (Campylobacter), an autoimmune disease that can cause paralysis, and reactive arthritis (Salmonella), which can cause acute, debilitating joint pain.
“In order to decrease the likelihood of these pathogens in the food chain, it is essential to analyze the raw material, the environment where the food is produced, and food products at different manufacturing stages; for example niches where Salmonella could be harbored in the environment that could cause cross-contamination,” says Claudia Narvaez, PhD, professor of food science at the University of Manitoba, Canada.
Advances in laboratory and onsite testing equipment are allowing manufacturers to more easily and economically sample their raw ingredients, environment and facilities, and finished products for evidence of bacterial contamination, thus greatly reducing the potential for a recall, or worse. These developments include time-of-flight mass spectrometry, bacteriophage-based assays, novel biosensors, as well as advances in traditional techniques, such as polymerase chain reaction (PCR).
“Test methods continue to improve, with many methods now available that give results within 24 hours of sample receipt by the lab,” explains Timothy Freier, PhD, vice president for scientific affairs and microbiology at Mérieux NutriSciences (North America). But he also urges caution. “With these faster turn-around times, test methods are walking the line between incubation time and detection capabilities, so careful validation of these ultra-rapid methods is crucial,” Dr. Freier tells Food Quality & Safety magazine.
Accurately detecting and eliminating pathogens is increasingly essential for industry because advances in whole genome sequencing (WGS) are allowing public health agencies and government regulators to identify and trace foodborne contamination, such as Salmonella, back to specific growers and processing plants with increasing accuracy, faster and more cheaply than ever before.
“This is raising the bar for the food industry, as new food-illness associations are found,” Freier adds. “A combination of ingredient testing, finished product testing, and environmental monitoring are typically needed to control this hazard.”
Illnesses and Deaths
While exact numbers remain unknown, the CDC has estimated that about 48 million people in the U.S. get sick from a foodborne illness, 128,000 are hospitalized, and 3,000 die annually. The World Health Organization notes that Campylobacter is the world’s most common foodborne bacterial cause of diarrhea, responsible for more than 95 million illnesses and 21,000 deaths annually, according to a 2015 report.
In 2016, surveillance from labs in 10 U.S. states confirmed about 24,000 foodborne infections, more than 5,500 hospitalizations, and nearly 100 deaths caused by nine enteric pathogens commonly transmitted through food, according to the CDC’s latest Foodborne Diseases Active Surveillance Network (FoodNet) report, published in April.
Among the bacteria, Campylobacter and Salmonella led the pack, being responsible for 8,547 and 8,172 illnesses, respectively. The remaining pathogens were distant finishers, from Shigella (number three on the list with 2,913 illnesses) to the parasite Cyclospora (number nine with only 55 illnesses). While Listeria was close to the bottom in terms of prevalence (127 cases) it was the most virulent of all, with 97 percent of its victims requiring hospitalization and 13 percent of them dying.
Because FoodNet collects data from public health departments in 10 states, representing only 15 percent of the U.S. population, the nationwide numbers are much larger. Additionally, the actual number of foodborne illnesses always exceeds the number reported because many people who get sick do not seek, or necessarily require, medical treatment.
Not only does the FoodNet report “provide important information about which foodborne germs are making people sick in the United States,” says Robert Tauxe, MD, director of CDC’s Division of Foodborne, Waterborne, and Environmental Diseases, but “it also points out changes in the ways clinicians are testing for foodborne illness and gaps in information as a result.”
In particular, FoodNet counts infections diagnosed both by traditional, culture-based methods as well as those diagnosed using newer, culture-independent diagnostic tests (CIDTs). CIDTs, such as
immunoassays and nucleic-acid amplified tests, can be faster and easier than traditional culture-based methods, which also require use of trained personnel. CIDTs can identify a general bacteria type within hours without having to culture, or grow the pure bacteria strain (or isolate) in a laboratory, a process that typically takes days.
But without the isolate, public health scientists are unable determine the DNA subtype (“fingerprint”), its resistance pattern, or other characteristics necessary to detect outbreaks, track antibiotic resistance, monitor disease trends, and ultimately prevent outbreaks, CDC says.
For instance, PulseNet, the CDC-run network that connects public health and food regulatory agency laboratories, relies on the collection of DNA fingerprints of bacteria taken from sick patients to identify local and multistate outbreaks. The growing use of CIDTs is endangering PulseNet’s effectiveness. “Without a DNA fingerprint of the bacteria, CDC and public health labs will not be able to find, monitor, and prevent foodborne disease outbreaks, track antibiotic resistance, or follow trends to know if prevention policies are working,” CDC says.
Problems with Culturing
Regulatory bodies in both U.S. and the European Union are emphasizing the reduction of Campylobacter and Salmonella while increasing testing requirements, says Mike Clark, International PCR group manager, Food Science Division, Bio-Rad Laboratories. “Industry must be equipped to respond to these changes with testing solutions that are fit-for-purpose,” he tells Food Quality & Safety.
PCR techniques are based on amplification of the DNA of target pathogens. As the cost and complexity of genetic testing began to come down in the 1980s, PCR became commonplace in government and in company labs. Today, it remains among the most widely used approaches to detecting foodborne bacteria.
But because PCR also requires culturing, identification can still take many days. Advanced PCR tests, including quantitative and real-time PCR, can produce results more quickly by using probes and primers designed to target highly-conserved regions of the target genome.
“Many laboratories are using conventional methods combined with molecular methods for detecting these two foodborne pathogens,” Dr. Narvaez explains. For example, PCR is often combined with immunomagnetic separation that uses antibody-antigen interactions to detect very low levels of pathogens. “These are probably among the most-used methods used by industry and academia, and are approved by regulatory agencies,” she explains.
However, even these are not fast enough because they can take upwards of 24 hours, including enrichment, to produce definitive results. “Scientists are working on developing detection methods that can be sensitive and specific but also faster, ideally less than one hour with no enrichment, to obtain definitive results,” Dr. Narvaez says.
New Technologies and Approaches
Among the many developments in laboratory and onsite testing are advances in established approaches such as time-of-flight mass spectrometry; the invention of novel biosensors and assays using bacteriophages, enzymes, antibodies, nucleic acids, cell receptors, or polymers; nanotechnology-based sensors; and other rapid detection methods. The following are summaries of a few of these approaches.
MALDI-TOF. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry can determine the unique proteomic fingerprint of a bacterium relatively quickly and inexpensively. It compares the bacterial protein profile obtained from a culture to a library of known patterns. “Typically, no more than an isolated colony from a culture plate or a small aliquot from a broth is required,” explains Daniele Sohier, PhD, business development manager, industrial microbiology and diagnostics at Bruker Daltonik GmbH. “The entire method takes only a few minutes for a single sample, with results up to 24 hours faster than traditional methods,” she tells Food Quality & Safety.
Bacteriophage-based assays. Bacteriophages are viruses that infect bacteria. Because they are highly specific, interact quickly, and are harmless to humans, bacteriophages can be incorporated into novel assays and biosensors to detect, and in some cases even eliminate, foodborne pathogens.
Novel biosensors. These biosensors use biological elements, such as small molecules, proteins, or cells attached to a sensor surface to recognize or bind to specific targets or components of bacteria. Detection methods include label-free sensors, immunosensors, fluorescence-based, and carbon-nanofiber sensors.
Rapid microbial detection methods. These typically use fluorescent DNA markers to identify pathogens rapidly and accurately. These culture-independent platforms use fluorescent in situ hybridization, fluorescent microagglutination, and filter cytometry. Other rapid approaches include low-cost test strips to indicate the presence of a particular pathogen within hours. For example, paper- or film-based assays using stencil-printed carbon electrodes are able to detect E.coli and other bacteria within 4-12 hours.
WGS Still King
Despite these and other advances, WGS or next-generation sequencing, remains the gold standard for pathogen detection because of its high precision. As the cost declines, officials expect small WGS sequencers to proliferate among state and local public health agencies, as well as among private labs and manufacturing companies.
“We are looking at some very small sequencers that could fit in the pocket,” says Marc Allard, PhD, CFSAN’s research area coordinator for genomics. “We could have a lab in a briefcase that could go out to the consumer safety officer and actually do field testing. This is the future vision,” he says.
But because of its current complexity, cost, and other requirements, the food industry has largely steered clear of WGS. “The science and technology behind WGS are new and might seem a bit more complicated than the ones that have been in use by the industry for many years,” said Behzad Imanian, PhD, WGS project leader at the Institute for Food Safety and Health (IFSH) at Illinois Institute of Technology.
The amount of data produced by WGS can be “overwhelming,” Dr. Imanian told an IFSH symposium in May. The data analysis requires a proficiency in bioinformatics, which could be problematic for industry, while data interpretation “is far from simple, even for trained bioinformaticians,” he added. Thus, while U.S. and international food safety regulators are increasingly embracing WGS, the food industry has been reluctant. A notable exception has been the Consortium for Sequencing the Food Supply Chain, an initiative started in 2015 by IBM Research and Mars Inc. Recently joined by Bio-Rad Laboratories, the consortium is sequencing the genetic material of food and soil samples in order to create a “microbial baseline” to better understand the factors behind contamination and foodborne disease.
In 2011, the CDC, FDA, and USDA’s Food Safety and Inspection Service established the Interagency Food Safety Analytics Collaboration (IFSAC) to improve coordination of federal food safety analytic efforts and address cross-cutting priorities for food safety data collection, analysis, and use. In addition to Campylobacter and Salmonella, IFSAC’s efforts have been directed at E. coli and Listeria monocytogenes.
During its first five years, IFSAC developed a new food categorization scheme, which CDC now uses to classify food implicated in outbreaks, as well as an agreed-upon method for estimating sources of foodborne illnesses. In its latest strategic plan, IFSAC outlines three goals for 2017-2021: 1) improve the use and quality of new and existing data sources, 2) improve analytic methods and models, and 3) enhance communications about its analytic products.
- Data sources. Current sources, such as for foodborne illness outbreaks, are valuable but also incomplete and inconsistent. To address this, IFSAC will acquire additional data sources such as regulatory sampling data and WGS information. IFSAC will also work with state and local public health and regulatory agencies to obtain more and better outbreak reporting.
- Analytic methods. While there’s been progress in applying new methods and models for attributing foodborne illnesses, no best data sources or approaches have been identified. IFSAC will expand its scientific exchanges to identify gaps, develop ways to incorporate sporadic illness surveillance data, and integrate multiple data sources into estimates and analyses.
- Enhance communications. Because foodborne illness attribution is complex and constantly changing, effective communication with health practitioners, academics, industry, and the public is vitally important. IFSAC intends to improve and expand these relationships.
“It is always positive to see different parts of the government collaborating on food safety issues, although, as always, the issues are lack of funding and lack of time to move things forward,” says David Acheson, MD, founder and CEO of the Acheson Group and a former FDA associate commissioner for foods. “But we should always be looking to do more because there is always more to do,” he says.