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Comments about “Modified Polymerase Chain Reaction Distinguish between Live and Dead Bacteria”

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The blog about PCR and distinguishing between live and dead bacteria have drawn some comments as shown below:
Gerold Schwarz You might have a look for Reagent D – we established a protocol for Enterobacteriaceae in instant milk formula and Yeast and Mold in dairy products – it works similar utilizing a halogen light in addition source to eliminate dead cells before amplification.
 Gerold Schwarz Send the following additional Information:
 By: Dr. Gerold Schwarz, Produktmanager BIOTECON Diagnostics GmbH
 

Elimination of DNA form dead cells prior a PCR-Setup:

Reagent D is designed for the rapid elimination of DNA from dead cells to avoid false-positive PCR results. The reagent contains a light sensitive substance which can penetrate the cell membranes of dead cells, whereas the outer membrane compartments of living cells can actively protect their cytosolic compartments.
  1. After a brief incubation of a freshly prepared enrichment culture with reagent D.
            
  1. The complete Assay is exposed 5 minutes to a high-power halogen light source.
 
  1. Isolate/ extract DNA
  1. Run PCR
After incubation with light the DNA is irreversibly linked and amplification is blocked.  
 
  1. Final PCR Results and no or low false positive rates.
We have tested and validated the procedure for our foodproof Enterobacteriaceae plus Cronobacter Detection Kit and the foodproof Yeast & Mold Quantification LyoKit and third with the foodproof Vibrio Detection LyoKit.

Other Comments:

Relating to the original Blog:
Angela Aucoin This could be invaluable in reducing or eliminating triple re-examination costs of false positive environmental samples
Peter Ball Similar methods using propidium monoxide and ethidium monoxide have been published multiple times and are well known to most working with QPCR.
 
Francesc Codony Iglesias  The dynamic approach in vPCR has been suggested in the past , mainly by Dr Soejima working with different chemical compounds based on Pt and Pd. In the patent appointed by Ruth the authors also are following the dynamic approach, with a phenantridinium dimer. Although the dye interaction is quite strong, it’s not irreversible therefore during PCR denaturation this DNA will be available for amplification. For this purpose I opine that photo-reactive phenantridiniums are better reagents because their reagents can be activated by light and the binding to DNA is irreversible. Otherwise during sampling transport and handling, it’s quite critical in some clinical and food applications, the reagent will remain active affecting post sampling damaged cells. Regarding the patent, probably the inventive novelty can be refuted during future examinations.
 
John Mackay Use of PMA etc is patented – does this method vary sufficiently? Although doesn’t seem to need cross-linking. Patent describes a comparison among the dyes.
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Rapid Methods for Pathogen Detection in the FSMA Era

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Food online just published my guest column “Rapid Pathogen Detection Methods — What To Consider” Below are exerts from this blog:

Why Rapid Methods?

Rapid methods of pathogen testing have been gaining acceptance in the food industry. Recent advances in technology result in faster detection and identification of pathogens, more convenient, more sensitive, more reproducible, and more specific than conventional methods. The main reasons for their adaptation are because faster results mean:
  • Faster intervention and corrective actions
  • Fewer lost lots or reduced amount of product in a contamination event
  • Faster reaction to a problem
  • Improved throughput and reduced warehouse space
  • Decreased manufacturing cycle through faster release of inventory
  • Ability to link strains of pathogens to a specific case
  • Accelerates root cause analysis
  • Rapid pathogen testing can be useful in preventing an outbreak of illness
For ready-to-eat products, the new FSMA proposal requires environmental pathogens to be controlled. As a result, environmental pathogen testing is required in some segments of the industry. In most cases, the cost of assay materials increases with new methods. However, the operational and financial benefits far outweigh the expense.

Available Methods

It is important to remember, in most foods, rapid methods still lack sufficient sensitivity and specificity for direct testing of the food. Therefore, the foods still need to be enriched in a culture media before the rapid method analysis. New methods include antibody-based assays, genetic amplification methods, and newer sensor development methods.  The Food OnLine article discusses in more details:
  • Growth-based methods
  • Immunological-based methods
  • Molecular detection methods
  • Biosensor devices
  • Whole Genome Sequencing (WGS)
 

What About FSMA?

FSMA requires food producers, processors, manufacturers, and service providers to certify their products are free from pathogens, such as Listeria, Salmonella, and pathogenic E. coli. The testing results obtained must use valid pathogen testing protocols.
Typically, all methods for pathogen testing are validated by vendors through an organization, such as AOAC International  , MicroVal, or AFNOR, either by an independent laboratory or a more stringent multi-laboratory study. However, according to FSMA, all microbiology methods utilized must be proven to be adequate for the particular products tested. Validation is required to demonstrate the method is equivalent to the reference method (for the matrices validated). Method verification will demonstrate it achieves an acceptable level of precision and accuracy, and there are no matrix effects or interference when utilized for the particular product(s) of the company.

Laboratory Accreditation

FSMA mandates laboratory accreditation with the objective to align commercial laboratories with government labs. This would assist the acceptance of analytical data, improve the efficiency of government labs, and support the testing of food imports. The FDA establishes an accredited third-party certification body that must be used in a laboratory accreditation. The accreditation is done to the ISO 17025 standard, or equivalent. These accredited labs will report results of public health concern directly to the FDA. The agency is required to establish a registry of accrediting bodies and accredited laboratories that includes laboratory contact information. Laboratories must be accredited for the particular sampling or analytical testing methodologies used to analyze their particular products. The new rules are placing greater emphasis on laboratory expectations, technical competence, and use of validated methods, and thereby, improved consistency across the industry in producing reliable data. According to Tom Wechler, lab accreditation is not inconsequential. A sizable initial investment is required in order to put systems in place and provide proper training for staff. The review fee for accreditation can run $15,000 or more and, once accredited, labs can expect additional ongoing costs for staffing, management and overall compliance.

Pathogen Testing Market Size

The demand for microbiological testing in the food industry is higher than ever before. Pathogen testing seems to account for over 50 percent of the entire microbiological testing market and its growth rate is three times greater than that of the total market. For more details go to Food OnLine article.
 

Difficult Choices

While the new methods offer advantages in technology, speed, and accuracy, they represent an incremental progress, rather than a revolution. It seems like every so often we hear about a new better methodology coming to market.
With the large number of competitors in the pathogen testing arena, it is hard to differentiate among the technologies and find the clear winners. Every small progress of one technology is soon followed by a competitor that makes it slightly better.
With over 40 different assays on the market it is increasingly more difficult for companies to choose a new rapid method and to decide to invest in it. This might be one of the reasons that laboratories. Small- and mid-sized food plant labs are closing, and looking to contract testing labs for their pathogen testing. The extensive validation requisite as part of FSMA, as well as the increased training and documentation requirement, is driving smaller manufacturers toward contract laboratories. Some of the difficult choices include:
  • Internal (In-House Testing Lab) Or External Testing Lab Internal laboratories can yield faster results. With faster results comes faster access to data. However, increasingly sophisticated and price competitive contract laboratories offer a good alternative. Furthermore, food company customers — including global food retail and food service companies — are requesting analytical results provided by an accredited third-party lab rather than the food plant itself.
     
  • Standard Methods Or New Methods? If New, Which New Method Should You Use? Newer and more rapid method are generally more sensitive, specific, time-efficient, labor-saving, and reliable than conventional methods and are currently more frequently used. Most of the U.S. market has moved to newer methods, while close to 50 percent of the European market is using the newer methods. Technology continues to evolve at a faster pace and the next generation assays are being developed.
Currently, numerous technologies are under development, pushing the limits of current methodologies. Some are trying to move current technologies forward, such as digital PCR, next generation sequencing. One essential step is the separation or concentration of the organisms from the sample prior to the utilization of rapid methods.
One technology attracting interest is Magnetic nanoparticles (MNPs) due to its super-paramagnetic property and large surface-to-volume ratio as excellent ligand attachment. There is a need for automation and the removal of manual steps that slow down the assay time. We expect constant progress in new method development over the next couple of years.
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Modified Polymerase Chain Reaction Distinguish Between Live and Dead Bacteria

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DNA-based diagnostic, such as Polymerase Chain Reaction (PCR) tends to overestimate the number of live cells because it will also measure the DNA from dead cells. This is because of the relatively long perseverance of DNA after cell death (e.g. up to 3 weeks). DNA extracted from a sample can originate from any cell, regardless of its metabolic state. Consequently, most DNA-based diagnostics cannot distinguish between live and dead bacteria, and this is a major drawback of the techniques.
After processes such as pasteurization or disinfections, dead bacteria might be present. While the dead bacteria present no hazard it can still be detected by PCR. Injured cells are virulent and may or may not be detected by standard procedures. PCR offers a more rapid and sensitive method than culture-based techniques, but the major limitation is the lack of differentiation of DNA from live or dead bacteria. In food matrices and the environment, DNA can be very stable and persist for extended periods of time, and therefore, it is desirable to have DNA-based assays that can identify only viable organisms.
A study by Tuskegee University researchers (Drs. Temesgen Samuel, Woubit S. Abdela, and Tsegaye Habtemariam with Yehualaeshet) allows the separation between live and dead bacteria. “PCR has been developed decades back, and the time spent for conventional PCR and our PCR — we call it viability PCR — protocol is the same,” Yehualaeshet said . “The novel aspect of our patent is that we developed a modified sample preparation, which enables the PCR to detect only viable, or live, bacteria.”
“During the sample preparation for PCR, we used a safe compound which will be ideal as a routine detection protocol for the presence of viable organisms. This invention will be mainly beneficial, but not limited, to the food industry to monitor biological decontamination, disinfection or the sanitization process.” That’s important, Dr. Yehualaeshet said, because the risk of contamination and disease comes from live bacteria. “If a detection method could not differentiate the dead from the live bacteria, then there is always a risk of false positive alarm”.
The research team from Tuskegee just got a United States Patent no. 9434976, for the rapid and more reliable detection of viable foodborne, pathogens and other infectious microbes using modified Polymerase Chain Reaction sample preparation. It provides a method of detecting the presence of a live microbe in a sample. The method comprises of:
  1. Isolating the microbe from the culture;
  2. Adding Gel Red™ dye to the isolated microbe from step (a);
  3. Extracting DNA from the microbe after step (b);
  4. Performing PCR on the DNA from step (c);
  5. Analyzing PCR results from step (d) for the presence or absence of amplified DNA using real time PCR and further gel electrophoresis confirmation; and
  6. Correlating the presence of amplified DNA from step (e) with the presence of live bacteria in the test sample. It may be desirable to further confirm that no viable bacteria were present by culturing on an appropriate media after heat and isopropyl alcohol inactivation of the culture.
The key ingredient used in the assay is Gel Red™ a DNA-intercalating chemical that is highly selective in penetrating only into dead bacteria (not live). The penetrating chemical binds with the DNA and blocks amplification of the targeted gene. It is claimed to be extremely stable and environmentally safe fluorescent nucleic acid dye.
“If there is no live organism after sterilization, it means the chemical used and the sterilization process works well. Therefore, the best detection technology should be able to detect only viable bacteria.”
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Multi State Outbreak of Listeriosis due to Soft raw Milk Cheeses

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The recall

The Center of disease Control (CDC) and the US Food & Drug Administration (FDA) are investigating a multistate outbreak of Listeria monocytogenes infections in raw milk cheeses.  Six people from four states were hospitalized, and two people from Connecticut and Vermont died. One illness was reported in a newborn.
The FDA found Ouleout cheese contaminated with L. monocytogenes. Epidemiological and laboratory data indicates that the source of the infections is the cheese made by Vulto Creamery of Walton, NY. Whole Genome Sequencing (WGS) performed on clinical isolates from all six ill people shows that the isolates are closely related genetically. This close genetic relationship provides additional evidence that people in this outbreak became ill from a common source.
The same strain of Listeria was identified in samples taken from three intact wheels of Ouleout cheese collected from Vulto Creamery. As a result on March 7, 2017, Vulto Creamery recalled all lots of Ouleout, Miranda, Heinennellie, and Willowemoc soft wash-rind raw milk cheeses.
The Connecticut Department of Public Health collected leftover cheeses from the deceased person’s home in Connecticut. The outbreak strain of Listeria was identified in a leftover cheese that the family identified as Ouleout cheese from Vulto Creamery.
The New York Division of Milk Control and Dairy Services collected three intact wheels of Ouleout cheese from the Creamery during a joint inspection with FDA. The outbreak strain of Listeria was identified in samples taken from the three wheels of cheese. Investigations concerning this outbreak are ongoing, and more cases are believed to become linked in the future.
The Company recalled cheeses of Ouleout, Miranda, Heinennellie, and Willowemoc soft wash-rind raw milk cheeses. The cheeses were distributed nationwide, with most being sold at retail locations in the northeastern and Mid-Atlantic States, California, Chicago, Portland, Oregon, and Washington, D.C.
On March 11 the FDA had increase the recall to include all the lots of the previously recalled cheeses and four additional cheeses.  As a result Vulto Creamery has recalled the following eight cheese items: Heinennellie, Miranda, Willowemoc, Ouleout , Andes, Blue Blais, Hamden & Walton Umber due to potential contamination of Listeria monocytogenes

Creamery History

Jos Vulto, an artist in trade, came to the United States from the Netherlands in 1990.  According to the company’s website, before Vulto started the business, he “had been making cheese in his apartment, in Brooklyn NY, for about five years, aging it under the Brooklyn sidewalk. He had never set out to become a cheese maker but after half a year of experimenting and favorable reception of some of his creations, he started to explore the possibility of starting a creamery.”
Expanding his business, he moved to Walton, N.Y., in the Catskills in 2012, closer to the source of his milk. Ouleout was an instant hit when it came out a few years ago. Mr. Vulto quickly earned a reputation among cheese lovers. When Vulto started creating Miranda in 2012, his aim was to make something that evoked his immediate geography.
During aging, the cheese are washed with Meadow of Love absinthe, made by Delware Phoenix Distillery (down the street from the creamery) from a variety of herbs, many grown in New York State. “I wanted something hyperlocal,” Vulto says.  “When Miranda is at its best, the influence of the absinthe wash is forward without being overwhelming,”. “The flavor is a complex but well-balanced swirl of brothy, meaty, and earthy, with nutty and grassy notes and a hint of fried onions on top.”
While getting accolades for the cheeses, the operation of manufacturing these cheeses was amateurish, with 3 part time employees and handmade operation, with limited controls over manufacturing.

The Lawsuit 

The widow of Richard Friedman, Vermont resident Veronica Friedman, has filed suit against Vulto Creamery after their cheese tainted with Listeria hospitalized her husband and led to his death. Ms. Friedman is alleging wrongful death as well as emotional and financial damage.
The Friedman’s purchased raw milk cheese that was manufactured by Vulto Creamery, cheese contaminated with Listeria, sometime in early October. Mr. Friedman’s symptoms began to take hold around October 11, 2016, when he went to the Emergency Room at Brattleboro Hospital. He was transferred to Dartmouth-Hitchcock medical center on October 12, where he stayed for a week and a half before being transferred to Mt. Ascutney Rehab in Windsor, Vermont. On October 31, at the rehab facility, Richard Friedman suffered a massive stroke, and was airlifted back to Dartmouth-Hitchcock where he died on November 2, 2016. The stroke was caused by the Listeriosis infection.

Advocates of Raw Milk

Advocates of raw milk believe that pasteurization destroys much of the beneficial bacteria in raw milk, enzymes and micro nutrients found in raw milk, and that the risk of bacterial contamination is very low given today’s modern, well-managed dairies. Others simply prefer the taste and freshness of locally produced milk over the longer shelf life and additives in commercial milk.
Some consumers say raw milk has more flavor and makes better cheese. Others choose unpasteurized milk as part of a broader shift away from processed foods, which are increasingly seen as unhealthy.
Raw milk has garnered increasing interest and attention nationwide in the last 10 years or so, as a small, but growing, segment of nutrition-conscious Americans choose it over commercially produced and pasteurized milk for taste and health reasons. 
Raw milk products are illegal in 20 US states, can be obtained from farms in 25 states and are available in shops in 13 states. EU countries make their own laws but products made with raw milk must be labeled. About a fifth of French cheese is made using raw milk.
Europeans have eaten raw milk cheese for hundreds of years. In France, for example, 15 percent of its cheese is made of unpasteurized milk, according to French Agricultural Statistics (http://www.agreste.agriculture.gouv.fr/IMG/pdf_primeur264.pdf). The thinking is that when milk is cooked, or pasteurized, many of the flavor-rich enzymes are destroyed.
In the United States, regulations on raw milk cheese are less stringent than in Europe, where more steps are required to ensure that there is no contamination.

The FDA and CDC position on Raw milk Cheeses

The FDA claims that there is a 50- to 160-fold increase in the risk of listeriosis from a serving of soft-ripened raw-milk cheese, compared with cheese made from pasteurized milk.
A mild heat treatment that kills 99.9% of the bacteria (3 log10 reduction) in bulk raw milk before cheese-making, would reduce the mean risk approximately 7-fold to 10-fold, compared with the baseline estimate for raw-milk cheese. This intervention is not full milk pasteurization.
Properly applied, full milk pasteurization kills all bacteria in raw milk. Testing the bulk milk used to make raw-milk cheese reduces the risk approximately 27-fold to 37-fold, but is less effective than testing raw-milk cheese lots, and still results in higher risk than the baseline risk estimate for pasteurized-milk cheese.
According the FDA, an analysis by the Centers for Disease Control and Prevention (CDC), between 1993 and 2006 more than 1500 people in the United States became sick from drinking raw milk or eating cheese made from raw milk. In addition, CDC reported that unpasteurized milk is 150 times more likely to cause foodborne illness and results in 13 times more hospitalizations than illnesses involving pasteurized dairy products.
Raw milk-related outbreaks are more common in states that allow the legal sale of raw milk for people to drink than in states that do not allow its sale. In addition, raw milk sales in one state can lead to outbreaks in neighboring states.
As reported before, this recall comes on the coattails of the cheese distributed by Deutsch Käse Haus of Middlebury, Indiana that included products sold under various brand names, including Sargento, Taylor Farms, Country Fresh, Choice Farm, MDS Foods, Meijer, Sara Lee, Guggisberg Cheese, Biery Cheese , and Saputo and others. These were all pasteurized milk cheeses. However in that case, no one has been reported sick in the outbreak.