Monday, 15 July 2019

An interview with Dr. Tim Sandle

Dr Tim Sandle is currently Head of Microbiology and Sterility Assurance at Bio Products Laboratory Limited. Here he talks to the Microbiology Society about his current role, his area of research and the importance of antimicrobial resistance (AMR) as a health issue. He also explains why he joined the Microbiology Society and offers advice for anyone thinking about a career change.

You are currently Head of Microbiology and Sterility Assurance: tell us more about your role with Bio Products Laboratory Limited.

I am responsible for heading up four departments. One is associated with supporting the manufacturing areas in terms of assessing cleanrooms for levels of microorganisms in the air and on surfaces for assessing product bioburden, microbial levels in water, screening samples for bacterial endotoxin and verifying that the finished product is sterile. The second area is associated with the development of novel microbial methods, the qualification of equipment, and dealing with regulatory submissions. The third area is to do with risk assessments, carried out in order to lower microbial contamination risks in process areas and to investigate when high microbial levels are recovered. The fourth area is linked to proactive practices to improve hygiene and to support new technologies. My role is to ensure these different entities connect together and to develop appropriate policies and standards in order to enhance sterility assurance.

Why did you choose to become a microbiologist?

I was always interested in biology: as a child, I was encouraged by my grandfather to take an interest in the natural world. I started off with an interest in biological sciences in general and was encouraged by a teacher to consider the importance of microbiology in health and disease.

Do you have any advice for anyone thinking about a career change and making a brave move from academia to industry?
The key attraction with industry is the ability to research and develop life-saving medicines and see these come to fruition. However, it is a different working experience and there are different types of pressures (these days both academia and industry are subject to increasing cost and time demands). Certainly, in industry there is a need to produce and release on schedule, otherwise this creates financial complexities. However, the work is very varied and there remains opportunities to engage in research and to produce papers. I’ve certainly managed to continue to contribute to peer-reviewed papers and book chapters. There also remains the opportunity to present at conferences.

Tell us about your biggest professional achievement(s) so far.

Some of the recent research I’ve been undertaking has concerned a partly overlooked issue of whether organisms that are resistant to antimicrobials have enhanced resistance to biocides. Although there is no direct evidence that organisms can acquire resistance to disinfectants, organisms that are resistant to antimicrobials may be harder to kill with the disinfectants commonly used in the pharmaceutical or healthcare setting. There is some evidence of this with some organisms, which calls for a renewed focus on aspects of disinfectant efficacy, like the minimum inhibitory concentration.

Tell us about your area of research?

The research is mostly applied. Over the past few years I’ve been working with microbiologists in Saudi Arabia, principally Dr Vijayakumar Rajendran, to determine the frequency of biocide resistant genes (e.g. qacA, qacE and cepA) in multidrug resistant bacteria, such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, and to correlate the presence or absence of resistant genes with biocides susceptibility. We’ve written several papers looking at different organisms and different biocides, assessing whether organisms that are antimicrobial resistant are also more resistant to common disinfectants. The research may have an impact on how pharmaceuticals and healthcare works, such as the need to reassess minimum inhibitory concentrations.

What have you done to try to maximise the impact of your research?

The research is ongoing and the full implications have yet to be realised; however, the research is showing a new dimension to the antimicrobial issue. The main thing in terms of impact is kicking-off discussion about overlooked areas in relation to AMR, which should help to encourage other researchers to consider different perspectives. Outside of this, I publicise where I can the importance of taking steps to reduce AMR, especially when different international campaigns are taking place. Social media provides a great outlet for this.

How important is AMR as a health issue?

It is an issue of great importance. Humans face the very real risk of a future without antibiotics. The implications of this are that life expectancy could fall due to people dying from diseases that are readily treatable today. In the last two decades, the rate at which bacteria are becoming resistant to current antibiotic treatments has substantially increased. For example, this trend is threatening the ability of medical staff to carry out routine operations or transplants in the future.

In your opinion, which areas of research are likely to have greatest impact on tackling AMR in the future?

Scientists have a role in addressing AMR even if they are not directly involved with AMR research or practices. This is by helping to promote best practices, such as avoiding the mis-prescribing of antibiotics to patients or though seeking better practices and alternatives to antibiotics in terms of rearing animals. One key research area that will have the greatest impact is in the search for new antimicrobials. There are some interesting ones in development, such as Dalvance, an intravenous drug that can treat skin and soft tissue infections; Oritavancin, a lipoglycopeptide with bactericidal activity against Gram-positive bacteria; and Teixobactin, a peptide-like secondary metabolite found in some bacteria that kills some Gram-positive bacteria and which has received the most media attention. The search for new antimicrobials, however, needs to continue, and the spectrum of searching needs to extend to areas of low human contact, such as deep in caves or parts of the oceans.

Do you have any advice for early career scientists who’d like to work in AMR?

First, research into AMR is a long process and there are many routes that do not lead to anything tangible. Patience is important. Second, potential candidate drugs to address AMR can come from the most unlikely of places, so keeping an open mind is also important.

The Microbiology Society is often seen as a Society for academics. What would you say to disperse this myth? What are the main benefits of being a member?

The Microbiology Society provides topical material for microbiologists in all sorts of occupations, not just academia. In recent years there has been a variety of different topics that connect what is being researched in universities to what needs to be developed by industry in order to meet healthcare demands – the hunt for new antimicrobials being a prime example. The Microbiology Society is so varied, and this richness leads to a range of different subject matter that enhances knowledge across both academia and industry. The sharing of ideas across these two sectors is to the benefit of all professional microbiologists. It provides an important arena for networking and sharing ideas across a range of different microbiological disciplines. It also plays a vital role in promoting the interaction between microbiologists and the general public, helping to educate and to engage.

And finally, why does microbiology matter?

Microbiology matters because it impacts across every aspect of society, from food production to global warming (such as toxic algal blooms); to the development of new medicines through biotechnology; for protecting the manufacture of medicines from contamination; and, of course, in protecting people from disease and with fighting diseases. Through being involved with any of these fields, you can make a difference.

Are you a member and interested in sharing stories about your research journey? Email March 2019


Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 14 July 2019

Artificial intelligence used to identify bacteria accurately

Microscopes enhanced with artificial intelligence (AI) could help clinical microbiologists diagnose potentially deadly blood infections and improve patients' odds of survival, according to microbiologists at Beth Israel Deaconess Medical Center (BIDMC).

The scientists demonstrated that an automated AI-enhanced microscope system is "highly adept" at identifying images of bacteria quickly and accurately. The automated system could help alleviate the current lack of highly trained microbiologists, expected to worsen as 20 percent of technologists reach retirement age in the next five years.

"This marks the first demonstration of machine learning in the diagnostic area," said senior author James Kirby, MD, Director of the Clinical Microbiology Laboratory at BIDMC and Associate Professor of Pathology at Harvard Medical School. "With further development, we believe this technology could form the basis of a future diagnostic platform that augments the capabilities of clinical laboratories, ultimately speeding the delivery of patient care."

Kirby's team used an automated microscope designed to collect high-resolution image data from microscopic slides. In this case, blood samples taken from patients with suspected bloodstream infections were incubated to increase bacterial numbers. Then, slides were prepared by placing a drop of blood on a glass slide and stained with dye to make the bacterial cell structures more visible.

Next, they trained a convolutional neural network (CNN) -- a class of artificial intelligence modeled on the mammalian visual cortex and used to analyze visual data -- to categorize bacteria based on their shape and distribution. These characteristics were selected to represent bacteria that most often cause bloodstream infections; the rod-shaped bacteria including E. coli; the round clusters of Staphylococcus species; and the pairs or chains of Streptococcus species.

"Like a child, the system needed training," said Kirby. "Learning to recognize bacteria required a lot of practice, making mistakes and learning from those errors."

To train it, the scientists fed their unschooled neural network more than 25,000 images from blood samples prepared during routine clinical workups. By cropping these images -- in which the bacteria had already been identified by human clinical microbiologists -- the researchers generated more than 100,000 training images. The machine intelligence learned how to sort the images into the three categories of bacteria (rod-shaped, round clusters, and round chains or pairs), ultimately achieving nearly 95 percent accuracy.

Next, the team challenged the algorithm to sort new images from 189 slides without human intervention. Overall, the algorithm achieved more than 93 percent accuracy in all three categories. With further development and training, Kirby and colleagues suggest the AI-enhanced platform could be used as fully automated classification system in the future.

In the meantime, Kirby suggests automated classification can ameliorate the shortage of human technologists by helping them work more efficiently, "conceivably reducing technologist read time from minutes to seconds," he said.

While human technologists routinely provide highly accurate diagnoses, demand for these highly skilled workers exceeds supply in the United States. Nine percent of lab technologists remain unfilled, and that number is expected to dramatically increase as technologists of the Baby Boomer generation begin to retire in droves, according to a 2014 survey from the American Society for Clinical Pathology.

What's more, these images can be sent remotely, bringing the highest level expertise anywhere the internet reaches. That's critical, as rapid identification and delivery of antibiotic medications is the key to treating bloodstream infections, which can kill up to 40 percent of patients who develop them. Each day a patient goes untreated is linked with an increased risk of mortality.

In addition to its clinical uses, the new tool could also have applications in microbiology training and research, Kirby noted.

"The tool becomes a living data repository as we use it," he said. "And could be used to train new staff and ensure competency. It can provide unprecedented level of detail as a research tool."


Kenneth P. Smith, Anthony D. Kang, James E. Kirby. Automated Interpretation of Blood Culture Gram Stains using a Deep Convolutional Neural Network. Journal of Clinical Microbiology, 2017; JCM.01521-17 DOI: 10.1128/JCM.01521-17

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Saturday, 13 July 2019

Paper stickers to monitor pathogens are more effective than swabs

Using paper stickers to collect pathogens on surfaces where antisepsis is required, such as in food processing plants, is easier, and less expensive than swabbing, yet similarly sensitive.

"The porous structure of paper seems able to collect and accumulate [bacterial] contamination," said first author Martin Bobal, technical assistant, Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department for Farm Animal and Public Health in Veterinary Medicine, The University of Veterinary Medicine, Vienna, Austria. "This requires mechanical contact, for example by hand, or by splashed liquids."

In the study, the investigators, who specialize in monitoring cheese production, chose to target the organism Listeria monocytogenes, a pathogen that commonly contaminates raw milk and other raw dairy products, including soft cheeses such as Brie, Camembert, and Feta. They used qPCR, a method of quantifying DNA samples to determine the numbers of these bacteria, as well as of Escherichia coli.

Surfaces in food processing plants must be cleaned regularly. Unlike swabs, artificially contaminated stickers provided a record of contamination that took place over at least two weeks, despite washing, flushing with water, or wiping with Mikrozid, an alcohol-based disinfectant, to simulate cleansing practices. "Recovery [of DNA] from the stickers was rather variable, at around 30%, but did not distinctly decrease after 14 days of storage," the report stated. "This suggests the possibility of sampling over two weeks as well."

In a proof of concept experiment, the researchers placed stickers at multiple locations that frequently undergo hand contact -- such as on light switches and door handles -- for one to seven days. Both bacterial species were detected repeatedly from these stickers.
Unlike stickers, swabbing is impractical on complex surfaces, such as door handles, light switches, and other fomites (objects likely to be contaminated with, and spread infectious organisms) and does a poor job of taking up bacteria from dry surfaces, according to the report.

"In the food production facility, conventional swabbing as a standard method can only expose a momentary snapshot," the investigators wrote. "For example, it is not possible to reconstruct information about yesterday's status after cleansing has been performed. In addition, when moistened swabs or contact-plate sampling methods are used, they bring with them growth medium into a supposedly clean environment, making subsequent disinfection necessary."

The investigators showed that plain paper stickers could trap not only bacterial pathogens and related DNA, but dead, and viable but non-culturable pathogens, which also can pose a threat to public health.

"A major advantage of stickers is in handling: they are easy to distribute and to collect," the authors concluded. "We put the stickers directly into the DNA-extraction kit's first protocol step. We did not encounter any inhibition or loss of information during DNA-extraction, nor during qPCR," said Mr. Bobal.


Martin Bobal, Anna Kristina Witte, Patrick Mester, Susanne Fister, Dagmar Schoder, Peter Rossmanith. A novel method for sampling and long-term monitoring of microbes using stickers of plain paper. Applied and Environmental Microbiology, 2019; DOI: 10.1128/AEM.00766-19

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Friday, 12 July 2019

New Liposome Treatment for Community-acquired Pneumonia

The findings are of significance for pharmaceutical companies and the medical sector. This is in the context of a time of great struggle for antibiotic companies given the increase in instances of antibiotic resistant bacteria. What is of particular global concern is the acceleration of resistance. U.S. Centers for Disease Control and Prevention (CDC) data finds that many high-income countries are entering a “post-antibiotic era.”

Tim Sandle has written an article for BioPharma Trends on the subject of liposomes. Here is an extract:

"CAL02 is composed of liposomes engineered to entrap and neutralize a large panel of bacterial toxins, particularly those medically identified as causing severe complications. A liposome is a spherical vesicle having at least one lipid bilayer. In medical research, liposomes are considered one of the most versatile and promising drug-carrier devices."

To read the article, see:

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Thursday, 11 July 2019

New dispersion method to kill biofilm bacteria for wound care

Biofilms are a structured community of bacterial cells that are adherent to inert or living surfaces. What makes these structures special is that living within these biofilm communities makes its resident bacteria resistant to antibiotics. A research team led by Karin Sauer, professor of biological sciences at Binghamton University, demonstrated that two important human pathogens, P. aeruginosa and S. aureus, need pyruvate to form these structured biofilm communities that are inherently resistant to antibiotics. In turn, the research team demonstrated that removal of pyruvate induces a physiological change in biofilm bacteria that has two consequences: 1) it causes them to disassemble the biofilm structure in a process referred to as biofilm dispersion; and 2) it renders biofilm bacteria more susceptible to antibiotics.

Biofilm infections are almost impossible to treat by conventional antibiotic therapy. In that regard, these findings are noteworthy, Sauer said. Inducing biofilm dispersion by depleting pyruvate is an add-on therapy that maximizes the effectiveness of conventional antibiotics in killing biofilms. That this novel therapeutic strategy works was apparent as the combination treatment (inducing biofilm dispersion in addition to conventional antibiotic therapy) was significantly more effective than treatment with antibiotics alone or even with the antimicrobial cream silver sulfadiazine, which is considered the gold standard in wound care.

What this means for wound care is that pyruvate depletion can improve the anti-biofilm activity of conventional antibiotic therapy (which by itself is not working so well), to better treat infected wounds and, ultimately, improve wound healing.

Given that pyruvate depletion not only disperses already established biofilms, but also prevents the formation of antibiotic-resistant biofilms by the two principal pathogens associated with wound infections, pyruvate depletion can also be used to prevent biofilm-related wound infections.


James Goodwine, Joel Gil, Amber Doiron, Jose Valdes, Michael Solis, Alex Higa, Stephen Davis, Karin Sauer. Pyruvate-depleting conditions induce biofilm dispersion and enhance the efficacy of antibiotics in killing biofilms in vitro and in vivo. Scientific Reports, 2019; 9 (1) DOI: 10.1038/s41598-019-40378-z

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Wednesday, 10 July 2019

Standard for Disinfectants and Sterilants (TGO 54)

Previously in this blog we mentioned the Australian TGA consultation for its Standard for Disinfectants and Sterilants (TGO 54). The consultation period has now been completed and the TGA has made the new Therapeutic Goods Order 104: Standard for Disinfectants (TGO 104) to replace the previous TGO 54 'Standard for Disinfectants and sterilants' which sunsets on 1 April 2019.

The TGA has incorporated stakeholder feedback from consultation, about the proposed new TGO:

  • Updated sections of the previous TGO 54 'Standard for Disinfectants and Sterilants' and clarifies the requirements for hard surface disinfectants;
  • The labelling requirements of the previous and TGO 37 'General Requirements for Labels for Therapeutic Devices' (which sunset on 1 October 2018); and
  • Standards and requirements within the Guidelines for the evaluation of disinfectants.
  • As a result, these regulatory requirements are now contained within one TGO. 

Stakeholder feedback will also inform TGA's review of:
  • the disinfectants pages of the TGA website, to ensure the application processes for inclusion in the Australian Register of Therapeutic Goods (ARTG) are clear for all current and potential sponsors of disinfectants; and
  • the guidance documents for exempt and listed disinfectants (which will be published in late April in response to requests from stakeholders for further amendments). In the interim, the previous guidance documents provide support to sponsors for understanding the TGO.
  • In response to requests from stakeholders, the TGA intends to consult separately on a Standard for sterilants and disinfectants of medical devices during 2019. In the interim, sponsors may continue to comply with the appropriate sections of TGO 104 (which refers to the Guidelines for the evaluation of disinfectants) as 'state of the art'.

The TGA also intends to amend the definition of hospital grade disinfectant to more accurately reflect these products are used outside of a medical setting, at the next review of the regulations.

For details see – TGA

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Tuesday, 9 July 2019

GDP Office Based Evaluation and Risk Assessment programme (OBERA)

The MHRA GDP Inspectorate is embarking on a pilot of a new inspection approach that will impact holders of a Wholesale Dealer’s Licence (WDA(H)) whose main activities operate from a head office supplied from a number of ‘satellite’ facilities. For the companies selected, their satellite sites will be assessed remotely using information provided by the company in a standardised format.

The Office Based Evaluation and Risk Assessment (OBERA) is targeted at companies that operate from a single head office location, where the majority of the wholesale activity takes place, with a number of satellite sites which perform a very limited range of GDP activity.

Inclusion in the programme will be dependent upon the head office of the company passing an on-site 'Gateway Inspection’.

For the purposes of the pilot, companies with over 100 sites on their Wholesale Dealer’s Licence will be allocated a Gateway Inspection first. These companies will be contacted shortly, with the Gateway Inspections scheduled to commence during spring 2019.

For further details, see MHRA -

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 8 July 2019

Disinfectant efficacy testing for bacterial endospores against hydrogen peroxide

Efficacy is demonstrated through performance testing to show that the disinfectant is capable of reducing the microbial bioburden in either suspension (planktonic state) or from cleanroom surfaces to an acceptable level.

The disinfectant efficacy validation should provide documented evidence that the disinfectant demonstrates bactericidal, fungicidal, and/or sporicidal activity necessary to control microbial contamination in the facility. The greater challenges are around sporicidal disinfectants.

To assess the qualification of sporicidal agents, Tim Sandle has written a newpeer reviewed paper. The abstract reads:

Effective cleaning and disinfection of pharmaceutical and healthcare facilities requires effective practices and appropriate biocides. Application is typically through the use of two biocides in rotation. The expectation is that one disinfectant is sporicidal, not least because the presence of spore-forming bacteria poses a contamination risk due to the ability of these organisms to survive harsh environmental conditions. An example sporicide is hydrogen peroxide. It is incumbent upon each user to assess the selected sporicide for efficacy; however, developing a suitable test is not straightforward. This paper provides an approach that can be adopted for developing a sporicidal efficacy test against bacterial endospores, using hydrogen peroxide as the test sporicidal disinfectant.

The reference is:

Sandle, T. (2019) Disinfectant efficacy testing for bacterial endospores against hydrogen peroxide, Chemico Oggi (Chemistry Today), 37 (2): 60-65

To view a copy, see:

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 7 July 2019

Virulence factor of the influenza A virus mapped in real-time

Image: NativeAntigen

One of virulence factors found in the influenza A viruses is hemagglutinin (HA). Researchers at Kanazawa University have recently studied the structure of HA of avian influenza virus, H5N1, using high-speed atomic force microscopy (HS-AFM) and the findings are essential for developing therapeutic approaches against influenza A viruses in future.

HA is initially synthesized by host cells in its precursor form that known as HA0. Conversion of HA0 to HA is depending on the pathogenicity of influenza A viruses: extracellular conversion for low pathogenic influenza A viruses and intracellular conversion for highly pathogenic influenza A viruses. Therefore, understanding the structure and properties of HA0 is paramount to deciphering HA. Richard Wong and his research team thus sought out to scrutinize HA0 under the microscope. Recombinant HA0 protein of H5N1 was visually analyzed by HS-AFM system developed by Kanazawa University.

Both HA0 and HA exist in homotrimeric forms and conversion of HA0 to HA does not significantly modify the homotrimeric structure. Therefore, it is sensible to use HA as a template to generate HA0 HS-AFM simulation images. Acidic endosomal environment is the critical factor for HA to induce fusion between viral membrane and endosomal membrane in order to release viral materials into host cells. To elucidate the acidic effect on HA0, it was first exposed to an acidic environment. The trimer of HA0 turned out to be very sensitive to the acidic solution and expanded considerably. When conformational changes of hemagglutinin were measured in real-time using HS-AFM, the team found that its area was larger, and its height shorter. Acidic environment essentially made the molecule flatter and more circular, as compared to its original counterpart. This change in conformation was, however, reversible as the structure reverted back to its original form upon neutralization.

"Our pilot work establishes HS-AFM as an inimitable tool to directly study viral protein dynamics, which are difficult to capture with low signal-to-noise techniques relying on ensemble averaging, such as cyro-EM and X-ray crystallography," says lead author of the study Dr Kee Siang Lim. "With high scanning speed and a minimally invasive cantilever, we predict that HS-AFM is feasible to reveal the flow of irreversible conformational changes of HA2 induced by low pH, which is mimicking the true biological events that occur when HA enters a host endosome, in future study."

This study paved the way for investigating biological events within viruses in real-time. The authors state the importance of HS-AFM for this research: "Our work establishes HS-AFM as an inimitable tool to directly study viral protein dynamics, which are difficult to capture with low signal-to-noise techniques relying on ensemble averaging, such as cyro-EM and X-ray crystallography," explains Dr Richard Wong, senior author of the study.

See: Kee Siang Lim, Mahmoud Shaaban Mohamed, Hanbo Wang, Hartono, Masaharu Hazawa, Akiko Kobayashi, Dominic Chih-Cheng Voon, Noriyuki Kodera, Toshio Ando, Richard W. Wong. Direct visualization of avian influenza H5N1 hemagglutinin precursor and its conformational change by high-speed atomic force microscopy. Biochimica et Biophysica Acta (BBA) - General Subjects, 2019; DOI: 10.1016/j.bbagen.2019.02.015
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Saturday, 6 July 2019

How rod-shaped bacteria grow long, not wide

The slender, rod-shaped Bacillus subtilis is one of the best-studied bacteria in the world, a go-to system for exploring and understanding how bacteria grow, replicate, and divide. One of its outstanding mysteries has been how it manages to keep its precise diameter while growing and and getting bigger end-to-end.

This week, a team led by Ethan Garner of Harvard University describes the opposing and balanced enzymatic actions that keep B. subtilis from bulging wide while it builds up its inner cell wall and elongates. The study, in Nature Microbiology, is a collaboration with microscopy developer Rudolf Oldenbourg of the Marine Biological Laboratory (MBL).

"I had been impressed by Rudolf's work for many years and always hoped that I (or someone) would introduce polarization microscopy to bacterial cell biology," Garner says. This paper was his opportunity.

With polarization microscopy, scientists can visualize the orientation of individual molecules in a live cell, and how that orientation may change over time. "Polarization microscopy was key to this project," Garner says, giving his team essential and hard-to-obtain information on the orientation of material that B. subtilis adds to its cell wall as it grows.

"As I have been giving talks on this work, the bacterial community has been incredibly impressed by this [polarization microscopy] assay," Garner says. "There are many other bacteria that people want to explore with it."

Oldenbourg, a senior scientist at MBL, is happy to oblige. "We are standing ready to support the bacteria research community through the OpenPolScope Resource at MBL," he says.

See: Michael F. Dion, Mrinal Kapoor, Yingjie Sun, Sean Wilson, Joel Ryan, Antoine Vigouroux, Sven van Teeffelen, Rudolf Oldenbourg, Ethan C. Garner. Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems. Nature Microbiology, 2019; DOI: 10.1038/s41564-019-0439-0

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Friday, 5 July 2019

Detecting bacteria in space

Until now, relatively little was known about the different types of microbes found on the space station. The new approach enables researchers to identify and map different species inside the ISS, which will ultimately help safeguard astronauts' health and be key to future long-term space travel.

It will also have applications in the realms of environmental management and health care.

"The new methodology provides us spectacular snapshots of the bacterial world in space and the possibilities of applying this method to explore new microbiome environments are really exciting," said Nicholas Brereton, a researcher at UdeM's Institut de recherche en biologie végétale.

The challenge of maintaining cleanliness within space environments was first documented on the Russian MIR space station, where conditions eventually deteriorated so much that mould became widespread. On the ISS, space agencies have been trying to reduce the amount of microbial growth in the station since it was first launched in 1998.

Strict cleaning and decontamination protocols are now in place to maintain a healthy ISS environment; in orbit, crew members regularly clean and vacuum the space station's living and working quarters. But as resupply missions arrive carrying a range of material including food, lab equipment, live plants and animals, new bacteria species are continually being added.

Combined with existing human bacteria, and also because no windows can be opened, the build-up of bacteria inside the cramped quarters can be significant.

"Scientists have a well-documented understanding of broad bacterial families on the ISS, but now we've discovered a more diverse bacterial ecosystem that we ever expected," said Emmanuel Gonzalez, a metagenomic specialist at McGill. "It's an exciting step forward in understanding the biosphere that will accompany humans into extra-terrestrial habitats."

Although the microbial characterization method was piloted in space, its applications will be far broader, say the scientists behind the technology. Researchers can replicate this approach to address many challenges and environments, including in oceans and soils It is already being applied to human diseases and microbiomes.

See: Emmanuel Gonzalez, Frederic E. Pitre, Nicholas J. B. Brereton. ANCHOR: a 16S rRNA gene amplicon pipeline for microbial analysis of multiple environmental samples. Environmental Microbiology, 2019; DOI: 10.1111/1462-2920.14632

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Thursday, 4 July 2019

Light and nanotechnology prevent bacterial infections on medical implants

The insertion of a medical implant in a patient's body carries alongside the risk of bacterial contamination during surgery and subsequent formation of an infectious biofilm over the surface of the surgical mesh. Such biofilms tend to act like a plastic coating, impeding any sort of antibiotic agent to reach and attack the bacteria formed on the film in order to stop the infection. Thus, antibiotic therapies, which are time-limited, could fail against these super resistant bacteria and the patient could end up in recurring or never-ending surgeries that could even lead to death. As a matter of fact, according to the European Antimicrobial Resistance Surveillance Network (EARS-Net), in 2015 more than 30,000 deaths in Europe were linked to infections with antibiotic-resistant bacteria.

In the past, several approaches have been sought to prevent implant contamination during surgery. Post-surgery aseptic protocols have been established and implemented to fight these antibiotic-resistant bacteria but none have entirely fulfilled the role of solving this issue.

In a recent study published in Nano Letters and highlighted in Nature Photonics, ICFO researchers Dr. Ignacio de Miguel, Arantxa Albornoz, led by ICREA Prof. at ICFO Romain Quidant, in collaboration with researchers Irene Prieto, Dr. Vanesa Sanz, Dr. Christine Weis and Dr. Pau Turon from the major medical device and pharmaceutical device company B. Braun, have devised a novel technique that uses nanotechnology and photonics to dramatically improve the performance of medical meshes for surgical implants.

Through an ongoing collaboration since 2012, the team of researchers at ICFO and B. Braun Surgical, S.A., developed a medical mesh with a particular feature: the surface of the mesh was chemically modified to anchor millions of gold nanoparticles. Why? Because gold nanoparticles have been proven to very efficiently convert light into heat at very localized regions.

The technique of using gold nanoparticles in light-heat conversion processes had already been tested in cancer treatments in previous studies. Even more, at ICFO this technique had been implemented in several previous studies supported by the Cellex Foundation, thus being another salient example of how early visionary philanthropic support addressed at tackling fundamental problems eventually leads to important practical applications. For this particular case, in knowing that more than 20 million hernia repair operations take place every year around the world, they believed this method could reduce the medical costs in recurrent operations while eliminating the expensive and ineffective antibiotic treatments that are currently being employed to tackle this problem.

Thus, in their in-vitro experiment and through a thorough process, the team coated the surgical mesh with millions of gold nanoparticles, uniformly spreading them over the entire structure. They tested the meshes to ensure the long-term stability of the particles, the non-degradation of the material, and the non-detachment or release of nanoparticles into the surrounding environment (flask). They were able to observe a homogenous distribution of the nanoparticles over the structure using a scanning electron microscope.

Once the modified mesh was ready, the team exposed it to S. aureus bacteria for 24 hours until they observed the formation of a biofilm on the surface. Subsequently, they began exposing the mesh to short intense pulses of near infrared light (800 nm) during 30 seconds to ensure thermal equilibrium was reached, before repeating this treatment 20 times with 4 seconds of rest intervals between each pulse. They discovered the following: Firstly, they saw that illuminating the mesh at the specific frequency would induce localized surface plasmon resonances in the nanoparticles -- a mode that results in the efficient conversion of light into heat, burning the bacteria at the surface. Secondly, by using a fluorescence confocal microscope, they saw how much of the bacteria had died or was still alive.

For the bacteria that remained alive, they observed that the biofilm bacteria became planktonic cells, recovering their sensitivity or weakness towards antibiotic therapy and to immune system response. For the dead bacteria, they observed that upon increasing the amount of light delivered to the surface of the mesh, the bacteria would lose their adherence and peel off the surface. Thirdly, they confirmed that operating at near infrared light ranges was completely compatible with in-vivo settings, meaning that such a technique would most probably not damage the surrounding healthy tissue. Finally, they repeated the treatment and confirmed that the recurrent heating of the mesh had not affected its conversion efficiency capabilities.

As ICREA Prof at ICFO Romain Quidant comments, "the results of this study have paved the way towards using plasmon nanotechnologies to prevent the formation of bacterial biofilm at the surface of surgical implants. There are still several issues that need to be addressed but it is important to emphasize that such a technique will indeed signify a radical change in operation procedures and further patient post recovery."

As Director of Research and Development of B. Braun Surgical, S.A. Dr. Pau Turon explains, "our commitment to help healthcare professionals to avoid hospital related infections pushes us to develop new strategies to fight bacteria and biofilms. Additionally, the research team is exploring to extend such technology to other sectors where biofilms must be avoided."

See: Ignacio de Miguel, Irene Prieto, Arantxa Albornoz, Vanesa Sanz, Christine Weis, Pau Turon, Romain Quidant. Plasmon-Based Biofilm Inhibition on Surgical Implants. Nano Letters, 2019; 19 (4): 2524 DOI: 10.1021/acs.nanolett.9b00187

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Wednesday, 3 July 2019

Novel genome editing method for filamentous fungi

Image: William.W.Mangin-1

A team led by Dr Arazoe and Prof Kuwata has devised simple and quick techniques for gene editing (target gene disruption, sequence substitution, and re-introduction of desired genes) using CRISPR/Cas9 in the rice blast fungus Pyricularia (Magnaporthe) oryzae, a type of filamentous fungus. Spurred on by encouraging results, the researchers surmise, "Plants and their pathogens are still coevolving in nature. Exploiting the mutation mechanisms of model pathogenic fungi as a genome editing technique might lead to the development of further novel techniques in genetic engineering."

The working component of the CRISPR/Cas9 system binds to the target gene region (DNA) and causes a site-specific double-stranded break (DSB) in the DNA. Effective binding of this component requires a certain "motif" or "pattern" called the protospacer-adjacent motif (PAM), which follows downstream of the target gene region.

Most genome editing techniques require DSBs induced at the target site, which trigger DNA "repair" pathways in the host. Homologous recombination (HR) is a mechanism for repair of DSBs, and it is useful because it adds complementary sequences. However, the underlying methodology is laborious, and its efficiency conventionally depends on external factors such as the host properties as well as PAMs. HR can be divided into two pathways: "noncrossover" (gene conversion) and "crossover" type. Crossover-type repairs are known to occur in cells that undergo meiosis. However, the understanding of their role in cells that undergo mitosis is limited, and such information on filamentous fungi is virtually unavailable. It is this gap in knowledge that the researchers were looking to address.

In their study, the researchers first created a vector (gene delivery system) based on CRISPR/Cas9 to confirm crossover-type HR in the recipient gene region in the rice blast fungus.

Then, to check gene targeting or "sequence substitution," they created a "mutant" vector, optimized for single crossover-type HR, for targeted disruption of the host gene that encodes scytalone dehydratase (SDH), a protein involved in melanin formation. This vector was introduced into the vector containing the gene for hygromycin B phosphotransferase (hph), which confers resistance to the antibiotic hygromycin B. The researchers speculated that the single crossover-type HR would insert the entire vector along with hph into the target site. The mutants with disrupted SDH gene would be identified as white colonies (owing to loss of melanin) on a medium containing hygromycin B. The researchers found that the number of hygromycin B-resistant white colonies dramatically increased by using the CRISPR/Cas9 vector, which means that the CRISPR/Cas9 system is effective in inducing single crossover-type HR. The greatest benefit of this technique is that it needs extremely short homologous sequences (100 base pairs; which is really small in molecular biology).

The researchers also used a similar strategy to check whether gene introduction (or "knock in") is possible via single crossover-type HR using a CRISPR/Cas9 vector. They used the green-fluorescent protein (GFP) gene, which is widely used as a "reporter" gene to make host cells glow fluorescent green when inserted into their genome. They speculated that single crossover HR would result in introduction of GFP into the recipient sequence. Indeed, they found that use of the CRISPR/Cas9 vector gave rise to green fluorescent colonies on hygromycin medium. These findings show that the CRISPR/Cas9 system can be used for efficient "one-step" gene knock-in.

This research points towards a surprising fact that, perhaps, PAMs are not all that necessary for CRISPR/Cas9 gene editing in fungi. Hailing the success of the research, the team states, "We have found that filamentous fungi have unique genomic characteristics, wherein crossovers are frequently induced, even in somatic cells, by cleaving the target DNA. We used these characteristics to disrupt the target DNA and to introduce "reporter" genes. We also succeeded in increasing the efficiency and speed of the knock-in, using a single-step process. This technology overcomes the restriction posed by PAMs -- which is one of the biggest disadvantages of the CRISPR/Cas9 system -- and enables more flexible genome editing, which has been difficult in previous studies on filamentous fungi."

Finally, when asked about the broader applications of this research, Dr Arazoe and Prof Kuwata eloquently state, "Rice blast fungus is an important pathogen that causes destructive disease of rice, which is the staple food of the country. The CRISPR/Cas9-based genome editing technique developed in our study can speed up molecular biological research on this pathogen, ultimately contributing to stable food supply and plant-based food safety. Also, this technique is applicable to other filamentous fungi widely used in industry -- especially in the bioprocessing, food, and fermentation industries."

See: Tohru Yamato, Ai Handa, Takayuki Arazoe, Misa Kuroki, Akihito Nozaka, Takashi Kamakura, Shuichi Ohsato, Tsutomu Arie, Shigeru Kuwata. Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus. Scientific Reports, 2019; 9 (1) DOI: 10.1038/s41598-019-43913-0

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Tuesday, 2 July 2019

The powers of bacteria visualized in real time

The global spread of antibiotic resistance is a major public health issue and a priority for international microbiology research. In a new paper, researchers report on filming the process of antibiotic resistance acquisition in real time, discovering a key but unexpected player in its maintenance and spread within bacterial populations.

In his paper to be published in the journal Science, Christian Lesterlin, Inserm researcher at Lyon's "Molecular Microbiology and Structural Biochemistry" laboratory (CNRS/Université Claude Bernard Lyon 1), and his team were able to film the process of antibiotic resistance acquisition in real time, discovering a key but unexpected player in its maintenance and spread within bacterial populations.

The researchers chose to study the acquisition of Escherichia coli resistance to tetracycline, a commonly used antibiotic, by placing a bacterium that is sensitive to tetracycline in the presence of one that is resistant. Previous studies have shown that such resistance involves the ability of the bacterium to expel the antibiotic before it can exert its destructive effect using "efflux pumps" found on its membrane. These specific efflux pumps are able to eject the antimicrobial molecules from the bacteria, thereby conferring on them a certain level of resistance.

In this experiment, the transmission of the DNA from one specific "efflux pump" -- the TetA pump -- was observed between a resistant bacterium and a sensitive bacterium using fluorescent marking. Thanks to live-cell microscopy, the researchers just had to track the progression of the fluorescence to see how the DNA of the "pump" migrated from one bacterium to another and how it was expressed in the recipient bacterium.

The researchers revealed that in just 1 to 2 hours, the single-stranded DNA fragment of the efflux pump was transformed into double-stranded DNA and then translated into functional protein, thereby conferring the tetracycline resistance on the recipient bacterium.

In their video, the transfer of DNA from the donor bacteria (green) to the recipient bacteria (red) is revealed by the appearance of red localization foci. The rapid expression of the newly acquired genes is revealed by the production of green fluorescence in the recipient bacteria.

See: Sophie Nolivos, Julien Cayron, Annick Dedieu, Adeline Page, Frederic Delolme, Christian Lesterlin. Role of AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid transfer. Science, 2019; 364 (6442): 778 DOI: 10.1126/science.aav6390

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 1 July 2019

Sandle's Pharmaceutical Microbiology Dictionary

Pharmaceutical Microbiology, an applied branch of Microbiology, focused on study of micro-organisms associated with the manufacture of pharmaceuticals, primarily in minimizing the numbers in a process environment; ensuring that the finished product is sterile and excluding those specific strains that are regarded as objectionable from starting materials and water. The discipline is also associated with drug development, including the application of biotechnology. This dictionary provides definitions and descriptions of the leading terms association with pharmaceutical microbiology and related fields. The dictionary is designed to assist students and those who do not work directly in the field to understand the terminology; and as an aide-memoire for more experienced practitioners.

The book is available as an e-book or as a paperback via Amazon.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

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