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A New Technology Can be Used Instead of Antibiotics to Kill Superbugs

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  Dr. Timothy Lu, an associate professor in biological engineering at the Massachusetts Institute of Technology, found a new potential way to kill superbugs with a DNA editor called CRISPR-Cas9. The Wall Street Journal reported that Dr. Lu said: “is is basically a molecular scissor” that can snip bacterial genes that make bacteria drug-resistant, killing the bug in the process.  The technology combines bacteriophages and CRISPR-Cas9 to target drug-resistant genes.

What is CRISPR?

CRISPR is used to edit or delete genes from living cells.  “CRISPR” means Clustered Regularly Interspaced Short Palindromic Repeats. They are the characteristic of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology.
CRISPR-Cas9 can be programmed to target portions of genetic code and edit DNA at exact locations. It allows researchers to modify genes in living organisms permanently. This technology can be used to remove the genes that make the bacteria drug-resistant, and in the process, it can also kill the bacteria.

The New Technology to Eliminate Drug-Resistant Bacteria

Dr. Lu is studying ways to eliminate superbugs with CRISPR-Cas9. He is contemplating combining the CRISPR-Cas9 technology with bacteriophages, and engineering the bacteriophages to attack only bacteria with drug-resistant genes. They were successful in including the CRISPR-Cas9 into a bacteriophage that was designed to attack a drug-resistant E. coli (Nature Biotechnology, 2014 Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases,  R. J. Citorik, M. Mmee, and T. K. Lu). The new technology has the advantage of being a more targeted approach.
Numerous hurdles need to be overcome before this technology can be used against superbugs, including the demonstration that in humans, bacteriophages are safe and effective to use. Another concern is that the CRISPR can deviate from the target, thereby slicing the wrong genes. However, there is a race among scientists to find new applications for this novel technology.
One major concern is that CRISPER can veer off target, slicing away the wrong genes with potentially harmful effects, scientists say. There are also fears of unknown effects due to the use of CRISPER to modify bacteria. Regardless of the promise of this technology, any potential therapy is years away. Nevertheless, many other scientists are trying to harness this novel technology for a variety of applications.

Can a similar technology be used in food plants to eliminate pathogenic bacteria from the environment?

Bacteriophages have been recommended for rapid detection of food-borne pathogens as well as a natural food preservative (Front Microbiol. 2016; 7: 474). Phage cocktails were created for the treatment of foods contaminated with various pathogens (Campylobacter jejuni, Cronobacter sakazakii, E. coli O157:H7, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, and Vibrio spp). Numerous other studies report that phages may be useful for controlling specific food pathogen. However, there is no widespread use of bacteriophages to control pathogens.
Several of bacteriophage-based applications have been approved for pre-harvest control of food pathogens in livestock and poultry. Another application is the decontamination of surfaces in food-processing facilities (Neha Bhardwaj, Sanjeev K. Bhardwaj, Akash Deep, Swati Dahiya and Sanjay Kapoor, 2015. Lytic Bacteriophages as Biocontrol Agents of Foodborne Pathogens. Asian Journal of Animal and Veterinary Advances, 10: 708-723.) 
Using the new advanced technology described above might improve the stability of the bacteriophages and improve their ability to attack the bacteria. As a result, it might gain more traction in the food industry.
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Sample6 Pathogen DETECT Platform Acquired by IEH

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Sample6 has two main products: DETECT, the pathogen detection system for the detection of Listeria monocytogenes in environmental samples, and CONTROL a food safety software package. Sample6 businesses were split into two. The pathogen detection system (DETECT) was acquired by IEH while Sample6 will continue with its CONTROL software.
PRNews reported that Sample6 and IEH had jointly announced that IEH is acquiring the DETECT platform while Sample6 will continue to pursue the CONTROL software.

The Pathogen DETECT System

The principle of the technology is described in an article entitled Advancing bacteriophage-based microbial diagnostics with synthetic biology by Lu et al. (Lu TK, Bowers J, Koeris MS.2013).
An engineered phage designed to interact with the target pathogens (i.e., Listeria monocytogenes, or Salmonella), makes the bacteria produce a large amount of the reporter enzyme. After a few hours, the bacterial cells go through a lysis step, and the reported enzyme is detected.  The enzyme introduced by the phage makes the bacteria produce a biolumination compound that glows.  The Biolumination signal is detected by the system. It is an enrichment free system capable of detecting one cfu/swab in 4 hours.
The CEO of Sample 6, Dr. Michael Koeris said: “IEH’s resources and reach will allow for a more rapid deployment of the groundbreaking in-shift, on-site technology, as well as the successful launch of the high-throughput platform into the central and 3rd party laboratory market worldwide.”

CONTROL Software

Sample6 CONTROL is environmental monitoring software, allowing to schedule, monitor and report environmental program data. It allows gaining an insight into the effectiveness of the environmental monitoring system.
Automated scheduling can be obtained by the system and results from any test method can be easily entered into the CONTROL software. In the event of a nonconforming or presumptive positive test result, a corrective action is generated.
The system provides the reporting tools necessary to evaluate the performance of the environmental monitoring plan and to make adjustments based on historical and real-time data.

IEH Laboratory and Consulting Group

The company owns more than 95 laboratories, combining consulting with accredited testing laboratory. IEH is serving the food and pharmaceutical industries, providing services in a variety of disciplines. The company is lead by Dr. Mansour Samadpour.
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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.