How do immune responses control microbial gene expression?

Commensal microbiota provide protection from bacterial infections. However, when the body's control mechanisms are disrupted, microbial gene expression may be altered, ultimately changing bacterial behavior and localization.

High levels of bacterial flagellar protein are associated with gut mucosal barrier breakdown. Using mice deficient in Toll-like receptor 5 (TLR5), researchers found that innate and adaptive immunity interact to modulate the microbiome’s production of flagella, and the flagella of gut commensal bacteria stimulate TLR5 (Cullender, T.C. et al. (2013) Cell Host Microbe 14, 571).

Posted by Tim Sandle

World Sepsis Day

September 13 is World Sepsis Day

World Sepsis Day is an international day of action and awareness-raising, supported by organisations around the world. It is coordinated internationally by the Global Sepsis Alliance, a collaborative group of non-profit organisations.

Sepsis is a life threatening condition that arises when the body’s response to an infection injures its own tissues and organs. It can be caused by something as simple as a cut or insect bite, or an infection like pneumonia. It is also a risk following surgery, or for women who have just given birth. It causes a range of diseases and deaths worldwide.

For this reason, World Sepsis Day is promoted on September 13 each year in order to raise awareness. It is is an initiative of the Global Sepsis Alliance and its founding members, all of whom are non-profit organizations.

According to the campaign organization: "Every few seconds someone dies of sepsis. Prevent it. Spot it. Treat it – beat it. For that we campaign to reduce sepsis cases by 20% by 2020".

To give an indication of the extend of the problem, in the U.S. Dr. Liu, from the Kaiser Permanente Northern California, has determined that with U.S. hospitals:

a) Sepsis contributed to 1 in every 2 to 3 deaths, and most of these patients had sepsis at admission.

b) Patients with sepsis, normal blood pressure, and measured lactate levels of less than 4 mmol/L (n = 15 095) comprised 55.9% (95%CI, 53.6%-58.1%) of sepsis deaths. Surprisingly, patients with initially less severe sepsis made up the majority of sepsis deaths. The majority of individuals who died with sepsis presented to the hospital with the illness. This contradicts the belief of policymakers and some healthcare authorities that sepsis results primarily from hospital-acquired infections.

One way of dealing with problems of sepsis is in developing appropriate diagnostic tools. According to Pharmaceutical Microbiology: "Diagnostics play a significant role in identifying sepsis in a patient. Surviving Sepsis Campaign guidelines recommend obtaining blood cultures - the gold standard - for recovering the pathogen causing infection before antimicrobial therapy is initiated. Accurate and fast results help physicians to determine the appropriate antimicrobial therapy to cure the patient."

Whilst diagnosis is important, a lack of international agreement on appropriate guidelines is arguably hampering efforts to address sepsis on a global scale. Professor Sandra Peake, of The Queen Elizabeth Hospital in England, says that guidelines continue to be debated among clinicians and researchers: "In 2004, international guidelines were introduced for the resuscitation of patients with sepsis, but even now we see variation in practices within and between countries on treatment approaches.

"The debate will continue internationally for some time yet, but in reality the mainstay of treatment for patients with sepsis is antibiotics, intravenous fluids, drugs to support the heart and maintain the blood pressure, and surgery, if needed, on an affected area. Whether or not there is one specific or uniform resuscitation approach remains to be seen."

Therefore, it remains an issue that treatment practices for patients hospitalized with sepsis will continue to vary because of individual differences between hospitals and countries.

You can sign up to support World Sepsis Day here.

Posted by Tim Sandle

First crystal structure of the C. difficile surface protein

Clostridium difficile is a major problem as an aetiological agent for antibiotic-associated diarrhea. The mechanism by which the bacterium colonizes the gut during infection is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. This study provides some insights that may help in developing a new type of drug to treat the infection.

Researchers reported the first crystal structure of the C. difficile surface protein Cwp84. This cysteine protease enzyme is found on the surface of the bacterium and assists with production of the microbe's surface-layer, which is likely to play an essential step in the colonisation of the gut. The enzyme cleaves a single polypeptide (surface-layer protein A; SlpA) into low- and high-molecular-weight subunits.

Scientists have identified three critical regions in a mutant of the enzyme that could represent novel targets for drugs to attack C. difficile by blocking maturation of its surface layer during colonisation.

For further details, see:

William J. Bradshaw, Jonathan M. Kirby, Nethaji Thiyagarajan, Christopher J. Chambers, Abigail H. Davies, April K. Roberts, Clifford C. Shone, K. Ravi Acharya. The structure of the cysteine protease and lectin-like domains of Cwp84, a surface layer-associated protein fromClostridium difficile. Acta Crystallographica Section D Biological Crystallography, 2014; 70 (7): 1983 DOI: 10.1107/S1399004714009997

Posted by Tim Sandle

'Revolution' biomotor discovered in many bacteria

Scientists at the University of Kentucky, led by nano-biotechnologist Peixuan Guo, have made an important discovery into the operation of biomotors, the molecular machines used by viruses and bacteria in the packaging of DNA. Biomotors function similarly to mechanical motors but on a nano-scale.

The researchers have reported on the discovery of a new, third class of biomotor, unique in that it uses a "revolution without rotation" mechanism. Rotation is the turning of an object around its own axle, as the Earth does every 24 hours. Revolution is the turning of an object around a second object, as the Earth does around the sun.

The reference for the research is:

Cell & Bioscience 2014, June, 4:30: www.cellandbioscience.com/content/4/1/30

 Posted by Tim Sandle

Candida glabrata

A group of researchers at the Max F. Perutz Laboratories has created one of the three world's largest gene libraries for the Candida glabrata yeast, which is harmful to humans. Molecular analysis of the Candida glabrata fungus mutations led to the discovery of 28 new genes that are partly responsible for the yeast's tolerance of common drugs.

The working group led by Karl Kuchler at the Max F. Perutz Laboratories (MFPL) - a research and training centre run jointly between the University of Vienna and the Medical University of Vienna at the Vienna Biocenter Campus - coordinated an international study cooperation aimed at researching new tolerance and virulence genes in Candida glabrata. During this process, genetic methods were used to generate one of the three world's largest libraries of "knock-out fungi". More than 600 fungus mutations were created from which a single gene was specifically removed.

The findings represent a new step in the discovery and characterisation of Candida glabrata resistance genes, laying the foundations for the development of new anti-fungal medications.

For reference see: "Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes" – Tobias Schwarzmüller, et al

PLoS Pathogens 10: e1004211. DOI: 10.1371/journal.ppat.1004211

Posted by Tim Sandle

Challenges of GMP and GXP in India

Pharmig are hosting the following free-to-view presentation 'Challenges of GMP and GXP in India'.

GMP and regulatory issues affecting pharmaceuticals and healthcare in India. The presentation covers Good Manufacturing Practices, and includes national and global (e.g. FDA) requirements.

The presentation has been prepared by Nasir  Ansari  of  Piramal  Enterprises  Limited  .

Challenges of GMP and GXP in India 

Posted by Tim Sandle

A Tale of Two Species - Blue Blood

Horseshoe crabs' blue blood, which contains copper, not iron, is prized by the biomedical community for its ability to detect bacteria in human medicines. It's just one of the amazing qualities of the 350-million-year-old evolutionary marvel detailed in "Crash: A Tale of Two Species."

"Crash: A Tale of Two Species" aired on PBS Sunday, March 20, 2011 at 8pm as part of the 28th season of the Peabody and Emmy award-winning series produced by Thirteen in association with WNET.ORG for PBS. Major support provided by Canon U.S.A. Inc.

Here is a fascinating excerpt:

Horseshoe crab amebocytes coagulate around as little as one part in a trillion of bacterial contamination. Even better, the reaction takes 45 minutes, not two days as with mammalian equivalents. Coagulan, the chemical that makes this possible, is used for testing medical equipment and vaccines prior to use, without which many more people would die from infections. Unfortunately, coagulan synthesis is in its infancy so a quarter of a million crabs are harvested each year for their blood, as shown in this video:

Posted by Tim Sandle

Assessment of Culture Media in Pharmaceutical Microbiology

Culture media is of fundamental importance for most microbiological tests: to obtain pure cultures, to grow and count microbial cells, and to cultivate and select microorganisms. Without high-quality media, the possibility of achieving accurate, reproducible, and repeatable microbiological test results is reduced. A microbiological culture medium is a substance that encourages the growth, support, and survival of microorganisms. 


Culture media contains nutrients, growth promoting factors, energy sources, buffer salts, minerals, metals, and gelling agents (for solid media). Culture media has been used by microbiologists since the nineteenth century. Even with the increased use of rapid methods the majority of techniques found in the pharmaceutical quality control laboratory require growth media. For the assessment of culture media, no one definitive standard exists. In light of this, this article presents some considerations for designing the testing regime and for the selection and control of microorganisms.

This is the basis of a new article by Tim Sandle for American Pharmaceutical Review, addressing issues relating to microbiological culture media.

To view the article see APR.



Posted by Tim Sandle

Cleanroom Management in Pharmaceuticals and Healthcare

Cleanroom Management in Pharmaceuticals and Healthcare

Edited by Tim Sandle and Madhu Raju Saghee.

"Everything you need to know about the operation and management of cleanrooms."

In 26 Chapters and over 600 pages this book provides a unique tool to help you achieve regulatory compliance. It first creates a foundation in history and established practice and then helps you understand how state of the art technology and engineering solutions can deliver the best practice and so provide reliable systems performance.

An essential read for practitioners in cleanroom technology: engineers, microbiologists, quality assurance, healthcare practitioners and pharmaceutical professionals.

For further details see: cleanrooms

Contents:

1. Introduction by Tim Sandle and Madhu Raju Saghee
2. History and development of cleanrooms by Tim Sandle
3. Cleanroom standards and GMP requirements by Mark Hallworth
4. Design and construction of pharmaceutical cleanrooms by Alexander Fedotov
5. Air handling systems for the protection of pharmaceutical manufacturing processes by Hans Schicht
6. Cleanrooms in hospitals by Alexander Fedotov
7. Commissioning and qualification of cleanrooms by Kevin Beauchamp and Miroslav Tonovski
8. Cleanroom certification and ongoing compliance by TimSandle and Madhu Raju Saghee
9. Fundamentals of pharmaceutical isolators by Brian Midcalf, John Neiger and Tim Sandle
10. The choice of isolators: A risk-based decision by Didier Meyer
11. Validation concepts in pharmaceutical aseptic application isolators by Rajesh Thempadiyil
12. Risk-based product and occupational exposure control in multi-product facilities by Julian Wilkins
13. Future of aseptic processing by James L Drinkwater
14. Aseptic process simulations/media fills by Marco Budini
15. Microbial risk management during aseptic manufacture by Tim Eaton
16. Airflow studies and airflow mapping by Tim Sandle,Marco Budini and T Rajesh
17. Cleanroom contamination sources and control measures by Eric Strauss
18. Particle counters and particle counting by Tony Harrison
19. Environmental monitoring in cleanrooms by Tim Sandle and Madhu Raju Saghee
20. Cleaning and disinfection practices by Tim Sandle and Madhu Raju Saghee
21. Cleanroom clothing byMatts Ramstorp
22. Quality assurance in hospital pharmacies by Richard Bateman
23. Building Management Systems for cleanroom process parameters monitoring and control by Sunil Chand Singhai and Rajesh Thempadiyil
24. Energy management and sustainable cleanrooms by Nigel Lenegan and Ulla Thomsen
25. Auditing cleanroom operations by Tim Sandle and MadhuRaju Saghee
26. Developments in cleanroom technology by Tim Sandle and Madhu Raju Saghee

Published by Euromed

Posted by Tim Sandle

Are bacteria growing less susceptible to common antiseptics?

Chlorhexidine gluconate (CHG) has been increasingly used in hospitals in light of recent evidence that daily antiseptic baths for patients in intensive care units (ICUs) may prevent infections and stop the spread of healthcare-associated infections. However, the impact of this expanded use on the effectiveness of the disinfectant is not yet known.

In a recent study, investigators compared bacterial resistance between cultures from patients in eight ICUs receiving daily antiseptic washes to patients in 30 non-ICUs who did not bathe daily with CHG. Bacterial cultures obtained from patients with regular antiseptic baths showed reduced susceptibility to CHG when compared with those from patients who did not have antiseptic baths. Regardless of unit protocol, 69 percent of all bacteria showed reduced CHG susceptibility, a trend that requires vigilant monitoring.

The investigators caution that the clinical implications of their findings remain unclear. For example, antibiotic susceptibility tests are commonly used to determine whether patients will respond to antibiotic treatment. A similar correlation between antiseptic susceptibility and response to an antiseptic are not as well defined. Identifying particular bacteria and settings in which these bacteria will not respond to antiseptic agents used in hospitals is an important next step.

The reference is:

Nuntra Suwantarat, Karen C. Carroll, Tsigereda Tekle, Tracy Ross, Lisa L. Maragakis, Sara E. Cosgrove, Aaron M. Milstone. High Prevalence of Reduced Chlorhexidine Susceptibility in Organisms Causing Central Line–Associated Bloodstream Infections. Infection Control and Hospital Epidemiology, 2014; 35 (9): 1183 DOI: 10.1086/677628

Posted by Tim Sandle

Tests to diagnose invasive aspergillosis

The fungal infection invasive aspergillosis (IA) can be life threatening, especially in patients whose immune systems are weakened by chemotherapy or immunosuppressive drugs. IA is difficult to diagnose and it is caused by the fungus Aspergillus fumigatus.

A recent study compared three diagnostic tests and found that the combination of nucleic acid sequence-based amplification (NASBA) and real-time quantitative PCR (qPCR) detects aspergillosis with a high degree of accuracy.

For this, researchers evaluated the diagnostic performance of two nucleic acid amplification assays (qPCR and NASBA) and one antigen detection method (galactomannan enzyme-linked immunosorbent assay [GM-ELISA]) using blood samples collected from 80 patients at high risk of IA.

Data analysis showed that the combination of NASBA and qPCR led to 100% specificity and 100% positive predictive value (the probability that subjects truly have the infection).

The reference is:

Lipeng Wang, Yunyan He, Yun Xia, Xiaoyan Su, Huijuan Wang, Shumei Liang. Retrospective Comparison of Nucleic Acid Sequence-Based Amplification, Real-Time PCR, and Galactomannan Test for Diagnosis of Invasive Aspergillosis. The Journal of Molecular Diagnostics, 2014; DOI: 10.1016/j.jmoldx.2014.05.001

Posted by Tim Sandle

DNA reactive (mutagenic) impurities in pharmaceuticals

Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk (M7)

This ICH guideline emphasizes considerations of both safety and quality risk management in establishing levels of mutagenic impurities that are expected to pose negligible carcinogenic risk. It outlines recommendations for assessment and control of mutagenic impurities that reside or are reasonably expected to reside in final drug substance or product, taking into consideration the intended conditions of human use. Implementation of M7 is encouraged after publication; however, because of the complexity of the guideline, application of M7 is not expected (with certain exceptions) prior to 18 months after ICH publication.

For details, see ICH

Posted by Tim Sandle

Pharmacy Compounding of Human Drug Products

Pharmacy Compounding of Human Drug Products Under Section 503A of the Federal Food, Drug, and Cosmetic Act

This guidance announces FDA’s intention with regard to enforcement of section 503A FD&C Act to regulate entities that compound drugs, now that section 503A has been amended by Congress to remove the advertising and solicitation provisions that were held unconstitutional by the U.S. Supreme Court in 2002. Several parts of section 503A require rulemaking and consultation with a Pharmacy Compounding Advisory Committee to implement. This guidance explains how the provisions will be applied pending those consultations and rulemaking. This guidance also describes some of the possible enforcement actions FDA can bring against individuals or firms that compound drugs in violation of the FD&C Act.

For details, see FDA

Posted by Tim Sandle

Keeping surfaces microbe free

In order to achieve satisfactory disinfection, a good quality disinfectant is required. An ideal disinfectant should have a high inactivating capacity for a wide range of viruses, such as HIV and hepatitis, as well as being effective against bacteria, including tuberculosis. It should be safe to use and suitable for frequent application. Disinfectants are typically supplied as pre-saturated wipes which may be alcohol-based or non-alcohol based. This article considers the key requirements for a surface disinfectant and examines the comparative advantages of alcohol and non-alcohol wipes.

This is the basis for an article examining good disinfection procedures for dental practices. The article has been written by Tim Sandle and it has been published in the magazine The Dentist.

The reference is:

Sandle, T. (2014) Keeping surfaces microbe free, The Dentist, June 2014, pp60-61

If you are interested in reading a copy, please contact Tim Sandle.

Posted by Tim Sandle

Approaching the Selection of Rapid Microbiological Methods

Rapid microbiological method technologies aim to provide more sensitive, accurate, precise, and reproducible test results when compared with conventional, growth-based methods. Rapid methods normally involve some form of automation, and the methods often capture data electronically. With several different technologies available on the marketplace, the microbiologist has a difficult, and sometimes expensive, choice to make in selecting the optimal method.

Tim Sandle has written a paper for the Journal of Validation Technology. The paper provides an overview of the topic.

This paper, whilst addressing some of the emerging technologies, is not so much about the different rapid microbiological methods that are available; it is more concerned with the considerations that need to be considered for their selection. As such, the paper provides some advice for the microbiologist to consider when drawing up a rationale for the selection of a rapid or alternative microbiological method.

The reference is:

Sandle, T. (2014) Approaching the Selection of Rapid Microbiological Methods, Journal of Validation Technology, Vol. 20, Issue 2, Jun 2014. Published on-line.

For further details see: IVT or contact Tim Sandle

Posted by Tim Sandle