Microbiology is Recognized with New Emojis

The Unicode Consortium today announced it has finalized a new set of 157 emoji. This brings the total number of approved emojis to 2,823.

The new Emoji 11.0 set is fixed and final, and includes the data needed for vendors to begin working on their emoji fonts and code ahead of the release of Unicode 11.0, scheduled for June 2018. The new emoji typically start showing up on mobile phones in August or September.

So, what does this have to do with microbiology or rapid methods? Believe it or not, there will be six (6) brand new laboratory and microbiology-related emojis including a lab coat, goggles, Petri dish, test tube, DNA molecule and finally, a microbe (which looks like E. coli on steroids!). These will be great additions to the microscope emoji which has been quite lonely for all these years!

Here is a sneak peek at the new emojis coming later this year. Please click on the image for a larger view.

Biosensors Detect Harmful Bugs in the Lungs of Cystic Fibrosis Patients

Pseudomonas aeruginosa are strains of bacteria that are widely found in the environment. Pseudomonas is a major cause of lung infections in people with cystic fibrosis. The bacteria thrive in moist environments and equipment, such as humidifiers and catheters in hospital wards, and in kitchens, bathrooms, pools, hot tubs and sinks.

Pseudomonas infections are known as opportunistic. This means the bacteria only cause infections when a person has CF or another condition that weakens the body's immune system.

Pseudomonas is one of the most common bacteria found in people with CF. About half of all people with CF have Pseudomonas. More than 60 percent of adults with CF have Pseudomonas.

The number of people with CF with the bacteria has been decreasing, however.Once Pseudomonas is established in the airways, it's very difficult to eliminate. But aggressive treatment can delay the development of long-term infection.

The rapid detection of Pseudomonas in CF patients is also paramount in treating patients. And scientists at the Imperial College London are doing just that. The following text provides an overview of their work.

Biosensors and the detection of Pseudomonas

A team of Imperial researchers has developed a tool which 'lights up' when it detects the chemical signature of harmful bacteria in the lung.

In a clinical first, the group from the Department of Medicine used the tools, called cell-free biosensors, to test samples of sputum (phlegm) from patients with cystic fibrosis (CF).

Biosensors are based on engineered DNA circuits, designed to detect changes in their environment, such as the presence of chemicals, or changes in pH or temperature.

These tools, which harness the biological machinery inside cells, can be used to quickly spot chemical traces of active microbial colonies in samples from the lung and could help to accurately diagnose bacterial infections in vulnerable patients.

In a small, proof-of-concept study, the team found that their biosensors could accurately detect markers of Pseudomonas bacteria – a leading cause of chest infections in people with weak immune systems or chronic conditions, such as CF – and were as sensitive as existing chemical diagnostic tests but could potentially be cheaper and easier to use.

Rapid diagnostic tests

The researchers are hopeful they could eventually develop their cell-free sensors into a range of rapid diagnostic tests which could be used either at home, GP surgeries or in hospital clinics or even in remote areas of the world with limited access to hospital diagnostics, at a fraction of the price of existing tests.

Professor Paul Freemont, co-founder and co-director of The Centre for Synthetic Biology and Innovation at Imperial, said: “The driving force behind this research is to show that these tools work and could be used to detect particular diagnostic markers associated with infection.”

He added: “By applying an engineering approach to biology, these systems could be altered to sense for any microbe we choose. The possibilities for public health and cost-savings for health systems could be considerable.”

Biosensors have emerged through the growing field of synthetic biology, with scientists tweaking living cells to respond to certain conditions, such as the presence of a chemical compound.

The engineered DNA circuit: when the bacterial molecules (AHL) are present they trigger the production of a coloured protein (GFP)

At the heart of the technique are lengths of engineered DNA which, when inserted into a living cell, act as a circuit. If the right signal – such as a specific chemical compound – is present, then the circuit is switched ‘on’ and the cell produces a signal in the form of a coloured output, in this case a green fluorescent protein. If the substrate is not present, then the circuit remains ‘off’ and there is no signal.

In the latest study, published this week in the journal ACS Synthetic Biology, researchers report on using a ‘cell-free’ form of sensor to test clinical samples for the first time. Instead of being contained within the membrane of a cell, the engineered DNA circuit and cellular machinery of their sensors are free-floating in a solution.

The team engineered their sensor’s circuit to respond to a molecule produced specifically by the bacterium Pseudomonas aeruginosa. These bacteria release a chemical signature in order to communicate with bacteria around them and to sense how many of them there are. As they replicate, more cells release the signature, and so the concentration of the signal increases, giving the bacteria an idea of the state of their population.

Previous studies have revealed levels of this same P. aeruginosa signature were higher in hospitalised patients than those with stable condition.

Samples taken from the lungs of patients, either with or without P. aeruginosa infection, were screened by adding them to tiny wells containing solutions of the biosensor. After four hours the samples were tested for green fluorescent protein – a sign that the bacterial signature was present.

Analysis revealed that the cell-free biosensor was able to detect the bacterial signature with the same accuracy as existing diagnostic tests, called liquid chromatography tandem mass spectrometry (LC-MS/MS). The biosensors were able to detect the concentration of the signal within a close range to LC-MS/MS.

Taking the technology forward

Currently, a major limitation for the cell-free sensors is the amount of time taken to prepare samples before testing, with lung sputum samples needing to be ‘cleaned up’ with the removal of biological materials which could interfere with the results.

In addition, as the DNA is synthetic – engineered in the lab – it would need to be disposed of carefully, with concerns around potential to contaminate the environment or pass to other organisms.

However, according to the researchers, the approach could offer significant cost-savings compared to the existing LC-MS/MS detection method. If suitably scaled up, using similar cell free biosensors could work out to be a fraction of the cost per sample.

Professor Jane Davies of the NHLI and an honorary consultant paediatrician at the Royal Brompton Hospital said: “Pseudomonas is the major infection in patients with cystic fibrosis and is closely linked to the severe lung disease which develops over time leading to premature death.

“At the moment, we only have opportunities to detect infection when patients come to clinics, perhaps every 2-3 months. A point of care test could transform the way in which we monitor patients, allowing us to treat early and personalise therapies.”

The group is now exploring how to take the technology forward and develop working prototypes, as well as a test to detect the signal in a patient’s urine. If the biosensor solution could be freeze-dried, this could even potentially take the form of a credit card-sized paper-based test, which would be affordable and ready for use in the field.

Loren Cameron, a PhD student in Freemont’s lab and one of the study authors, said: “Now we have shown these types of biosensors can be used with clinical samples, the next step is to refine our approach. We hope that in future, tools like this could be used to help test for the presence or severity of bacterial infections.” Ms Cameron is one of five postgraduate students funded as part of the Cystic Fibrosis Trust’s Strategic Research Centre for Pseudomonas infection.

Ke Yan Wen, a graduated PhD student from Freemont’s lab and also one of the lead authors, said: "We believe that cell-free systems are a promising platform for developing biosensors, since they are easy to use, produce a rapid response, and can be stored long-term in normal conditions.”

Source: Imperial College London

My Opinions on Sage Products Rapid Method Warning Letter (and a reply to Tim Sandle's Blog)

Tim Sandle published a recent blog post concerning a (FDA) Warning Letter related to an inadequate validation of a rapid microbiological method by Sage Products Inc. Tim's post reviews the warning letter findings and poses a number of follow up questions.

My opinions are as follows. Tim's original blog post is reproduced after my opinion. Tim's blog on this topic may be viewed by clicking here.

The FDA warning letter may be viewed by clicking here.


MY OPINION

This was an unfortunate event that should, in no way, dissuade firms from implementing rapid methods. To support my opinion, I need to provide clarity around the warning letter points, which are presented below in quotation marks.

“Your firm failed to establish and document the accuracy, sensitivity, specificity, and reproducibility of its test methods. Specifically, you use the [redacted] method to screen for microbiological contamination in drugs produced entirely at your facility and those manufactured under contract. This [redacted] screening method [redacted] for microbiological examination of your liquid drug products is not adequate for its intended use. You attempted to validate your [redacted] microbial detection method, but were not able to demonstrate that it could reliably and repeatedly determine whether objectionable microorganisms were present in your drugs.”

This tells me two things. First the rapid method was not adequately validated to demonstrate equivalency to the compendial method, which is a requirement in USP and the other compendia.

“After receiving three consumer complaints for discoloration of this product, you initiated testing of your retains using both the modified U.S. Pharmacopeia (USP) microbiological limits method and the [redacted] method. Both analyses found microbial contamination. Notably, the USP modified method [redacted] found an exceedingly high microbial count of over 57,000 CFU/ml, and also identified Burkholderia cepacia, an objectionable microorganism, in this product lot.”

“Had your firm been utilizing a screening method capable of consistently detecting B. cepacia, these products may not have been released in the first instance.”

Obviously! It appears the “validated” rapid method did not detect the presence of microorganisms in the original sample (which is why it was apparently released) and as such, there was no positive response to illicit confirmatory testing of an objectionable, such as B. cepacia.

“FDA informed you that your [redacted] method [redacted] has not been adequately validated for detecting the presence of microorganisms, including the presence of B. cepacia. In a subsequent meeting on November 30, 2016, FDA advised you to use a verified compendial method for all bulk drug solutions and finished product microbiological testing until you could further assess the suitability of the [redacted] method.”

This statement could mean a number of things. An alternative method, including a rapid method, must be equivalent to the compendial method. For nonsterile drugs, which I assume is the case with this warning letter, you must demonstrate the finished product is within aa appropriate quantitative specification (the reason why we perform USP 61) and does not contain any objectionable or specified microorganisms (which is why we perform USP 62 and additional tests for organisms that may not be specifically covered in the compendia). Some of the reasons why the rapid method could not detect microorganisms, including B. cepacia, could be fond in the next FDA statement.

“It specifies a [redacted] dilution factor. USP <62> requires a 1:10 dilution factor. Your dilution factor is [redacted] times greater than the USP method and provides insufficient detectability to rule out the presence of objectionable microorganisms and unacceptable total counts.”

Well, this is an issue regardless of whether the method used was USP 62 or an alternative method. Diluting out the test sample more than what is prescribed in the compendia may dilute out organisms that could be present. This was a mistake that should have been avoided.

Furthermore the rapid method it not account for the enrichment step called for in USP <62>. Also “it does not include the scraping step during sample preparation, which your submitted laboratory data indicates is required to validate organism recovery.”

Again, the same sample preparation steps must be performed in an alternative method, unless you can show a different procedure gives equivalent results to the compendial or original sampling and sample preparation strategy.

There was also a comment about the use of stressed organism as part of method validation (a controversial point within pharmaceutical microbiology): “it lacks evidence that small numbers of various microorganisms, including those that are injured and stressed, can be reliably recovered. Specifically, sample effect (defined by your firm as the inhibitory effect of a sample on the growth of various microorganisms) data for B. cepacia was collected using a fresh-grown culture, not a stressed organism.”

This was an interesting finding. The use of stressed organisms fueling the validation of rapid methods has been suggesting in PDA Technical Report #33 (TR33) and up until recently, the prior version of Ph. Eur. 5.1.6. Obviously, the FDA continues to require the use of stressed organisms when validating an alternative or rapid microbiological method because organisms normal found in finished product, especially if the product is preserved, will be stressed to some extent.

The final charge was that the method validation did not “establish potential sample interference factors (e.g., enhancing or quenching) for each product formulation.”

This tells me the firm did not perform method suitability on the product to eliminate the potential for false positives and false negatives. This is a current requirement in the USP, Ph. Eur. and TR33 and if Sage did not perform these tests during their rapid method validation studies, this is a significant issue.

There was also concern that the replacement method, unlike the USP method, did “not provide for potential speciation of the detected microbial contamination in the [redacted] initial screening test.”

Some rapid methods are based on the growth of microorganisms and may provide CFUs for subsequent analysis, including confirmation of an objectionable organisms and/or microbial identification. However, other rapid methods may not be based on growth and as such, follow up analysis of positive results may not be possible unless a separate sample was tested specifically for this purpose. Because eh exact rapid method under dispute is not disclosed in the warning letter, it is not possible to conclude the capabilities of the alternative method at this time.

Tim Sandle poses the following questions in his original post on this subject, based on the Sage warning letter. I will answer each in turn:

Q. Does the validation of methods require the use “stressed” organisms?

A. Depending on the application, the use of stressed organisms should be considered. Firms do not have to test dozens of stressed organisms but should select relevant strains and use a stressing condition that is defendable. Novartis published a number of papers on stressing organisms when they validated a rapid sterility test (see the reference page at rapidmicromethods.com for downloads or links to the papers). IN any case, firms should discuss their validation plans with the relevant regulatory authority to understand if the use of stressed organisms is expected.

Q. Does validation always need to be against each product formulation (not just the strongest concentration of the most inhibitory ingredient)?

A. If method suitability testing takes into consideration the greatest (and least) challenge to the alternative method, then you may be able to justify bracketing formulations to support these studies. But this will depend on the actual formulations intended for testing. Again, discuss your strategy with the regulators up front.

Q. How are representative samples built into the validation process?

A. Samples should be selected for validation studies identical to what will be tested routinely (by the rapid method). Consideration for sample preparation, dilutions, etc. should also be noted and as required by the compendia or regulatory expectations.

Q. Does a list of objectionable organisms need to be prepared ahead of the method validation? Or is this just a B cepacia issue?

A. Firms should understand what organisms should be detected by the method, based on a product’s intended use and patient population. This should be done regardless of whether an alternative method will be validated or if the standard compendial method is utilized.

TIM'S ORIGINAL POST

In July 2017 Sage Products Inc. received a warning letter from the U.S. Food and Drug Administration (FDA). Within the warning letter was a concern about the implementation of rapid microbiological method, where the method had been used to replace a compendial method.

In the warning letter, the FDA state “Your firm failed to establish and document the accuracy, sensitivity, specificity, and reproducibility of its test methods.” This relates to the use of a rapid method.

Specifically: “You use the (b)(4) method to screen for microbiological contamination in drugs produced entirely at your facility and those manufactured under contract. This (b)(4) screening method (b)(4) for microbiological examination of your liquid drug products is not adequate for its intended use. You attempted to validate your (b)(4) microbial detection method, but were not able to demonstrate that it could reliably and repeatedly determine whether objectionable microorganisms were present in your drugs.”

The FDA are concerned about the comparative findings from the USP method and the replacement rapid method: “After receiving three consumer complaints for discoloration of this product, you initiated testing of your retains using both the modified U.S. Pharmacopeia (USP) microbiological limits method and the (b)(4) method. Both analyses found microbial contamination. Notably, the USP modified method (b)(4) found an exceedingly high microbial count of over 57,000 CFU/ml, and also identified Burkholderia cepacia, an objectionable microorganism, in this product lot.”

It seems that the firm elected to run with the results from the alternative method, despite the USP method finding an objectionable microorganism. This led the FDA to state: “Had your firm been utilizing a screening method capable of consistently detecting B. cepacia, these products may not have been released in the first instance.” Part of the reason was “because [the company] failed to include B. cepacia on the list of objectionable organisms.”

The use of the alternative method had also gone against FDA advice: “FDA informed you that your (b)(4) method (b)(4) has not been adequately validated for detecting the presence of microorganisms, including the presence of B. cepacia. In a subsequent meeting on November 30, 2016, FDA advised you to use a verified compendial method for all bulk drug solutions and finished product microbiological testing until you could further assess the suitability of the (b)(4) method.”

The concerns the FDA had with the rapid method were:

“It specifies a (b)(4) dilution factor. USP <62> requires a 1:10 dilution factor. Your dilution factor is (b)(4) times greater than the USP method and provides insufficient detectability to rule out the presence of objectionable microorganisms and unacceptable total counts.”

Furthermore the rapid method it not account for the enrichment step called for in USP <62>. Also “it does not include the scraping step during sample preparation, which your submitted laboratory data indicates is required to validate organism recovery.”

There was also a comment about the use of stressed organism as part of method validation (a controversial point within pharmaceutical microbiology): “it lacks evidence that small numbers of various microorganisms, including those that are injured and stressed, can be reliably recovered. Specifically, sample effect (defined by your firm as the inhibitory effect of a sample on the growth of various microorganisms) data for B. cepacia was collected using a fresh-grown culture, not a stressed organism.”

The final charge was that the method validation did not “establish potential sample interference factors (e.g., enhancing or quenching) for each product formulation.”

There was also criticism about the sampling and sample plan used to ensure the tested sample was representative of the lot. There was also concern that the replacement method, unlike the USP method, did “not provide for potential speciation of the detected microbial contamination in the (b)(4) initial screening test.”

The implications from the FDA letter, as well as signaling a pertinent lesson when implementing a rapid method that needs to be equivalence to or better than the rapid method, are:

• Does the validation of methods require the use “stressed” organisms?
• Does validation always need to be against each product formulation (not just the strongest concentration of the most inhibitory ingredient)?
• How are representative samples built into the validation process?
• Does a list of objectionable organisms need to be prepared ahead of the method validation? Or is this just a B cepacia issue?

If you have views on this, please add a comment.

Thanks to Nigel Halls for the heads-up about this warning letter and its considerable implications.

Longitude Prize Bacterial Dx Competition Announces Seed Funding Winners

On average antibiotics add 20 years to each person’s life. The development of antibiotics has been vital to our survival, yet the rise of antimicrobial resistance is threatening to make them ineffective in the future. The World Health Organization estimates that antibiotics treatments add an average of 20 years to all of our lives. But in the 80 years since the discovery of penicillin, our overuse of antibiotics has put pressure on bacteria to evolve resistance, leading to the emergence of untreatable superbugs that threaten the basis of modern medicine.

Clinicians often prescribe broad spectrum antibiotics to sick patients because doctors have to act quickly on imperfect information. These methods put selective pressure on microbes to evolve resistance to antibiotics. Prescriptions from clinicians are just one way in which people access antibiotics, however. There are many routes to these drugs and they vary around the world.

Radical change is needed to address the global problem of growing anti-microbial resistance, to ensure a healthcare system that can sustainably control and treat infections.

We cannot outpace microbial evolution. A new broad-spectrum antibiotic, if applied with current methods, would eventually meet new forms of resistance. The overall solution involves a long-term path towards a more intelligent use of antibiotics enabling a future of more effective prevention, targeted treatments and smart clinical decision support systems.

The Longitude Prize is a £10m prize fund that will reward a competitor that can develop a point–of–care diagnostic test that will conserve antibiotics for future generations and revolutionise the delivery of global healthcare. The test must be accurate, rapid, affordable and easy to use anywhere in the world.

Run by Nesta and supported by Innovate UK as funding partner, the Longitude Prize competition recently announced 13 winners for the second round of seed funding.

Each winning team received awards between £10,000 ($13,210) and £25,000 ($37,927) for their research proposals. Funding for the second round draws on a £250,000 grant from Merck & Company. The groups come from Australia, Belgium, India, Israel, The Netherlands, the US, and the UK.

Teams are at various stages in test developments, ranging from proof of concept to initial clinical validation to fabrication of components for prototypes. They are attempting to diagnose infections including Escherichia coli, urinary tract infections, and sepsis.

Many teams are working on technology that will detect the susceptibility of bacteria to antibiotics, which will support specific antibiotic treatments for bacterial infections and minimize the spread of drug resistance.

Here is an overview of the Discovery Award Winning Teams:

• ID Genomics, a Seattle-based company, is developing "bacterial fingerprinting" technology that will reduce prescription errors and overuse of broad-spectrum antibiotics by improving the speed and accuracy of the antibiotic prescribing process. ID's Clonet rapid diagnostic test determines the bacterial fingerprint of different strains of the main pathogens in urinary tract infections (UTIs) in less than 30 minutes, which is then matched against the company's reference database of thousands of bacterial strains and their profiles. A clinician can then prescribe the antibiotic best suited to their infection.   
• Coris BioConcept, a Belgium-based company, specializes in developing, manufacturing, and marketing rapid diagnostic tests based on strip chromatography with the use of colloidal gold particles, latex microspheres, or fluorescent dyes (ICT). The ICT range detects infectious disease including enteric and respiratory pathogens, and could potentially be used to confirm if a bacteria is resistant or sensitive to carbapenem antibiotics.  
• Drug and Diagnostics for Tropical Disease is developing a test to determine if an antibiotic can still bind to its targeted protein or not, providing an answer within minutes without specialized equipment and allowing a doctor to test a patient in the office.
• A consortium called EPDAL:Bradford and Lincoln plans to design an efficient and inexpensive diagnostic test to identify the most significant gram-negative pathogens. On the premise that protein chemistry is the same in the structure of gram-negative bacteria (GNB) or proteins, the company will exploit the ability of protein elements to select different color complexing molecules and use differential staining to organize bacteria into separate categories based on their protein elements and color complex-elution properties.
• Embryyo, an Indian company, is working on a diagnostic device that will facilitate in early detection of bacterial or viral infection on the bloodstream, at the onset of a pathogenic infection when pathogen counts are very low. Based on microfluidic cell separation and flow focusing microdevices, the device will potentially aid in the differential diagnosis of bacterial versus viral infections.
• Encompass Consortium, based in Australia, is developing a method to determine antimicrobial susceptibility with the accuracy and speed needed to influence a physician's initial choice of antibiotics. The company aims to miniaturize and automate its first-generation flow cytometry-assisted antimicrobial susceptibility tests (FAST) for near point-of-care use.
• Going Against the Flow, another Australian company, is working on a point-of-care test that will detect the activation of neutrophils to allow early detection and treatment of sepsis. Instead of detecting infection directly, the test will rely on the body's sensitive and quick response to bacterial infections using original yet simple methods to detect the early response to neutrophils.
• Microplate-Strathclyde Biomedical Engineer and SIPBS's approach will bring together expertise to develop a rapid diagnostic test for antimicrobial susceptibility.
• Module Innovations, an Indian company, is developing USense, a diagnostic platform based on color changing polymers for rapid microbial detection.
• OxTB, Cambridge and Oxford, is working on a bedside test that uses whole-genome sequencing (WGS) to determine the presence of Mycobacterium tuberculosis in a clinical sample, predict drug susceptibility, and inform disease surveillance by demonstrating the genetic relationships to previously witnessed strains.
• Prismatix, an Israeli-based company, has developed a method of phase-shift reflectometric interference spectroscopic measurements (PRISM) that monitor bacterial activity silicon-based microstructures in real time.
• RAPDIF, based out of The Netherlands, plans to improve the diagnosis of febrile diseases in sub-Saharan Africa. It aims to develop a diagnostic tool that can identify bacterial infections.
• Rapid AMR Detection Team, from the University of Bristol, is developing an optical technique to quickly detect whether bacteria are alive or not, relying on optical detection of the state of bacteria after injecting antibiotics and not relying on bacterial growth.

Source: The Longitude Prize Challenge

Inclusion of Rapid Test Results Leaves Gaps in CDC Foodborne Illness Data

For the first time, the CDC’s yearly report on foodborne illnesses in the U.S. included infections that were identified only with rapid diagnostic tests, which can speed up treatment for patients but lead to important gaps in data, researchers said.

Culture-independent diagnostic tests (CIDTs) are increasingly being used by clinicians to detect enteric infections because they produce results more quickly than traditional culture methods, researchers from the CDC and other public health facilities across the country wrote in MMWR. However, used alone, these tests tell only part of the story, leaving out important information on pathogen subtypes and antimicrobial resistance, and making it difficult to monitor trends or link infections to outbreaks, the researchers said.

Such information can be obtained only if a reflex culture is performed on the CIDT-positive specimen, the researchers said. But according to their report, which compared surveillance data for 15% of the U.S. population from 2013 to 2016, a large proportion of positive CIDT results do not receive these much-needed cultures.

“We need foodborne-illness trend data to monitor progress toward making our food supply safer,” Robert Tauxe, MD, MPH, director of the CDC’s Division of Foodborne, Waterborne, and Environmental Diseases, said in a statement. “It’s important that laboratories continue to do follow-up cultures on CIDT-positive patients so public health officials can get the information needed to protect people from foodborne illness.”

Potential for false-positive results

Including CIDT results in the CDC data for 2016 raised the incidence rates for six of the most reported bacterial foodborne illnesses. However, interpreting these increases is complicated, the researchers said.

As an example, they noted that the incidence of culture-confirmed infections with Campylobacter — the most commonly reported bacterial foodborne illness — was “significantly lower” in 2016 compared with the average over the previous 3 years. However, a “slight but not significant” increase occurred when infections that were diagnosed only through CIDTs were included.

Further, Campylobacter is one of the pathogens for which antigen-based CIDTs are often used. These tests can produce a large number of false-positive results, leading to skewed estimates of incidence, the researchers said.

“When interpreting incidence and trends in light of changing diagnostic testing, considering frequency of testing, sensitivity, and specificity of these tests is important,” they wrote. “The observed increases in incidence of confirmed or CIDT positive–only infections in 2016 compared with 2013 to 2015 could be caused by increased testing, varying test sensitivity, an actual increase in infections, or a combination of these reasons.”

FoodNet data

The researchers looked at preliminary 2016 data from the CDC’s Foodborne Disease Active Surveillance Network, also called FoodNet, which monitors cases of nine foodborne disease at 10 sites in Connecticut, Georgia, Maryland, Minnesota, New Mexico, Oregon, Tennessee and certain counties in California, Colorado and New York, and compared the incidence rates with data from 2013 to 2015.

The sites reported 24,029 foodborne infections in 2016, including 5,512 hospitalizations and 98 deaths. Campylobacter was the leading cause of bacterial foodborne illness with 8,547 reported infections, followed by Salmonella (8,172), Shigella (2,913), Shiga toxin-producing Escherichia coli (1,845), Cryptosporidium (1,816), Yersinia (302), Vibrio (252), Listeria (127) and Cyclospora (55).
Salmonella typhimurium infections, which often are linked to beef and poultry, decreased 18% in 2016 compared with the average incidence for 2013 to 2015. The reduction is possibly due to regulatory action by the USDA to reduce Salmonella contamination in poultry and vaccination of chicken flocks, according to a CDC news release.

The CDC said increases in Yersinia, Cryptosporidium, and Shiga toxin-producing E. coli infections were likely due to the increased use of CIDTs.

“This report provides important information about which foodborne germs are making people sick in the United States,” Tauxe said. “It also points out changes in the ways clinicians are testing for foodborne illness and gaps in information as a result.”

The researchers said previous analyses have indicated that the number of foodborne infections in the U.S. “far exceeds those diagnosed” and that CIDTs might be making them “more visible.” But they said more tools are needed to accurately interpret FootNet surveillance data in light of these changes to testing practices.

“FoodNet is collecting more data and developing these tools,” they wrote. “With these, FoodNet will continue to track the needed progress toward reducing foodborne illness.” – by Gerard Gallagher

Reference

Marder EP, et al. MMWR Morb Mortal Wkly Rep. 2017;doi:10.15585/mmwr.mm6615a1

New Paper on Using Statistics When Validating Rapid Microbiological Methods

From 2010 to 2013, European Pharmaceutical Review published a very successful series on rapid microbiological methods (RMM) that included such topics as how to demonstrate a rapid method is equivalent to a conventional or pharmacopoeial method. PDA Technical Report #33, the recently revised USP chapter <1223> and the proposed changes to Ph. Eur. Chapter 5.1.6 all recommend the use of statistical analysis when validating rapid methods and establishing equivalence. However, many microbiologists find the concepts of statistical analysis and hypothesis testing intimidating and, in some cases, unapproachable. Once and for all, it was time to demystify statistics and provide a framework for embracing the mathematical models required when validating novel RMM technologies. 

To accomplish this, I have teamed up with two of the industry’s leading experts on statistical analysis and the validation of rapid microbiological methods: Edwin R. van den Heuvel, PhD (Eindhoven University of Technology) and David Roesti, PhD (Novartis Pharmaceuticals).

Our paper, The Role of Statistical Analysis in Validating Rapid Microbiological Methods, has recently been published in this month’s European Pharmaceutical Review. Clicking on the publication title (above) will give you online access to the paper (this is free to view but you may need to sign up for access). Alternatively, please copy and past the following address into your web browser:

http://www.europeanpharmaceuticalreview.com/46555/european-pharmaceutical-review-magazine/past-issues/issue-6-2016/rmms-supplement/

This comprehensive article discusses the concepts of statistics and hypothesis testing, including:

• How to formulate a null and alternate hypotheses as it relates to rapid method validation 
• Identifing the appropriate statistical test
• How to calculate a p-value
• Comparing the p-value with a chosen alpha level

Next we, dive into equivalence and non-inferiority tests, which we believe is the preferred strategy for demonstrating a rapid method is as good as and/or no worse than the existing microbiological method you wish to replace. The article specifies how to select lower and upper equivalence levels and also provides case studies on how to utilize these statistical tools.

EMA Promotes the Use of Rapid Microbiological Methods for Water Testing

In 2005, the European Medicines Agency (EMA) explored the regulatory consequences of implementing an alternative method for rapid control of microbiological quality of water for injection (WFI) and Purified water. Discussions between the Quality Working Party, the Good Manufacturing Practices and Good Distribution Practices Inspectors Working Groups and the Committees for Human and Veterinary Medicinal Products concluded Since it is expected that the water will continue to meet Ph. Eur. specifications, if tested, no change to dossier requirements* (variations) would be involved and therefore no regulatory impact on individual products would normally be anticipated.

• *However, this will depend on the level of detail in the original dossiers concerned.

Following discussions over the last 2-3 years around the revision of the European Pharmacopoeia (Ph. Eur.) WFI monograph (0169), the Water Working Party concluded that there was evidence to support a revision of the monograph, which proposes to take account of current manufacturing practices using methods other than distillation for producing water of injectable quality.

The Ph.Eur. monograph (Monograph 169) was revised to include, in addition to distillation, reverse
osmosis (RO) coupled with suitable techniques, for the production of WFI.

In order to provide clarification and guidance in relation to the use of reverse osmosis in the manufacture of WFI and also to provide more detailed guidance on the control of biofilms, the Water Working Party developed a draft set of Questions and Answers that were sent out for public consultation on August 5, 2016, with an end of consultation date (i.e., the deadline for comments) of November 4, 2016. The draft Q&A is intended to provide preliminary guidance until such time the on-going revision of Annex I of the GMP guide is complete.

One of the questions posed by the working party is, “What testing should be employed during initial qualification and routine operation sampling?”

In answering this question, the working party states:

• Use of rapid microbiological methods should be employed as a prerequisite to the control strategy to aid with rapid responses to deterioration of the system.” 

• Methods to be considered should include, “rapid endotoxin testing – use of more sensitive and point of use test methods,” and “quantitative microbiological test methods – in line with Ph.Eur. 5.1.6 monograph Alternative Methods for control of Microbiological Quality. 

• Due consideration should be given to employing alternate methods for the rapid quantitative determination of the contamination levels existing within the water system. The validation of such system should be in line with the above referenced monograph.

As such, EMA is encouraging the industry to employ rapid microbiological methods for the quantitative assessment of viable organisms and the presence of endotoxin in WFI and purified water. Additionally, the use of such methods should be validating according to Ph. Eur. chapter 5.1.6.

In my opinion, this is long-overdo step in the right direction. Technologies for the rapid analysis of pharmaceutical grade water have been available for years, and a number of firms who have experienced water system-related contamination events could have benefited from using a rapid method to quickly detect, and most importantly, remediate issues that have directly impacted their manufacturing facilities and/or finished product.

The full Q&A draft may be viewed at:

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/08/WC500211657.pdf.

Comments on the draft are due by November 4, 2016.

CRISPR Aids New Zika Paper-Based Diagnostic Test

The rapid spread of Zika virus into South America and its link to fetal neurodevelopmental issues have underscored the need for fast, low-cost diagnostics for use in endemic regions. Now, a collaborative team of scientists from Wyss Institute for Biologically Inspired Engineering, Massachusetts Institute of Technology, Boston University, Arizona State University, Cornell University, University of Wisconsin-Madison, Broad Institute, and the University of Toronto have developed a cell-free, paper-based platform that can host synthetic gene networks and help reliable diagnosis of Zika-infected patients.

This new test, which can distinguish Zika from the very similar dengue virus within a few hours, can be stored at room temperature and read with a simple electronic reader, making it practical for widespread use.

"One of the key problems in the field is being able to distinguish what these patients have in areas where these viruses are co-circulating," explained coauthor Lee Gehrke, Ph.D., professor of microbiology and immunobiology at Harvard Medical School.

"We [now] have a system that could be widely distributed and used in the field with low cost and very few resources," added senior study author James Collins, Ph.D., professor of medical engineering and science in MIT's Department of Biological Engineering and Institute for Medical Engineering and Science (IMES).

The findings from this study were published recently in Cell in an article entitled “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.”

Currently, patients are diagnosed by testing for reactive antibodies against Zika in their bloodstream or by looking for pieces of the viral genome in a patient's blood sample using PCR. However, these tests can take days or weeks to yield results. Furthermore, the antibody test cannot discriminate accurately between Zika and dengue.

The researcher team created a cluster of diagnostic measuring devices on a freeze-dried piece of paper the size of a stamp that employs toehold sensors and isothermal RNA amplification—coupled to a CRISPR based module. Activated by the amplified sample, the diagnostic sensors programmed into the paper provide an extremely sensitive, low-cost, programmable assay that provides rapid results.

The new test is based on technology that Dr. Collins and colleagues previously developed to detect the Ebola virus. In October 2014, the researchers demonstrated that they could create synthetic gene networks and embed them on small discs of paper. These gene networks can be programmed to detect a particular genetic sequence, which causes the paper to change color.

The sensors, embedded in the paper discs, can detect 24 different RNA sequences found in the Zika viral genome, which, like that of many viruses, is composed of RNA instead of DNA. When the target RNA sequence is present, it initiates a series of interactions that turns the paper from yellow to purple, which can be visualized by eye. The researchers also developed an electronic reader that makes it easier to quantify the change, especially in cases where the sensor is detecting more than one RNA sequence.

The investigators tested the device with samples taken from monkeys infected with the Zika virus because samples from human patients affected by the current Zika outbreak have been difficult to obtain. Amazingly, they found that in these samples, the device could detect viral RNA concentrations as low as 2 or 3 parts per quadrillion.

“The diagnostic platform developed by our team has provided a high-performing, low-cost tool that can work in remote locations," noted lead study author Keith Pardee, Ph.D., assistant professor at the University of Toronto. "We have developed a workflow that combines molecular tools to provide diagnostics that can be read out on a piece of paper no larger than a postage stamp. We hope that through this work, we have created the template for a tool that can make a positive impact on public health across the globe."

"Our synthetic biology pipeline for rapid sensor design and prototyping has tremendous potential for application for the Zika virus and other public health threats, enabling us to rapidly develop new diagnostics when and where they are needed most," Dr. Pardee added.

The researchers have envisioned that this approach could also be adapted to other viruses that may emerge in the future. Dr. Collins now hopes to team up with other scientists to develop the technology further for diagnosing Zika.

"Here we've done a nice proof-of-principle demonstration, but more work and additional testing would be needed to ensure safety and efficacy before actual deployment," Dr. Collins said. "We're not far off."

Source: Genetic Engineering & Biotechnology News

African Scientists a Step Closer to Develop Rapid Test for TB

Tuberculosis ranks alongside HIV/AIDS as a leading cause of death worldwide. According to the World Health Organisation, 1.5 million people died from TB in 2014. The challenges in tackling the disease include the facts that people are tested too late and that the turnaround for most tests is long. To remedy this a point-of-care rapid diagnostic test for TB has been developed by a multinational team of scientists led by researchers at Stellenbosch University in South Africa. One of its co-inventors, Professor Gerhard Walzl, spoke to The Conversation Africa’s health and medicine editor Candice Bailey.

How have TB tests been done up until now and what are the challenges?

There are three main tests that are currently in use.

A culture test – the most sensitive – requires people to produce a sputum sample that is sent to a centralised laboratory where a culture test is done. A positive result shows up after ten days. A confirmed negative result takes up to 42 days.

The problem with this test is that it is only available in centralised laboratories, which means patients must make several trips to a hospital or health facility to get their results. And it is very expensive.

Then there is the sputum microscopy test. This is widely used in Africa. It requires the sputum slides of each patient to be individually checked.

The test is inexpensive. But it is labour intensive, which means that only a limited number of smear tests can be assessed a day. In addition, it only has a 60% sensitivity rate.

On top of this, the test poses particular challenges for children and for people living with HIV.

In the case of young children, samples need to be taken from their stomachs as they cannot follow instructions to produce a good quality sputum sample. This requires the use of a nasal tube, which is not pleasant for the child or the health-care worker.

The test also isn’t effective for people living with HIV. This is because their sputum often has low levels of the bacteria, which can lead to a false negative test result.

There is also a molecular test that detects bacterial DNA in the sputum sample. This test only takes two hours to produce a result and although it speeds up the detection of TB, it is not widely available to people in rural areas as instruments are placed in a centralised manner.

How will your test change this?

If our test is accepted after clinical trials are completed it will be able to provide almost immediate results. People will be able to be diagnosed and start treatment in a single visit to a health-care facility.

The test is done with blood obtained from a finger prick and can make a TB diagnosis in less than an hour. The diagnostic test is a hand-held, battery-operated instrument that will measure chemicals in the blood of people with possible TB. This test will not have to be done in a laboratory and health-care workers will be able to perform it with minimal training.

It is a low-cost screening test and has the potential to significantly speed up TB diagnosis in resource-limited settings.

At what stage is the test?

The test is still in development. We have patented the biosignature, which identifies the levels of chemicals in the blood of a patient. A biosignature consists of a combination of chemicals and indicates a disease state. This signature was discovered by African scientists. The inventors included South African, Cameroonian and Ethiopian scientists.

The test’s accuracy and efficacy will be tested in five African countries over the next three years. We will recruit 800 people who have TB symptoms from Namibia, the Gambia, Uganda, Ethiopia and South Africa.

Clinical research sites will be set up or strengthened in all five countries. And participating countries will be able to use the data generated from this project.

We are still trying to improve the signature by adding additional markers. In addition, we would like to optimise and fine tune the device to enable it to measure the signature on a strip similar to a pregnancy test or a glucose test strip.

Why is the test important for South Africa?

South Africa has the highest TB rates in the world. Each year between 450,000 and 500,000 people develop TB. This gives the country an incidence rate of 834 infections for every 100,000 people. On the rest of the continent, the incidence rate is between 300 and 600 infections for every 100,000 people. In China the incidence is 68 for every 100,000 people and in most European countries it is less than ten for every 100,000 people.

One of the challenges in South Africa is that people in remote areas with high TB incidence still do not benefit from newer developments in TB testing. As a result they face long diagnostic delays and often need to come back to clinics on several occasions before they are diagnosed.

This test will mean that health-care workers with minimal training can use the test at grassroots level and get immediate access to screening test results.

It would also reduce the cost of testing for TB. Our test would initially cost US$2.50 per test. With commercialisation that price could drop significantly. Currently the culture test costs $45 per test while the DNA sample test costs $12 per test.

How does this test fit into the bigger picture of dealing with TB?

The test would be used best as a screening test. This is because it can identify people who need further investigation and can screen out those who don’t. So far we have been able to identify 70% of patients who do not need further testing.

The World Health Organisation has identified a screening test as important for high-prevalence areas, for those who are in contact with people who have TB, those living with HIV, homeless people, immune-compromised people and those living in areas with poor access to diagnostic services.

Source: The Conversation

White House and ASM Microbiome Research Project Includes Rapid DNA Sequencing

The White House, along with the American Society of Microbiology (ASM), announced the formation of several initiatives designed to further knowledge of the microbiome's benefit to human life and planetary function.

And in an additional concerted effort to advance microbial research, a diverse group of scientists writing today in mBio advocated for new technologies, systems, and philosophical approaches to understanding and harnessing the microbiome.

National research initiative

The White House's Office of Science and Technology Policy (OSTP) today announced the genesis of an interdisciplinary National Microbiome Initiative (NMI). The program will boost federal support of research that attempts to understand the function of microorganisms that live on or in all living species and are necessary to the well-being of living creatures and ecosystems.

Imbalanced microbiomes caused by human disruption and environmental degradation contribute to a host of issues, including chronic and infectious diseases, the spread of harmful microbes, reduced agricultural yields, and excesses of atmospheric carbon, the OSTP said.

The NMI will focus on encouraging comparative study of microbiomes across ecosystems to arrive at a better understanding of the conditions under which microscopic life thrives. The initiative's objectives were distilled during a year-long fact-finding process and will support interdisciplinary research, develop technologies that organize and increase access to microbial data, and expand the microbiome workforce through education and citizen science, the OSTP said.

The federal government will invest more than $121 million in the NMI, adding to the 2012 to 2014 investment of $922 million in microbiome research. The OSTP is partnering with groups including the Bill and Melinda Gates Foundation, the University of Michigan, and The BioCollective, LLC, to advance understanding of the microbial interface between humans and agricultural practices, the effect of the microbiome on Type 1 diabetes, and the establishment of a microbial data and sample bank, the White House said. Stakeholders will provide an additional $400 million in funding.

"We expect that by accelerating progress in this important field, the NMI will deliver considerable benefits to our planet and those who inhabit it," said Jo Handelsman, PhD, OSTP associate director for science.

Multisector collaboration and funding

Despite growing scientific knowledge about the ubiquity of microscopic life and the benefits that microorganisms confer to human and animal health, little is known about how microbes function and communicate.

In response to this gap in knowledge, the ASM is partnering with the American Chemical Society, the American Physical Society, and the Kavli Foundation to offer $1 million in research support as part of the Kavli Microbiome Ideas Challenge, the ASM said.

Researchers in all scientific disciplines are invited to submit ideas for experimental research into how microbial life forms communicate, interact within communities, and regulate health and bodily processes with multicellular hosts, the ASM said.

"This initiative is an important step to expand our understanding of the pivotal role of microbes in the ecosystem," said Lynn Enquist, PhD, ASM president.

The future of microbiome research

An interdisciplinary group of scientists writing today in mBio propose additional collaborative research that reflects the microbiome's effects, not only on human health, but on all aspects of the environment.

Microbes have shaped the planet and its atmosphere for more than 3.5 billion years, yet scientific research has only recently prompted "the realization that our species, Homo sapiens, is at least as microbial as human in terms of cell numbers and much more so in terms of genetic potential," the authors said.

The microbiome affects numerous systems necessary for life, including the atmospheric oxygen-carbon cycle, soil quality, and human digestive and immune system function. Disruptions or imbalances to microbial life can result in environmental degradation and temperature change, increases in human and animal disease, and the evolution of microbes that can cause harmful infections.

"Our planet's natural biomes and those that we manage for food and fuel will likely experience conditions beyond their contemporary climate boundaries, and our current understanding of the sensitivities of their microbial components limits our ability to predict how they will respond," the authors said.

The authors advocate for cross-sector, collaborative efforts to advance understanding of microbial function and its benefits to human and ecosystem health that go beyond microbial DNA sequencing and move toward an understanding of function in microbial communities and in different physical environments.

The authors' recommendations for research priorities include development of technology for rapid microbial DNA sequencing, understanding of genetic diversity, and analysis of gene expression and function; establishment of a microbial data and sample reference catalog; advances in imaging technology that can penetrate soil and water without damaging microbes (eg, silicon-based sensor arrays); and better studies that evaluate microbial function and ecosystem dependencies.

A growing global population, increased exposure to new microbes in urban environments, and the threat of resource shortages and climate change makes these areas of study more crucial than ever, the authors said. Though they encourage cross-sector work to discover solutions to global challenges, the authors acknowledge that the most important partnership is the one between human and animal life and the microbiome.

"This intermingling of genes and functions across the tree of life continues, allowing multicellular organisms to adapt more rapidly to new environments, using the versatility of their microbial partners," the authors said.

MIT Develops a Rapid, Paper-Based Zika Diagnostic Test

A new paper-based test developed at MIT and other institutions can diagnose Zika virus infection within a few hours. The test, which distinguishes Zika from the very similar dengue virus, can be stored at room temperature and read with a simple electronic reader, making it potentially practical for widespread use.

“We have a system that could be widely distributed and used in the field with low cost and very few resources,” says James Collins, the Termeer Professor of Medical Engineering and Science in MIT’s Department of Biological Engineering and Institute for Medical Engineering and Science (IMES) and the leader of the research team.

An outbreak of the Zika virus that began in Brazil in April 2015 has been linked to a birth defect known as microcephaly. Many infected people experience no symptoms, and when symptoms do appear they are very similar to those of related viruses such as dengue and chikungunya.

Currently, patients are diagnosed by testing whether they have antibodies against Zika in their bloodstream, or by looking for pieces of the viral genome in a patient’s blood sample, using a test known as polymerase chain reaction (PCR). However, these tests can take days or weeks to yield results, and the antibody test cannot discriminate accurately between Zika and dengue.

“One of the key problems in the field is being able to distinguish what these patients have in areas where these viruses are co-circulating,” says Lee Gehrke, the Hermann L.F. von Helmholtz Professor in IMES and an author of the paper.

Collins, Gehrke, and colleagues from Harvard University’s Wyss Institute for Biologically Inspired Engineering and other institutions described the new device in the May 6 online edition of Cell. The paper’s lead authors are Melissa Takahashi, an IMES postdoc; Dana Braff, an MIT graduate student; Keith Pardee, an assistant professor at the University of Toronto and former Wyss Institute research scientist; Alexander Green, an assistant professor at Arizona State University and former Wyss Institute postdoc; and Guillaume Lambert, a visiting scholar at the Wyss Institute.

Paper-based detection

The new device is based on technology that Collins and colleagues previously developed to detect the Ebola virus. In October 2014, the researchers demonstrated that they could create synthetic gene networks and embed them on small discs of paper. These gene networks can be programmed to detect a particular genetic sequence, which causes the paper to change color.

Upon learning about the Zika outbreak, the researchers decided to try adapting their device to diagnose Zika, which has spread to other parts of South and North America since the outbreak began in Brazil.

“In a small number of weeks, we developed and validated a relatively rapid, inexpensive Zika diagnostic platform,” says Collins, who is also a member of the Wyss Institute.

Collins and his colleagues developed sensors, embedded in the paper discs, that can detect 24 different RNA sequences found in the Zika viral genome, which, like that of many viruses, is composed of RNA instead of DNA. When the target RNA sequence is present, it initiates a series of interactions that turns the paper from yellow to purple.

This color change can be seen with the naked eye, but the researchers also developed an electronic reader that makes it easier to quantify the change, especially in cases where the sensor is detecting more than one RNA sequence.

All of the cellular components necessary for this process — including proteins, nucleic acids, and ribosomes — can be extracted from living cells and freeze-dried onto paper. These paper discs can be stored at room temperature, making it easy to ship them to any location. Once rehydrated, all of the components function just as they would inside a living cell.

The researchers also incorporated a step that boosts the amount of viral RNA in the blood sample before exposing it to the sensor, using a system called NASBA (nucleic acid sequence based amplification). This amplification step, which takes one to two hours, increases the test’s sensitivity 1 million-fold.

Julius Lucks, an assistant professor of chemical and biomolecular engineering at Cornell University, says that this demonstration of rapidly customizable molecular sensors represents a huge leap for the field of synthetic biology.

“What’s really exciting here is you can leverage all this expertise that synthetic biologists are gaining in constructing genetic networks and use it in a real-world application that is important and can potentially transform how we do diagnostics,” says Lucks, who was not involved in the research.

Distinguishing viruses

The team tested the new device using synthesized RNA sequences corresponding to the Zika genome, which were were then added to human blood serum. The researchers showed that the device could detect very low viral RNA concentrations in those samples and could also distinguish Zika from dengue.

The researchers then tested the device with samples taken from monkeys infected with the Zika virus. (Samples from human patients affected by the current Zika outbreak are very difficult to obtain.) They found that in these samples, the device could detect viral RNA concentrations as low as 2 or 3 parts per quadrillion.

The researchers envision that this approach could also be adapted to other viruses that may emerge in the future. Collins now hopes to team up with other scientists to further develop the technology for diagnosing Zika.

“Here we’ve done a nice proof-of-principle demonstration, but more work and additional testing would be needed to ensure safety and efficacy before actual deployment,” he says. “We’re not far off.”

The research was funded by the Wyss Institute for Biologically Inspired Engineering, MIT’s Center for Microbiome Informatics and Therapeutics, the Defense Threat Reduction Agency, and the National Institutes of Health.

SOURCE: MIT News

Laser Tool Effective at Identifying Mutant Listeria Bacteria

The folks at Purdue University continue to develop next generation technologies for the detection of pathogenic bacteria. The latest involves the use of light scattering.

A Purdue University-developed laser tool already effective in quickly detecting harmful bacteria has been shown to detect mutant varieties of listeria - and in the same amount of time.

The BARDOT (pronounced bar-DOH') laser scans bacteria colonies looking for unique patterns that each bacterium makes. When the light penetrates a bacteria colony, it produces a scatter pattern that can be matched against a library of known bacteria patterns to identify a match. The system can identify bacteria such as salmonella, listeria, bacillus, vibrio and E. coli within 24 hours.

Now, Arun Bhunia, professor of food microbiology, and Atul Singh, research scientist, have shown that BARDOT (acronym for "bacterial rapid detection using optical scatter technology") can pinpoint small genetic mutations in listeria just as quickly, significantly reducing the time it would take scientists to identify those mutations in bacterial strains used for research. Their study was published in the journal Applied and Environmental Microbiology.

"This is a versatile microbiology tool, and we wanted to see if we can use it for mutant strains," Bhunia said. "This is a really powerful tool to help researchers find those mutant strains much easier on a petri plate. You can avoid the laborious techniques required to screen or detect these mutant strains."

Scientists use mutant bacteria to understand biology of pathogens and how they can combat outbreaks in food that can cause illnesses or death. Current methods of identifying mutants can take several days, whereas BARDOT can do the same work in less than a day.

Singh said he visualized the changes in bacteria using the laser system.

"It's like if you compare a wild type that is a normal bacteria, you get a scatter pattern. And then if you delete a certain gene, you get a new picture," he said.

The reverse was also true. By restoring the deleted gene, the BARDOT system recognized the bacteria as a regular wild type of strain.

Bhunia said his lab will continue to study BARDOT's ability to identify mutants of other bacteria and build libraries so the tool can be used for that work. He will also test the system's ability to identify mutant bacteria from natural settings such as from contaminated food.

The U.S. Department of Agriculture funded this study through the Center for Food Safety Engineering.

ABSTRACT

Virulence Gene-Associated Mutant Bacterial Colonies Generate  Differentiating Two-Dimensional Laser Scatter Fingerprints

Atul K. Singh, Lena Leprun a, Rishi Drolia, Xingjian Bai,
Huisung Kim , Amornrat Aroonual, Euiwon Bae, Krishna K. Mishra, Arun K. Bhunia

In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid detection using optical scattering technology) could be used to directly screen colonies of Listeria monocytogenes, a model pathogen, with mutations in several known virulence genes, including the genes encoding Listeria adhesion protein (LAP; lap mutant), internalin A (ΔinlA strain), and an accessory secretory protein (ΔsecA2 strain). Here we show that the scatter patterns of lap mutant, ΔinlA, and ΔsecA2 colonies were markedly different from that of the wild type (WT), with >95% positive predictive values (PPVs), whereas for the complemented mutant strains, scatter patterns were restored to that of the WT. The scatter image library successfully distinguished the lap mutant and ΔinlA mutant strains from the WT in mixed-culture experiments, including a coinfection study using the Caco-2 cell line. Among the biophysical parameters examined, the colony height and optical density did not reveal any discernible differences between the mutant and WT strains. We also found that differential LAP expression in L. monocytogenes serotype 4b strains also affected the scatter patterns of the colonies. The results from this study suggest that BARDOT can be used to screen and enumerate mutant strains separately from the WT based on differential colony scatter patterns.

SOURCE: Purdue University