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How Listeria monocytogenes can survive in extreme environmental conditions

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It is difficult to eliminate L. monocytogenes from post processing contamination of food production lines since this pathogen is common in various environments outside processing plants, and can endure in food processing environments.  It is one of the main concerns in environmental monitoring due to its ability to survive strict cleaning conditions and remain in the plant environment for months or even years.
Listeria monocytogenes has better survival ability than most other food pathogens, resulting in the colonization of Listeria in food processing environment. L. monocytogenes is capable of adapting to a variety of stress conditions, including pH variations, cold temperature, low water activity, high salt concentration, and different sanitizers such as quaternary ammonium compounds, sodium hypochlorite, and peracetic acid.
In the recent past, researchers have identified several hypervariable (easily changeable regions) regions of the bacterial genome called Genetic Insert Stress Survival Islet 1 (SSI-1). This genetic region exists in some other bacteria. Different genetic sequence inserts are utilized by the bacteria to help tolerate acidic conditions, bile salts, pH fluctuations, salt concentration, low water activity, temperature variations, etc. The SSI-1 is a five-gene islet that contributes to the growth of L. monocytogenes in sub-optimal conditions. However, SSI-1 does not explain the survival of L. monocytogenes during food sanitation conditions that are alkaline and highly oxidative.
In a recent publication by Harter et al., Sep 2017, it was reported that by looking at neighboring gene sequences to SSI-1, they identified a new stress survival islet 2 (SSI-2). SSI-2 is predominantly present in L. monocytogenes ST121 strains and is responsible for survival in alkaline conditions and oxidative conditions present in food processing environment.
Their study showed that SSI-2 is involved in a different stress response than SSI-1. The prevalence of SSI-1 is similar between clinical isolates and strains isolated from food and food processing environments.  SSI-2 strains are mostly present in L. monocytogenes strains isolated from food and food processing environments (84%), and not from clinical isolates.
SSI-2 is mainly contained in strains of ST121, while SSI-1 is present in diverse ST strains. The CC121 are prevalent in isolates from food and processing environment and are very rare among clinical isolates. ST121 strains persist for months in food processing environment, due to their ability to survive the oxidative and alkaline conditions, potentially resulting in contamination of the environment. The authors speculate that SSI-2 seems to have developed in response to the cleaning regime of food processing, because of their much higher prevalence in this environment.
SSI-2 contains two genes (lin0464 and lin0465) that support survival under alkaline and oxidative conditions. One gene is a transcriptional regulator directing the entrance of the second gene which is responsible for protease activity (breaking down proteins during oxidative stress). The broken proteins can be eliminated from the cell relieving the stress.
The SSI-2 are called “stress survival islet,” since both genes help the survival under stress conditions. Under stress conditions, mRNA production increases, as is the increase in transcription of the putative protease gene.
Harter et al. hypnotize that elemental horizontal gene transfer from L. innocua is most plausibly integrated into the L. monocytogenes genome to create the SSI-2.  This is because the two strains are more closely related than other strains of Listeria, and coexist in the same ecological niches.
L. monocytogenes ST121strains containing the SSI-2 genes survive the alkaline and oxidative stresses during cleaning and sanitation procedures. The oxidizing agents (e.g., chlorine dioxide, sodium hypochlorite, hydrogen peroxide) are frequently applied to kill bacteria on surfaces but can be survived by these strains of L. monocytogenes.
Progress has been made to better understand the genetic reasons for the survival of L. monocytogenes in food processing plants. To better understand the survival mechanisms of Listeria, and for the development of new strategies for prevention, these studies are essential.
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A Novel Concentration Device for the Detection of Food Pathogens

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Nature published in July 2017, an article by Gwangseong KimHoratiu Vinerean & Angelo Gaitas on a simple novel device to concentrate and detect food pathogens (immunocapturing method). The technique has the potential of being used for both clinical applications and food testing.

 System Set-Up

The technique employs polymer (polydimethylsiloxane) tubes (1.02 mm in diameter) coated with an antibody. The test sample is circulating through the antibody coated tubes. The re-circulating of liquid media containing the bacteria through the antibody conjugated tubes result in the capturing of the pathogens by the conjugated antibodies.
Several tubes can be used with different antibodies in each, thereby allowing the capture of different pathogens. Alternatively, several identical tubes can be used to increase the efficiency of the capturing.
As a result, the pathogens present in the sample are concentrated and accumulated in the tubes. This concentration step results in a higher concentration of the pathogens in a small volume of liquid.

Results

The results show that in larger volumes of 100-250 mL and small starting bacterial numbers of anywhere from 1 to 10 CFU anywhere from 55%-91% of bacteria were captured inside the tubes within 6-7 hours.
Ground chicken and ground beef were used as matrices to demonstrate the ability of the immuno-capturing method.  25 CFU of Salmonella typhimurium in 25 grams of ground meat was used to show the systems ability to work with real foods. The product was diluted 1:10 in 225 ml of buffered peptone water (BPW) or Romer Labs Primary enrichment media supplemented with phage. After 5-7 hours Salmonella was detected from these samples, representing significant time savings over the traditional methodology.
The two food matrices tested did not clog the 1mm tubes. To test larger volumes of samples required in food pathogens, long (120 cm) antibody coated tube was split into four 30 mm tubes.  The 250 ml sample was circulated approximately 10 times in the 7-hour experiment.
Use of Molecular Methods: The STyphimurium DNA was directly extracted from the concentration tubes by inserting DI water in the tube and heating to 100 °C for 10 minutes.  Other methods for DNA extraction were also tested.  Detection of the presence of the pathogens was done using either microscope fluorescence imaging or RT PCR.  10 µm from the content can be directly used for RT PCR without further purification steps.
Use of Lateral flow devices: have a higher limit of detection than PCR, and therefore requires longer enrichment time. However, they are low cost and easy to use. Therefore they also were tested with the immunocapturing method.
As shown below, 25 cfu of S. typhimurium in 25 gram of ground meat could detect in 14 hours with traditional enrichment, and in 9 hours when using the Romer Primary enrichment medium with phage. These time frames are significantly lower than the traditional methodology (36-44 hours).
(b) Positive results using Neogen Reveal 2.0 Salmonella strip in 14 hours in non-selective media.(c) Positive result using Romer Labs RapidChek SELECT Salmonella strip in 14 hours in non-selective media, (d) Positive result using Romer Labs RapidChek SELECT Salmonella strip in 9 hours in selective media

Bottom-line

There is certainly a need for a faster method to find food pathogens because it allows for faster intervention and faster corrective action. It allows to link pathogen strains to specific cases and can be useful in preventing outbreaks and illnesses.
This novel method can allow for results from food matrices in less than a single shift. However, the technology is currently in prototype stage and will need to be developed to a full commercial product.
The inventors of the technology are currently seeking funding to finish the commercialization of the product. They expect the product to be commercially available in the next two years.
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Salmonella in Papaya Sickened 47 People in 12 States

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Findings of the Papaya Outbreak

 An investigation of a multistate outbreak of Salmonella Kiambu has being conducted by the CDC, FDA and public health officials in several states. As of last Friday (7/21/17), 47 individuals from 12 states have been infected with Salmonella Kiambu, the outbreak strain.
Whole genomic sequencing (WGS) shows that the Salmonella Kiambu isolated from the infected people is genetically closely related, and therefore, are more likely to share the same source of infection.
More than third of the affected people were hospitalized, and one death was reported in New York City. The epidemic chart below shows the number of people who became ill each day. Illnesses that happened after June 23, 2017might not been reported yet because it takes 2-4 weeks for the data to be reported.
In Maryland, a cluster of illness was identified. Several ill people reported eating papayas purchased from the same grocery store. Epidemiologic and laboratory data indicated that yellow Maradol papayas were a likely source of this outbreak.
Among 58% of the people with available information, were of Hispanic ethnicity. 44% of sickened people interviewed reported eating papaya within days before the illness, significantly higher than Hispanics eating papaya in the general population (16%).
 
From the samples collected from ill people, Salmonella Kiambu and Salmonella Thompson was isolated. Clusters, like the one discovered in Maryland, provided a critical clue to the source of the outbreak, and as a result, the papaya was identified as the main source of the infection.
The Maryland Department of Health collected papayas at a Baltimore retail location and found 3 of the 5 yellow papayas that they tested confirmed to be contaminated with Salmonella. As a result, they warned the public not to buy Caribeña’s yellow Maradol papayas.
The majority of the cases occurred in New York (13) and New Jersey (12), followed by Virginia (6), Maryland (5), Pennsylvania (4)  and a single case in Iowa, Kentucky, Louisiana, Massachusetts, Minnesota, Texas, and Utah. 
The FDA s advising consumers not to eat Caribeña brand Maradol papayas because of their link to the Salmonella outbreak. Maradol papayas are green before they ripen and turn yellow, so consumers should not eat Caribeña brand regardless of the color.
According to the FDA, the distribution pattern of Caribeña brand Maradol papayas does not fully explain all of the illnesses. Therefore other firms might have distributed contaminated Maradol papayas as well. All the farms producing this type of papayas are only in Mexico.

Papaya as the Source of Past Salmonella Outbreak

Between May 12 and August 18, 2011, an investigation by CDC and the FDA, in collaboration with public health officials in Texas, Illinois, Georgia, and other states examining a multistate outbreak of Salmonella Agona infections linked to whole fresh papayas imported from Mexico.
A total of 106 individuals infected with the outbreak strain of Salmonella Agona were identified between January 1 and August 25, 2011, in 25 states. The states involved were:  Arkansas (1), Arizona (4), California (8), Colorado (1), Georgia (8), Illinois (18), Indiana (1), Kentucky (1), Louisiana (2), Massachusetts (1), Minnesota (3), Missouri (3), Nebraska (2), Nevada (1), New Jersey (1), New Mexico (3), New York (9), Ohio (1), Oklahoma (1), Pennsylvania (2), Tennessee (1), Texas (25), Virginia (2), Washington (5), and Wisconsin (2). Ten patients were hospitalized, but no deaths were reported.
Epidemiological data, traceback investigations, and laboratory data linked this outbreak to eating fresh whole papayas imported from Mexico by Agromod Produce, Inc. of McAllen, Texas.  
Of the ill people, 57% reported consuming papayas in the week before illness onset. This was significantly different compared with results from a survey of healthy people in which 11% of the Hispanic/Latino ethnicity and 3% of non-Hispanic/Latino ethnicity reported consuming papaya in the 7 days before they were interviewed. 
The FDA investigation found two papaya samples contaminated with Salmonella Agona. The FDA’s evidence showed a widespread problem that prompted the FDA to issue a countrywide import alert for papayas from Mexico.  

What Can Be Done?

Some Salmonella outbreaks in the recent past are due to fruits and vegetables. The outbreaks can be due to the ability of Salmonella to attach or internalize into fruits.  Survival and multiplication of Salmonella on fresh fruits is considerably increased once the protective epidermal barrier has been broken either by physical damage due to punctures or bruising or by degradation by plant pathogens. 
Environmental factors such as contaminated water used to irrigate and wash produce crops have been implicated in a large number of outbreaks.  FSMA is designed to help minimize the risk of illness from foodborne pathogens in fresh fruits, and include requirements for water quality, employee hygiene, and equipment and tool sanitation. Hopefully, these new FSMA rules will be able to reduce Salmonella incidents in fresh fruits.
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A New Method for the Detection of Salmonella in Powdered Dairy Products

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The Journal of Dairy Sciences reports that a team of researchers from China (Zhao et al. J. Dairy Sci. 100:3480–3496  May 2107) developed a new method for the detection of Salmonella in infant powdered milk.
The developed method is claimed to be rapid, specific, and sensitive. It is is based upon loop-mediated isothermal amplification technique combined with a lateral flow dipstick (LAMP-LFD) as the detection step.

Loop-Mediated Isothermal Amplification Technique (LAMP)

LAMP is a powerful new nucleic acid amplification method that detects very low levels of DNA. The method amplifies a few copies of target DNA with high specificity, efficiency and rapidity. The method uses a set of 4 specifically designed primers that recognize 6 distinct sequences of target DNA, and a DNA polymerase.
The cycling reaction can result in the accumulation of 109 fold of copies in less than 1 hour. The method is claimed to be more specific and less susceptible to interference than PCR, it is very fast, without the need of denaturing step.
 

Target Genes

The target gene invA encodes a Salmonella invasion protein and is thus considered a virulence gene located on Salmonella pathogenicity island (SPI), and is used frequently for the detection of Salmonella. The SPI4 region includes genes from siiA to siiF that are important for adhesion to polarized epithelial cells, and plays an important role in Salmonella pathogenicity.
The authors claim that this is the first attempt to use LAMP and the siiA gene to detect Salmonella.

 Lateral Flow Dipstick (LFD)

Lateral flow immunoassays dipsticks are used routinely to detect pathogens in food. Lateral flow dipstick use a sandwich type ELISA and the majority use polyclonal antibody as a capture antibody and a monoclonal antibody as the detection antibody. The antibodies are fixed on a hydrophobic membrane in immobilized in lines. Their role is to react with the analyte bound to the conjugated antibody. Recognition of the sample analyte results in an appropriate response on the test line, while a response on the control line indicates the proper liquid flow through the strip.
In the LAMP-LFD assay LFD strip is inserted into a tube that allows the strip to be immersed in the amplified sample. The sample migrates through the conjugate pad, which contains antibodies specific to the target analyte and are conjugated to colloidal gold and latex microspheres. The sample, together with the conjugated antibody bound to the target analyte, migrates along the strip into the detection zone
A number of researchers have combined LAMP with LFD. In this combination the LFD is soaked in LAMP amplified sample and the liquid travels by capillary action across the membrane to react with the antibodies and provide a color band.

Elimination of carryover Contamination

The high sensitivity of LAMP can become its largest potential disadvantage because trace left over material can be amplified and detected, causing false positive results, after several times of detection in the same place. Therefore, there is a need to eliminate any contamination from previous LAMP reactions.
To reduce incidence of LAMP contamination, the authors applied propidium monoazide (PMA) to eliminate carryover contamination of LAMP. The appropriate concentration of PMA diluted in water was applied to the working environment of any contaminated area and adequate light exposure conditions were used to complete the decontamination process.
 

Results

A very specific and conserved Salmonella target gene siiA was used to establish the LAMP-LFD detection method for Salmonella in powdered Infant formula.
In this study, the limit of detection of the LAMP-LFD for inoculated powdered infant formula, without enrichment was 2.2 cfu/g, which is 100x lower than the limit of detection for most PCR methods. A pure culture study of 21 Salmonella strains (with limited number of serotypes), and 60 inoculated samples of powdered infant formula yielded all positive results.  31 non-Salmonella strains (75% gram positive), including 20 non inoculated samples all yielded negative results.
 While more testing of this method is required, the reported method seems to be very rapid, specific, and sensitive for the detection of Salmonella in powdered infant formula.  PMA needs to be used to eliminate the LAMP carryover contamination.
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MIT Developed Novel Pathogen System Based on Janus Emulsions

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Rapid methods for 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. Many new methods are available involving antibody-based assays, genetic amplification methods, and newer sensor development methods. 
However, the industry is always looking for faster, simpler and cost effective new methods. An article in ACS Central Science describes the work of researchers at Massachusetts Institute of Technology (MIT) that are developing a new method for pathogen detection, utilizing Janus emulsions. The team is lead by Timothy Swager and Qifan Zhangis the lead author.
The test is based on the analysis of liquid droplets (Janus droplets, or Janus emulsion) that are powerful liquid phase sensing particles. These droplets are formed from two equally sized hemispheres. One half is composed of a fluorocarbon and the other from a hydrocarbon.
The fluorocarbon is denser than the hydrocarbon when droplets sit on a surface, therefore the fluorocarbon orients to the bottom. When the different hemispheres are functionalized to have orthogonal physical and biochemical properties, they can be used as sensors. Consequently Janus particles with covalently modified surfaces have been used for sensing applications.
From above the droplets are transparent but when viewed sideways they appear opaque. This property relates to the way that light passes through the droplet, and it is the path of light that can be adapted to make the sensor.
Building on this the scientists at MIT developed a surfactant molecule that contains mannose sugar to form the top half of the droplet surface. These molecules are capable of binding to a protein called lectin. Lectin is a protein that can bind specifically to certain sugars and cause agglutination of particular cells, and it is found on the surface of strains of E. coli.
The emulsion assay uses the carbohydrate surfactant molecule, which self-assembles at the droplet surfaces during the emulsification process. Therefore, no further element is required for bacterial recognition. These changes in the alignment of the Janus droplets are used for the detection of analytes.  The droplets are capable of binding to specific bacterial proteins. The mannose surfactant functionalized emulsion assay described in this work was designed specifically for E. coli as a model system.  
 
Whenever E. coli is present the droplets attach to the Lectin proteins. This causes the droplets to clump together causing light to scatter in many directions. The Janus emulsion assay enables detection of E. coli bacteria at a concentration of 104 cfu/mL.   The figure below shows the effect of the agglutination process.
On the left, Janus droplets are viewed from above. After the droplets encounter their target, they clump together (right). Credit: Qifan Zhang
The intrinsic optical lensing behavior of the Janus droplets also enables both qualitative and quantitative detection of protein and E. coli bacteria. The qualitative assay is very simple and can be scanned with a  Smartphone. To demonstrate the simplicity of the agglutination assay for qualitative results, the researchers placed inside a Petri dish QR barcode (Quick Response Code two-dimensional barcode)
As seen in the figure above when E. coli are present, the droplets clump together and the QR code can’t be read.( Credit: Qifan Zhang)
To precisely quantify the degree of agglutination, the researchers implemented an image processing program to calculate the percentage of area covered by agglutinated Janus emulsions and to evaluate the differences in optical intensity of the images before and after exposure to ConA (concanavalin A, serves as a functional substitute for E. coli bacteria). The program uses the adaptive threshold algorithm to distinguish areas with higher transparency (pristine Janus emulsions) from the opaque regions (agglutinated Janus emulsions).
The MIT team plans to create droplets customized with more complex sugars that would bind to different bacterial proteins. In this paper the researchers used a sugar that binds to E. coli, but they expect that they could adapt the sensor to other pathogens.
The researchers are now working on optimizing the food sample preparation so they can be placed into the wells with the droplets. They also plan to create droplets customized with more complex sugars that would bind to different bacterial proteins. The team leader, Savagatrup says “You could imagine making really selective droplets to catch different bacteria, based on the sugar we put on them”.
The researchers are also trying to improve the sensitivity of the sensor, which currently is similar to existing techniques but has the potential to be much more sensitive, they believe. They hope to launch a company to commercialize the technology within the next year and a half.
Explaining a clear advantage of the technology, one of the lead scientists, Professor Timothy Swager, said: “What we have here is something that can be massively cheaper, with low entry costs. The sensor has been tested out with multiple samples of the infective bacterium and the results are sufficiently successful for the sensor to be considered for commercialization”
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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.