LAL roundtable

Tim Sandle has participated in an endotoxin / LAL discussion, published in American Pharmaceutical Review. The discussion addresses key issues like low endotoxin recovery, together with future developments in LAL testing.

Here is an extract: “Dr. Tim Sandle: I think the difference between purified LPS and natural endotoxin complexes is established, and they will behave differently under conditions of the test.

The issue of low endotoxin one is important, although the subject is causing some confusion for LAL users. It would be good to get a consensus between the providers of LAL regents together with a clear signal from the regulators.”

The roundtable discussion appears in:

LAL Roundtable, American Pharmaceutical Review, 19(6): 47-51

The feature can be accessed here: APR

Posted by Dr. Tim Sandle

Design and Construction Standards to Build a Cleanroom

To maintain and assure quality, there are specific standards that apply to these spaces and guide the process of constructing and operating the rooms. While adhering to these requirements makes designing and constructing a cleanroom more complex than conventional construction projects, the standards serve as a blueprint for the successful completion of a cleanroom that will meet the exacting demands of the end-user.

Matt Strong has written an interesting article for Controlled Environments titled “Applying Design and Construction Standards to Successfully Build a Cleanroom.”

Here is an extract: “The first step in the cleanroom design process involves creating the Utility Matrix (UM), which outlines detailed specifications on each piece of equipment used in the cleanroom. This document, which is crucial to the cleanroom design, must be created and approved with the participation of the cleanroom operator/user. The UM can be developed directly by the operator, if the expertise exists in-house, or an outside consultant can be brought in to assist in development of the cleanroom layout and process flow. Once it’s completed, designers will use the UM to design the specific support systems required for the cleanroom. The UM remains an important document even after facility construction and should be continually updated throughout the life of the cleanroom.”

The article can be accessed here.

Posted by Dr. Tim Sandle

In silico molecular typing methods for Staphylococcus aureus

Currently, many different methods have been developed for subspecies typing of Staphylococcus aureus. However, there is no “ideal” one because of its disadvantages and thus there is no commonly accepted guidelines for the selection of the optimal typing method for epidemiological investigations. The aim of the study was an in silico comparison of widely used typing methods, including Multilocus sequence typing (MLST), Staphylococcus aureus Protein A (spa) typing, Pulsed-field gel electrophoresis (PFGE), Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) and whole genome multilocus sequence typing (wgMLST), in terms of discriminatory power and coefficients congruence. Quantitative assessment of discriminatory power and correlation of five typing methods: wgMLST, PFGE, MLST, MLVA and spa typing was performed, by targeting the different sites of the Staphylococcus aureus genome (which had evolved at different rates).

This is the basis of a new article of interest. The reference is:

Babenko, A., Turmuhambetova, A., Sandle, T. and Chesca, A. (2016) In silico comparison of molecular typing methods for characterization of Staphylococcus aureus, Acta Medica Mediterranea, 32: 1021-1027

The article can be accessed here.

For details, please contact Tim Sandle



Posted by Dr. Tim Sandle

New bacteria groups discovered underground

One of the most detailed genomic studies of any ecosystem has revealed an underground world of microbial diversity. The research has added dozens of new branches to the tree of life. The bacterial find comes from scientists who reconstructed the genomes of more than 2,500 microbes from sediment and groundwater samples collected at an aquifer in Colorado.

The scientists netted genomes from 80 percent of all known bacterial phyla, a remarkable degree of biological diversity at one location. They also discovered 47 new phylum-level bacterial groups, naming many of them after influential microbiologists and other scientists. And they learned new insights about how microbial communities work together to drive processes that are critical to the planet's climate and life everywhere, such as the carbon and nitrogen cycles.

Between the 47 new bacterial groups reported in this work, and 35 new groups published last year has led to a doubling of the number of known bacterial groups.

For further details see:

Karthik Anantharaman, Christopher T. Brown, Laura A. Hug, Itai Sharon, Cindy J. Castelle, Alexander J. Probst, Brian C. Thomas, Andrea Singh, Michael J. Wilkins, Ulas Karaoz, Eoin L. Brodie, Kenneth H. Williams, Susan S. Hubbard, Jillian F. Banfield. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nature Communications, 2016; 7: 13219 DOI:10.1038/ncomms13219

Posted by Dr. Tim Sandle

Mass starvation looming for Siberian reindeer

One of the world’s largest concentration of reindeer is to be found in the Yamal Peninsula in northwestern Siberia. A new report presents worrying evidence that the reindeer are threatened with starvation.

In the region of Russia, hundreds of thousands of reindeer are to be found. Many are herded by the indigenous Nenets people. For 2016-2017 a new report suggests that many of the reindeer are threatened due to a scarcity of food.

The report comes from the International Union for Conservation of Nature, and it has declared the reindeer to be vulnerable. Since 1990 there has been a steady decline in the population, with a 20 percent decline (around 80,000 animals) over the past twenty-five years.  This is due to scarcity of food and the root cause is climate change.

The largest decline occurred in 2013, when 61,000 reindeer starved to death on the peninsula. This occurred due to atypically thickly layers of snow and ice blocking and obscuring access to food - lichen and other vegetation.

The cause of the unusual ice and snow was attributed to retreating sea ice in the adjoining Barents and Kara Seas, which adjoin the peninsula. The researchers put the sequence of events into a flow diagram, which has been summarized by Laboratory Roots as:

Warming → Sea ice decline → Increased precipitation and winter temperatures → [Rain-on-snow] events → Reindeer mortality.

Essentially, warming temperature causes the melting of ice, which leads to high levels of evaporation and humidity. These temperature effects cause increased rainfall. The rain soaks the snowy ground, and when this is followed by a fall in temperature, the snow freezes leading to a thick layer of ice forming. The layer can prove too thick for the reindeer to smash through.

According to the lead researcher, Dr. Bruce Forbes: “Reindeer are used to sporadic ice cover, and adult males can normally smash through ice around 2 centimeters thick. But in 2006 and 2013, the ice was several tens of centimeters thick”

The concern is that a similar event will occur this coming winter, based on climate predictions. This is based on reducing ice in both the Arctic Sea and the Kara Sea, as collated by the National Snow and Ice Data Center, which is linked to the University of Lapland in Finland.

The findings are published in journal Biology Letters. The paper is titled “Sea ice, rain-on-snow and tundra reindeer nomadism in Arctic Russia.”

Posted by Dr. Tim Sandle

European Antibiotic Awareness Day: Update

On European Antibiotic Awareness Day (18th November), the European Association of Hospital Pharmacists (EAHP) has issued a call for all citizens to become students in prudent use. An update from Hospital Pharmacy Europe:

Speaking in Brussels, at an event organised by the European Centre for Disease Prevention and Control, Tajda Miharija Gala, EAHP Vice-President and policy lead on antimicrobial resistance (AMR), said"Working together at the European level to tackle the cross-border health threat of antimicrobial resistance, a relentless focus must be placed on achieving prudent use of antibiotics.

Succeeding in this endeavour has many components, including continuing to educate the public about the importance of appropriate use of antibiotics. Hospital pharmacists, alongside other healthcare professionals, need to assist patients in becoming students of prudent use, via direct counselling. In addition to this, all health systems can still achieve more in respect to antimicrobial stewardship in hospitals. Within multi-disciplinary teams the hospital pharmacist can lead on matters such as reviewing antibiotic serum levels and duration of therapy, advising on the cessation or amendment of inappropriate antibiotic treatments, educating other healthcare professionals on the restricted use of certain antibiotics, and counselling patients on their antibiotic therapy.

Combatting antimicrobial resistance involves everyone, including those in other sectors such as farming and veterinary care. We therefore encourage the European Commission to be bold in defining the roles of different professions within the next EU Action Plan on AMR. A team works best when there is total clarity on individual responsibilities."

Posted by Dr. Tim Sandle

Antimicrobial Resistance in the Environment

The class of chemicals characterized as biocides are used in all parts of society from home to hospital, to farms and industry. The presence of biocides selects for genes in microorganisms that can protect against their lethal effects. These biocide resistance genes are often the same genes as antibiotic resistance genes (i.e. cross-resistance), or they can be co-located on plasmids, for example, which means when biocides are present the microorganism will also co-select for antibiotic resistance genes (i.e. co-resistance).

Dr. Andrew C Singer has written an interesting blog post on antimicrobial resistance. Here is an extract:

“The problem with antimicrobial resistance (AMR), globally, is the combination of: 1) increased prevalence of antibiotic resistance; 2) rapid spread of AMR due to global travel; 3) antibiotic misuse; and 4) too few new antimicrobials in development. National, regional and global AMR Action Plans have been drafted to tackle many of these problems. Thorough reviews, such as the O’Neill AMR Reviews, provide a useful overview of these challenges and some mitigation measures. However, symptomatic of the O’Neill Reviews and AMR Action Plans is their under appreciation of the role that the environment plays in the selection, spread and transmission of AMR. Discussions of the environment are typically limited to the pharmaceutical manufacturing plants as a source of antibiotics and the role that sewage and farm run-off can play in dissemination of antibiotics. The discussions on these issues are superficial and narrow in scope.

Current AMR Action Plans and the O’Neill Reviews see antibiotics as the primary driver of AMR; hence, all mitigating measures are focused solely on reducing their use and release into the environment. This vision of the challenge of AMR is not helpful as it omits other AMR drivers that could be, on their own, more important than antibiotics for selecting, maintaining and spreading AMR in the environment, let alone as a collective group of AMR drivers.”

The full post can be accessed here.

Posted by Dr. Tim Sandle

The Science Of Christmas

This book looks at many of the traditions of Christmas and takes an amusing, sideways look st the science behind Christmas. Just how fast will Santa need to travel to deliver gifts to all the children? Where did tinsel come from and is it safe? Why do reindeer have red noses? And more!

Posted by Dr. Tim Sandle

Mold Monitoring and Control in Pharmaceutical Manufacturing Areas

Tony Cundell has written an important article on fungi for American Pharmaceutical Review.

The introduction reads:

“Recent high profile product recalls associated with mold contamination has resulted in more attention from the FDA to fungal isolation in environmental monitoring and product testing in the pharmaceutical industry. Companies need to anticipate these FDA concerns especially with respect to the upcoming regulatory inspections and institute remediation when mold is found in their products and manufacturing facilities to protect patient safety.”

In the article, Cundell reviews fungal isolation media and enumeration methods for cleanrooms, as well as identification strategies.

The article can be accessed here.

Posted by Dr. Tim Sandle

Spatiotemporal microbial evolution on antibiotic landscapes

An interesting research paper, considering a new way to examine the development of antibiotic resistance:

A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. Laboratory experiments, however, often study evolution in well-mixed systems. Here, we introduce an experimental device, the microbial evolution and growth arena (MEGA)–plate, in which bacteria spread and evolved on a large antibiotic landscape (120 × 60 centimeters) that allowed visual observation of mutation and selection in a migrating bacterial front. While resistance increased consistently, multiple coexisting lineages diversified both phenotypically and genotypically. Analyzing mutants at and behind the propagating front, we found that evolution is not always led by the most resistant mutants; highly resistant mutants may be trapped behind more sensitive lineages. The MEGA-plate provides a versatile platform for studying microbial adaption and directly visualizing evolutionary dynamics.

For further details, see Science.

Posted by Dr. Tim Sandle

First-generation Cephalosporins show Promise in Tuberculosis Therapy

Tuberculosis (TB) is the most deadly infectious disease in the world with treatment spanning for up to 6 months; however, in case of MDR-TB, it can even go up to 2 years. Research shows that the cost of developing a new drug is quite huge. Now, one research published in Scientific Reports shows that first generation cephalosporins could be a potential treatment for TB.  Read more 

Posted by Dr. Tim Sandle

New test for detecting prions in urine

A new test may detect the presence of abnormal proteins indicative of a deadly prion disease known as Creutzfeldt-Jakob Disease (CJD). By adapting an established blood test for variant CJD, researchers at the University College London say the new test accurately picks up on prions in urine, which could enable early detection and treatment.

Modifications to a blood test showed that it can be adapted to urine samples. The team tried the test in 162 people – a group that consisted of healthy controls, people with CJD, and people with other types of neurodegenerative diseases. They found the test to have 100 percent specificity (no false positives). The sensitivity to CJD was lower, at 40 percent. However, the team noted that this was a marked increase from a previous test in which the sensitivity to vCJD was at 7.7 percent.

For further details, see Laboratory Roots

Posted by Dr. Tim Sandle

What role will innovation and regulation play in tackling AMR?

News from the Royal Society of Biology:

A panel of expert speakers and delegates from across the life sciences gathered as part of Biology Week to debate how regulation and innovation can help tackle the antimicrobial resistance crisis.

Professor Mark Fielder, an advisor for the Clinical and Laboratory Sciences Institute and AMR expert, said in the USA up to 70% of antibiotic consumption was non-human because the drugs increase animal growth and meat yields by 10%.

“We have AMR moving between the animal and human world and we don’t look at that enough. We need to take a ‘one health view’ – think of the human, the environment and the animal, instead of thinking of them separately.”

The use of antibiotics as growth promoters was banned in the EU in 2006, but Professor Fielder said that the veterinary sector was ‘not as tightly controlled’ as the human health and medicine sector because practices are private. “Surveillance is expensive but it needs to happen to keep a lid on this and keep that lid shut tight.”

John Broughall, from Antibiotic Research UK, explained how the Yorkshire-based charity is screening old antibiotics and other compounds in the hope of finding ‘antibiotic resistance breakers’ – drugs which can disrupt organisms’ resistance mechanisms. This could lead to existing drugs being co-administered alongside failing antibiotics to boost their efficacy, a potential solution which would cost a fraction of the cost of developing a new antibiotic.

Tamar Ghosh, lead of the Longitude Prize, explained how the £10m fund will be awarded to scientists who within five years can develop a rapid, affordable, point of care diagnostic to reduce overuse of antibiotics. Last year the general public voted for the prize to focus on antimicrobial resistance from six shortlisted challenges to humanity, and it has so far attracted 205 teams from 39 countries.

“What’s great about those teams is that they are not all led by who you’d expect,” said Ghosh. “They’re not all microbiologists – there’s a great mix from industry, academia, people have come out of retirement, people are resurrecting old ideas. They are forming new consortia and new collaborations of engineers, chemists, physicists, social scientists.”

But Tamar Ghosh said that a diagnostic on its own 'is not going to solve the problem if people are not aware of it, if we don’t use the diagnostic collectively, and act on the result.'

The discussion moved between many related topics, such as the availability of online antibiotics, the pressure on GPs to prescribe antibiotics without proper diagnosis, the use of synthetic biology and phage therapy in novel antibiotics, and the thorny issue of why developing new antibiotics is not worth the investment for pharmaceutical companies.

“The payback period for an antibiotic is something like 23 years,” said the debate’s chair, Professor Jeff Cole, vice-president of the European Federation of Biotechnology. “As the O’Neill report concluded, you have to break the link between sales and research and development – although he did not come up with a solution, other than creating a global fund.”

The panel heard from the floor about how access to antibiotics has been restricted with some success in countries like Norway. But John Broughall made the point that many countries do not have the infrastructure to govern the use of antibiotics. “We can do whatever we want here but that resistance is only a six hour flight away,” he said.

Tamar Ghosh added the salient point that many more people die from a lack of access to antibiotics than from resistance issues, a factor which will undoubtedly make global cooperation on the issue difficult.

Source: RSB

Posted by Dr. Tim Sandle

Removal of Endotoxin from Protein in Pharmaceutical Processes

Bacterial endotoxin is the lipopolysaccharide component of the cell wall of Gram-negative bacteria, together with other cellular material that combines to form an endotoxin complex. Endotoxin is pyrogenic and it presents a risk to patients who are administered intravenous and intramuscular preparations.1Thus bacterial endotoxins pose a risk to many pharmaceutical processes and, where not controlled, to the finished products. There are different methods for endotoxin removal. These include depyrogenation,2 such as dry-heat processes applied to glassware, and rinsing,3 as might be applied to closures. These areas receive reasonable coverage within the pharmaceutical sector. Areas that are actively discussed within biotechnology fields but which receive less attention in the wider pharmaceutical context are steps to remove endotoxin bound to protein. This article considers some of the biotechnological applications for endotoxin removal.

The article reference is:

Sandle, T. (2016) Removal of Endotoxin from Protein in Pharmaceutical Processes, American Pharmaceutical Review, 19 (8): 1-5 

For further details see: Endotoxin


Posted by Dr. Tim Sandle

New FDA Q7 guidance (APIs)

The U.S. Food and Drug Administration has issued a new guidance document “Q7 Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients - Guidance for Industry.”

This guidance applies to the manufacture of APIs for use in human drug (medicinal) products. It applies to the manufacture of sterile APIs only up to the point immediately prior to the APIs being rendered sterile. The sterilization and aseptic processing of sterile APIs are not covered by this guidance, but should be performed in accordance with GMP guidances for drug (medicinal) products as defined by local authorities.

This guidance covers APIs that are manufactured by chemical synthesis, extraction, cell culture/fermentation, recovery from natural sources, or any combination of these processes.

According to the document:

“This document is intended to provide guidance regarding good manufacturing practice (GMP) for the manufacturing of active pharmaceutical ingredients (APIs) under an appropriate system for managing quality. It is also intended to help ensure that APIs meet the quality and purity characteristics that they purport, or are represented, to possess.

In this guidance, the term manufacturing is defined to include all operations of receipt of materials, production, packaging, repackaging, labeling, relabeling, quality control, release, storage and distribution of APIs and the related controls. In this guidance, the term should identifies recommendations that, when followed, will ensure compliance with CGMPs. An alternative approach may be used if such approach satisfies the requirements of the applicable statutes.”

The document can be accessed here: FDA

Posted by Dr. Tim Sandle

Rifampicin and antibiotic resistance

A common antibiotic called rifampicin, used to treat tuberculosis, leprosy, and Legionnaire's disease, is becoming less effective as the bacteria that cause the diseases develop more resistance.

One of the mechanisms leading to rifampicin's resistance is the action of the enzyme Rifampicin monooxygenase.

Pablo Sobrado, a professor of biochemistry in the College of Agriculture and Life Sciences, and his team used a special technique called X-ray crystallography to describe the structure of this enzyme. They also reported the biochemical studies that allow them to determine the mechanisms by which the enzyme deactivates this important antibiotic.

The enzyme, Rifampicin monooxygenase, is a flavoenzyme -- a family of enzymes that catalyze chemical reactions that are essential for microbial survival. These latest findings represent the first detailed biochemical characterization of a flavoenzyme involved in antibiotic resistance.

Details on the latest research can be found in the following papers:

Heba Abdelwahab, Julia S. Martin Del Campo, Yumin Dai, Camelia Adly, Sohby El-Sohaimy, Pablo Sobrado. Mechanism of Rifampicin Inactivation in Nocardia farcinica. PLOS ONE, 2016; 11 (10): e0162578 DOI:10.1371/journal.pone.0162578

Li-Kai Liu, Heba Abdelwahab, Julia S. Martin Del Campo, Ritcha Mehra-Chaudhary, Pablo Sobrado, John J. Tanner. The Structure of the Antibiotic Deactivating,N-hydroxylating Rifampicin Monooxygenase. Journal of Biological Chemistry, 2016; 291 (41): 21553 DOI:10.1074/jbc.M116.745315

Posted by Dr. Tim Sandle

Insight into Pseudomonas aeruginosa survival mechanism

The bacterium Pseudomonas aeruginosa can thrive in environments as different as the moist, warm tissue in human lungs, and the dry, nutrient-deprived surface of an office wall. Such adaptability makes it problematic in healthcare.

According to Blanca Barquera: "These organisms are able to live everywhere, under conditions with an enormous variety of food supply, salt levels, temperature, acid/base level, and oxygen level. And we have to ask -- how can they do this?"

She adds: “In order for the organisms to survive in so many different environments, the interior of the cell must remain a hospitable place for the biochemistry of life, regardless of what happens outside. And there are proteins in the membrane that are responsible for this."

Transport proteins make up the active interface between the cell and the environment, and among the most important of these proteins are those which transport ions -- atoms or molecules with a net positive or negative electrical charge -- into and out of the cell, Barquera said. Ion transport proteins maintain favorable concentrations of ions inside the cell, and also are at the heart of energy production. Transport of positively charged hydrogen and sodium ions, called cations, create gradients that provide energy for diverse cellular processes, such as cell motility, import of nutrients, and extrusion of chemicals that are toxic to the cell.

In the current project, "Control of Na+ and H+ transport in bacterial adaptation," researchers will seek to understand how transport proteins that move hydrogen and sodium cations through the cell membrane allow Pseudomonas to adjust its metabolism to different environmental conditions.

Researchers will look at several transport proteins including NQR, which moves sodium from the interior to exterior of the cell; NUO, which moves protons from the interior to the exterior of the cell; and sodium/proton anti-porters, which exchange ions to maintain constant pH and ionic concentrations inside the cell.

For further details see: Rensselaer Polytechnic Institute (RPI)

Posted by Dr. Tim Sandle

UN agrees to take action on antimicrobial resistance

A statement from the Royal Society for Biology, in relation to the United Nations agreeing to take action on antimicrobial resistance

“The Learned Society Partnership on Antimicrobial Resistance (LeSPAR) welcomes news of global political and pharmaceutical industry support for actions to tackle the threat of resistant infections.

The G7 and G20 summits and the UN General Assembly have now agreed proactive steps to ensure collaboration between nations, accepting the recommendations of the WHO Global Action Plan on AMR and the UK AMR Review. In particular they have agreed:

Support for all countries to design and implement national action plans

Initiatives to stimulate research and development of new antimicrobial products

Public awareness and engagement activities

In line with these agreements, the pharmaceutical industry has published a roadmap with an emphasis on public-private partnership. This guides both the development of new drugs and the management of access to antimicrobials, where and when they are needed.

LeSPAR will continue to work with our collective community of experts to ensure actions are taken in support of these resolutions.”

Posted by Dr. Tim Sandle