Saturday, 14 December 2019

COPAN Advancing Artificial Intelligence (A.I.) and Digital Plate Reading for Microbiology


A new study titled “Automated Detection of Streptococcus pyogenes pharyngitis using COLOREX Strep A CHROMagar and WASPLab™ Artificial Intelligence Chromogenic Detection Module Software ” was recently published on the Journal of Clinical Microbiology (JCM).

The study is the first-in-its class peer-reviewed publication on PhenoMATRIX™ used with COLOREX Strep A culture media for the automatic detection of Group A Strep (GAS). Group A Strep (GAS) pharyngitis is an infection caused by Streptococcus pyogenes, responsible for 20% to 30% of sore throats in children between 5 and 15 years old. Pharyngeal swab samples were collected from pediatric patients with suspected pharyngitis at the Children’s Hospital Los Angeles (CHLA).

The samples were analyzed using COPAN’s Chromogenic Detection Module (CDM), which is part of the PhenoMATRIX™ suite of A.I. algorithms. The Chromogenic Detection Module is designed to accurately detect and differentiate Strep A on chromogenic agar allowing the laboratory to provide faster results than with traditional culture methods and manual reading. Any suspected positive Group A Strep growth detected by the algorithm was confirmed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry.

All specimens in this study were collected using COPAN ESwab™, which enabled specimens to be processed in parallel using a molecular assay. The study concluded that “PhenoMATRIX™ CDM software and COLOREX Strep A Agar can improve detection of GAS from throat specimens and compared favorable to molecular testing.” This breakthrough publication is the first of its kind using PhenoMATRIX™ with the novel COLOREX™ chromogenic media for the automatic detection of Group A Strep. It reflects the advancements in Artificial Intelligence technology occurring in Microbiology laboratories and the way they are impacting laboratory medicine favorably.

Microbiology is one of the most labor-intensive disciplines within the clinical laboratory field, and its role is of vital importance to overall healthcare. It is well established that laboratory professionals are asked to do more with fewer resources and to constantly demonstrate the value of laboratory medicine in clinical outcomes.

“As the demand for laboratory testing increases and reimbursement rates and qualified personnel dwindle at a rapid speed, COPAN is investing in developing comprehensive A.I. solutions to support and amplify the laboratories’ ingenuity and maximize the use of their resources,” stated Norman Sharples, CEO of COPAN Diagnostics, Inc. “Having worked in a clinical laboratory in the past, it is fascinating to see Microbiology at an interesting juxtaposition where traditional techniques, like planting and streaking on culture media, combine with incredible advances in Artificial Intelligence and digital plate reading. COPAN is staying at the forefront of this new exciting frontier for Microbiology” added Sharples.
Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Friday, 13 December 2019

Pricing Agreement Makes TB Prevention Affordable for High-Burden, Low-, Lower-Middle and Upper-Middle Income Countries



A new volume-based agreement announced today by Unitaid, the Global Fund and Sanofi will drastically lower the price of rifapentine (Priftin®), a critically important drug used to prevent tuberculosis (TB). The agreement with Sanofi brings Priftin® 150mg tablets down from €13.60/pack of 24 tablets (current GDF access price) to a net discounted ex-factory price of €4.62/pack of 24 tablets [1] (a 66% discount). The discounted price will be available to the public sectors of low-income countries, lower-middle income countries, and upper-middle income countries with a high burden of TB and TB/HIV.

The agreement comes at a time when Aurum Institute and its partners prepare to roll-out a short regimen for preventing TB—three months of rifapentine and isoniazid treatment (3HP)—to 12 high-burden TB countries. The agreement lowers a patient treatment course of Priftin® from $45 to $15. Priftin® is currently the only quality-assured rifapentine formulation available for short-course treatment of latent TB infection.

“In 2018, an estimated ten million people contracted tuberculosis, an unimaginable number,” said Gavin Churchyard, founder and CEO of the Aurum Institute. “We know how to prevent TB disease in some of the most vulnerable people, but innovations in prevention will only sit on the shelf if the price puts the solution out of reach. Now that we have a short-course TB preventive therapy that is affordable, it’s time for the world to come together to make sure that these people can live healthy and productive lives.”

TB can lie dormant for decades before it strikes; this is called “latent TB infection.” People with latent infection—almost a quarter of the globe—have no symptoms, are not contagious and most of them don’t know they are infected. If left untreated, latent infection can develop into active TB, the form of TB that makes people sick and is capable of being transmitted from one person to another.

Treatment of TB infection is referred to as TB preventive therapy (TPT) and is one of the most powerful ways to prevent TB. The goal of TPT is to prevent people who are already infected with the TB bacterium from falling ill with active TB disease.

In March 2019, study results presented at the Conference on Retroviruses and Opportunistic Infections (CROI) showed that weekly administration of rifapentine and isoniazid for three months (3HP) in adults with HIV taking dolutegravir (DTG) was well-tolerated, with no need for dose adjustment. These findings are significant given that people living with HIV are at high risk of developing TB and are 20 to 37 times more likely to move from latent infection to active TB. They also allowed tenofovir/lamivudine/dolutegravir (TLD) transition countries to move forward with co-administration of 3HP and DTG. But until now, the price of rifapentine remained an obstacle for low- and middle-income countries to implement this innovation.

“Today’s agreement will kick-start the global roll-out of more effective TB preventive therapy,” Unitaid Executive Director Lelio Marmora said. “It is in emphatic, tangible response to the call to action made last year at the high-level meeting of the United Nations General Assembly on the fight against tuberculosis.”

The 3HP regimen offers a shorter, safer alternative to the earlier standard of care—isoniazid preventive therapy (IPT)—in which people take isoniazid every day for between six and 36 months. In February 2018, the World Health Organization (WHO) released consolidated guidelines for the treatment of latent TB infection that recommend the use of 3HP for people living with HIV and contacts of TB cases of any age in high TB burden settings.

Priftin® is already on the list of WHO prequalified products and has received marketing authorization in numerous countries including the United States, South Africa, the Philippines and Indonesia.

The Aurum Institute, with its partners under the IMPAACT4TB project, has been facilitating the regulatory approval of 3HP products in project countries. IMPAACT4TB will support 3HP uptake in 12 high-burden countries, which represent 46% of the global TB burden and 84% of the current IPT uptake. IMPAACT4TB will initiate the first order under this agreement of 325,000 patient packs in Malawi, Mozambique, Zimbabwe, Kenya, Tanzania, Ethiopia, Cambodia, Indonesia and Ghana. In high TB burden countries where Priftin® is not yet registered, an importation waiver to use it may be obtained through the Global Fund, the Global TB Drug Facility, WHO and other international development agencies.



“With this new price in hand, we are now ready to start 400,000-600,000 people on 3HP across 12 countries,” added Professor Churchyard. “The next few years will test our political resolve—if we truly want to stifle the TB pandemic, we need to make sure preventive treatment gets to those people who are most at risk.”

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Thursday, 12 December 2019

Cigarette smoke makes MRSA more drug-resistant


Cigarette smoke can make MRSA bacterial strains more resistant to antibiotics, new research from the University of Bath has shown. In addition cigarette smoke exposure can make some strains of Staphylococcus aureus -- a microbe present in 30-60% of the global population and responsible for many diseases, some fatal -- more invasive and persistent, although the effect is not universal across all strains tested.

The researchers believe the stress cigarette smoke causes to S. aureus sparks an emergency 'SOS' response, which increases the rate of mutation in microbial DNA, resulting in hardy and persistent variants better able to resist antibiotics.

Previous studies had attributed smokers' increased susceptibility to infection to the damaging effects of smoke on our immune system, but this study shows that it may also be changing the DNA and characteristics of pathogenic microbes as well.

In a series of lab-based experiments the researchers exposed six reference strains of the most important 'superbug' Methicillin- Resistant S. aureus (MRSA) clones to cigarette smoke.

The strains were known to cause conditions ranging from skin infections to pneumonia and endocarditis and were chosen for their clinical relevance and genetic diversity. Although not all responded to cigarette smoke in the same way some, including those known to cause invasive infections, showed increased resistance to the antibiotic rifampicin and increased invasiveness and persistence. Resistance to other antibiotics is also likely to be affected.
The study links these changes to the emergence of Small Colony Variants (SCVs) -- hardy sub-populations that are adapted to harsh conditions. SCVs have been linked to chronic infections in smokers in previous research.

The scientists are now interested in studying how air pollution, from diesel exhaust fumes and other sources, might affect the microbes in our nasal passages as many of the pollution compounds are the same as in cigarette smoke.


See:

Alicia Lacoma, Andrew M. Edwards, Bernadette C. Young, José Domínguez, Cristina Prat, Maisem Laabei. Cigarette smoke exposure redirects Staphylococcus aureus to a virulence profile associated with persistent infection. Scientific Reports, 2019; 9 (1) DOI: 10.1038/s41598-019-47258-6

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Wednesday, 11 December 2019

New Form of Lyme Disease Detected by State-of-the-Art Test


IGeneX Laboratory Publishes Results of Novel Line Immunoblot Testing for Tick-borne Relapsing Fever

A novel test for a new form of Lyme disease was described in a report published in the prestigious medical journal Healthcare (Basel) (https://www.mdpi.com/2227-9032/7/4/121/pdf). The new test detects exposure to tick-borne relapsing fever, a Lyme-related disease transmitted by ticks that is spreading worldwide.

Lyme disease is a tick-borne infection caused by Borrelia burgdorferi, a type of corkscrew-shaped bacteria known as a spirochete (pronounced spiro’keet). Recently the Centers for Disease Control and Prevention (CDC) announced that Lyme disease is much more common than previously thought, with over 400,000 new cases diagnosed each year in the United States. That makes annual new cases of Lyme disease in this country about four times more common than new cases of HIV/AIDS, tuberculosis and syphilis combined.

Recently another form of tick-borne disease caused by relapsing fever Borrelia has been recognized in the United States and internationally. Until now there has been no reliable test for exposure to this family of Borrelia spirochetes. The new study describes a sensitive and specific test called a line immunoblot developed by IGeneX Laboratory that detects antibodies against relapsing fever Borrelia. IGeneX had previously developed a line immunoblot test for exposure to Lyme spirochetes related to Borrelia burgdorferi (https://www.mdpi.com/2227-9032/6/3/99).

The current study was a collaborative effort by an international team of scientists. Researchers included Jyotsna S. Shah, Song Liu, Iris Du Cruz, Akhila Poruri and Ranjan Ramasamy from IGenex Laboratory in Milpitas, CA; clinicians Мariia Shkilna, Mykhaylo Korda, Ivan Klishch, Stepan Zaporozhan, Kateryna Shtokailo and Mykhaylо Andreychyn from Ternopil National Medical University in Ukraine; biochemist Rajan Maynard from Stanford University, Palo Alto, CA; and internist Raphael Stricker from Union Square Medical Associates in San Francisco, CA.

“Our findings demonstrate the complexity of Lyme disease,” said Dr. Shah, the lead author of the study who is President and Laboratory Director of IGeneX. “The new test shows exposure to another form of the disease that cannot be detected with currently available Lyme testing.”

In the study, the line immunoblot test was used to detect antibodies against relapsing fever Borrelia in well-characterized serum samples from patients in Australia, Ukraine and the United States. The novel test showed high sensitivity for these antibodies, and there were very few false-positive results.

“Line immunoblot testing offers a reliable method to detect exposure to relapsing fever Borrelia,” said Dr. Shkilna, who treats Lyme disease in Ukraine. “The test can demonstrate exposure in individual patients and help us understand the global spread of tick-borne relapsing fever.”

Dr. Stricker pointed to the implications of the new test for Lyme disease diagnosis and treatment. “Lyme disease is not a simple infection anymore,” he said. “We need to pay attention to new forms of the disease, and the line immunoblot test is a good start.”

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Tuesday, 10 December 2019

Bringing remaining Over-The-Counter medically important antimicrobial drugs under veterinary oversight




The U.S. Food and Drug Administration released draft guidance for industry (GFI) #263 to explain the recommended process for voluntarily bringing remaining approved animal drugs containing antimicrobials of human medical importance (i.e., medically important) under the oversight of licensed veterinarians by changing the approved marketing status from over-the-counter (OTC) to prescription (Rx).

This is part of the FDA’s Five-Year Plan for Supporting Antimicrobial Stewardship in Veterinary Settings and builds upon the momentum generated by the successful implementation of GFI #213. Under GFI #213, animal drug sponsors worked in collaboration with FDA over a 3-year period to voluntarily change OTC medically important antimicrobials used in the feed or drinking water of food-producing animals to VFD/Rx marketing status and eliminated the use of these products for production purposes (e.g., growth promotion).

See: https://www.fda.gov/animal-veterinary/cvm-updates/fda-releases-draft-guidance-bringing-remaining-approved-over-counter-medically-important

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 9 December 2019

Microbiology Roundtable


American Pharmaceutical Review has run another in its Microbiology Roundtable features. Here is an extract from Tim Sandle:

Q. In general, what are some of the current critical issues/trends facing pharmaceutical manufacturers in regards to microbiology testing and remediation?

Sandle:  One of the biggest issues is with time-to-result, which is affected by the (largely) continued dependency upon culture-based methods and which is symptomatic by the slow take-up of rapid microbiological methods. Being able to obtain data faster, enables better responses.

While rapid methods will undoubtedly help, it remains that contamination control and good design are the most important considerations. There is little value testing if a given process has not been correctly designed to minimize the ingress of contamination, Of the different routes in, the main one remains people and the way they behave. Be it sterile or non-sterile manufacturing, designing systems and putting in place barriers to reduce the opportunity for personnel to get close to the product are paramount.

The reference is:

Microbiology Roundtable (2019) Michael Reynier, Jordi Iglesias, Tony Cundell, Suzanne Williams, Frank Panofen, Paula Peacos, Tim Sandle, Quinton Inglet, Jonathan Swenson, American Pharmaceutical Review, pp86-91

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 8 December 2019

Switch that kills inactive HIV isolated


Using genetic sequencing, University of California San Diego School of Medicine researchers have identified a principal cellular player controlling HIV reproduction in immune cells which, when turned off or deleted, eliminates dormant HIV reservoirs.

"This is one of the key switches that the HIV field has been searching for three decades to find," said Tariq Rana, PhD, professor of pediatrics and genetics at UC San Diego School of Medicine. "The most exciting part of this discovery has not been seen before. By genetically modifying a long noncoding RNA, we prevent HIV recurrence in T cells and microglia upon cessation of antiretroviral treatment, suggesting that we have a potential therapeutic target to eradicate HIV and AIDS."

HIV spreads through certain bodily fluid attacking the immune system and preventing the body from fighting off infections. If left untreated, the virus leads to the disease AIDS.
Antiretroviral therapy is used to prevent and treat HIV, allowing patients to live long and healthy lives. However, the medication does not cure patients. Instead, the virus remains inactive in the body. If therapy is discontinued, the virus awakens and multiplies rapidly.

Rana and colleagues report the first genome-wide expression analysis of long noncoding RNA (lncRNA) in HIV-infected macrophages -- specialized immune cells that promote tissue inflammation, stimulate the immune system and rid the body of foreign debris. In general, lncRNAs do not encode the recipe for proteins the way other RNAs do, but instead help control which genes are turned "on" or "off" in a cell.

The team described how a single lncRNA dubbed HIV-1 Enchanced LncRNA (HEAL) is elevated in people with HIV. HEAL appears to be a recently emerged gene that regulates HIV replication in immune cells, such as macrophages, microglia and T cells.
Using a combination of genomic, biochemical and cellular approaches, they found that silencing HEAL or removing it with CRISPR-Cas9 prevented HIV from recurring when antiretroviral treatment was stopped. Additional research to confirm these effects in animal models will be performed.


"Our results suggest that HEAL plays a critical role in HIV pathogenesis," said Rana. "Further studies are needed to explain the mechanism that leads to HEAL expression after an individual is infected by HIV, but this finding could be exploited as a therapeutic target."

See:

Ti-Chun Chao, Qiong Zhang, Zhonghan Li, et al. The Long Noncoding RNA HEAL Regulates HIV-1 Replication through Epigenetic Regulation of the HIV-1 Promoter. mBio, 2019; 10 (5) DOI: 10.1128/mBio.02016-19

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Saturday, 7 December 2019

Long-term disruption of gut microbes after antibiotics


Trillions of microbes in the intestine aid human health, including digestion of breast milk, breaking down fiber and helping control the immune system. However, antibiotic treatment is known to disrupt the community structure of these microbes -- 500 to 1,000 bacterial species that have a mainly beneficial influence.

A study at the University of Alabama at Birmingham now has tracked this disruption at the level of a strain of microbes replacing another strain of the same species in 30 individuals -- all of them young, healthy adults who would be expected to have stable microbial communities.

The UAB study used bioinformatic tools to analyze a previously described study of 18 individuals who had been given a single antibiotic, cefprozil, for a week. Their fecal samples were collected at pre-treatment, at the end of antibiotic treatment and at three months post-treatment.

The UAB study also analyzed previously described data of 12 individuals who were given a combination of three antibiotics -- meropenem, gentamicin and vancomycin -- for four days. Their fecal samples were collected at pretreatment; at end of treatment; and at four, 38 and 176 days post-treatment. Six control individuals who did not receive antibiotics were also analyzed.

In general, the UAB researchers found that strains of the 10 most abundant species remained stable in controls. In the single antibiotic treatment individuals, 15 of 18 individuals had transient new strains post-treatment that, in turn, were replaced by the original strain by three months post-treatment.


In contrast, the triple-antibiotics individuals showed a significant increase of new strains that persisted as long as six months after treatment, as compared to the single antibiotic and the control individuals. Furthermore, the fraction of transient strains was also significantly higher in the multiple antibiotics individuals. This suggested a long-term change to an alternative stable microbiome state. These changes were not due to a difference in growth rates.

See:

Hyunmin Koo, Joseph A. Hakim, David K. Crossman, Ranjit Kumar, Elliot J. Lefkowitz, Casey D. Morrow. Individualized recovery of gut microbial strains post antibiotics. npj Biofilms and Microbiomes, 2019; 5 (1) DOI: 10.1038/s41522-019-0103-8

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Friday, 6 December 2019

Immune response to influenza


It is estimated that influenza (flu) results in 31.4 million outpatient visits each year. New research from the University of Minnesota Medical School provides insights into how the body can protect itself from immunopathology during flu.

"One of the reasons people feel bad during flu and some people die from flu isn't actually the virus replication itself, but it is the immune system's attempt to control the virus that causes that damage," said lead author Ryan Langlois, PhD, assistant professor of Microbiology and Immunology at the Medical School. "That immune response, called immunopathology, is a very serious complication of flu."

Many people who get the flu recover in under two weeks because the immune system is able to clear the virus, leaving no trace of it in the body. Traditional theory thought this was accomplished by T cell-mediated killing of all infected cells. Several years ago, however, Langlois genetically engineered a flu virus that could permanently label infected cells, which led to the discovery that some infected cells do survive clearance.

The new study published in PLOS Pathogens examines why some infected cells evaded T cell-mediated killing in the lungs of a mouse model. Langlois and his team identified where the virus was in the lung and what types of cells it was in. They found that the formerly infected cells are able to clear the virus from the cell quick enough so that no remnants of the virus remained. Because T cells can't kill what they can't see, T cells need to see a virus in a cell to kill that cell.


After clearance, no virus exists in the lung, but the cells that used to be infected remain. The team found that those "survivor cells" actually divide and replenish themselves at a faster rate than uninfected cells.

"One can imagine that if T cells killed every infected cell, like people once thought they did, whole airways could be lost," Langlois said. "This study lends more data to the idea that preventing immunopathology is incredibly important, and it allows us to better understand the basic mechanisms of how the body regulates itself to prevent it."

See:

Jessica K. Fiege, Ian A. Stone, Rebekah E. Dumm, Barbara M. Waring, Brian T. Fife, Judith Agudo, Brian D. Brown, Nicholas S. Heaton, Ryan A. Langlois. Long-term surviving influenza infected cells evade CD8 T cell mediated clearance. PLOS Pathogens, 2019; 15 (9): e1008077 DOI: 10.1371/journal.ppat.1008077

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Thursday, 5 December 2019

Bacteria must be 'stressed out' to divide


A new study from EPFL scientists has found that bacteria use mechanical forces to divide, along with biological factors. The research, led by the groups of John McKinney and Georg Fantner at EPFL, came after recent studies suggested that bacterial division is not only governed by biology, but also by physics. However, this interplay is poorly understood.

Most bacteria are rod-shaped cells that multiply by doubling their length and dividing in the middle to yield two "daughter cells." Mechanisms that control these processes in space and time are critical for survival. The importance of these mechanisms becomes even clearer, given how pervasive bacteria are in everyday life, and how ubiquitous their use is in biotechnology.

The scientists studied bacteria that are very similar to the human pathogen that causes tuberculosis, which kills more people than any other infectious disease. To study the growth and division dynamics of these "mycobacteria" the scientists built a special instrument that combines optical and atomic force microscopy (AFM) to image and manipulate cells at the size scale of molecules.

The data showed that mycobacterial cell division requires mechanical forces in addition to previously identified division molecules (enzymes). Before a cell divides, there is a progressive build-up of mechanical stress in the cell wall, right at the point where the cell will divide.

The build-up eventually culminates in a millisecond-fast splitting of the cell into two new cells. Remarkably, when the researchers physically pressed on the bacteria with an ultra-sharp AFM needle, they caused instantaneous and premature cell division. "This experiment proves that physics is essential for this important biological process," says Georg Fantner.
But where is the biological part of the story? When a bacterial cell divides the two daughters must separate, a process mediated by enzymes that dissolve the molecular connections between them. The investigators found that this essential process could be bypassed by pressing on the nascent division site using the AFM needle.


"Our work demonstrates that biological enzymes and mechanical forces 'collaborate' to bring about the separation of daughter cells in bacterial cell division," says John McKinney.


See:

Pascal D. Odermatt, Mélanie T. M. Hannebelle, Haig A. Eskandarian, Adrian P. Nievergelt, John D. McKinney, Georg E. Fantner. Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division. Nature Physics, 2019 DOI: 10.1038/s41567-019-0679-1

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Wednesday, 4 December 2019

DNA-reeling bacteria yield new insight on how superbugs acquire drug-resistance


A new study from Indiana University has revealed a previously unknown role a protein plays in helping bacteria reel in DNA in their environment -- like a fisherman pulling up a catch from the ocean.

The discovery was made possible by a new imaging method invented at IU that let scientists see for the first time how bacteria use their long and mobile appendages -- called pili -- to bind to, or "harpoon," DNA in the environment.


By revealing the mechanisms involved in this process, the study's authors said the results may help hasten work on new ways to stop bacterial infection.
"The issue of antibiotic resistance is very relevant to this work since the ability of pili to bind to, and 'reel in,' DNA is one of the major ways that bacteria evolve to thwart existing drugs," said Ankur Dalia, an assistant professor in the IU Bloomington College of Arts and Sciences' Department of Biology, who is senior author on the study. "An improved understanding of this 'reeling' activity can help inform strategies to stop it."

The act of gobbling up and incorporating genetic material from the environment -- known as natural transformation -- is an evolutionary process by which bacteria incorporate specific traits from other microorganisms, including genes that convey antibiotic resistance.
The need for new methods to stop bacterial infection is growing since overuse of existing antibiotics, which speeds how quickly infectious organisms evolve to outsmart these drugs, is causing the world to quickly run out of effective treatments. By 2050, it's estimated that 10 million people could die each year from antimicrobial resistance.

Although they may look like tiny arms under a microscope, Dalia said, pili are actually more akin to an erector set that is quickly put together and torn down over and over again. Each "piece" in the structure is a protein sub-unit called the major pilin that assembles into a filament called the pilus fiber.

"There are two main motors that had previously been implicated in this polymerization and depolymerization process," added Jennifer Chlebek, a Ph.D. student in Dalia's lab, who led the study. "In this study, we show that there is a third motor involved in the depolymerization process, and we start to unravel how it works."

The two previously characterized "motors" that control the pili's activity are the proteins PilB, which constructs the pili, and PilT, which deconstructs it. These motors run by utilizing ATP, a source of cellular energy. In this study, IU researchers showed that stopping this process, which switches off the power to PilT, does not prevent the retraction of the pili, as previously thought.

Instead, they found that a third motor protein, called PilU, can power pilus retraction even if PilT is inactive, although this retraction occurs about five times more slowly. The researchers also found that switching off power to both retraction proteins slows the retraction process to a painstaking rate of 50 times slower. An unaltered pilus retracts at a rate of one-fifth of a micron per second.

Moreover, the study found that switching off PilU affects the strength of pilus retraction, which was measured by collaborators at Brooklyn College. The study also showed that PilU and PilT do not form a "hybrid" motor, but instead that these two independent motors somehow coordinate with one another to mediate pilus retraction.

"While the PilU protein had previously been implicated in pilus activity, its exact role has been difficult to determine because cells that lack this protein generally only have very subtle effects," Chlebek added. "Our observation that PilU can support pilus retraction in a mutant strain, when we threw a wrench in the PilT motor, was the key to unlocking how this protein aids in the depolymerization of pili."

The ability to precisely measure the pili's retraction rate -- and therefore precisely measure the impact of altering the proteins that affect this process -- was made possible by the ability to see pili under a microscope, which was not possible until the breakthrough imaging method invented at IU.

"The ability to fluorescently dye the pili was huge," Dalia said. "It allowed us to not only see the pili's activity but also measure it in ways which simply would not have been possible in the past."


Next, Chlebek aims to learn more about how the pili still retract when the power is switched off to both retraction motors, as well as explore how these insights could apply to understanding pili activity in other strains of bacteria.

See:

Jennifer L. Chlebek, Hannah Q. Hughes, Aleksandra S. Ratkiewicz, Rasman Rayyan, Joseph Che-Yen Wang, Brittany E. Herrin, Triana N. Dalia, Nicolas Biais, Ankur B. Dalia. PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili. PLOS Genetics, 2019; 15 (10): e1008448 DOI: 10.1371/journal.pgen.1008448

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Tuesday, 3 December 2019

Bacterial resistance to ceftolozane-tazobactam examined


French investigators have described development of resistance to one of the last resort therapies used to treat extremely drug-resistant Pseudomonas aeruginosa. That resistance arose in a single patient over a scant 22 days. They subsequently identified the single nucleotide mutation in P. aeruginosa that caused the resistance.

Unexpectedly, the mutation partially re-sensitized P. aeruginosa to antimicrobials that have long been in use -- carbapenems and piperacilline-tazobactam -- to which the bacterium had been fully resistant.

That peculiar finding might prove beneficial for the treatment of extremely drug-resistant P. aeruginosa, by enabling use of piperacilline-tazobactam and carbapenems in such cases, as their minimum inhibitory concentrations (MIC) "decrease significantly," said principal investigator Leurent Dortet, PharmD, PhD. Nonetheless, he cautioned that clinicians would need to proceed with caution "since other resistance mechanisms might be present."
Dr. Dortet said that using higher doses of ceftolozane-tazobactam to begin with might limit the appearance of the mutation by killing the pathogens more rapidly. Dr. Dortet is Associate Professor of Microbiology, University Paris-Saclay, France.


To determine the mechanism responsible for causing this resistance, the team analyzed P. aeruginosa clinical isolates, both susceptible and resistant, that had been collected from this patient during the infection, and performed whole genome sequencing on these. That enabled the investigators to identify the single mutation in a gene that encodes a natural enzyme, cephalosporinase. (Overexpression of cephalosporinase causes resistance to nearly all antimicrobials of the ?-lactam family.)

Modeling the mutant enzyme in silico confirmed its role as the cause of resistance to ceftolozane-tazobactam, and resensitization to carbapenems and piperacilline-tazobactam.
"Our results demonstrated that resistance to this novel molecule can occur rapidly during treatment," said Dr. Dortet. He noted that at the time the investigators discovered the mutation, the antibiotic, ceftolozane-tazobactam, had only been in clinical use for a couple of years.

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Monday, 2 December 2019

Visual inspection of particulate matter


Each final container of all parenteral preparations shall be inspected to the extent possible for the presence of observable foreign and particulate matter.

In relation to this a new article of interest has been issued:

This article highlights the key inspectional focal areas in visual inspection processes to guide the reader to re-assess their procedures and practices for enhancing visual inspection process. Each container of liquid parenteral product is required to be inspected for evidence of visible particles and any containers which are seen to be contaminated must be rejected. In addition, containers are also examined for flaws, cracks, misplaced seals etc. These are material issues which could compromise the integrity of the containers and therefore of the sterility of its contents. For staff tasked with the inspection, the inspector will check whether they can detect different particles (or microbial growth) based on their training and may esquire if frequent eye tests are carried out.

The reference is:

Saghee, M.R., Sandle, T. and Das, P. (2019) Regulatory inspection of sterile facilities – the focal points. Part 1 – Visual inspection of particulate matter, GMP Review, 18 (1): 13-18

Foe details, contact Tim Sandle



Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

Sunday, 1 December 2019

Pharmaceutical Microbiology Resources is 10 years old


Today marks an anniversary of sorts. This website - Pharmaceutical Microbiology Resources - is ten years old, and with over 3 million reads.

I began blogging and posting interesting research and my own thoughts on all things pharmaceutical microbiology (with the odd general science story or healthcare related snippet thrown in for good measure) due to a general lack of information. My biggest frustration was not finding out about new standards or publications of interest.

Over the past ten years, pharmaceutical microbiology has grown strongly and it continues to evolve into a respected and knowledgeable profession, distinct from other types of microbiology.

The evolution of pharmaceutical microbiology, outside the narrow of some academic interpretations (where the focus was often only on sterilization or antimicrobial efficacy) was driven by the voluminous and insightful work of Scott Sutton and those other pioneers who put together the Pharmaceutical Microbiology Forum in the U.S. and those other innovators who came up with the idea of Pharmig in the U.K. (which was driven by the energies of Poly Hajipieris) and continued with equal energy and commitment by Maxine Moorey and committee.

Over the years a number of individuals have made a significant contribution (based on those I've cited most often in my own writings) - Tony Cundell, Jeanne Moldenhauer, Luis Jimenez, Ed Tidswell, Ziva Abraham, Karen McCullough, Jim Agalloco, Jim Ackers, Nigel Halls, Michael Miller - and there are, of course, many others.

Promotion of pharmaceutical microbiology is evident with the PDA and PHSS and their journals, plus magazines like American Pharmaceutical Review, European Pharmaceutical Review and Cleanroom Technology.

For more in-depth analysis, Davis Healthcare International - operated by the pioneering Amy Davis - has issued a number of titles relating to pharmaceutical microbiology and related areas. I'm proud to have contributed to several DHI publications.

I'm aiming to continue with this site for a few more years and I welcome any contributions, industry news, links to articles of interest and so on.

Thank you for your support over the past decade.

Tim

Here are the ten most popular posts over the past ten years:

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology

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