
Nanotechnology and Microbiology

Updated routinely by Dr. Michael J. Miller, our RMM blog will keep you informed of new and noteworthy technologies, reviews of recent publications and presentations, upcoming conferences and training events, and what's changing in the RMM world. You can also follow our blog posts in the Rapid Microbiology Methods LinkedIn group.
Dangers of Diarrhoea
Children are at higher risk of contracting diarrhoea than adults due to their underdeveloped immune systems and they are likely to be affected for longer periods. In the Western world, most cases are easily treated but it is a different story in developing countries, where infection rates are higher and death is a common outcome.
Acute diarrhoea is generally caused by bacterial, viral, or parasitic infection and one of the key bacteria is diarrhoeagenic Escherichia coli, which can enter the body via contaminated food. However, it is not simply one strain that is responsible. In foods, four main categories can cause diarrhoea: enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorraghic (EHEC) and enteroinvasive (EIEC) E. coli.
Each of these categories contains similar but individual DNA, which proved a blessing for a team of Chinese scientists who have exploited the differences in a novel detection method. Lichun Cui from the Northeast Forestry University, Harbin, with colleagues from the Northeast Agricultural University, Harbin, and the Heilongjiang and Hainan Entry-Exit Inspection and Quarantine Bureaus, devised a procedure based on denaturing HPLC that could detect single or mixed E. coli infections in food.
Denaturing HPLC
In denaturing HPLC, mismatches in the DNA bases of double-stranded DNA allow their separation provided certain criteria are met. At elevated temperatures, the hetero and homo duplex chains unwind, or denature, to their individual strands which are resolved on the HPLC column.
The HPLC stationary phase must be inert and electrically neutral. Under these conditions, DNA cannot bind due to its inherent negative charge but the addition of an ion pairing agent to the mobile phase changes the properties and binding is achieved via electrostatic interactions.
The hetero duplexes are denatured to a greater extent than the homo duplexes, so that they are retained less strongly on the column and elute first. So, pairs of hetero and homo chains are observed in the HPLC chromatograms.
In the case of E. coli, the researchers employed a poly(styrene-divinylbenzene) column and added triethylammonium acetate to the mobile phase for ion pairing. Separation was effected with a gradient of acetonitrile.
DNA was extracted from E. coli bacteria and subjected to polymerase chain reaction (PCR) amplification using unique primers based on the conserved regions of each of the four bacteria. Sufficient sample was generated for HPLC analysis and only the expected products were produced, as proven by agarose gel electrophoresis. The average size of the products was 220, 300, 330 and 500 base pairs for ETEC, EPEC, EIEC and EHEC, respectively.
Bacterial Strains Detected Together in Food
The HPLC separations were carried out at 50°C under non-denaturing conditions, which produced a single peak for each amplified fragment. They eluted at different retention times over 4-9 minutes, allowing them to be distinguished from each other.
The PCR products from all four E. coli categories were then mixed together for HPLC, confirming that they can be separated and distinguished using their retention times.
The critical step in the process is the specificity towards each category. This was assured by subjecting the genomic DNA from 34 bacterial strains to the same amplification and analysis process using the unique primers. Only the ETEC, EPEC, EIEC and EHEC strains and their isolates gave positive results.
The novel procedure was used to test 189 samples of faecal matter from patients as well as 690 import and export food samples, including beef, pork, chicken, sausages and milk. Blind testing was carried out and the results were compared with those from the conventional method which involves bacterial culture over 2-3 days and biochemical reactions.
A total of 60 positive samples were identified with all four strains being detected. The results were in perfect agreement with the conventional method, confirming the validity of the approach. However, the PCR denaturing HPLC method is much faster and provides a valid alternative. It could also replace the basic PCR assay which requires a gel electrophoresis step.
The multiplex method is capable of detecting all four strains in one procedure, so can identify one or more of the infections in contaminated food and in patient faeces, permitting rapid detection and diagnosis and reducing the time before appropriate action can be taken.
The original article appears here. Image by Renjith Krishnan.
“A rapid pathogen screener is a device capable of detecting microorganisms in near real-time, without the use of cultured colonies or a traditional microbiology lab,” PhD student Monika Weber told Security Management. Weber leads the development team.
Development of the device started as an assignment while under instruction of Prof. Mark Reed, the Harold Hodgkinson professor of engineering and applied science. The assignment was to develop a device that had marketable potential.
“We were thinking about what kind of device would be beneficial….We started asking doctors what areas of medical science could be improved,” Weber said.
Doctors told them that bacteria diagnostics would be a promising focus area because of the current time and costs of culture growth – which can sometimes take days or require separate labs. Their comments resonated with Weber, who recently became lactose intolerant because of a bacterial infection. Testing using the alpha screen is 10 to 50 times faster than the current methods—culture growth and polymerase chain reaction. The current cost of one test is estimated at $1-20 times less expensive than current testing methods. Weber says a quicker diagnosis could have prevented her condition.
Weber’s isn't the first endeavor focused on rapid bacteria detection. Researchers in the past have spent years developing similar technologies, but Weber says variations of the alpha screen are being developed to address all areas that have the need for bacteria detection.
The low cost of testing using the alpha screen will be beneficial to developing countries, Weber said—countries where a timely diagnosis could mean the difference between life and death. The device will allow doctor’s offices to make diagnoses in real-time.
For the food industry, the alpha screen has very promising applications as well, she said. “We hope that the alpha-screen technology…will help prevent disease outbreaks, like the 2011 Listeria outbreak in the U.S. and the E. coli outbreak in Germany. We anticipate that alpha-screen will have dramatic implications for domestic and international food security,” she said.
The alpha screen is still in its development stages. The basic version is a battery-powered device about the size of a coin. It can only detect one or two types of bacteria. “However, to meet the needs of prospective customers we have also designed a larger unit, which could detect simultaneously over 100 different types of bacteria,” Weber said.
The team is working to raise funds for further development and prospective commercialization. Earlier this month, they moved $20,000 closer to that goal after winning the Create the Future design contest. The contest awarded prizes to recognize research efforts in engineering focused on benefitting humanity, the environment, and the economy. More than 900 product ideas from 50 countries were entered.
Weber said she hopes the publicity for their work will pay off for the project.
“We hope to attract people and institutions that would sponsor research and investors to move the project along,” Weber said.
Weber’s isn’t the first endeavor focused on rapid bacteria detection. Researchers in the past have spent years developing similar technologies.
During last year’s PDA Micro meeting, the USP stated that they were going to update/revise the existing USP 1223 informational chapter and provide additional guidance with respect to the use of alternative micro methods. First, we must be reminded that the use of RMMs as a replacement for existing methods is nothing new, as the USP provides guidance on the validation of alternative methods. More importantly, USP micro methods are intended to be referee tests (i.e., adjudicative) for the analysis of monograph products and compendial articles, and they were not intended to be QC release assays or in-process tests. Actually, USP referee tests were not intended to be used as QC assays without modification, and this modification is the responsibility of the method user.
USP <1223>, the informational chapter that provides guidance on validating alternative or rapid methods, is under revision. Some of the changes from the current version may include enhanced guidance on method selection and qualification, and more specific content than what is currently provided. Additionally, the committee is concerned that RMM implementation is being held up because users are too concerned at arriving at a perfect definition of method equivalence. Therefore, we may see some changes in how the USP recommends how to determine whether an alternative method is as good (or better) than a compendial method that is current in use.
Next, the expert committee is now exploring the development of a new referee sterility test; however, the referee method cannot be sourced from a single, patented technology. The committee will initially y will focus on biologics (cytotherapy/regenerative medicine products) and radiopharmaceuticals. And they will get support from the USP biologics committee as well as scientists from CBER (please see my August blog posts for additional guidance form the FDA on the use of RMMs for sterility testing).
Finally, the committee plans on providing validation-useful information for the development and validation of HPLC methods for antibiotic assays for products where micro methods are still being employed.The opening keynote address is being presented by a world-renowned microbiologist and subject matter expert in rapid methods for the food industry, Dr. Daniel Y.C. Fung. Dr. Fung is Industry Professor, Food and Science, at Kansas State University. His presentation focused on Global Developments of Rapid Methods and Automation in Microbiology: A Thirty Year Review and Predictions into the Future.
Rapid methods and automation in microbiology is a dynamic area of technological advancement sustaining a stream of emerging technologies. Rapid microbial methods continue to offer unique opportunities for improving product quality assurance and economy of quality control and manufacturing operations. Almost ten years ago, improvements in microbial isolation, rapid detection, characterization, and enumeration lead to his prediction “…companies that aren’t converting to rapid methods won’t be in business in 10 years…”
Dr. Fung reviewed the use of rapid methods within the food and medical sectors since the 1960’s. Methods have included modifications of traditional, growth-based procedures using conventional medium, including a double tube agar method Dr. Fung developed himself. In this procedure, growing C. perfringens was able to be viewed within a few hours. And over the years, more automated systems were being introduced. For example, impedance microbiology procedures have been around for more than 30 years, as well as methods for the detection of ATP.
Immunological dip-sticks then came on the market, which provided results on the presence of food-borne pathogens in as early as 10 minutes. Today, we can utilize a wide variety of molecular and nucleic amplification systems, including automated, real-time PCR, as well as novel biosensors, microarrays and nanosensors.
Within the food processing sector, it was projected that more than 740 million micro tests were performed in 2008 by more than 40,000 food processing plants, and it is estimated that the worldwide market for micro testing is more than $2 billion. And the market for food microbiology testing continues to grow, year over year. For example, the rate of growth of micro testing from 2008 to 2010 was more than 6%. But Dr. Fung also stated that the use of rapid methods can also provide considerable cost savings, depending on the method being utilized.
The take home message from Dr. Fung’s keynote is that the number of microbiology assays associated with the monitoring of food will continue to increase, especially in light of recent contamination events, and that rapid technologies will play a very important role in protecting the world’s food supplies.
When asked what the pharmaceutical industry can learn from the food industry (in terms of the adoption of rapid methods), Dr. Fung stated that the expectations for microbiological safety is much higher in the pharmaceutical industry than in the food industry, and that we can benefit greatly from the implementation of rapid methods. Interestingly, the food industry looks up to the pharma industry for guidance on excellence in microbiology testing. Their perception is that we pharma microbiologists strive for perfection, and that we are always looking at ways to implement new technologies. Unfortunately (from my point of view), our industry has been extremely slow to adopt rapid methods for a number of reasons, and that the food industry is actually well ahead of where we are today. This will be a topic of discussion during my rapid methods presentation tomorrow afternoon.
Keynote Address: Global Developments of Rapid Methods and Automation in Microbiology: A Thirty Year Review and Predictions into the Future. Daniel Y.C. Fung, PhD, Industry Professor, Food and Science, Kansas State University
A Rapid and Automated Prototype System for the Microbiological Monitoring of Sterile Pharmaceutical Environments. Michele J. Storrs-Mabilat, PhD, Global Scientific Partnerships Manager, Industrial Microbiology Division, bioMerieux, Inc.
Advanced Microbiological Systems—A Case Study on Validating a Microbial ID System to Meet the New Regulatory Requirements for Part 11. Gene Zhang, PhD, Principal Scientist, Bayer Healthcare Pharmaceutical
The Benefits of Developing an In-house Disinfectants Qualification Process: Understanding, Responsiveness, and Control. Eric Myers, Senior Supervisor - QC Microbiology, Pfizer, Inc., Claudio D. Denoya, PhD, Adjunct Professor, Department of Molecular and Cell Biology University of Connecticut
Single-Molecule Technology for Broadband Detection and Identification of Bacteria. Rudolf Gilmanshin, PhD, Vice President, Advanced Platform Research, Pathogenetix
Rapid Micro Project in Chiesi Pharmaceuticals Group: Development and Qualification of Alternative Method for the Release of Non-sterile and Sterile Products. Alessio Fantuzzi, PhD, Microbiological Project Supervisor, Chiesi Pharmaceutical, Michele Bosi, Quality Control Manager, Chiesi Pharmaceutical
Debunking the Myth’s Surrounding Rapid Microbiological Methods and Their Impact on Pharmaceutical Manufacturing and the Quality of Medicinal Products. Michael J. Miller, PhD, President, Microbiology Consultants, LLC.