Use of Acclimatized Microorganisms in Validation Studies

For conventional culture based microbial methods and with many rapid and alternative microbiological methods, the way that method suitability is demonstrated is with the recovery of known populations of microorganisms. For years these were recognized cultures drawn from an approved culture collection. This test panel has generally become broadened to include environmental isolates, although this inclusion is not universally accepted and there remain some debate as to what this entails.

As an expansion upon this, some microbiologists, and also some regulators, have put forward the view that at least some of the microorganisms used for method verification should be ‘acclimatized’; that is closer to the state that they are in the environment from which a test sample is drawn. Acclimatized may also be stressed or even where the organism is damaged.

The reason for considering the inclusion of such ‘stressed’ organisms in studies is because organisms that have gone through a stress response maybe more difficult to recover (and, as an aside, harder to remove, inactivate or kill -to adapt an old aphorism: “what does not kill them makes them stronger”).

An example is with testing samples of water. In this context, the argument runs, challenge organisms should not be laboratory cultures grown on nutritious agar, but organisms held, for a period of time, in water (which will be a low nutrient environment and one subject to osmotic forces). This step will add robustness to method qualification and show that organisms can be recovered from given environmental niches.

This paper looks at how acclimatization might be achieved, in the context of method verification. The paper begins by looking at the objectives of method verification and then considers the appropriateness of environmental isolates in expanding microbial test panels. The paper then considers the how it can be ensured that environmental isolates are not simply facsimiles of laboratory cultures but are instead rendered to a closer approximation of the organism in its natural state in the environment.

The reference is:

Sandle, T. (2019) Use of Acclimatized Microorganisms in Validation Studies, Journal of Validation Technology, 25 (3): http://www.ivtnetwork.com/article/use-acclimatized-microorganisms-validation-studies

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Major class of viruses reveals complex origins

A new study examines the evolutionary dynamics of circular Rep-encoding single-stranded (CRESS) DNA viruses. The findings show that this broad class of single-stranded DNA viruses, which infect all three cellular domains of life, have acquired their genetic components through complex evolutionary processes not traceable to a single ancestral event. Rather, viruses are obsessive borrowers, appropriating genetic material from many sources, including bacterial, archaeal and eukaryotic cells as well as circular parasitic replicons, known as plasmids, and other mobile genetic elements, such as transposons.

When a group of mobile elements -- like CRESS DNA viruses -- arise from more than a single common evolutionary ancestor or ancestral group, they are known as polyphyletic. The phenomenon is common in the viral world, presenting both challenges and opportunities for researchers, as the definitions, taxonomies and evolutionary trajectories of this vast domain are reconsidered, with the help of powerful new techniques.

A better understanding of the promiscuous sharing of genetic information between different viruses and cell-derived genetic snippets may one day improve efforts to control these parasitic entities, some of which have had devastating effects on human wellbeing and crop yield.

Such explorations also hold the potential to shed new light on the origins of earth's earliest life, and resolve the question of how cell-based life came to co-exist with the planet's staggering array of viruses (the virome).

Recent research into environmental genomics has shown that the most abundant biological entities on earth are viruses, with virus particles outnumbering cells by one to two orders of magnitude. They display extraordinary diversity and have adapted themselves to virtually all earthly environments. They may also be considered the most successful biological players in terms of their growth potential, abundance, biodiversity, adaptability and impact.

Viruses consist of nucleic acid -- either RNA or DNA -- surrounded by a protective shell, known as the capsid. The job description of every virus is simple: enter a living cell, hijack its metabolic machinery and make progeny.

Viruses differ markedly from cells belonging to the bacterial, eukaryotic and archaeal realms, particularly in terms of their modes of replication. While all cellular life relies on double-stranded DNA inheritance, viruses can be single- or double-stranded and make use of either DNA or RNA as their genetic material. Further, their genomes can be either circular or linear, consisting of single or multiple molecules. Viruses lack a single common ancestor and indeed, not a single gene is conserved across the entire virome, making viruses a sort of genetic collage.

Among the viruses illuminated through viral metagenomics are the CRESS DNA viruses. Once believed to be rare, such viruses have since been uncovered in soils, deep-sea vents, Antarctic lakes and ponds, wastewater samples, oceans and hot springs. CRESS DNA viruses are part of a vast and diverse viral supergroup that is of critical importance, both medically and economically.

CRESS DNA viruses can be identified through a specific protein enzyme, known as Rep. This protein plays a crucial role in the genome replication mechanism common to CRESS DNA viruses as well as diverse circular plasmids found in bacteria and archaea. Researchers have recently noted that the rep gene is conserved in all CRESS DNA viruses. Among their biological tasks is the cutting and rejoining of single-stranded DNA segments -- activity essential to the replication mechanism known as rolling circle replication.

The rolling circle process begins when the Rep protein nicks one of the strands in the dsDNA form of the viral genome, initiating the replication sequence. The loose single strand created by the nick is elongated with the help of a host DNA polymerase, using the un-nicked strand as a template.

Eventually, the newly synthesized single strand of DNA completely dissociates from the original double-stranded form and its ends are joined together into a new single-stranded circle, with the help of Rep. A complementary strand can then form, creating a new double-stranded unit (See Figure 1). The process allows for the rapid synthesis of multiple copies of circular DNA.

Recombination of various functional modules from distinct viral and plasmid groups, derived from across the virosphere is a ceaseless process that is constantly generating new ssDNA viruses. The current study examines sequence similarities between various CRESS DNA viruses and non-viral replicons, such as plasmids, combined with phylogenetic tools used to explore their evolutionary relationships.

The results reveal three distinct evolutionary events contributing to the genetic composition of CRESS-DNA viruses. An intriguing kinship appears to exist between CRESS-DNA viruses and rolling circle plasmids found in bacteria, archaea and some eukaryotes. The new results help to illuminate the expanding galaxy of ssDNA viruses that replicate using the rolling-circle mechanism, among these, the CRESS-DNA viruses.

See:

Darius Kazlauskas, Arvind Varsani, Eugene V. Koonin, Mart Krupovic. Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids. Nature Communications, 2019; 10 (1) DOI: 10.1038/s41467-019-11433-0

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Special enzyme drives new class of antibiotics

Understanding how antibiotic scaffolds are constructed in nature can help scientists prospect for new classes of antibiotics through DNA sequencing and genome mining. Researchers have used this knowledge to help solve the X-ray crystal structure of the enzyme that makes obafluorin -- a broad spectrum antibiotic agent made by a fluorescent strain of soil bacteria. The new work is from Washington University in St. Louis and the University at Buffalo.

A multi-part enzyme called a nonribosomal peptide synthetase produces the highly reactive beta-lactone ring that is responsible for obafluorin's antimicrobial activity. These chemicals could be used as next-generation antibiotics for humans, or even to benefit the agriculture sector.

The new work provides a useful road map that shows how individual protein domains in the ObiF1 enzyme are stitched together in three-dimensional space. An enzyme's structure is fundamental to almost every function it performs.

Obafluorin is made by a fluorescent strain of soil bacteria that forms biofilms on plant roots. Like penicillin, obafluorin has a four-membered ring -- sometimes called an enchanted ring. A four-membered ring puts strain on bond angles that carbon prefers to adopt. But because a four-member ring is unstable, these molecules are also short-lived and difficult to make. For example, it took years for chemists to learn how to synthesize penicillin from chemicals and then figure out how fungi make it. This ultimately led to the global production of penicillin by fermentation.

Researchers were able to fast-track the discovery process using genetics to zero in on the biosynthetic machinery that bacteria use to make obafluorin, and then to reconstruct that multi-step, enzyme-catalyzed process in the laboratory.

The result is a comprehensive, detailed molecular structure at 3 Angstrom resolution that allows one to identify the atoms in the protein chain, see their location and points of contact along the chain, and determine how the pieces are assembled to produce useful molecules from start to finish.

One particular component -- something called an MbtH-like protein, or MLP, because it was first identified in a related system to produce mycobactin in the bacteria that causes tuberculosis -- was shown to play a critical role in facilitating protein-to-protein interactions between catalytic domains.

See:

Dale F. Kreitler, Erin M. Gemmell, Jason E. Schaffer, Timothy A. Wencewicz, Andrew M. Gulick. The structural basis of N-acyl-α-amino-β-lactone formation catalyzed by a nonribosomal peptide synthetase. Nature Communications, 2019; 10 (1) DOI: 10.1038/s41467-019-11383-7

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

How bacteria beat immune systems

The evolution of more severe infections is not necessarily driven by bacteria multiplying faster, new research shows. Humans and animals can develop resistance to harmful bacteria (pathogens) over time or with antibiotics or vaccines, and it is usually assumed that pathogens respond by multiplying faster.

The new study shows pathogen virulence and replication rates can evolve separately. The researchers  believe that, once resistance spreads in host species, virulence may be driven by other means such as by manipulating host immune systems.

The research examined the spread of bacteria called Mycoplasma gallisepticum among house finches -- a rare example of a well-studied host-bacteria evolution where humans have not intervened with antibiotics or vaccines.

The study shows that pathogens can evolve to become more virulent without increasing their rate of replication. The researchers hypothesise that the increase in virulence that observed in this study was driven by an improved ability of the pathogen to manipulate the host immune system in order to generate the symptoms necessary for its transmission.

For example, if trying to kill the pathogen inevitably leads to more virulent infections, it might be worth trying to slow down pathogen evolution by combining treatments that both eliminate the pathogen and prevent it manipulating host immune systems.

Some populations of house finches have been exposed to Mycoplasma gallisepticum for more than 20 years, while others have not -- and have therefore not developed resistance.

In the study, carried out in Arizona and supported by Arizona State University and Auburn University, 57 finches from previously unexposed populations were exposed to the pathogen.

The findings show virulence has increased consistently over more than 150,000 bacterial generations since outbreak (1994 to 2015).

By contrast, while replication rates increased from outbreak to the initial spread of resistance (1994 to 2004), no further increases have occurred subsequently (2007 to 2015).

See:

Luc Tardy, Mathieu Giraudeau, Geoffrey E. Hill, Kevin J. McGraw, Camille Bonneaud. Contrasting evolution of virulence and replication rate in an emerging bacterial pathogen. Proceedings of the National Academy of Sciences, 2019; 201901556 DOI: 10.1073/pnas.1901556116

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Revisiting Fosfomycin, a “Forgotten” Antibiotic

In the ongoing fight against antibiotic resistance, NIAID supports studies of new therapeutics, as well as new uses for older, established antibiotics. In a new study, NIAID-supported researchers are investigating whether fosfomycin, an older antibiotic, can be safe and appropriate for treating lung infections when delivered through an IV, rather than taken by mouth.

As bacteria evolve and develop resistance to available antibiotics, healthcare providers’ arsenal of effective drugs shrinks. Some researchers are taking a second look at older, established antibiotics, hoping that these “forgotten” medicines may still be effective when applied to infections in new ways or in new combinations. NIAID is currently supporting one such study, which is investigating an older antibiotic, fosfomycin, given via an intravenous (IV) infusion. The trial is part of a larger effort to identify new tools for fighting stubborn bacterial infections.

Fosfomycin is a broad-spectrum antibiotic that has been used by healthcare professionals in the United States for more than 45 years. It is usually given by mouth to treat uncomplicated urinary tract infections. In Europe, the IV formulation of fosfomycin is used to treat different types of infections, including serious multi-drug resistant infections. Because the intravenous formulation of the drug is delivered directly into the bloodstream, it reaches the target organs and begins fighting infections faster than the oral version, an important consideration for difficult-to-treat drug-resistant infections.

For further details see: https://www.niaid.nih.gov/news-events/revisiting-uses-antibiotics

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Toxin responsible for Legionella growth identified

A team of scientists led by EMBL group leader Sagar Bhogaraju and Ivan Dikic of Goethe University, Frankfurt, discovered that the toxin SidJ in Legionella bacteria enforces a unique modification on human proteins and helps legionella grow inside human cells. SidJ hijacks human protein Calmodulin to its own advantage in one of the classic examples of pathogenic bacteria exploiting the human molecular machinery and turning it against us. This makes SidJ an ideal target to curb Legionella infection. The results have been published in Nature.

Legionella – a complex bacterium

Pneumonia resulting from exposure to Legionella – although uncommon and affecting only 1 in 100,000 in Europe – has a higher than 10% fatality rate. The pathogenic bacterium Legionella pneumophilahas more than 300 toxins that it uses to infect humans. Once the aerosols containing the bacteria are inhaled, Legionella enters the lungs where it starts infecting human cells, causing pneumonia.

Legionella toxins especially target the innate immune pathways facilitating the survival of the bacteria inside human cells and allowing the replication of the bacteria. Due to the large number of toxins it is difficult to see the effects of deleting one or multiple of these toxins on the Legionella infection capacities. This is further complicated by the fact that several toxins with similar functions exist inside the bacteria. This makes Legionella hard to target with specific drugs.

Focus on the SidJ toxin

Researchers from EMBL Grenoble and the Goethe University in Frankfurt have now studied the toxin SidJ in detail. It is an important toxic protein of Legionella that gets injected into the human cytoplasm and enables the successful infection and replication of the bacteria. In contrast to the other toxins in Legionella, the deletion of SidJ alone leads to a considerable growth defect of the bacteria in human cells. This makes SidJ one of the most important toxins of Legionella and an attractive target to curb Legionella infection.

While SidJ has been studied in the field for already more than a decade, the precise function of it remained unknown until today. “SidJ has no sequence similarity to any of the proteins with a known function. We had to resort to standard biochemical methods and mass spectrometry to determine its function”, explains Bhogaraju. “While working out its mechanism proved to be challenging, it was also very exciting!”

In particular, the missing detailed molecular study of the toxin hindered the development of drugs that can target SidJ. The work by multidisciplinary scientists of Bhogaraju and Dikic groups now describes the molecular function of this protein in detail, elucidates its importance for Legionella infection and provides the identity of the human proteins that are targeted by SidJ.

Toxin at work

The group showed that SidJ possesses protein glutamylation activity: it attaches the amino acid glutamate to a target protein as post-translational modification. “This kind of activity is a first for bacterial proteins”, says Ivan Dikic, Director of the Institute of Biochemistry II at Goethe University. SidJ glutamylates many human proteins that are involved in tackling microbial infections and innate immunity. In order to do this, SidJ interacts with the human protein Calmodulin – a highly conserved multifunctional intermediate calcium-binding messenger protein. “Legionella has cleverly evolved to use Calmodulin to trigger SidJ's activity and as a result prevents SidJ's activation before the infection in the human body takes place”, says Dikic.

The cryogenic electron microscopy structure of SidJ interacting with human Calmodulin also revealed that the toxin has a kinase domain fold. "This is a both interesting and important find, as the kinase fold is druggable" says Michael Adams, a PhD student in Bhogaraju group.

Start of a long way to therapeutic usage

The outcome of the study is going to prime many studies in the future, further dissecting the mechanism of SidJ mediated glutamylation. Importantly, since the researchers found that SidJ has a kinase fold, this discovery will initiate the search for a drug molecule with potential therapeutic effects.

“While our work doesn’t have a direct pharmaceutical application, our results on the structural and functional characteristics of one of the most important toxins of Legionella, will lead to future studies aiming to target this protein for therapeutic uses”, says Sagar Bhogaraju.

Source article:

Sagar Bhogaraju, Florian Bonn, Rukmini Mukherjee, et al. Inhibition of SidE ubiquitin ligases through SidJ/Calmodulin catalyzed glutamylation. Nature, published on 22 July 2019. DOI: 10.1038/s41586-019-1440-8

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

New formula to combat Bacterial Vaginosis

An all-female team of doctors has developed a revolutionary new formula to aid the millions of women around the world suffering from Bacterial Vaginosis following extensive research and gene engineering.

The condition, caused by excessive production of bacteria, can result in vaginal discharge and an unpleasant odour, making sufferers uncomfortable and self-conscious. Conventional treatments focus on the use of antibiotics, which fail to address the problem completely and can lead to a reoccurrence in more than 50% of cases during a twelve-month period.

Dr Edita Misti, Head of Research and Development and Founder of the ProBV formula® comments “After talking to many women, we found that besides being a truly unpleasant experience, Bacterial Vaginosis still doesn’t have the proper treatment methods.

“There is a great need for more effective methods for Bacterial Vaginosis treatment assistance and prevention. In addition to their lack of effectiveness, drugs frequently used to treat the condition have potentially serious side-effects. Notably, the use of these medications by pregnant women can be harmful to their foetus. My team of female doctors have been inspired to come together to find a more permanent solution to something which many in the medical community overlook.”

ProBV formula® contains an antibody fragment capable of specifically neutralising vaginolysin, a toxin secreted by the pathogenic bacteria, Gardnerella Vaginalis. It was born as a collaboration between scientists and gynecologists.

Available in a number of different formats including premium intimate hygiene washes, ovules and gel, the ProBV formula® is the result of hundreds of hours of research and gene engineering. The previously undiscovered treatment method effectively tackles bacterial vaginosis without harming the body or causing unpleasant side effects. 

It has received much attention in the medical community and is already being heralded a modern breakthrough from those looking for a long-term solution to Bacterial Vaginosis.

See: www.probvformula.com

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

A novel mechanism of action of ketoconazole on multidrug-resistant Staphylococcus aureus

A new research paper of interest:

A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus

Background: The rapid emergence of antimicrobial resistance among Gram-positive organisms, especially staphylococci, has become a serious clinical challenge. Efflux machinery and biofilm formation are considered two of the main causes of antimicrobial resistance and therapy failure.
Aim: Our study aims to evaluate the antibiofilm and efflux pump inhibitory activity of the antifungal ketoconazole against multidrug-resistant (MDR) Staphylococcus aureus.

Methods: Ketoconazole was tested for its effect on the following: minimum inhibitory concentrations (MICs) of ciprofloxacin, norfloxacin, levofloxacin, and ethidium bromide (EtBr) by the broth microdilution method, the efflux of EtBr by NorA-positive MDR S. aureus, and the relative expression of NorA, NorB, and NorC efflux pump genes. Docking studies of ketoconazole were performed using 1PW4 (glycerol-3-phosphate transporter from Escherichia coli which was the representative structure from the major facilitator superfamily).

Results: Ketoconazole significantly decreased the MICs of levofloxacin, ciprofloxacin, norfloxacin, and EtBr (a substrate for efflux pump) by 8 to 1024-fold (P<0.01) and decreased the efflux of EtBr. Furthermore, a time-kill assay revealed that combinations of levofloxacin with ketoconazole or carbonyl cyanide m-chlorophenylhydrazone showed no growth for the tested strains after 24 h in comparison to the effect of levofloxacin alone. Docking studies and the ability of ketoconazole to diminish the relative expression of NorA gene in comparison to control (untreated strains) confirmed its action as an efflux pump inhibitor. 

Conclusion: The findings showed that the antifungal ketoconazole has no antibacterial activity but can potentiate the activity of the fluroquinolones against MDR S. aureus via inhibiting efflux pump and biofilm formation in vitro.

The reference is:

Abd El-Baky RM, Sandle T, John J, Abuo-Rahma GE, Hetta HF (2019) A novel mechanism of action of ketoconazole: inhibition of the NorA efflux pump system and biofilm formation in multidrug-resistant Staphylococcus aureus, Infection and Drug Resistance, 12: 1703-1708

To access see: https://www.researchgate.net/publication/333679872_A_novel_mechanism_of_action_of_ketoconazole_inhibition_of_the_NorA_efflux_pump_system_and_biofilm_formation_in_multidrug-resistant_Staphylococcus_aureus 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

BP and USP formalize partnership to strengthen quality of medicines and public health

The British Pharmacopeia (BP) and the United States Pharmacopeia (USP) formalized their long-standing partnership to strengthen the quality of medicines and improve public health around the world, in an agreement signed on Friday, July 26, at USP in Rockville, MD. Pharmacopoeial quality standards help drug manufacturers and regulatory agencies ensure medicines quality.

The formal Memorandum of Understanding establishes a framework for cooperative activities, including developing drug product monographs, information sharing, and expanding collaboration to new areas. The organizations intend to exchange scientific staff and participate in joint events.

See: https://www.usp.org/news/bp-and-usp-formalize-partnership-to-strengthen-quality-of-medicines-and-public-health

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Development of new antibiotics encouraged with new pharmaceutical payment system

The new trial will be led by the U.K. National Institute for Health and Care Excellence (NICE) and NHS England and NHS Improvement. It will test a ‘subscription’ style model that pays pharmaceutical companies upfront for access to drugs based on their usefulness to the NHS.

This will make it more attractive for companies to invest the estimated £1 billion it costs to develop a new drug, as they can be reassured they will still be paid for the drug even though it may be stored for reserves. Currently, drugs companies are paid by volume of antibiotics sold, while the NHS is trying to reduce their use to prevent antimicrobial resistance (AMR).

Low returns on investment in development mean industry is reluctant to invest in the research and clinical trials necessary to bring new antibiotics to market.

NICE and NHS England and NHS Improvement are calling for companies to identify products to be considered for the initial phase of the test.  The work will be evaluated from the start and findings will be shared with the rest of the world so that other healthcare systems can test similar models.

See: https://www.gov.uk/government/news/development-of-new-antibiotics-encouraged-with-new-pharmaceutical-payment-system

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Can mathematics help us understand the complexity of our microbiome?

For nearly a century, evolutionary biologists have probed how genes encode an individual's chances for success -- or fitness -- in a specific environment.

In order to reveal a potential evolutionary trajectory biologists measure the interactions between genes to see which combinations are most fit. An organism that is evolving should take the most fit path. This concept is called a fitness landscape, and various mathematical techniques have been developed to describe it.

Like the genes in a genome, microorganisms in the gut microbiome interact, yet there isn't a widely accepted mathematical framework to map the patterns of these interactions. Existing frameworks for genes focus on local information about interactions but do not put together a global picture.

"If we understand the interactions, we can make predictions about how these really complex systems will work in different scenarios. But there is a lot of complexity in the interaction networks due to the large number of genes or species. These add dimensions to the problem and make it tricky to solve," said Ludington.

So, Ludington began talking to mathematician Michael Joswig of the Technical University in Berlin.

"Michael thinks natively in high dimensions -- many more than four. He understood the problem right away," said Ludington.

Joswig and Ludington then joined with Holger Eble of TU Berlin, a graduate student working with Joswig, and Lisa Lamberti of ETH Zurich. Lamberti had previously collaborated with Ludington to apply a slightly different mathematical framework for the interactions to microbiome data. In the present work, the team expanded upon that previous framework to produce a more global picture by mapping the patterns of interactions onto a landscape.

"In humans, the gut microbiome is an ecosystem of hundreds to thousands of microbial species living within the gastrointestinal tract, influencing health and even longevity," Ludington explained. "As interest in studying the microbiome continues to increase, understanding this complexity will give us predictive power to engineer it."

But the sheer diversity of species in the human microbiome makes it very difficult to elucidate how these communities influence our physiology. This is why the fruit fly makes such an excellent model. Unlike the human microbiome, it consists of only a handful of bacterial species.

"We've built a rigorous mathematical framework that describes the ecology of a microbiome coupled to its host. What is unique about this approach is that it allows a global view of a microbiome-host interaction landscape," said Ludington. "We can now use this approach to compare different landscapes, which will let us ask why diverse microbiomes are associated with similar health outcomes."

READ MORE: Skin microbiome summit showcases scientific developments for treating skin conditions

See:

Holger Eble, Michael Joswig, Lisa Lamberti, William B. Ludington. Cluster partitions and fitness landscapes of the Drosophila fly microbiome. Journal of Mathematical Biology, 2019; DOI: 10.1007/s00285-019-01381-0

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

NOW IS THE TIME for Animal Welfare in Pharma

Symposium - Animal Welfare in Pharma Join us at a symposium to celebrate the 60th anniversary of the 3Rs guiding principles (Reduction, Refinement and Replacement) for ethical use of animals in lab testing. Experts from the pharma industry will present ongoing initiatives to apply the 3Rs principles throughout the pharma process: from the drug development to production and batch release; there are many ways to avoid the use of animals.

NOW IS THE TIME for Animal ... 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

EU and US reach a milestone in mutual recognition of inspections of medicines manufacturers

With the recognition by the US Food and Drug Administration (FDA) of Slovakia, the European Union and the United States have now fully implemented the mutual recognition agreement (MRA) for inspections of manufacturing sites for certain human medicines in their respective territories.

Each year, EU national authorities and the FDA inspect many production sites of medicinal products in the EU, the US and elsewhere in the world, to ensure that these sites operate in compliance with good manufacturing practice (GMP). Under the MRA, EU and US regulators will now rely on each other’s inspections for human medicines in their own territories and hence avoid duplicative work. As a result of the MRA, both the EU and the US will be able to free up resources to inspect facilities in other countries.

The MRA is underpinned by robust evidence on both sides of the Atlantic that the EU and the US have comparable procedures to carry out GMP inspections for human medicines. Since May 2014, teams from the European Commission, the EU national competent authorities, EMA and the FDA have been auditing and assessing the respective supervisory systems. With the positive assessment of Slovakia, this process has now concluded for GMP inspectorates covering human medicines.

From now on, a batch testing waiver will also start to apply. This means that the qualified persons in EU pharmaceutical companies will no longer have to carry out quality controls for products manufactured in and imported from the US when these controls have already been carried out in the US. 

The MRA implementation work will continue with a view to expanding the operational scope to veterinary medicines, human vaccines and plasma-derived medicinal products.

See:  https://www.ema.europa.eu/en/news/eu-us-reach-milestone-mutual-recognition-inspections-medicines-manufacturers

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Microbial community with small diversity cleans up algal blooms

Algae blooms regularly make for pretty, swirly satellite photos of lakes and oceans. They also make the news occasionally for poisoning fish, people and other animals. What's less frequently discussed is the outsize role they play in global carbon cycling. A recent study now reveals surprising facts about carbon flow in phytoplankton blooms. Unexpectedly few bacterial clades with a restricted set of genes are responsible for a major part of the degradation of algal sugars.

Algae take up carbon dioxide (CO2) from the atmosphere and turn the carbon into biomass while releasing the oxygen back to the atmosphere. Fast algal growth during phytoplankton blooms leads to a massive transfer of carbon dioxide into algal biomass. But what happens to the carbon next?

"Once the algae die, the carbon is remineralized by microorganisms consuming their biomass. It is thus returned to the atmosphere as carbon dioxide. Alternatively, if the dead algae sink to the seafloor, the organic matter is buried in the sediment, potentially for a very long time," explains first author Karen Krüger from the Max Planck Institute for Marine Microbiology in Bremen. "The processes behind the remineralization of algal carbon are still not fully understood."

Thus, Krüger and her colleagues investigated microorganisms during spring algal blooms in the southern North Sea, at the island of Heligoland. They specifically looked at the bacterial use of polysaccharides -- sugars that make up a substantial fraction of the algal biomass. Together with colleagues from the Max Planck Institute, the University of Greifswald and the DOE Joint Genome Institute in California, Krüger carried out a targeted metagenomic analysis of the Bacteroidetes phylum of bacteria, since these are known to consume lots of polysaccharides. In detail, the scientists looked at gene clusters called polysaccharide utilisation loci (PULs), which have been found to be specific to a particular polysaccharide substrate. If a bacterium contains a specific PUL, that indicates it feeds on the corresponding algal sugar.

"Contrary to what we expected, the diversity of important PULs was relatively low," says Krüger. Only five major polysaccharide classes were being regularly targeted by multiple species of bacteria, namely beta-glucans (such as laminarin, the main diatom storage compound), alpha-glucans (such as starch and glycogen, also algal and bacterial storage compounds), mannans and xylans (typically algal cell wall components), and alginates (mostly known as slimy stuff produced by brown macroalgae). Of these five substrates, only two (alpha- and beta-glucans) make up the majority of substrates available to the bacteria during a phytoplankton bloom. This implies that the most important polysaccharide substrates released by dying algae are made up of a fairly small set of basic components.

"Given what we know of algal and bacterial species diversity, and the enormous potential complexity of polysaccharides, it came as no small surprise to see such a limited spectrum of PULs, and in only a relatively small number bacterial clades," co-author Ben Francis from the Max Planck Institute for Marine Microbiology sums up in an accompanying comment. "This was especially unexpected because previous studies suggested something different. An analysis of more than 50 bacterial isolates -- i.e. bacteria that can be cultured in the lab -- that our working group carried out in the same sampling region revealed a much broader diversity of PULs," he adds.

During the course of the algal bloom, the scientists observed a distinct pattern: In early bloom stages, fewer and simpler polysaccharides dominated, while more complex polysaccharides became available as the bloom progressed. This might be caused by two factors, Francis explains: "First, bacteria will in general prefer easily degradable substrates such as simple storage glycans over biochemically more demanding ones. Second, more complex polysaccharides become increasingly available over a blooms' course, when more and more algae die."

This study provides unprecedented insights into the dynamics of a phytoplankton bloom and its protagonists. A fundamental understanding of the bulk of glycan-mediated carbon flow during phytoplankton bloom events is now within reach. "Next, we want to dig deeper into processes underlying the observed dynamics," says Krüger. "Moreover, it will be interesting to investigate polysaccharide degradation in habitats with other carbon sources, such as the Arctic Seas or the sediment."

See:

Karen Krüger, Meghan Chafee, T. Ben Francis, Tijana Glavina del Rio, Dörte Becher, Thomas Schweder, Rudolf I. Amann, Hanno Teeling. In marine Bacteroidetes the bulk of glycan degradation during algae blooms is mediated by few clades using a restricted set of genes. The ISME Journal, 2019; DOI: 10.1038/s41396-019-0476-y



Posted by Dr. Tim Sandle, Pharmaceutical Microbiology