Wednesday, 31 May 2017

Newly-discovered gene that makes bacteria resistant to the most powerful antibiotics

A disturbing new gene that can make normal bacteria resistant to the strongest antibiotics has been detected in Canada, it had been revealed.

From The Independent:

The gene, MCR-1, is notable because it produces a chemical that makes bacteria resistant to colistin, a powerful antibiotic that has fallen out of favour due to its dangerous side effects, but one that is still used as a last-ditch medicine when all other remedies have failed.

The mobilized colistin resistance (mcr-1) gene confers plasmid-mediated resistance to colistin, one of a number of last-resort antibiotics for treating gram negative infections. mcr-1 is capable of horizontal transfer between different strains of a bacterial species and after discovery in November 2015 in E. coli (strain SHP45) from a pig in China it has been found in Escherichia coli, Salmonella enterica, Klebsiella pneumonia, Enterobacter aerogenes, and Enterobacter cloacae.As of 2017 it has been detected in more than 30 countries on 5 continents in less than a year.

If the newly-discovered gene were to make bacteria invincible to colistin, the sickest patients could find themselves in even more trouble.

As the Toronto Star reports, MCR-1 was first reported in British medical journal The Lancet in November last year, after Chinese scientists discovered a number of samples of the E-coli bacteria containing the MCR-1 gene on meat and hospital patients.

The noted that the gene likely originated in farm animals, due to the large amounts of antibiotics given to livestock in order to stop illness.

As the Star notes, MCR-1 has also been detected in bacteria samples gathered across the world, in Asia, Africa and even the UK.

Now, it has appeared in Canada, once in a patient in Ottowa and two in samples of ground beef sold in Ontario.

Posted by Dr. Tim Sandle

Tuesday, 30 May 2017

How Gonorrhea manipulates the female reproductive tract

Gonorrhea is a widespread sexually transmitted disease caused when Neisseria gonorrhoeae bacteria infect the normally protective inner lining of human genital tissues. In women, the opening of the uterus, known as the endocervix, serves as a primary infection site for N. gonorrhoeae. However, the strategy used by N. gonorrhoeae to penetrate the lining of the endocervix has been unclear.

To investigate this mechanism, Liang-Chun Wang of the University of Maryland, College Park, and colleagues needed to develop an alternative to the mouse models normally used to study gonorrhea, since they have been inadequate for this purpose. The team developed a new model using tissue samples obtained from the human endocervix.

The researchers infected the endocervix tissue, as well as lab-grown cells of the same type as those that line the endocervix, with N. gonorrhoeae. They then employed a variety of molecular and imaging techniques to examine the infection mechanism.

The results demonstrate that N. gonorrhoeae penetrates the endocervix lining by interfering with a normally protective process. Usually, infected cells in the lining can be shed and disposed of without breaking the tight connections between cells that keep the lining uncompromised. N. gonorrhoeae appears to be able to break these connections and induce cell shedding, opening paths for penetration without reducing its ability to adhere to and invade the cells of the lining.


Liang-Chun Wang, Qian Yu, Vonetta Edwards, Brian Lin, Jessica Qiu, Jerrold R. Turner, Daniel C. Stein, Wenxia Song. Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation. PLOS Pathogens, 2017; 13 (4): e1006269 DOI: 10.1371/journal.ppat.1006269

Posted by Dr. Tim Sandle

Monday, 29 May 2017

Establishing a Contamination Control Strategy for Aseptic Processing

To assess the risks of non-sterility each organization should develop a contamination control strategy. This requires an assessment, acknowledgement and remediation process for contamination risks. A contamination control strategy will be multifaceted and complex; as a means of addressing some of the basics and in raising some points for consideration, this article discusses the key starting points to be included in contamination control strategy for aseptically produced products.

In relation to this, Tim Sandle has written an article for American Pharmaceutical Review. The reference is:

Sandle, T. (2017) Establishing a Contamination Control Strategy for Aseptic Processing, American Pharmaceutical Review, 20 (3): 22-28

To view the article, click here.

Posted by Dr. Tim Sandle

Sunday, 28 May 2017

Biosimilars (EDQM News)

EDQM Biosimilars: Ph. Eur. monographs are flexible and evolving standards During a seminar co-organised with the European Medicines Agency (EMA), the EDQM clarified further the role that Ph. Eur. monographs play in the assessment of biosimilars. As public standards for the quality of medicines in Europe, monographs ensure the quality of biosimilar and other biotherapeutic products, but compliance with them is not sufficient for demonstrating biosimilarity.

However, while Ph. Eur. monographs provide specifications in the form of tests and acceptance criteria for all medicines, they are dynamic documents that can be adapted to scientific progress.

Dr Peter Richardson, Head of Quality at the EMA, provided information on EU legislation in the field of biosimilars, and Dr Niklas Ekman, Senior Researcher at the Finnish Medicines Agency (FIMEA) shared his experience as an assessor.

See: biosimilars seminar from which a video recording of the EDQM-EMA session on biosimilars can be viewed

Posted by Dr. Tim Sandle

Saturday, 27 May 2017

Synthetic biologists engineer inflammation-sensing gut bacteria

Synthetic biologists at Rice University have engineered gut bacteria capable of sensing colitis, an inflammation of the colon, in mice. The research points the way to new experiments for studying how gut bacteria and human hosts interact at a molecular level and could eventually lead to orally ingestible bacteria for monitoring gut health and disease.

The research, published in a new study in Molecular Systems Biology, involved a series of breakthroughs in the lab of Jeffrey Tabor, assistant professor of bioengineering and of biosciences at Rice, and key contributions from collaborators Robert Britton and Noah Shroyer at Baylor College of Medicine. Tabor's team, including lead co-author and postdoctoral researcher Kristina Daeffler, identified the first genetically encoded sensor of a novel biomarker linked to inflammation, inserted the genes for the sensor into a well-studied gut bacterium and collaborated with Shroyer and Britton to use the engineered bacteria to detect colon inflammation in mice.

"The gut harbors trillions of microorganisms that play key roles in health and disease," Tabor said. "However, it is a dark and relatively inaccessible place, and few technologies have been developed to study these processes in detail. On the other hand, bacteria have evolved tens of thousands of genetically encoded sensors, many of which sense gut-linked molecules. Thus, genetically engineered sensor bacteria have tremendous potential for studying gut pathways and diagnosing gut diseases."

Synthetic biologists like Tabor specialize in programming single-celled organisms like bacteria in much the same way an engineer might program a robot. In particular, Tabor's team is working to develop bacterial sensors that can detect disease signals in the gut. Like electrical engineers who build circuits from wires and electronic components, Tabor's team uses genetic circuits to program single-celled creatures to carry out complex information processing.

Previous work has suggested that alterations to the gut microbiota, genetic predisposition and other environmental factors may play key roles in inflammatory bowel disease, a condition that includes Crohn's disease and ulcerative colitis and which affects as many as 1.6 million Americans.

"Based on a number of previous studies, we hypothesized that the molecule thiosulfate may be elevated during colitis," Daeffler said. "It has been difficult for scientists to study this link because there aren't tools for reliably measuring thiosulfate in living animals. Our first goal in this project was to engineer such a tool."

From the outset of the project in 2015, Daeffler said, the idea was to use sensor bacteria, in this case an engineered form of Escherichia coli, to sense thiosulfate and related sulfur-containing compounds that may also be biomarkers of colitis. There were well-understood methods for programming E. coli to produce a fluorescent green protein in response to specific stimuli, but there were no known genes -- in any organism -- that were used to sense thiosulfate, and few for the other compounds.

"There's a link between gut sulfur metabolism and inflammation, and we knew that we needed to be able to measure sulfur metabolites accurately to diagnose colon inflammation," she said.

Tabor said study co-author Ravi Sheth, an undergraduate researcher in the group in 2015, used a computer program to identify potential sensors of thiosulfate and other sulfur compounds in the genome of Shewanella, a type of bacteria that live in marine sediment. Tabor's group believes that Shewanella likely breathe these molecules and use the sensors to turn on the proper enzymes in their presence.

Daeffler spent one year engineering E. coli to express the sensor genes, validate their function and optimize them to respond to the potential biomarkers by producing a green fluorescent protein signal. It took another year to prove that the system worked and detected colon inflammation in mice.

The researchers administered orally two drops containing about a billion sensor bacteria to both healthy mice and to mice with colitis. They measured the activity of the sensor bacteria in each group six hours later. The tell-tale green fluorescent protein showed up in the feces of the mice. Though it was not visible to the unaided eye, it could easily be measured with a standard laboratory instrument called a flow cytometer.

The team found that the thiosulfate sensor was activated in the mice with inflammation, and was not activated in the healthy mice. Furthermore, the researchers found that the more inflammation the mouse had, the more the sensor was activated.

Tabor said the study shows that gut bacteria can be outfitted with engineered sensors and used to noninvasively measure specific metabolites and that this result could open the door to many new studies that could help elucidate a wide range of gut processes.

Though it would likely take several additional years of development, and it remains unknown if thiosulfate is a biomarker of human colitis, the sensor bacteria might eventually be re-engineered to function as a diagnostic of human colitis, Tabor said. In particular, the green fluorescent protein could be replaced with an enzyme that makes a colored pigment.

"We'd like to develop a home inflammation test where a person prone to colitis flare-ups would eat yogurt that contained the engineered bacteria and see blue pigment in the toilet if they were sick," he said.

Tabor said such a test could reduce unneeded and costly trips to the doctor and unneeded colonoscopy procedures, which are both expensive and invasive. He said his team has begun collaborations with gastroenterologists at Baylor to achieve this goal.


Kristina NM Daeffler, Jeffrey D Galley, Ravi U Sheth, Laura C OrtizVelez, Christopher O Bibb, Noah F Shroyer, Robert A Britton, Jeffrey J Tabor. Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Molecular Systems Biology, 2017; 13 (4): 923 DOI: 10.15252/msb.20167416

Posted by Dr. Tim Sandle

Friday, 26 May 2017

EMA: risk based prevention of cross contamination in production (draft)

The European Medicines Agency has issued a new draft document, with the lengthy title of “Questions and answers on implementation of risk based prevention of cross contamination in production and ‘Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities’ (EMA/CHMP/CVMP/SWP/169430/2012).”

The document deals with highly hazardous products are those that can cause serious adverse effects at low doses and that therefore would benefit from a full toxicological assessment in order to derive a safe Health Based Exposure Limits. A health based exposure limit is a limit (permitted daily exposure (PDE) or equivalent) at which a product is regarded to be safe in humans. It can be based on either clinical dose or non-clinical safety data, depending on which would give the lower exposure limit.

Such products are:

  1. Genotoxic (specifically mutagenic) compounds that are known to be, or highly likely to be, carcinogenic to humans. Compounds of this group are easily identifiable, since genotoxicity would be related to the pharmacology, e.g. as DNA alkylating cytostatics, and their use is usually restricted to oncology indications with respective warning statements in the Summary of Product Characteristics.
  2. Compounds that can produce reproductive and/or developmental effects at low dosages, for example where evidence exists of such effects being caused by a clinical dose of <10 mg/day (veterinary dose equivalent 0.2 mg/kg/day) or dosages in animal studies of ≤1 mg/kg/day.
  3. Compounds that can produce serious target organ toxicity or other significant adverse effects at low doses, for example where evidence exists of such effects being caused by a clinical dose of <10 mg/day (veterinary dose equivalent 0.2 mg/kg/day) or dosages in animal studies of ≤1 mg/kg/day.
  4. Compounds with a high pharmacological potency i.e. recommended daily dose of <1 mg (veterinary dose equivalent 0.02 mg/kg)

Compounds with a high sensitising potential.

The document can be accessed here: EMA

Posted by Dr. Tim Sandle

Thursday, 25 May 2017

New assay for bioterrorism threat

Researchers in Spain are using RPA as the foundation for a highly sensitive and specific solid-phase optical assay that can detect the potential biowarfare agent, Y. pestis, in less than an hour. The enzyme-linked oligonucleotide assay (ELONA) approach developed by Ioanis Katakis and Ciara K. O’Sullivan, at the Universitat Rovira i Virgili’s Interfibio Research Group, and the ICREA (Catalan Institution for Research and Advanced Studies), uses conventional PCR primers to amplify both single- and double-stranded Y. pestis DNA. Their work provides proof of concept for applying RPA in a heterogeneous format, with one primer immobilized onto a solid surface. The researchers aim to further develop the technology into an integrated, portable lateral flow-type test device for the rapid amplification and detection of Y. pestis in resource limited and field settings.

For further details see:

Researchers uncover how bacteria burst our cells

Scientists based in Vienna unveil the complex molecular structure that causes lethal infections by Mycobacterium tuberculosis (Mtb). Their findings might have implications for potential therapies against antibiotic-resistant tuberculosis. The researchers have described the overall architecture of an assembly of proteins known as Type VII (T7SS) secretion systems found in a group of bacteria which cause diseases such as tuberculosis.

T7SS-systems play a key role in tuberculosis infections and might present important targets for much needed new drugs: blocking these systems could prevent the bacteria from bursting the host cells and could thus alleviate the infection.

In addition to the core body of T7SS, some of the proteins extend down into the bacterial cell. The team collected Small Angle X-ray Scattering (SAXS) data at the EMBL SAXS beamline on the DESY campus in Hamburg to help understand what they look like and how these parts of the secretion system might move. "We believe these arm-like proteins help to move the molecules of different shapes and sizes from the inside of the bacterial cell towards the pore of the secretion system for them to be transported out of the cell," says first author Kate Beckham from EMBL Hamburg.

Now further biochemical and genetic experiments will be carried out to support the structural data and to provide in vivo insights into the components required for assembly of the T7 secretion system.


Katherine S. H. Beckham, Luciano Ciccarelli, Catalin M. Bunduc, Haydyn D. T. Mertens, Roy Ummels, Wolfgang Lugmayr, Julia Mayr, Mandy Rettel, Mikhail M. Savitski, Dmitri I. Svergun, Wilbert Bitter, Matthias Wilmanns, Thomas C. Marlovits, Annabel H. A. Parret, Edith N. G. Houben. Structure of the mycobacterial ESX-5 type VII secretion system membrane complex by single-particle analysis. Nature Microbiology, 2017; 2: 17047 DOI: 10.1038/nmicrobiol.2017.47

Posted by Dr. Tim Sandle

Wednesday, 24 May 2017

Too much pressure: a behavioral approach to Data Integrity

The MHRA has published an interesting article on the behavioral factors that shape data integrity.

Data integrity refers to maintaining and assuring the accuracy and consistency of data over its entire life-cycle, and is a critical aspect to the design, implementation and usage of any system which stores, processes, or retrieves data.

Here is an extract:

“Implementing a quality culture and ensuring job satisfaction is easier said than done, but relatively simple actions taken by management can make big differences in everyday operations.  Informal senior management visits to the shop floor enable an understanding of operational issues which are invisible from the boardroom. The ability of an individual to justify their decision to manipulate data can be reduced by making sure that all employees have ‘visibility to the patient’ and understand the impact of their actions. Senior management has the power to fix problematic test methods from the beginning, upgrade outdated equipment and software, encourage open reporting of deviations, and reward good behavior rather than speed.”

The full article can be accessed here.

Tim Sandle’s article on data integrity for the microbiology laboratory can be read here.

Posted by Dr. Tim Sandle

Tuesday, 23 May 2017

How a beneficial gut microbe adapted to breast milk

Breast milk provides vital nutrients not only to infants, but also to beneficial microbes that inhabit the gastrointestinal tract. A new study shows that a bacterial species called Bifidobacterium longum has successfully adapted to the unique niche of the infant gut by producing an enzyme called LnbX, which enables this microbe to grow on a sugar that is abundant only in human milk.

Gut microbes in early life are thought to have long-lasting effects on human health, and studies have shown that diet strongly influences the composition of this population. For example, human milk sugars are known to selectively promote the growth of beneficial gut microbes such as Bifidobacteria, which prevent diarrhea and pathogenic infection in infants. One major component of human milk is a sugar called lacto-N-tetraose, which is virtually absent in the milk of other mammals. Bifidobacteria produce enzymes that break down this sugar, strongly suggesting that a symbiotic relationship recently evolved between these microorganisms and humans.

While investigating how this symbiotic relationship evolved, Katayama and co-senior study author Shinya Fushinobu of the University of Tokyo previously characterized LnbB and isolated LnbX -- enzymes that degrade lacto-N-tetraose in Bifidobacterium bifidum and Bifidobacterium longum, respectively. In the new study, the researchers set out to build on these findings by determining the X-ray crystal structure of the catalytic domain of LnbX. The crystal structure, in combination with mutation and pharmacological experiments, revealed that LnbX has a distinct structure and catalytic mechanism from LnbB and therefore belongs to a novel family of glycoside hydrolase enzymes called GH136.

For further details see:

Chihaya Yamada, Aina Gotoh, Mikiyasu Sakanaka, Mitchell Hattie, Keith A. Stubbs, Ayako Katayama-Ikegami, Junko Hirose, Shin Kurihara, Takatoshi Arakawa, Motomitsu Kitaoka, Shujiro Okuda, Takane Katayama, Shinya Fushinobu. Molecular Insight into Evolution of Symbiosis between Breast-Fed Infants and a Member of the Human Gut Microbiome Bifidobacterium longum. Cell Chemical Biology, 2017; DOI: 10.1016/j.chembiol.2017.03.012

Posted by Dr. Tim Sandle

Monday, 22 May 2017

Pharmig Guide to Cleanrooms

Pharmig has issued a new publication relating to cleanrooms. The guide covers good cleanroom design (together with suitable operational parameters), cleanroom specifications, microbial control, and classification according to the international cleanroom standard ISO 14644 together with EU GMP. The guide includes practical information, including reference to risk assessment.


Sandle, T. (2017) Pharmig Guide to Cleanroom Operation and Contamination Control, Pharmig, Stanstead Abbotts, UK

For further information, see: Pharmig

Also purchase:

Posted by Dr. Tim Sandle

Sunday, 21 May 2017

Giant viruses found in Austrian sewage

Giant viruses are characterized by disproportionately large genomes and virions that house the viruses' genetic material. They can encode several genes potentially involved in protein biosynthesis, a unique feature which has led to diverging hypotheses about the origins of these viruses. But after discovering a novel group of giant viruses with a more complete set of translation machinery genes than any other virus known to date, scientists at the U.S. Department of Energy Joint Genome Institute (DOE JGI), a DOE Office of Science User Facility, believe that this group (dubbed "Klosneuviruses") significantly increases our understanding of viral evolution. Thus the Klosneuviruses contradict the theory that viruses make up a distinct domain of life.
The predicted hosts for the Klosneuviruses are protists (single-celled eukaryotic (nucleus-containing) microorganisms) and while their direct impacts on protists are not yet worked out, these giant viruses are thought to have a large impact on these protists that help regulate the planet's biogeochemical cycles.

Scientists have been fascinated by giant viruses since 2003, when a group of French biologists led by Didier Raoult discovered the Mimiviruses. Since then, a handful of other giant virus groups have been found. The unique ability among them to encode proteins involved in translation (typically DNA to RNA to protein) piqued researchers' interests as to the origin of giant viruses. Since then, two evolutionary hypotheses have emerged. One posits that giant viruses evolved from an ancient cell, perhaps one from an extinct fourth domain of cellular life. Another -- a scenario championed by Koonin -- presents the idea that giant viruses arose from smaller viruses.

The discovery of Klosneuvirus supports the latter idea, according to Tanja Woyke, DOE JGI Microbial Genomics Program lead and senior author of the paper. "In this scenario, a smaller virus infected different eukaryote hosts and picked up genes encoding translational machinery components from independent sources over long periods of time through piecemeal acquisition," she said.

For further details see:

Frederik Schulz, Natalya Yutin, Natalia N. Ivanova, Davi R. Ortega, Tae Kwon Lee, Julia Vierheilig, Holger Daims, Matthias Horn, Michael Wagner, Grant J. Jensen, Nikos C. Kyrpides, Eugene V. Koonin, Tanja Woyke. Giant viruses with an expanded complement of translation system components. Science, 2017; 356 (6333): 82 DOI: 10.1126/science.aal4657

Posted by Dr. Tim Sandle

Pharmig News

The latest Pharmig News letter is now available.

Members will have received a copy, non-members can purchase a copy via the Pharmig website.

No. 67 – May 2017

In this issue:
  • Chairman’s Newsletter
  • Book Reviews
  • The Future Possibilities of Graphene for Microbiology
  • Microbiological Cleaning Validation (Disinfectant Validation)
  • Pharmig News Corner #35

Posted by Dr. Tim Sandle

Saturday, 20 May 2017

Maple syrup extract enhances antibiotic action

Antibiotics save lives every day, but there is a downside to their ubiquity. High doses can kill healthy cells along with infection-causing bacteria, while also spurring the creation of "superbugs" that no longer respond to known antibiotics. Now, researchers may have found a natural way to cut down on antibiotic use without sacrificing health: a maple syrup extract that dramatically increases the potency of these medicines.

To figure out how the extract makes antibiotics work better, the researchers investigated whether the extract changed the permeability of bacterial cells. The extract increased the permeability of the bacteria, suggesting that it helps antibiotics gain access to the interior of bacterial cells. Another experiment suggested that the extract may work by a second mechanism as well, disabling the bacterial pump that normally removes antibiotics from these cells.


American Chemical Society. "No more 'superbugs'? Maple syrup extract enhances antibiotic action.”

Posted by Dr. Tim Sandle

Friday, 19 May 2017

The most beautiful bacteria you’ll ever see

Synthetic biologist Tal Danino manipulates microorganisms in his lab to create eye-catching, colorful patterns. Here’s a look at the process he uses to turn “Oh, yuck” into “Oh, wow.”

TED are hosting a range of beautiful microbial images. These are from Synthetic biologist Tal Danino, who has a new project called Microuniverse. Here he produced a series of dazzling, abstract images created by different species of bacteria, each grown under different conditions for varying lengths of time.

TED (Technology, Entertainment, Design) is a media organization which posts talks online for free distribution, under the slogan "ideas worth spreading". Tal Danino is a synthetic biologistartist and assistant professor of biomedical engineering at Columbia University.

“The project is about getting to see this unseen universe that’s really small and all around us, every day.” 

The images can be accessed here: TED

Posted by Dr. Tim Sandle

European Pharmacopoeia 9th Edition (Supplement 9.1)

The 1st supplement to the 9th edition of the European Pharmacopeia became effective on 1st April 2017.

Of interest to readers:

Water for injections (0169)

Production: revision to include purification processes equivalent to distillation (such as reverse osmosis coupled with appropriate techniques) for producing water for injections (WFI), in addition to distillation; use of non-distillation technologies for the production of WFI requires that notice is given to the supervisory authority of the manufacturer before implementation.

A requirement for regular monitoring of total organic carbon has been added to further emphasize the specific test controls required in the Production section.

Posted by Dr. Tim Sandle

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