Friday, 31 January 2020

Vaccinating against inflammatory diseases


Targeted immunization against bacterial flagellin, a protein that forms the appendage that enables bacterial mobility, can beneficially alter the intestinal microbiota, decreasing the bacteria's ability to cause inflammation and thus protecting against an array of chronic inflammatory diseases, according to a new study.

The findings suggest this approach offers a way to vaccinate against diseases associated with chronic inflammation of the digestive tract, a group of diseases that includes inflammatory bowel diseases, as well as obesity and metabolic syndrome.

The intestinal tract is colonized by billions of bacteria and other microorganisms that play numerous beneficial roles, but improperly controlled microbiota can lead to chronic inflammatory diseases. Previous studies have shown the intestinal microbiota are associated with inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, and diseases characterized by low-grade inflammation of the intestinal tract, such as obesity and metabolic syndrome.

Therapeutic options have focused on lessening the inflammatory response and have often overlooked the contribution of the intestinal microbiota. The researchers wanted to determine if a targeted immune response could be used to beneficially shape the intestinal microbiota and protect against inflammatory diseases. Previously, they found that a common feature of microbiotas associated with inflammation is an increased level of expression of flagellin by select microbiota members, which can drive bacteria to penetrate the intestinal mucosa and disrupt homeostasis.

The researchers immunized mice with flagellin to elicit an adaptative immune response and demonstrated targeted immunization against bacterial flagellin is sufficient to beneficially alter the composition and function of the intestinal microbiota. Anti-flagellin antibodies were produced and affected the microbiota by reducing its pro-inflammatory potential and ability to penetrate its host. These alterations were associated with protection against chronic inflammatory diseases.

Journal Reference:

Hao Q. Tran, Ruth E. Ley, Andrew T. Gewirtz, Benoit Chassaing. Flagellin-elicited adaptive immunity suppresses flagellated microbiota and vaccinates against chronic inflammatory diseases. Nature Communications, 2019; 10 (1) DOI: 10.1038/s41467-019-13538-y

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Thursday, 30 January 2020

The chemical conversation of the human microbiome


The microbial community populating the human body plays an important role in health and disease, but with few exceptions, how individual microbial species affect health and disease states remains poorly understood.

The identity and balance of bacterial species on human skin and mucosal surfaces influences a variety of disease conditions, ranging from digestive ailments to halitosis, bacterial vaginosis and eczema. The microbiome also aids immune development and the fight against pathogens. However, the human microbiome is incredibly diverse; the communities of bacteria, viruses, fungi and other tiny organisms differ according to the tissue where they live, and across human populations and individuals. It's unclear what constitutes a normal, healthy microbiome, much less how one might go about bringing a sick one back into balance.

A common approach to solving this problem is to culture an individual microbe in the lab and explore how it contributes to health or disease states. Unfortunately, it can be difficult to identify and isolate very rare species, or find the conditions necessary to support their growth outside their natural niche. To do this with every species would be a daunting task. Alternatively, scientists can examine the microbiome in situ, with the aim of describing its individual components and how they interact.

One way microbes communicate -- and do battle -- with each other and with human cells is through biologically active small molecules.

The researchers developed computer algorithms that can detect BGCs by analyzing and interpreting metagenomic sequencing data. Metagenomic sequencing data are composed of genetic sequences obtained from the tissues or excretions of hundreds of human subjects. Some metagenomic data sets are drawn from clinical samples taken from diverse populations, including persons in different states of health or disease, or people in different geographical locales. Intensive analysis is needed to make sense of the rich but often fragmentary information contained in these data sets.

The approach employed began by identifying genes essential for the synthesis of a particular molecule or chemical of interest, then using computational algorithms to sort through metagenomic data for similar (homologous) genetic sequences, and grouping these sequence fragments together. They then assessed the prevalence of each group in the human population, and used the grouped sequences to piece together full-length BGCs. Importantly, this approach allowed identification of novel BGCs even if they are extremely rare.

To validate this approach, the researchers investigated whether they could detect BGCs involved in the synthesis of type II polyketides. This class of chemicals, which includes the anti-cancer drug doxorubicin and several antibiotic drugs, was previously found in soil bacteria but had never before been found in bacteria of the human microbiome.

With this technology, it is now possible to mine our own microbiomes for drug discovery or novel biological interactions. What other treasures might this type of analysis reveal? 

Reference:

Yuki Sugimoto, Francine R. Camacho, Shuo Wang, Pranatchareeya Chankhamjon, Arman Odabas, Abhishek Biswas, Philip D. Jeffrey, Mohamed S. Donia. A metagenomic strategy for harnessing the chemical repertoire of the human microbiome. Science, 2019; eaax9176 DOI: 10.1126/science.aax9176

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Wednesday, 29 January 2020

New CRISPR system targets antibiotic-resistant genes


Taking advantage of powerful advances in CRISPR gene editing, scientists at the University of California San Diego have set their sights on one of society's most formidable threats to human health.

A research team led by Andrés Valderrama at UC San Diego School of Medicine and Surashree Kulkarni of the Division of Biological Sciences has developed a new CRISPR-based gene-drive system that dramatically increases the efficiency of inactivating a gene rendering bacteria antibiotic-resistant. The new system leverages technology developed by UC San Diego biologists in insects and mammals that biases genetic inheritance of preferred traits called "active genetics."

Widespread prescriptions of antibiotics and use in animal food production have led to a rising prevalence of antimicrobial resistance in the environment. Evidence indicates that these environmental sources of antibiotic resistance are transmitted to humans and contribute to the current health crisis associated with the dramatic rise in drug-resistant microbes. Health experts predict that threats from antibiotic resistance could drastically increase in the coming decades, leading to some 10 million drug-resistant disease deaths per year by 2050 if left unchecked.

The core of Pro-AG features a modification of the standard CRISPR-Cas9 gene editing technology in DNA. Working with Escherichia coli bacteria, the researchers developed the Pro-AG method to disrupt the function of a bacterial gene conferring antibiotic resistance. In particular, the Pro-AG system addresses a thorny issue in antibiotic resistance presented in the form of plasmids, circular forms of DNA that can replicate independently of the bacterial genome. Multiple copies of, or "amplified," plasmids carrying antibiotic-resistant genes can exist in each cell and feature the ability to transfer antibiotic resistance between bacteria, resulting in a daunting challenge to successful treatment. Pro-AG works by a cut-and-insert repair mechanism to disrupt the activity of the antibiotic resistant gene with at least two orders of magnitude greater efficiency than current cut-and-destroy methods.

Valderrama and Kulkarni, working in the UC San Diego labs of study coauthors Professors Victor Nizet and Ethan Bier, respectively, demonstrated the effectiveness of the new technique in experimental cultures containing a high number of plasmids carrying genes known to confer resistance to the antibiotic ampicillin. The system relies on a self-amplifying "editing" mechanism that increases its efficiency through a positive feedback loop. The result of Pro-AG editing is the insertion of tailored genetic payloads into target sites with high precision.

While Pro-AG is not yet ready for treating patients, "a human delivery system carrying Pro-AG could be deployed to address conditions such as cystic fibrosis, chronic urinary infections, tuberculosis and infections associated with resistant biofilms that pose difficult challenges in hospital settings," said Nizet, distinguished professor of Pediatrics and Pharmacy and the faculty lead of the UC San Diego Collaborative to Halt Antibiotic-Resistant Microbes (CHARM).

When combined with a variety of existing delivery mechanisms for spreading the Pro-AG system through populations of bacteria, the scientists say the technology also could be widely effective in removing, or "scrubbing," antibiotic-resistant strains from the environment in areas such as sewers, fish ponds and feedlots. Because Pro-AG "edits" its targets rather than destroys them, this system also enables engineering or manipulating bacteria for a broad range of future biotechnological and biomedical applications rendering them harmless or even recruiting them to perform beneficial functions.

Journal Reference:

J. Andrés Valderrama, Surashree S. Kulkarni, Victor Nizet, Ethan Bier. A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus. Nature Communications, 2019; 10 (1) DOI: 10.1038/s41467-019-13649-6

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Tuesday, 28 January 2020

How immune cells switch to attack mode


Macrophages have two faces: In healthy tissue, they perform important tasks and support their environment. However during an infection, they stop this work and hunt down the pathogens instead. Upon coming into contact with bacteria they change their metabolism drastically within minutes. This is shown by a new study under the leadership of the University of Bonn, which has now been published in the journal "Immunity." In the medium term, the results may lead to new vaccination strategies, but also to new approaches for combating autoimmune diseases.

Macrophages can practically "sniff out" intruders: Their cell surface contains numerous sensors, the Toll-like receptors. These work in a similar way to the olfactory receptors in the nose: They are activated when they encounter a specific chemical signal. The alarm they trigger then leads to a series of reactions inside the cell. "During this phase, macrophages initiate their inflammatory response," explains Mario Lauterbach, who is completing his doctorate at the Institute of Innate Immunity at the University of Bonn. "How they change their metabolism in the first few minutes and what the consequences are has been unclear so far."

There are different groups of Toll-like receptors, each of which responds to different "smells." These are molecules that have emerged as important danger signals in the course of evolution. Among these are the so-called lipopolysaccharides (LPS), important components of the bacterial cell wall. "We have now confronted macrophages with LPS and investigated what happens in the following minutes and hours," explains Lauterbach.

The scientists were able to show that the cell metabolism changes dramatically shortly after LPS contact: Macrophages immediately absorb more glucose from their environment -- but not primarily in order to obtain energy. Instead, they convert the sugar into so-called acetyl groups, which are small molecules related to acetic acid. These then serve as a kind of label in the cell nucleus: They are used to label genome sequences that are supposed to be read more intensively.

The DNA is actually a meter-long wafer-thin thread. It would, however, be difficult to store in this form. This is why it is rolled up on many small spools, the histones. Enzymes now attach the acetyl groups to certain parts of the histones. This process is stimulated by the increased acetyl group synthesis after the alarm is triggered, which ultimately loosens the coil of DNA and makes the corresponding genes more readable. "These include genes that are responsible for the release of inflammatory messengers or that improve the mobility of macrophages," explains Lauterbach.

It has long been known that the activation of Toll-like receptors alters the reading of genes. However, the mechanisms responsible for this differ from the one that has now been discovered. It is likely that this newly discovered mechanism allows the fine regulation of the genetic response. The results may therefore also provide new starting points, such as for improving the effectiveness of vaccinations. Toll-like receptors also play an important role in mediating the "learned" or aquired immune response. This arm of immunity increases the effectiveness of the defense mechanisms against infections that the body has already been through. Vaccinations strategies are also based on this principle.

In many diseases, such as rheumatism, diabetes or multiple sclerosis, the immune response is misdirected or too strong. "The mechanism we discovered might enable us to inhibit harmful inflammatory processes without suppressing the immune system too much," hopes Prof. Dr. Eicke Latz, head of the Institute of Innate Immunity. Instead of permanently hunting down (non-existent) invaders, the macrophages could concentrate again on their important tasks.

One reason why it was possible to shed light on the immune mechanism is the excellent cooperation between the University of Bonn, the TU Braunschweig and the LMU Munich. This success is also a result of the Cluster of Excellence ImmunoSensation, of which Latz is a member.


Reference:

Mario A. Lauterbach, Jasmin E. Hanke, Magdalini Serefidou, et al. Toll-like Receptor Signaling Rewires Macrophage Metabolism and Promotes Histone Acetylation via ATP-Citrate Lyase. Immunity, 2019; 51 (6): 997 DOI: 10.1016/j.immuni.2019.11.009

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Monday, 27 January 2020

Integrating Good Distribution Practice into the QMS

Good Distribution Practice (GDP) concerns the distribution processes for pharmaceutical products that results in medics and patients obtaining access to the medications required. For the pharmaceutical organization, the distribution process occurs both upstream and downstream. Upstream are the suppliers who create goods and services used in a manufacturer’s own operations, such as raw components or materials. The downstream supply chain efficiently distributes a company’s products or services to its customers. Each stage, both upstream and downstream, needs to be proactively managed to minimize quality, as well as financial, confidentiality, operational, reputational and legal risks.


Here is an extract:

These distribution processes concern supply chain, including cold supply chains (where required), and the tracking and tracing of medicines. Traceability includes ensuring that the required environmental controls are met, and that tampering or fraudulent activities are avoided, to the level that each induvial item can be traced from the completion of manufacture to its arrival with the end user (Marucheck et al, 2011). The distribution network for medicinal products is invariably complex and it involves many different parties at different stages. In addition to the challenges associated with this complexity and with protecting the product from being affected by environmental conditions, damage, or loss, there is also a threat from criminal activities centered on seeking to introduce falsified medicines into the supply chain (Bruinsma, and Bernasco, 2004).

GDP requirements are designed to codify and to structure the processes. These requirements bear close similarity to the requirements set out in Good Manufacturing Practice (GMP) regulations. The primary difference is that GDP covers the wholesale distribution of medicines, whereas GMP covers their manufacture.  There overlap between the two rest with the need to maintain product quality after a batch has been released from the manufacturing site, as well as the necessity to monitor and control complaints, address problems, and have a system in place to enact a recall.

In assessing the requirements for GDP, there are different national and supranational standards. In the US GMP is based on the Code of Federal Regulations 21 CFR 210/211, with additional guidance contained within USP chapter 1079 “Good Storage and Distribution Practices for Drug Products.” (USP, 2018) There is an additional USP chapter of interest, chapter 1197 “Good Distribution Practices for Pharmaceutical Excipients” (USP, 2018b). For Europe GDP is based on the Directive of the Board of the European Community 92/25/EEC regarding the wholesale distribution of drugs for human consumption, supported by guideline 2015/C 95/01 (European Commission, 2015), and the Falsified Medicines Directive (European Commission, 2011), which requires a unique identifier and an anti-tampering device to allow the verification of the authenticity of medicinal products. With the World Health Organization, the applicable text is Annex 5 of the WHO recommendations “good distribution practices for pharmaceutical products.” (WHO, 2010a) One commonality through these regulations and following on from items raised during pharmaceutical organization inspections, is with a focus on serialization. This has required for new strategies, processes, and technologies that allow for a business to, at any time, pinpoint the location and origin of any single drug.


A weak GDP system is one where there is a key disconnect between the manufacturer and the process that occurs once the product leaves the facility (Rees, 2013). An overarching area of regulatory concern is with the effectiveness of the incorporation of GDP into the Quality Management System (QMS), a system that applies for both wholesaler and broker. This chapter looks at Quality Risk Management in relation to GDP, covering areas like good distribution principles, the necessity of having Quality Technical Agreements in place, and measures to take appropriate corrective and preventative actions should deviations occur.


The reference is:

Sandle, T. (2019) Integrating Good Distribution Practice into the QMS. In Schmitt, S. (Ed.) Good Distribution Practice: A Handbook for Healthcare Manufacturers and Suppliers, Volume 1, DHI/PDA Books, River Grove, IL, USA, pp241-272

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 26 January 2020

Are herpes virus infections linked to Alzheimer's disease?


Researchers at Baylor College of Medicine report evidence that refutes the link between increased levels of herpes virus and Alzheimer's disease. In addition, the researchers provide a new statistical and computational framework for the analysis of large-scale sequencing data.

About 50 million people worldwide are affected by Alzheimer's disease, a type of progressive dementia that results in the loss of memory, cognitive abilities and verbal skills, and the numbers are growing rapidly. Currently available medications temporarily ease the symptoms or slow the rate of decline, which maximizes the time patients can live and function independently. However, there are no treatments to halt progression of Alzheimer's disease.

"Like all types of dementia, Alzheimer's disease is characterized by massive death of brain cells, the neurons. Identifying the reason why neurons begin and continue to die in the brains of Alzheimer's disease patients is an active area of research," said corresponding author Dr. Zhandong Liu, associate professor of pediatrics at Baylor and the Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital.

One theory that has gained traction in the past year is that certain microbial infections, such as those caused by viruses, can trigger Alzheimer's disease. A 2018 study reported increased levels of human herpesvirus 6A (HHV-6A) and human herpesvirus 7 (HHV-7) in the postmortem brain tissues of more than 1,000 patients with Alzheimer's disease when compared to the brain tissues of healthy-aging subjects or those suffering from a different neurodegenerative condition.

Presence of elevated levels of genetic material of herpes viruses indicated active infections, which were linked to Alzheimer's disease. In less than a year, this study generated a flurry of excitement and led to the initiation of several studies to better understand the link between viral infections and Alzheimer's disease.

Surprisingly, when co-author Dr. Hyun-Hwan Jeong, a postdoctoral fellow in Dr. Liu's group and others, reanalyzed the data sets from the 2018 study using the identical statistical methods with rigorous filtering, as well as four commonly used statistical tools, they were unable to produce the same results.

The team was motivated to reanalyze the data from the previous study because they observed that while the p-values (a statistical parameter that predicts the probability of obtaining the observed results of a test, assuming that other conditions are correct) were highly significant, they were being ascribed to data in which the differences were not visually appreciable.

Moreover, the p-values did not fit with simple logistic regression -- a statistical analysis that predicts the outcome of the data as one of two defined states. In fact, after several types of rigorous statistical tests, they found no link between the abundance of herpes viral DNA or RNA and likelihood of Alzheimer's disease in this cohort.

"As high-throughput 'omics' technologies, which include those for genomics, proteomics, metabolomics and others, become affordable and easily available, there is a rising trend toward 'big data' in basic biomedical research. In these situations, given the massive amounts of data that have to be mined and extracted in a short time, researchers may be tempted to rely solely on p-values to interpret results and arrive at conclusions," Liu said.

"Our study highlights one of the potential pitfalls of over-reliance on p-values. While p-values are a very valuable statistical parameter, they cannot be used as a stand-alone measure of statistical correlation -- data sets from high-throughput procedures still need to be carefully plotted to visualize the spread of the data," Jeong said. "Data sets also have to be used in conjunction with accurately calculated p-values to make gene-disease associations that are statistically correct and biologically meaningful."

"Our goal in pursuing and publishing this study was to generate tools and guidelines for big data analysis, so the scientific community can identify treatment strategies that will likely benefit patients," Liu said.

Journal Reference:

Hyun-Hwan Jeong, Zhandong Liu. Are HHV-6A and HHV-7 Really More Abundant in Alzheimer’s Disease? Neuron, 2019; 104 (6): 1034 DOI: 10.1016/j.neuron.2019.11.009

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Saturday, 25 January 2020

Four-level food web for gut microbes discovered


A new computational model suggests that the food web of the human gut microbiome follows a hierarchical structure similar to that of larger-scale ecosystems.

In the human gut, hundreds of species of microbes exchange nutrients in a complex food web. Large-scale food webs, such as those of tropical forests, typically follow a hierarchy in which energy flows from plants, to herbivores, to carnivores. Wang and colleagues wondered if the gut microbiome could be considered to follow a similar hierarchy, from microbes that consume nutrients in food eaten by the human host, to those that eat nutrients produced by the first microbes, and so on.

To address this question, the researchers developed a computational model that uses the known species of microbes in a person's gut to predict microbial metabolites -- the substances the microbes generate as part of their biological activities, and which may serve as nutrients for other gut microbes. The metabolite predictions generated by the model are in line with experimental data, providing support for its accuracy.

The new model indeed predicts a four-level hierarchy for the food web of the gut microbiome. This suggests that species composition systematically changes along the length of the gut. Near the entrance to the lower gut, one might find bacteria from the highest hierarchical level -- those that consume nutrients in food eaten by the human. Near the end of the gut, one might find bacteria from the lowest level.

The researchers are now working to refine their model by using a machine-learning approach to infer important competitive relationships between gut microbes. Doing so could improve the model's accuracy, potentially reducing the need for expensive measurements of metabolic profiles in research on gut function.

Journal Reference:

Tong Wang, Akshit Goyal, Veronika Dubinkina, Sergei Maslov. Evidence for a multi-level trophic organization of the human gut microbiome. PLOS Computational Biology, 2019; 15 (12): e1007524 DOI: 10.1371/journal.pcbi.1007524


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Friday, 24 January 2020

Identifying harmful bacteria based on its DNA


A new bacterial identification method, called ON-rep-seq, examines selective, strain-specific fragments of the bacterial genome, allowing the generation of results that earlier required DNA sequencing of the entire bacterial genome or tedious approaches like pulsed field gel electrophoresis, which previously has been the golden the standard for strain-level typing of microorganisms. Hence, the method has the potential to change the approach utilized for investigating food-based disease outbreaks by making analysis much less time- and cost consuming.

Today, bacterial detection and identification based on bacterial DNA requires expensive instrumentation and many hours of work by highly trained specialists. Let's imagine, for example, there is a suspected Salmonella outbreak. Usually in order to locate its origin, not only will investigators have to analyze many samples, but the analysis has to be precise in order to distinguish one bacterial strain from another.

The new method is based on nanopore sequencing, which is a new, real-time DNA sequencing approach "that will definitely revolutionize the future of DNA sequencing" according to Lukasz Krych.

The research project was carried out in collaboration with the polish company GenXone S.A., which helped to set up a bioinformatics pipeline that is needed to perform fast and efficient analysis of the sequencing data.

The smallest ever sequencer offered by Oxford Nanopore Technologies, called MinION, is a $999 hand-held, USB-powered device that became commercially available in 2015. A year later it was taken to the International Space Station, where it achieved the first DNA sequencing in history performed in zero-gravity conditions. Despite the indisputable revolution in DNA sequencing offered by MinION, it quickly became clear that the data generated with the device are still not perfect due to e.g. sequencing errors while the analysis remained relatively expensive to perform (app. $150 per bacterium).

The scientists from the Department of Food Science at the University of Copenhagen have found a way to utilize this technology to analyze hundreds of bacteria at a time, cutting costs to less than $2 per bacterium, while at the same time increasing the accuracy to more than 99%.

At the moment, there are several companies testing the method to implement in their systems for establishing rapid screening programmes for thousands of strains.

Journal Reference:

Łukasz Krych, Josué L. Castro-Mejía, Laura M. Forero-Junco, Daniel N. Moesby, Morten B. Mikkelsen, Morten A. Rasmussen, Maciej Sykulski, Dennis S. Nielsen. DNA enrichment and tagmentation method for species-level identification and strain-level differentiation using ON-rep-seq. Communications Biology, 2019; 2 (1) DOI: 10.1038/s42003-019-0617-x

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Thursday, 23 January 2020

'Upgraded' CRISPR tool developed


Columbia scientists have captured the first images of a new gene editing tool that could improve upon existing CRISPR-based tools. The team developed the tool, called INTEGRATE, after discovering a unique "jumping gene" in Vibrio cholerae bacteria that could insert large genetic payloads in the genome without introducing DNA breaks.

In the new study the researchers harnessed a Nobel Prize-winning technique called cryo-electron microscopy to freeze the gene editing complex in action, revealing high-resolution details about how it works.

The researchers used a technique called cryo-electron microscopy, which involves flash freezing a sample of the gene editing complex in liquid nitrogen and bombarding it with electrons. They then used the images they captured with the electron microscope to generate atomic resolution models of the INTEGRATE system.

The structural model reveals that the complex is made up of two main sections that are arranged in a helical filament. The larger portion, called Cascade, winds around and carries a guide RNA that it uses to scan the cell for a matching sequence in DNA. Once it locates and binds the target sequence, it threads the DNA strand through the TniQ "transposition" proteins that sit on the end of the complex and recruit other enzymes that help modify the DNA.

The scanning mechanism of INTEGRATE appears to work in a similar way to other well-studied CRISPR systems, some of which also contain a Cascade complex with guide RNA. However, unlike other CRISPR systems that use Cascade to target DNA for cutting, the function of Cascade within INTEGRATE is to target DNA for highly accurate insertion of genetic payloads.

Many researchers around the world now use CRISPR-Cas9 to quickly and cheaply make precise modifications to the genome of a cell. However, most uses of CRISPR involve cutting both strands of the target DNA, and the DNA break must then be repaired by the host cell's own machinery. Controlling this repair process is still a major challenge in the field, and undesired gene edits are often introduced inadvertently in the genome. Additionally, existing tools often perform poorly at inserting large genetic payloads in a precise fashion. Improving the accuracy of gene editing is a priority for researchers and is critical for ensuring the safety of therapies developed with this technique.

The new INTEGRATE system developed by the Sternberg lab can accurately insert large DNA sequences without relying on the cell's machinery to repair the strands. As a result, INTEGRATE could prove to be a more accurate and efficient way of making certain gene modifications than the original CRISPR-Cas system that is widely in use. The new tool could also help scientists perform gene editing in cell types with limited DNA repair activity such as neurons, where attempts to use CRISPR have been comparatively less successful.


In addition to informing future engineering efforts, the structures highlight a possible proofreading checkpoint. Existing CRISPR technologies often suffer from so-called "off-target effects," in which unintended sequences are promiscuously modified. The new structures reveal how Cascade and TniQ work together to ensure that only the correct "on-target" sequences are marked for DNA insertion. The researchers plan to further explore this checkpoint while developing the tool for new therapeutic approaches to disease.

Journal Reference:

Tyler S. Halpin-Healy, Sanne E. Klompe, Samuel H. Sternberg, Israel S. Fernández. Structural basis of DNA targeting by a transposon-encoded CRISPR–Cas system. Nature, 2019; DOI: 10.1038/s41586-019-1849-0
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Wednesday, 22 January 2020

Ranitidine containing medicinal products



At the request of the European Commission, the European Medicines EMA triggered a review of ranitidine medicines after tests showed that some of these products contained an impurity called Nnitrosodimethylamine (NDMA). NDMA is classified as a probable human carcinogen (a substance that could cause cancer) on the basis of animal studies.

Ranitidine medicines are used widely to reduce the production of stomach acid in patients with conditions such as heartburn and stomach ulcers. They are available over-the-counter and on prescription. Patients who have any questions about their current treatment can speak to their doctor or pharmacist. There are several other medicines used for the same conditions as ranitidine that could be used as an alternative.

Ranitidine belongs to a class of medicines known as H2 (histamine-2) blockers, which work by blocking histamine receptors in the stomach and reducing the production of stomach acid. It is used to treat and prevent conditions caused by excess acid in the stomach such as heartburn and stomach ulcers. Ranitidine-containing medicines are authorised by national authorities and are available as tablets and injectable formulations (EMA, 2019).

In 2018, NDMA and similar compounds known as nitrosamines were found in a number of blood pressure medicines known as ‘sartans’, leading to some recalls and to an EU review, which set strict new manufacturing requirements for these medicines. In 2019, a nitrosamine impurity has been detected in a few batches of pioglitazone from one company and in batches of ranitidine.

The chemical is present in some foods and in water supplies but is not expected to cause harm when ingested in very low levels.

The review of ranitidine medicines was initiated on 12 September 2019 at the request of the European Commission, under Article 31 of Directive 2001/83/EC. The review will be carried out by the Committee for Medicinal Products for Human Use (CHMP), responsible for questions concerning medicines for human use, which will adopt an opinion. The CHMP opinion will then be forwarded to the European Commission, which will issue a final legally binding decision applicable in all EU Member States. The EMA evaluated the data to assess whether patients using ranitidine are at any risk from NDMA and undertook to provide information about this (at the time of writing, no further information was made available) (EMA, 2019b).

The U.S. FDA has also been investigating NDMA and other nitrosamine impurities in blood pressure and heart failure medicines called Angiotensin II Receptor Blockers (ARBs) since last year. In the case of ARBs, the FDA has recommended numerous recalls as it discovered unacceptable levels of nitrosamines. According to the FDA, some ranitidine medicines, including some products commonly known as the brand-name drug Zantac, contain a nitrosamine impurity. The FDA is evaluating whether the low levels of NDMA in ranitidine pose a risk to patients (FDA, 2019a).

In 2019, the FDA announced that a voluntary recall of 14 lots of prescription ranitidine capsules distributed by Sandoz Inc., used to decrease the amount of acid created by the stomach, had taken place in relation to N-nitrosodimethylamine (FDA, 2019b).

EMA (2019a) EMA to provide guidance on avoiding nitrosamines in human medicines, European Medicines Agency, at: https://www.ema.europa.eu/en/documents/press-release/ema-provide-guidance-avoiding-nitrosamines-human-medicines_en.pdf  (accessed 6th November 2019)


EMA (2019b) EMA to review ranitidine medicines following detection of NDMA. European Medicines Agency, at: https://www.ema.europa.eu/en/documents/referral/ranitidine-article-31-referral-ema-review-ranitidine-medicines-following-detection-ndma_en.pdf (accessed 6th November 2019)

FDA (2019a) Statement alerting patients and health care professionals of NDMA found in samples of ranitidine, US Food and Drug Administration at: https://www.fda.gov/news-events/press-announcements/statement-alerting-patients-and-health-care-professionals-ndma-found-samples-ranitidine (accessed 6th November 2019)

FDA (2019b) FDA announces voluntary recall of Sandoz ranitidine capsules following detection of an impurity, US Food and Drug Administration at: https://www.fda.gov/news-events/press-announcements/fda-announces-voluntary-recall-sandoz-ranitidine-capsules-following-detection-impurity (accessed 6th November 2019)

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Tuesday, 21 January 2020

Microbiologists investigate the cleanliness of hospital washers



Microbiologists have found that many washing machines in hospital setting as are reservoirs of multidrug-resistant bacteria. In one case study pathogens, were transmitted regularly to newborns in a neonatal intensive care unit at a children's hospital.

The bacterium involved was a single clone of Klebsiella oxytoca. The study showed how the organisms were transmitted repeatedly to new babies in a ward located in a children's hospital. The transmission of the organism halted only when a ‘smoking gun’ washing machine was disassembled and removed from the hospital.

Microbiologists, as reported by the American Society for Microbiology, have demonstrated how resistance genes, as well as many water-borne microorganisms, can persist in domestic washing machines at reduced temperatures.

Klebsiella oxytoca is a Gram-negative, rod-shaped bacterium that is closely related to K. pneumoniae. Outbreaks of antibiotic-resistant Klebsiella oxytoca have occurred in multiple hospitals and ICUs throughout the world

With the hospital case study, standard screening protocols demonstrated the presence of the Klebsiella organism on infants in the ICU. Genetic comparative testing traced the source of the bacterium to the washing machine. In the course of the investigation, both incubators (used for babies born prematurely) and healthcare workers were ruled out as the sources of the contamination.

It appears that clothes, like knitted caps and socks, and blankets washed in the machine transmitted K. oxytoca from the washer to the infants. The residual water on the rubber mantle of the washer together with the final rinsing process (where unheated water is used) were found to contain the contaminant.

The infants in the intensive care units (ICU) were colonized, but not infected by K. oxytoca. However, the potential for a serious public health risk exists unless action is taken.

Lead researcher Dr. Martin Exner states: “We have proven for the first time that a washing machine can also spread antibiotic-resistant bacteria to humans.”


The research also carries implications for household washers, as well as those located in hospitals. This may relate to factors associated with energy management and environmental concerns. The water temperatures used in many domestic washing machines have been declining, ostensibly to save energy (and money). This was driven temperatures to regularly to be below 60°C (140°F). At such temperatures the water is less effective in terms of killing vegetative bacteria.

The study recommended that changes in washing machine design and processing are needed in order to prevent the accumulation of residual water leading to conditions favourable for microbial growth.

The case study has been reported to the journal Applied and Environmental Microbiology, with the research paper headed “The washing machine as a reservoir for transmission of extended spectrum beta-lactamase (CTX-M-15)-producing Klebsiella oxytoca ST201 in newborns.”

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 20 January 2020

Variability and the LAL Assay for Bacterial Endotoxin Detection


With biological tests all measurements susceptible to variations in analytical conditions should be suitably controlled as far as is practicable. Here the LAL assay has a relatively elevated level of variability even for a biological assay. This variation derives from three principle sources: reagents, product, and method / instrumentation. This article examines some of the reasons for this variation in relation to the test and the test reagents. The article also examines the coefficient of variation which is one way to examine for test variation.
In relation to the LAL assay, Tim Sandle has written a paper assessing assay control.

This article examines some of the reasons for this variation in relation to the test and the test reagents. The article also examines the coefficient of variation which is one way to examine for test variation.







The reference is:

Sandle, T. (2019) Variability and the LAL Assay for Bacterial Endotoxin Detection, Journal of GxP Compliance, 23 (5): 1-10: http://www.ivtnetwork.com/article/variability-and-lal-assay-bacterial-endotoxin-detection

For further details, please contact Tim Sandle



Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 19 January 2020

Collagen adhesion gene associated with bloodstream infections caused by MRSA



Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection.

A new paper of interest:

Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information.

WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria.

These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Saturday, 18 January 2020

Impact of vaccines on antimicrobial resistance


AMR is now considered a key threat to global health, leading to more mortality and increased healthcare costs threatening future conduct of routine medical procedures. Traditional approaches to address AMR include antibiotic stewardship, better hygiene/infection control, promoting antibiotic research and development, and restricting use for agricultural purposes.

A new article of interest:

Antibiotic use drives the development and spread of resistant bacterial infections. Antimicrobial resistance (AMR) has become a prolific global issue, due to significant increases in antibiotic use in humans, livestock and agriculture, inappropriate use (under-dosing and over-prescribing), and misuse of antibiotics (for viral infections where they are ineffective). Fewer new antibiotics are being developed.

While antibiotic development is declining, vaccine technology is growing. This review shows how vaccines can decrease AMR by preventing bacterial and viral infections, thereby reducing the use/misuse of antibiotics, and by preventing antibiotic-resistant infections. Vaccines are less likely to induce resistance. Some future uses and developments of vaccines are also discussed.

Vaccines, along with other approaches, can help reduce AMR by preventing (resistant) infections and reducing antibiotic use. Industry and governments must focus on the development of novel vaccines and drugs against resistant infections to successfully reduce AMR.

See: "Impact of vaccines on antimicrobial resistance."

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)

Friday, 17 January 2020

Ancient feces reveal how 'marsh diet' left Bronze Age Fen folk infected with parasites


New research published today in the journal Parasitology shows how the prehistoric inhabitants of a settlement in the freshwater marshes of eastern England were infected by intestinal worms caught from foraging for food in the lakes and waterways around their homes.

The Bronze Age settlement at Must Farm, located near what is now the fenland city of Peterborough, consisted of wooden houses built on stilts above the water. Wooden causeways connected islands in the marsh, and dugout canoes were used to travel along water channels.
The village burnt down in a catastrophic fire around 3,000 years ago, with artefacts from the houses preserved in mud below the waterline, including food, cloth, and jewellery. The site has been called "Britain's Pompeii."

Also preserved in the surrounding mud were waterlogged "coprolites" -- pieces of human faeces -- that have now been collected and analysed by archaeologists at the University of Cambridge. They used microscopy techniques to detect ancient parasite eggs within the faeces and surrounding sediment.

Very little is known about the intestinal diseases of Bronze Age Britain. The one previous study, of a farming village in Somerset, found evidence of roundworm and whipworm: parasites spread through contamination of food by human faeces.
The ancient excrement of the Anglian marshes tells a different story. "We have found the earliest evidence for fish tapeworm, Echinostoma worm, and giant kidney worm in Britain," said study lead author Dr Piers Mitchell of Cambridge's Department of Archaeology.
"These parasites are spread by eating raw aquatic animals such as fish, amphibians and molluscs. Living over slow-moving water may have protected the inhabitants from some parasites, but put them at risk of others if they ate fish or frogs."

Disposal of human and animal waste into the water around the settlement likely prevented direct faecal pollution of the fenlanders' food, and so prevented infection from roundworm -- the eggs of which have been found at Bronze Age sites across Europe.

However, water in the fens would have been quite stagnant, due in part to thick reed beds, leaving waste accumulating in the surrounding channels. Researchers say this likely provided fertile ground for other parasites to infect local wildlife, which -- if eaten raw or poorly cooked -- then spread to village residents.


"The dumping of excrement into the freshwater channel in which the settlement was built, and consumption of aquatic organisms from the surrounding area, created an ideal nexus for infection with various species of intestinal parasite," said study first author Marissa Ledger, also from Cambridge's Department of Archaeology.

See:

Marissa L. Ledger, Elisabeth Grimshaw, Madison Fairey, Helen L. Whelton, Ian D. Bull, Rachel Ballantyne, Mark Knight, Piers D. Mitchell. Intestinal parasites at the Late Bronze Age settlement of Must Farm, in the fens of East Anglia, UK (9th century B.C.E.). Parasitology, 2019; 1 DOI: 10.1017/S0031182019001021

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

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