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A Single Genetic Code Change Makes African Salmonella More Deadly

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A new study has identified a single genetic code change that allows Salmonella Typhimurium (ST313) to play a significant role in human bloodstream infections in sub-Saharan Africa.  The study was published in Proceedings of the National Academy of Sciences, 2018 (PNAS) [Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella Disa L. Hammarlöf, Carsten Kröger, Siân V. Owen, Rocío Canals, Lizeth Lacharme-Lora, Nicolas Wenner, Anna E. Schager, Timothy J. Wells, Ian R. Henderson, Paul Wigley, Karsten Hokamp, Nicholas A. Feasey, Melita A. Gordon and Jay C. D. Hinton; PNAS 2018] 
Invasive non-typhoidal Salmonellosis (iNTS) is killing approximately 390,000 people annually in sub-Saharan Africa. iNTS is caused by Salmonella that enters the bloodstream and spreads through the body. The African iNST is caused by a variant of Salmonella Typhimurium (ST313) that is antibiotic resistant, and affect people with deficient immune system. This Salmonella strain seems to be very similar to the Salmonella Typhimurium that causes gastrointestinal illness.
The team of scientists, led by Professor Jay Hinton at the University of Liverpool, used advanced genetic techniques to switch various single-nucleotide polymorphism (SNPs) to find the one responsible for the difference in the Salmonella strain between the antibiotic-resistant strain that can enter the bloodstream, and the one causing gastroenteritis.
The team analyzed hundreds of Salmonella genomes and showed that the Salmonella Typhimurium ST313 is closely related to the ST19 strains that cause gastroenteritis. The core genome of the two strains share over 4,000 genes, varies by only ~1,000 SNPs. They hypothesized that one or more of these SNPs are accountable for the difference between the ST313 and ST19.  A single nucleotide difference that was unique to the African ST313, was identified as the cause of the virulence of the strain and its ability to grow in the bloodstream.
Using transcriptomics (a type of RNA analysis), the scientists identified SNPs that affected the level of expression of important Salmonella genes. After studying 1000 different SNPs, they found the single nucleotide difference that is unique to the African ST313 strain which causes high expression of a virulence factor called PgtE that prevents Salmonella being killed in the bloodstream.  The virulence factor PgtE is an outer membrane protease Salmonella that causes diseases ranging from gastroenteritis to severe enteric fever.
There are thousands of SNP differences between different types of Salmonella; therefore it is surprising that a change of just one letter in the DNA sequence will cause such a profound difference in the disease-causing ability of the organism. Until now it has been hard to connect an individual SNP to the bacterial ability to cause a disease.
The researchers used animal models (chicken) to infect with bacteria with altered SNP to show that by removing it the organism lost its ability to cause the disease.

Summary

The research team from the Universities of Birmingham and Liverpool has identified a single-nucleotide polymorphism (SNPs), which helps the African Salmonella to survive in the human bloodstream.  SNPs represent a change in a single DNA letter between the two Salmonella strains. It seems to be the first link between an individual SNP and a disease.
The single SNP responsible for high levels of expression of the PgtE in the outer membrane protease was linked to the virulence of African S. Typhimurium ST313. The study has implications for bacterial genome-wide association studies, which they claim should clearly include a focus on noncoding regions of the genome. The study findings also emphasize the value of identifying all gene promoters in bacterial pathogens, to allow nucleotide differences to be correlated with the process of transcriptional initiation.
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UMass Amherst Developed a Low-Cost Chip to Detect Bacteria in Food and Water

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Rapid methods for the detection of pathogens have been gaining acceptance in the food industry. Recent advances in technology can result in faster detection of pathogens, more convenient, more sensitive methods.  We have seen many new alternative methods being proposed in the past couple of years. Below is an example of such a novel method.
According to a press release from University of Massachusetts Amherst (https://www.umass.edu/newsoffice/article/umass-amherst-food-scientists-are ) a team of scientists (including Lily He, Lynne McLandsborough, and Brooke Pearson) developed a low cost, rapid method for the detection of bacteria in food samples.
The assay steps include rinsing the fruit or vegetable, collecting the rinse water sample, placing the water to the chemical-based microchip that captures the harmful bacteria, and detecting the bacteria using a smartphone with a light microscope adaptor.  He said:” If there are harmful bacteria, it will be shown as visible dots, to indicate that you may have, for example, salmonella or Listeria.”
The Chip includes 3-mercaptophenylboronic acid (3-MBPA) that attracts and binds to any bacteria. Food particles, sugars, fat, and proteins can be washed away with a high-pH buffer.
There are two detection methods: (i) surface-enhanced Raman spectroscopy” (SERS) that relies on silver nanoparticles and the optical microscopy method. Both the optical method and the SERS mapping methods have a sensitivity of detection as few as 100 CFU/mL according to the publication (http://pubs.rsc.org/en/content/articlelanding/2017/ay/c7ay01596h#!divAbstract). The total assay time for the optical method is 1.25 hours and for SERS imaging 3 hours.
The method seems very attractive at first glance. It is straightforward and inexpensive. The use of a smartphone instead of a microscope is eye-catching. A lay person can use it without the need for a laboratory.
However, it seems to me that the method lack the desired selectivity as it detects all bacteria and not necessarily pathogenic bacteria. For pathogens, it lacks the desired sensitivity. While sensitivity of 100 CFU/mL is impressive, a system for pathogens needs to be 10,000 folds more sensitive.
There is a danger of prematurely promoting such rapid methods; it might take many years of research to obtain the desired specificity and sensitivity.
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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.