There’s a potential new tech in town. This one will help us with a common enemy: Salmonella.
Salmonella: The Details
Salmonella is the bacteria responsible for the illness salmonellosis, that according to the United States Centers for Disease Control and Prevention (CDC) is responsible for 1.2 million illnesses in the United States each year. About 1 million of these cases are caused by a food source, resulting in about 19,000 hospitalizations and around 380 deaths each year.
Salmonella belongs to the family Enterobacteriaceae, a facultative anaerobic (live and thrive in environments with or without oxygen) bacilli (rod shaped) that is predominantly motile (can move on its own). The genus Salmonella is made up of two species: enterica and bongori. All together there are over 2500 serotypes within the species. Most of the serotypes cause some form of gastroenteritis, though some such as S. Typhi, S. Paratyphi A, S. Paratyphi B, and S. Paratyphi C (typhoidal salmonellae) are responsible for enteric fever (can cause high fever, abdominal pain, and general malaise without diarrhea or vomiting).
While most people will only experience mild symptoms, some higher risk groups in the population in addition to the possibility to experience more severe symptoms are also of higher risk to becoming infected. The very young, the very old, and those with a compromised immune system are among the higher risk populations.
In general, symptoms of abdominal cramps, fever, and diarrhea begin between 12 and 72 hours after exposure with illness lasting for about 4 to 7 days. Expect to be sick for about a week, though normal bowel movements may not return for quite some time and additional complications may arise. Most will recover without treatment, though some may become severely dehydrated, requiring hospitalization. Often the illness will run its course if the bacteria stay inside the digestive tract. More serious complications may occur if the bacteria have an opportunity to leave the digestive tract and the infection can enter the blood stream or other body sites. Unless treated quickly with antibiotics, this type of infection may become fatal. A small number of those infected may develop a long-term illness known as reactive arthritis, causing joint pain, eye irritation, and painful urination. Reactive arthritis could last anywhere from months to years and can result in chronic arthritis.
Salmonella foodborne illness is serious business. With so many people becoming ill, the ability to identify Salmonella bacteria in clinical specimens, in food products, and in manufacturing environments is a key area of technology research. Traditional methods of culture are a primary diagnostic tool for identifying Salmonella, but new and faster methods are emerging. These new methods include genetic testing such as Polymerase Chain Reaction (PCR), Real-time PCR, and sequencing. Other methodologies include Immunomagnetic Separation (IMS), Mass Spectrometry, including matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Other fast tests including Hygiena InSite rapid test swabs and Menon Biosensors M2 technology to name a few.
Culture: The Gold Standard
State and Federal guidelines use traditional microbiological culture techniques as the gold standard. This testing methodology involves selective growth mediums, swabbing the sample onto the medium, and incubating for anywhere from a few days to weeks. If the bacteria grow, then the pathogen is present. This method can also be used to identify sensitivities to antibiotics. A series of growth media laced with different antibiotics allows the technician to report which antibiotics will work for the pathogen, and which ones will not.
While this is a strong diagnostic method, it often takes too long for effective monitoring applications. Particularly if the test is performed to detect pathogens present in food items with short shelf lives. While this slower but more accurate technology is great for clinical diagnostics and treatment, a faster protocol is necessary for prevention measures.
Pros: “Gold Standard”, Accurate
Cons: Slow Turn-around-time
PCR and Real-time Quantitative PCR (qPCR)
PCR and qPCR methodologies involve molecular analysis of DNA extracted from samples of food or human specimens. Research has identified short sections of DNA specific to Salmonella bacteria that are used as a template in a molecular reaction. This template serves as a target DNA sequence the test is based on. The reaction involves a replication process which allows an analyst to identify and measure DNA that matches the target template to indicate the presence of the pathogen.
This method is extremely fast and accurate results can be obtained in as little as a few hours. The downside is that it requires expensive equipment, trained personnel, and the sample must be cleaned up to provide a clean DNA sample into the PCR and/or qPCR reaction. Additionally, the method is DNA based, so there is no distinction between live and dead cells. Unlike culture, a positive result does not indicate whether the pathogen is alive or has been killed. With culture, if the bacteria reproduce, the cells are alive. With PCR and qPCR, a positive result indicates the presence of target DNA, which does not differentiate between alive or dead bacteria. Sample size is another limitation of PCR and qPCR. The limit of detection of qPCR is around 102 cells per reaction. Food that are naturally contaminated may not contain enough cells to generate a positive result.
Pros: Fast, Accurate
Cons: Cannot distinguish alive or dead
Enrichment Techniques to Solve Limit of Detection Issues
Solving the limit of detection issue is as simple as allowing the sample to sit for 6 to 24 hours in an enrichment medium. This allows bacteria to increase in number in a controlled environment so that it can be detected with methods where a limit of detection is a hurdle. Unfortunately, Salmonella will not be the only bacteria to grow in the enrichment medium. The sample must be cleaned up a bit to ensure proper results.
Enrichment techniques are particularly useful for methods that require a significant amount of the bacteria of interest to achieve reproducible results. This method is often employed in many detection techniques to ensure appropriate sample load.
A method known as Immunomagnetic Separation (IMS) allows selective binding to paramagnetic beads to achieve this cleanup process. This technology is used for Salmonella testing by adding anti-Salmonella antibodies bound to paramagnetic beads. A magnet is applied to the sample and everything that is not Salmonella attaches to the beads. The resulting sample should be mostly Salmonella bacteria. Allowing selection for Salmonella specific bacteria enables a shorter enrichment time to increase the sample size.
Pros: Selects for specific bacteria,
Cons: Requires specialized materials, equipment, and trained staff
Mass spectrometry is an identification method that relies on chemical makeup and size of a particular sample. In the case of bacteria identification, a fingerprint is obtained for a particular bacterial cell expressed proteins. The approach for this methodology doesn’t look at the organism, but the proteins associated with organism. Anywhere from tens to even hundreds of proteins can be measured in a single experiment.
Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a common used technique using mass spectrometry. Reference libraries allow technicians to compare reference spectra. Essentially, different parts and expressed proteins of the cells have different sizes and consequently travels through the spectrometer at different speeds. This creates a reproducible spectra image that allows technicians to identify the organism of interest.
Pros: Hundred of proteins can be measured per experiment.
Cons: Very expensive equipment
Next Generation Sequencing
Next-generation sequencing (NGS) or whole genome sequencing (WGS) involves sequencing the entire genome of bacteria in just a few hours. The data is compared to a public health database such as GenomTrakr to identify and connect the results of the sample with others already reported. This specificity can track down the exact source and connected samples in the outbreak.
In addition to identifying the pathogen of interest and traceability of the bacteria, characteristics of the bacteria, such as antibiotic resistance can be extrapolated. Genetic data can allow investigators to fairly accurately predict antimicrobial susceptibility patterns.
Whole genome sequencing allows for more accurate serotype classifications, particularly for serotypes of closely related isolates. Increased genetic data increases the resolution that separates similar looking bacteria from each other. Identifying specific serotypes is monumental is discovering related cases. This allows more information for the investigators to track down the source of the outbreak so that the issue can be mitigated and save more people from falling ill.
Pros: Fast Turn-around-time, Genetic information allows for characteristic extrapolation
Cons: Expensive equipment and requires highly trained technicians
Fast Screening Commercial Methods
Commercial companies are working on fast screening methods to help screen foodstuffs and monitor production and processing areas. Traditional methods often require packaging the sample, shipping it off to the laboratory, waiting on handling and testing times, then waiting on results. Time is precious when handling perishable products. Farms and food manufacturers want to get their products onto store shelves and into consumers hands as soon as possible. Quite literally, time is money. These fast kits help manufacturers and farmers save both time and money.
The Hygiena InSite Salmonella swab allows users to test their own samples using a color indicator test. The swab device is a large sponge swab pre-wetted with a chemical designed to neutralize the counter effects of any residual sanitizers. Additionally, it includes a pre-enrichment broth that allows the bacteria to reproduce to detectable numbers, allowing a smaller sample to reach the limit of detection.
The enrichment broth contains all of the nutrients that will allow the bacteria to grow and thrive to promote rapid growth. Current tests allow for the user to opt for a 6-hour or 24-hour pre-enrichment step. A 24-hour pre-enrichment kit requires fewer materials and less user manipulation than the more rapid 6-hour pre-enrichment kit. After the pre-enrichment phase has taken place, results may be obtained in 48 hours.
The test produces a color change to indicated a “presumptive positive” result. The color change is achieved by using a selective enrichment media in addition to the pre-enrichment media. As the Salmonella bacteria reproduce, the Salmonella Indicator Broth, PDX-SIB, changes from a purple to yellow color.
According to Hygiena, there are currently around 40 commercially available Salmonella detection methods. Hygiena sets themselves apart by allowing a testing method that does not require a skilled operator. Hygiena explains that adding the human element can be time-consuming and present the chance of errors at several stages of the process. Hygiena explains that the product InSite’s unique selling point is the “straightforwardness” of the product. There is a reduced risk for cross-contamination, and the active culture can be retained for additional verification or identification tests.
Pros: Easy identification of positive specimens, Retains active culture for future tests
Cons: Requires a two-step process with pre-enrichment
Menon Biosensors provides products driven by molecular biochemistry applications such as DNA analysis of biological pathogens. The M2 platform was previous classified by the United States government and offers both sensitivity and specificity while minimizing sample preparation.
M2 uses Nuclear Magnetic Resonance or NMR to detect pathogens such as Salmonella. This technology does not require the use of sample enrichment culture media. Removing this step decreases sample processing time by up to 24 hours compared to other methods that require pre-enrichment to meet limits of detection. Turn-around-time is greatly improved, allowing between 4 and 24 assays to be performed with results is as little as 1 to 2 hours. This system allows for high-throughput and early detection of pathogens found in potentially contaminated food products.
Laboratory tests have shown that cross-reactivity of other pathogens does not occur, enabling the technician to be sure that a positive result is indicative of a presumptive Salmonella contamination. At this time the Menon M2 technology operates in a seafood matrix. The Menon team is working on expanding to other food matrices such as poultry, eggs, dairy products, and more.
Cons: Only limited to seafood matrix for Salmonella detection at this time.
Choosing a Method
Choosing a diagnostic tool for the detection of Salmonella bacteria requires an understanding of the pros and cons of the available methods as well as the time constraints associated with particular need. For example, highly perishable foods and severely ill patients will need a faster turn-around time. Individuals with a potential antibiotic resistant infection need accuracy and highly selective results. As the need for sensitive and accurate timely results increases, more faster methods will make their way into the diagnostic sector.
MakeFoodSafe will continue to monitor new technologies as they become available.
By: Heather Van Tassell, Contributing Writer (Non-Lawyer)