Global Opportunity to Tackle Antibiotic Production Waste

Global policy makers gathered at The Hague in the Netherlands for the International Ministerial Conference on antimicrobial resistance (AMR).

Experts are calling for a greater focus on antibiotic production in order to tackle AMR and ensure the effectiveness of antibiotics in the future. Sustainable production is notably not a focus in the WHO’s AMR Global Action Plan, despite the fact that:

• There are 700,000 AMR related deaths each year predicted to rocket to 10 million deaths in 2050, more than all cancers combined
• Only 8 large R&D based and generic pharmaceutical companies set limits on antibiotics in their wastewater
• The amount of pharmaceutical effluent leaking into Europe’s waterways is estimated to increase by 2/3rds by 2050
• Antibiotics have been found in 65%of the world’s rivers, including the Thames where 5 antibiotics were found at up to 3 times the safe levels
• In Bangladesh waterways have been found to harbour 300 times the safe levels of antibiotics

Readers can download the media kit of three infographics (on AMR and health, AMR and the environment and an AMR timeline) here. Also included are case studies of sustainable manufacturing, the AMR industry alliance, and sustainable procurement practices in Sweden and Norway

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Autoclave packaging for efficient sterilisation

While steam and pressure are vital factors in the efficiency of the sterilisation process, the packaging of the items is pivotal to achieve a cost-efficient autoclave cycle. Cristina Masciola, communications manager at AM Instruments, explains

One of the most delicate phases in pharmaceutical production processes is autoclave sterilisation. However, the very concept of sterility is challenging. Arguably, to say that an object or environment is sterile doesn't mean the total absence of contamination, only that it contains the lowest concentration of possible contaminants. Consequently, such a condition needs to be addressed.

Sterility assurance level, or SAL for short, is used to express the probability of bacterial survival. It defines an area as sterile where 1 out of 1,000,000 units is contaminated (the bacterial count equals 10-6).

Sterilisation used to be defined as a process in which all possible life forms, including spores, were completely destroyed by passing an object through an autoclave that would allow the absence of germ proliferation. In time, it became evident that passage through an autoclave doesn't ensure sterility per se: a cycle in an autoclave is a process that must be considered in its entirety, from the preparation of the materials to their storage. None of these phases can be underestimated for a successful sterilisation cycle.

Autoclave: How it works

The autoclave consists of a steel container equipped with a door that can be hermetically sealed from the outside. Through special ductwork, the water vapour coming from a boiler is pumped into the autoclave. The air gradually leaves the equipment until only the pressurised steam remains inside.

The pressure inside the autoclave can reach anything between 6.9 x10-3 kPa (0.7 atm), which corresponds to a water vapour temperature of just over 115°C. Let's not forget that all forms of bacteria, moulds, and yeasts perish after only a few minutes of exposure to 100°C and that the most resistant spores (i.e. those of the tetanus bacillus) crumble at 115°C in 15 seconds. Good practice dictates that sterilisation is continued for 20 minutes to guarantee that all pathogenic and non-pathological forms of any kind are dead and therefore the material inside the autoclave is perfectly sterile.

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When operating an autoclave, the following conditions are required to combat bacteria and viruses that form spores effectively:

• The sterilising agent reaches all surfaces of the object to be sterilised. Some materials have a smooth, well-exposed structure, while others are much more articulated and therefore present challenging management
• The steam is in direct contact with the sterilised material. This step involves particular care during the loading phase of the autoclave
• A vacuum is created to move all the air initially present in the autoclave and then replace it with steam
• A well-designed control scheme for steam evacuation and cooling is implemented so that the load does not deteriorate 

Process efficiency

The efficiency of the sterilisation process depends on two factors: steam and pressure. Together, they ensure that enough heat is transferred into the organisms to kill them.

A series of negative pressure pulses are used to remove all possible air pockets, while steam penetration is maximised by applying a succession of positive pulses.

The relationship between temperature and pressure is generally the following: 

The specifications of the autoclave add another element to factor. Managers should consider both the type of materials and relative pressure loops of the item subject to sterilisation. The table below outlines the different criteria for consideration:

AM Instruments has developed a clear yet essential framework to understand the importance of the phases before and after the sterilisation cycle in an autoclave.

In fact, what has been said regarding the main characteristics of the machine is negated if the risks connected to the preparation of the material to be sterilised are not considered from the time of loading up to the final storage.

Materials in the spotlight

Let's start with the preparation of the material. The microbial charge on an object (bioburden) can be mild, medium or severe. For this reason, it must be effectively removed through decontamination and washing, both of which are essential preventive actions of the sterilisation process.


The subsequent packaging phase has specific purposes, such as:


• Allow the removal of air and, therefore, the penetration and contact of the sterilising agents with the surface of the object to be treated
• Reduce the risk of contamination of the sterilised material when the sterile package is open
• Preserve the sterility of the treated material until it's used
• Be free of toxicity

• Be practical, convenient and economical

On the other hand, "good" packaging depends on several factors:

• Material used
• Packaging methods
• Structure of the object to be packaged
• Storage

Medical paper is the most commonly used for this application, but is also the most at-risk type of material. Medical paper requires a high level of operation and handling with possible damage to the equipment. Plus, the coverage is irregular (a single layer covers some parts, two over others) affecting the passage of steam. Also, it's often difficult to remove. A high risk of fibre release combined with the possibility of tears and damage adds to the risks associated with the use of medical paper. Finally, a critical factor to consider in a production cycle is time; the operational time for packaging is high.

Autoclave topper before and after wrapping in medical paper

An effective and safe packaging method is one that meets specific requirements, such as:

• Designed to offer total security and resistance

• Customisable and easy to use

• Not influenced by autoclave sterilisation processes
• Resistant to tears and perforations
• Easy to apply and remove

• Minimal handling

• No taping if possible
• Low particle release

• Excellent microbiological barrier

• Uniform penetration of steam through the surface

With the above in mind, Tyvek material packaging systems provide a solution. The range is designed to make the preventive preparation operation not only safe for the autoclave cycles of the materials but also to minimise the operating times required by the cycle.

Unlike medical paper, the material in Tyvek generates very few airborne particles when opened or manipulated. This low-lint release property minimises the risk of introducing particulate matter into a clean environment.

A further advantage of using Tyvek material for sterilisable steam bags is that, unlike medical paper, Tyvek maintains its dimensional stability and high-quality visual appearance after steam sterilisation. Even in the most severe conditions in highly contaminated environments, Tyvek has proven to be highly resistant to the penetration of bacterial spores and other contaminating microorganisms.

Particle and bacteriological tests show that it performs better than other commercially available porous packaging materials, including medical paper.

Autoclave loading

Once the packaging has been carried out, autoclave loading represents an equally important step for the success of the sterilisation cycle. It must be performed in such a way that the steam can circulate freely and penetrate each package. Make sure the process follows the checklist below:

• The autoclave load must be evenly distributed and must not touch the internal walls
• The items to be sterilised must be arranged so that each surface is exposed to the sterilising agent for the expected temperature and time

• Special tools, such as containers and pipes, must be arranged with the opening facing downwards to prevent condensation and the formation of air bubbles

Upon completion of the sterilisation cycle, it's necessary to follow other procedures that ensure sterility is maintained: the cycle should be repeated in case of damaged packages or contact with wet surfaces, for example.

Pharmaclean by AM Instruments, the line and production facility launched by the Italian company to minimise the risk of microbiological and particulate contamination, meets the regulatory requirements, with Tyvek material packaging and sterilisation systems. The range, available in standard and custom-made formats, allows the operator minimum manipulation in total safety. The systems are located in A/C grade cleanrooms.

Autoclave topper before and after wrapping in Pharmaclean by AM instruments

With Pharmaclean, microbiological checks are carried out in accordance with Annex 1, and particle checks are performed in accordance with ISO 14644.

The stringent regulatory requirements, combined with the production rhythms of the pharmaceutical industry, make collaboration with suppliers increasingly important, to create optimal solutions that safeguard the product and do not slow down production cycles.

N.B. This article is featured in the July 2019 issue of Cleanroom Technology. The latest digital edition is available online.

Use of Hazard Analysis and Critical Control Points (HACCP) for EM locations

In the first part of a review of HACCP methodology (“Use of Hazard Analysis and Critical Control Points (HACCP) - Part 1: Assessing microbiological risks”) the application of HACCP as a tool for conducting microbiological risks assessments was discussed. This piece considered the approach taken to HACCP and the primary areas of microbiological concern to which the qualitative risk tool can be applied to.

This second example offers a case study of how HACCP can be applied to a practical setting: the identification of environmental monitoring locations, by considering, in the HACCP lexicon, monitoring locations as ‘critical control points’.

The selection of appropriate locations for environmental monitoring is a key aspect of any robust environmental monitoring program. If the locations are not in meaningful locations, the strength of the program is undermined. By ‘meaningful locations’ this means locations that reveal, through the data collected, an indication of potential product contamination. Contamination can arise through airborne deposition or by direct transfer, which signals the need for selecting locations that indicate this likelihood and the use of appropriate sampling methods to detect both airborne risks and surface risks (either fixed surfaces, on equipment, or on people). The HACCP approach provides a useful framework for such an assessment.

Tim Sandle’s follow up paper to his first HACCP article has been published. The reference is:

Sandle, T. (2019) Use of Hazard Analysis and Critical Control Points (HACCP) – Part 2: Determining Environmental Monitoring Locations, Journal of GxP Compliance, 23 92): 1-10

For further details, please contact Tim Sandle

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Revision of the Drinking Water Directive

On 1 February 2018, the European Commission published a proposal for a recast of the Directive on the quality of water intended for human consumption (the Drinking Water Directive). The proposal responds to the European Citizens’ Initiative, Right2Water, and builds on a fitness check which concluded that the 20-year old directive is fit for purpose, but needs updating. The main elements of the proposal consist of updating the water quality standards, introducing a risk-based approach to the monitoring of water, improving and streamlining the information provided to consumers, harmonising the standards for products in contact with drinking water, and imposing obligations to improve access to water.

In the European Parliament, the Committee on Environment, Public Health and Food Safety (ENVI) adopted its report on 10 September 2018. A plenary vote on the amendments, and on opening interinstitutional negotiations, took place on 23 October 2018. Although the Council reached a general approach on 5 March 2019, the Parliament concluded its first reading in plenary on 28 March 2019. Trilogue negotiations in view of reaching an early-second reading agreement could thus begin in the new parliamentary term.

For details, see: http://www.europarl.europa.eu/thinktank/en/document.html?reference=EPRS_BRI(2018)625179

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Electricity-conducting bacteria yield secret to tiny batteries

Credit: Edward H. Egelman

A new discovery about how strange bacteria that live in soil and sediment can conduct electricity. The bacteria do so, the researchers determined, through a seamless biological structure never before seen in nature -- a structure scientists can co-opt to miniaturize electronics, create powerful-yet-tiny  batteries, build pacemakers without wires and develop a host of other medical advances.
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Microbiologists had thought that Geobacter sulfurreducens conducted electricity through common, hair-like appendages called pili. Instead, a researcher at the University of Virginia School of Medicine and his collaborators have determined that the bacteria transmit electricity through immaculately ordered fibers made of an entirely different protein. These proteins surround a core of metal-containing molecules, much like an electric cord contains metal wires. This "nanowire," however, is 100,000 times smaller than the width of a human hair.

READ MORE: Fabric bacteria powered battery invented

This tiny-but-tidy structure, the researchers believe, could be tremendously useful for everything from harnessing the power of bioenergy to cleaning up pollution to creating biological sensors. It could actually serve as the bridge between electronics and living cells.

Geobacter bacteria play important roles in the soil, including facilitating mineral turnover and even cleaning up radioactive waste. They survive in environments without oxygen, and they use nanowires to rid themselves of excess electrons in what can be considered their equivalent to breathing. These nanowires have fascinated scientists, but it is only now that researchers at UVA, Yale and the University of California, Irvine, have been able to determine how G. sulfurreducens uses these organic wires to transmit electricity.

Journal Reference

Fengbin Wang, Yangqi Gu, J. Patrick O’Brien, Sophia M. Yi, Sibel Ebru Yalcin, Vishok Srikanth, Cong Shen, Dennis Vu, Nicole L. Ing, Allon I. Hochbaum, Edward H. Egelman, Nikhil S. Malvankar. Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers. Cell, 2019; 177 (2): 361 DOI: 10.1016/j.cell.2019.03.029

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

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.

See:

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

Kamari and Kinect Partner to Fund HIV Testing Programs in Africa

Kamari, a project building an ecosystem of mobile gaming and lotteries with existing licenses in over seven African countries, has partnered with Kinect, a blockchain-based health technology platform focused on advancing the United Nations Sustainable Development Goals (SDGs), to fund and promote new HIV testing programs in countries across the African continent.

Kinect and Kamari, both focused on development across Africa, have formed an official partnership with the intention of promoting health initiatives and increase the detection of infectious diseases, aimed to support the United Nations SDG #3: Good Health and Well Being. The two projects have proposed the establishment of a special, multi-country lottery to encourage untested males between the ages of 18 to 34 years to undergo an HIV test at participating clinics with the goal of promoting education and treatment for infected individuals.

"HIV infection is a serious issue in many African countries and the ongoing spread of the disease by untested individuals, has been incredibly difficult to address,” said Toby Carroll, Kinect Chairman. “We believe that this partnership with Kamari and the proposed lottery to incentivise those untested individuals to take their first test, will lead to increased education, treatment and hopefully reduce the spread of the disease.”

Kinect is a health focused technology company, which works in close alignment with Ministries of Health and large global charities across Africa, to improve the wellbeing of the African people. Kinect has also been operating in Mumbai, India for the last four months with its blockchain enabled electronic health record and patient incentivisation system working seamlessly to improve patient treatment programs.

Kamari is a new multi-country initiative with existing gaming, online casino, and lottery licenses across multiple African countries with the goal of building a united mobile gaming and lottery ecosystem for over one billion people. By integrating existing infrastructure and licensing with a standardized currency for gaming and payments, Kamari will instantly offer a better experience to hundreds of millions of people across Africa.

"Africa will be a multi-currency universe creating benefit and liquidity to all from the unbanked to the commercially astute,” said Dr. Christopher Cleverly, CEO of Kamari. “Kinect has a model that will thrive on an interoperable platform of shared values created through collaboration. This will be a game changer for medical data and application on the continent. "

Kinect recently formed a partnership with ZEU Crypto networks and its Canadian Stock Exchange listed company, St Georges Eco-Mining to develop its private permissioned blockchain that can integrate with major platforms including Cardano and EOS. ZEU Crypto Networks are investing US$2.45m into Kinect to enable development and expansion in existing operations.

Since 2016, Kinect has raised US$5.75m to bring the platform and business development to its current state and ensure successful expansion across Africa and other emerging economies with the greatest need.


Kinect and Kamari have entered into a US$2.5m coin swap to fund the initiative.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

New 3D microscope visualises fast biological processes better than ever

IMAGE: Tobias Wüstenfeld

Researchers from the European Molecular Biology Laboratory (EMBL) in Heidelberg have combined their expertise to develop a new type of microscope. The revolutionary new light-field microscopy system makes it possible to study fast biological processes, creating up to 200 3D images per second. Initial tests have already delivered new insights into the movement of blood cells in a heart.

“Many important biological processes occur in three dimensions and on millisecond timescales,” says Lars Hufnagel on the rationale for developing the new microscope. Capturing these fast processes is a big challenge in biology. And showing them not only in 2D but in 3D is – next to the needed high resolution – the second main aspect of modern microscopy.

The new light-field microscopy system developed by EMBL group leaders Lars Hufnagel, Robert Prevedel and their teams overcomes both hurdles at once. “Our new method allows us to study processes both in 3D and on timescales of 200 images per second,” says Robert Prevedel. Lars Hufnagel adds: “On top of that, it delivers up to ten times better, namely truly isotropic, resolution than classic light field microscopy.”

Previously developed microscopes, mostly based on light-sheet approaches, have also attempted to image fast biological processes but have only achieved much slower speeds than the new technique. As such, they were too slow to see dynamic processes within hearts and neuronal cells.

First feasibility study on fish heart

To demonstrate the capabilities of the new technique, the team studied the beating heart and blood flow in medaka – also known as Japanese rice fish – in real time. The medaka was used as it is a well understood model organism. In addition, blood cells move fast – up to one millimetre per second – which was a challenge for any existing microscope.

The images delivered by this test showed for the first time how individual blood cells move through the two heart chambers. “This opens up completely new possibilities,” says co-author Joachim Wittbrodtfrom the Centre for Organismal Studies at Heidelberg University. “In showing how genetic backgrounds or mutations have an effect on the dynamics of heartbeats, the new technology can be used to research heart defects.”

Interdisciplinary research and development

Constructing the new microscope was an interdisciplinary effort. The researchers within the two EMBL groups have backgrounds in various scientific fields: the multidisciplinary team comprised physicists, engineers, computer scientists and, of course, biologists.

“This new microscope demonstrates that EMBL is not only at the forefront of molecular biology research but also an important place to research and develop new technologies needed within the field,” says Hufnagel.

Next step: neurons

The study on the medaka heart was only the first test for the new microscope. Robert Prevedel is looking forward to using the microscope to study the activity and dynamics of neuronal cell populations in these animals. “Future camera developments can further increase the imaging speed. This would make our new microscope technique an attractive tool to study the dynamics within small neuronal networks on millisecond time scales in 3D,” concludes Prevedel.

Source article: Nils Wagner, Nils Norlin, Jakob Gierten, Gustavo de Medeiros, Bálint Balázs, Joachim Wittbrodt, Lars Hufnagel and Robert Prevedel: 'Instantaneous isotropic volumetric imaging of fast biological processes'. Nature Methods, published online on 29 April 2019. DOI: 10.1038/s41592-019-0393-z

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

New technology to improve tissue imaging

The issue: Even as digital pathology makes rapid advances worldwide -- with more physicians analyzing tissue images on "smart" computers to diagnose patients -- there are no reliable standards for the preparation and digitization of the tissue slides themselves.

That means poor quality slides get mixed in with clear and accurate slides, potentially confusing or misleading a computer program trying to learn what a cancerous cell looks like, for example.

Researchers from Case Western Reserve University are trying to change that.

Bioengineering researcher Anant Madabhushi and Andrew Janowczyk, a senior research fellow in Madabhushi's Center for Computational Imaging and Personal Diagnostics, have developed a program that they say will ensure the quality of digital images being used for diagnostic and research purposes.

The new tool incorporates a series of measurements and classifiers to help users flag corrupted images and help retain those that will aid technicians and physicians in their diagnoses.

The application is "open source" -- or free for anyone to use, modify and extend. It can be accessed through an online repository. It was developed by Janowczyk about 18 months ago after discovering what he believed to be a surprising number of poor-quality slides from the well-known Cancer Genome Atlas, home to more than 30,000 tissue slides of cancer samples.

See: Andrew Janowczyk, Ren Zuo, Hannah Gilmore, Michael Feldman, Anant Madabhushi. HistoQC: An Open-Source Quality Control Tool for Digital Pathology Slides. JCO Clinical Cancer Informatics, 2019; (3): 1 DOI: 10.1200/CCI.18.00157

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

New research shows weakness with antimicrobial paints

Antimicrobial paints offer the promise of extra protection against bacteria. But Northwestern University researchers caution that these paints might be doing more harm than good.

In a new study, the researchers tested bacteria commonly found inside homes on samples of drywall coated with antimicrobial, synthetic latex paints. Within 24 hours, all bacteria died except for Bacillus timonensis, a spore-forming bacterium. Most bacilli are commonly inhabit soil, but many are found in indoor environments.

"If you attack bacteria with antimicrobial chemicals, then they will mount a defense," said Northwestern's Erica Hartmann, who led the study. "Bacillus is typically innocuous, but by attacking it, you might prompt it to develop more antibiotic resistance."

Bacteria thrive in warm, moist environments, so most die on indoor surfaces, which are dry and cold, anyway. This makes Hartmann question the need to use antimicrobial paints, which may only be causing bacteria to become stronger.

Spore-forming bacteria, such as Bacillus, protect themselves by falling dormant for a period of time. While dormant, they are highly resistant to even the harshest conditions. After those conditions improve, they reactivate.

"When it's in spore form, you can hit it with everything you've got, and it's still going to survive," said Hartmann, assistant professor of civil and environmental engineering in Northwestern's McCormick School of Engineering. "We should be judicious in our use of antimicrobial products to make sure that we're not exposing the more harmless bacteria to something that could make them harmful."

One problem with antimicrobial products -- such as these paints -- is that they are not tested against more common bacteria. Manufacturers test how well more pathogenic bacteria, such as E. coli or Staphylococcus, survive but largely ignore the bacteria that people (and the products they use) would more plausibly encounter.

"E. coli is like the 'lab rat' of the microbial world," Hartmann said. "It is way less abundant in the environment than people think. We wanted to see how the authentic indoor bacteria would respond to antimicrobial surfaces because they don't behave the same way as E. coli."

See:

Jinglin Hu, Sarah B. Maamar, Adam J. Glawe, Neil Gottel, Jack A. Gilbert, Erica M. Hartmann. Impacts of Indoor Surface Finishes on Bacterial Viability. Indoor Air, 2019; DOI: 10.1111/ina.12558

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Cleanroom Particle Counting: Assessing Data for Trends and Patterns

Given the requirement for particle count trending it is surprising that there is little published on the subject and a paucity of examples for the reader to assess. While there are many different approaches that can be taken, improvements to quality reporting are driven through example. To partly address the shortfall of literature on particle count trending, this paper considers some different ways to assess particle data, in terms of routine assessments and where a statistical comparison of data is required. With the latter, this is less straightforward given that particle count data does not follow normal distribution.

In relation to this, Tim Sandle has written a new paper. The reference is:

Sandle, T. (2019) Cleanroom Particle Counting: Assessing Data for Trends and Patterns, Journal of GxP Compliance, 23 92): 1-10

For further details, please contact Tim Sandle

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Why certain strains of bacteria are associated with diabetic wounds that do not heal

About 10 percent of U.S. citizens have been diagnosed with diabetes, and one quarter of these patients will develop a wound that does not heal. In the worst case outcome, which occurs in up to 25 percent of these wound-developing patients, the wounds will require an amputation. Many patients who develop these ulcers may not notice the initial signs, since the high blood glucose of diabetes can lead to a lack of feeling and deformation of the feet. As a result, patients with diabetes commonly develop foot ulcers that may go unnoticed over time.

Current treatments are insufficient, meaning patients can live with these wounds for months or even years without healing. The mortality rate associated with diabetic foot ulcers is equivalent to that of breast cancer and prostate cancer combined -- higher than 70 percent when they lead to amputation.

"While wounds don't receive the attention of other diseases, they're incredibly common, and our study increases our understanding of how microbes impair or promote healing," said the study's senior author Elizabeth Grice, PhD, an associate professor of Dermatology. The lead author, Lindsay Kalan, PhD, now an assistant professor of Medical Microbiology and Immunology at the University of Wisconsin School of Medicine and Public Health, began this work as a post-doctoral researcher in Grice's lab.

Previous studies have used lower resolution techniques to catalogue the microbes that reside in chronic wounds. This study built on that research by using higher resolution DNA sequencing to identify specific species and subspecies and how they are related to patient outcomes. Researchers collected samples from 46 patient ulcers every two weeks for six months, or until the wound healed or was amputated.

S. aureus, a common and difficult-to-treat pathogen, was found in the majority of wounds, but researchers note the presence of the bacteria itself did not predict whether or not a wound would heal. However, the high resolution DNA sequencing showed certain strains of S. aureus were only in the wounds that did not heal over the course of the study. Further testing revealed that the "non-healing" strain was better equipped to cause tissue damage and evade antibiotic treatments. Researchers further validated their findings in mice.

They also noted that another common microbe found in diabetic wounds, Alcaligenes faecalis, was associated with quicker healing.

"It is possible there are bacteria that actually benefit the wound, and we can use what we learned in this study to develop new treatment strategies for non-healing wounds," Grice said. "We hope this research will eventually help identify patients at risk for bad outcomes and lead to treatment innovations that these patients desperately need."

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

On-chip drug screening for identifying antibiotic interactions in eight hours

A KAIST research team developed a microfluidic-based drug screening chip that identifies synergistic interactions between two antibiotics in eight hours. This chip can be a cell-based drug screening platform for exploring critical pharmacological patterns of antibiotic interactions, along with potential applications in screening other cell-type agents and guidance for clinical therapies.

Antibiotic susceptibility testing, which determines types and doses of antibiotics that can effectively inhibit bacterial growth, has become more critical in recent years with the emergence of antibiotic-resistant pathogenic bacteria strains.

To overcome the antibiotic-resistant bacteria, combinatory therapy using two or more kinds of antibiotics has been gaining considerable attention. However, the major problem is that this therapy is not always effective; occasionally, unfavorable antibiotic pairs may worsen results, leading to suppressed antimicrobial effects. Therefore, combinatory testing is a crucial preliminary process to find suitable antibiotic pairs and their concentration range against unknown pathogens, but the conventional testing methods are inconvenient for concentration dilution and sample preparation, and they take more than 24 hours to produce the results.

To reduce time and enhance the efficiency of combinatory testing, Professor Jessie Sungyun Jeon from the Department of Mechanical Engineering, in collaboration with Professor Hyun Jung Chung from the Department of Biological Sciences, developed a high-throughput drug screening chip that generates 121 pairwise concentrations between two antibiotics.

The team utilized a microfluidic chip with a sample volume of a few tens of microliters. This chip enabled 121 pairwise concentrations of two antibiotics to be automatically formed in only 35 minutes.

They loaded a mixture of bacterial samples and agarose into the microchannel and injected reagents with or without antibiotics into the surrounding microchannel. The diffusion of antibiotic molecules from the channel with antibiotics to the one without antibiotics resulted in the formation of two orthogonal concentration gradients of the two antibiotics on the bacteria-trapping agarose gel.

The team observed the inhibition of bacterial growth by the antibiotic orthogonal gradients over six hours with a microscope, and confirmed different patterns of antibiotic pairs, classifying the interaction types into either synergy or antagonism.

Professor Jeon said, "The feasibility of microfluidic-based drug screening chips is promising, and we expect our microfluidic chip to be commercialized and utilized in near future."

See: Seunggyu Kim, Fahim Masum, Ju-Kang Kim, Hyun Jung Chung, Jessie S. Jeon. On-chip phenotypic investigation of combinatory antibiotic effects by generating orthogonal concentration gradients. Lab on a Chip, 2019; 19 (6): 959 DOI: 10.1039/c8lc01406j

 Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

How E. coli clones take over human gut

Researchers from University of Birmingham investigated how and why a clone of E. coli called ST131 -- dubbed a 'superbug' because it is resistant to multiple drugs -- has become the major cause of drug resistant E. coli infections, but not so dominant that it has wiped out other clones that do not have multi-drug resistance.

Escherichia coli (E. coli) is a type of bacteria common in human and animal intestines, and forms part of the normal gut flora -- the bacteria that exist in the bowel. There are a number of different types of E. coli and, while the majority are harmless, some can cause serious food poisoning or infections including in the urinary tract or bloodstream.

The number of cases of E. coli have risen by 27% from 32,309 in 2012-13 to 41,060 in 2017-18. The rise has been linked to an increase in antibiotic resistant infections caused by so-called 'superbugs'.

The most globally dominant clone of E. coli that is resistant to multiple drugs is called ST131. Earlier research has shown that while ST131 emerged and rapidly spread in the late 1990s, it caused no more than 20% of clinical cases of E. coli once it had emerged on the scene. This is because of a type of evolutionary selection called negative frequency dependency selection (NFDS).

It is now known that while there are significantly dominant drug resistant clones of E. coli such as ST131 and other new ones are emerging all the time, it seems highly unlikely that any of them are ever going to become a completely dominant clone because this process called NFDS controls the balance across the whole E. coli population.

As part of the research the scientists also analysed almost 1,000 genome sequences of strains within ST131 to see if they could find any genetic patterns that may explain how this process happens. It was found that in the ST131 clone there was a lot of variation in the genes that are involved in allowing the bacteria to colonize in the human gut when compared to those in non-drug resistant bacteria that are very closely related to ST131.

The implications are that if a person is going to get a bloodstream or urinary infection from E. coli it usually comes after it has colonized in the gut, therefore we now know that genetically something has happened to this superbug which allows it to colonise the gut far more competitively than other E. coli.

See: Alan McNally, Teemu Kallonen, Christopher Connor, Khalil Abudahab, David M. Aanensen, Carolyne Horner, Sharon J. Peacock, Julian Parkhill, Nicholas J. Croucher, Jukka Corander. Diversification of Colonization Factors in a Multidrug-Resistant Escherichia coli Lineage Evolving under Negative Frequency-Dependent Selection. mBio, 2019; 10 (2) DOI: 10.1128/mBio.00644-19

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

The mystery of antimicrobial frog secretions

The Bombina variegata frog, also known as Yellow Bellied Toad, inhabits the forests, grasslands, wetland, and aquatic habitats across Central Europe. Their skin secretions contain antimicrobial agents -- called Bominin H2 and H4 -- that play a key role in protecting the species against infection.

Bombinin H2 and H4 are antimicrobial peptides (AMPs) -- or host defense peptides -- that play an important function in immune response. They have attracted attention for their ability to inhibit Leishmaniasis -- a highly infectious and potentially fatal tropical disease that has affected an estimated 20 million people worldwide, with 1.3 million new cases and 20,000 to 30,000 deaths reported each year.

H4 is an isomer of H2 -- they share the same formula but the atoms in the molecule are arranged differently -- with H4 having a naturally occuring D-amino acid at the end of the molecular chain. In terms of its antimicrobial properties, H4 is more potent than H2, but until now, the reason has remained an unsolved biological mystery.

In order to gain a better understanding of the molecular mechanism that drives the antimicrobial activity of Bombinin H2 and H4 peptides and what makes H4 more effective than H2 in this regard, the authors conducted electrophysiological experiments on a lipid bilayer membrane that replicated the lipid membrane surrounding cells or microorganisms The results were then analyzed using existing AMP models to determine how efficient these antimicrobial peptides are at disrupting the cell membrane of microbes.

The team found that H2 and H4 peptides inhibit microbial activity by making holes in the cell membrane of microorganisms, causing ions to leak out of the cell, which ultimately kills them. The efficiency of this anti-microbial activity is affected by ion permeability (how fast ions leak out of the cell), the speed of pore formation, and the size of the pores formed.

The results indicate that the peptides' ability to transform into another molecule with the same atomic composition but with atoms arranged differently facilitates faster pore formation. While H2 forms larger pores than H4, H4 forms pores more rapidly. A mixture of H2/H4, meanwhile, forms medium-sized pores at a slower rate than H4, but the presence of the D-amino acid enhances the binding affinity to the lipid membrane, thereby improving its disruptive abilities.

In terms of what this means, think of it like a field of different sized pit traps; larger traps take longer to dig, but can trap more animals than a smaller pit. On the other hand, one can dig many smaller pits in the same time it takes to dig just a few large ones. Digging medium sized pit traps and adding bait or a lure that would attract animals to the pit, would be the most effective approach of all.

Unravelling the molecular mechanism that facilitates antimicrobial activity of these peptides can help us better understand how the defense system of the frog has evolved, and how this can be used to fight microbial infections of medical importance.

The ultimate goal is to use this mechanism to develop better antimicrobial agents, especially antimicrobial agents that are effective against antibiotic-resistant bacteria.

Reference:

Yusuke Sekiya, Keisuke Shimizu, Yuki Kitahashi, Akifumi Ohyama, Izuru Kawamura, Ryuji Kawano. Electrophysiological Analysis of Membrane Disruption by Bombinin and Its Isomer Using the Lipid Bilayer System. ACS Applied Bio Materials, 2019; 2 (4): 1542 DOI: 10.1021/acsabm.8b00835

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology