Tag Archives: Technology

University of Louisville researchers receive $5.8 million to prevent immune system dysregulation

2 June, 2023 Admin Leave a comment

Reviewed by Megan Craig, M.Sc.Jun 2 2023

Researchers at the University of Louisville have received $5.8 million in two grants from the National Institutes of Health to expand their work to better understand and prevent immune system dysregulation responsible for acute respiratory distress, the condition responsible for serious illness and death in some COVID-19 patients. A separate $306,000 NIH Small Business Innovation Research grant supports early testing of a compound developed at UofL as a potential treatment.

The three grants combined total $6.1 million.

During the pandemic, health care providers worked tirelessly to treat patients who became seriously ill with COVID-19. Some of those patients developed severe lung disease known as acute respiratory distress syndrome (ARDS) due to an excessive response of the immune system often called cytokine storm.

As they treated these critically ill patients, physicians and other providers at UofL Health shared their clinical insights and patient samples with researchers at UofL to discover the cause of the immune system overresponse.

At one time we had over 100 patients with COVID in the hospital. Once they were on a ventilator, mortality was about 50%. We were looking at this issue to see why some people would do well while some developed bad lung disease and did not do well or died.”

Jiapeng Huang, an anesthesiologist with UofL Health and professor and vice chair of the Department of Anesthesiology and Perioperative Medicine in the UofL School of Medicine

The UofL researchers, led by immunologist Jun Yan, discovered that a specific type of immune cells, low-density inflammatory neutrophils, became highly elevated in some COVID-19 patients whose condition became very severe. This elevation signaled a clinical crisis point and increased likelihood of death within a few days due to lung inflammation, blood clotting and stroke. Their findings were published in 2021 in JCI Insight.

With the new NIH funding, Yan is leading research to build on this discovery with deeper understanding of what causes a patient’s immune system to respond to an infection in this way and develop methods to predict, prevent or control the response.

“Through this fruitful collaboration, we now have acquired NIH funding for basic and translational studies and even progress toward commercialization of a potential therapy,” Yan said. “That’s why we do this research – eventually we want to benefit the patients.”

Yan, chief of the UofL Division of Immunotherapy in the Department of Surgery, a professor of microbiology and immunology and a senior member of the Brown Cancer Center, will lead the new research, along with Huang and Silvia M. Uriarte, university scholar and professor in the Department of Oral Immunology and Infectious Diseases in the UofL School of Dentistry.

“COVID-19 continues to spotlight the impactful synergy between the clinical and research teams at the University of Louisville,” said Jason Smith, UofL Health chief medical officer. “Innovation is in the DNA of academic medicine. We collaborate to provide each patient the best options for prevention and treatment today, while developing the even better options for tomorrow.”

In addition to two research grants of $2.9 million each awarded directly to UofL, a $306,000 grant to a startup company will support early testing of a compound developed in the lab of UofL Professor of Medicine Kenneth McLeish that shows promise in preventing the dangerous cytokine storm while allowing the neutrophils to retain their ability to kill harmful bacteria and viruses. The compound, DGN-23, will be tested by UofL and Degranin Therapeutics, a startup operated by McLeish, Yan, Huang, Uriarte and Madhavi Rane, associate professor in the Department of Medicine.

“This is one more example of how UofL has led the charge in finding new and innovative ways to detect, contain and fight COVID-19 and other potential public health threats,” said Kevin Gardner, UofL’s executive vice president for research and innovation. “This team’s new research and technology could help keep people healthy and safe here and beyond.”

The knowledge gained through these studies may benefit not only COVID-19 patients, but those with other conditions in which immune dysregulation can occur, such as other types of viral and bacterial pneumonia and autoimmune diseases, and patients undergoing cancer immunotherapy and organ transplantation.

The grants

Grant 1 – $2.9 million, four-year grant to UofL. Investigators will study the new subset of neutrophils Yan identified to better understand how they contribute to acute respiratory distress and clotting. They also will determine whether a novel compound will prevent these complications. They will use lab techniques and studies with animal models that allow for manipulation of certain conditions that cannot be done in human subjects.

Grant 2 – $2.9 million, five-year grant to UofL. This work examines a more comprehensive landscape to characterize different subsets of neutrophils and measure their changes over the course of COVID-19 disease progression and how neutrophils contribute to immune dysfunction.

Grant 3 – $306,000, one-year grant to Degranin Therapeutics and UofL for early testing of DGN-23, a compound developed at UofL, to determine its effectiveness in preventing or reducing immune dysregulation.

Source:

University of Louisville

Microbiology

New tool shows early promise to help reduce the spread of antimicrobial resistance

25 May, 2023 Admin Leave a comment

Reviewed by Megan Craig, M.Sc.May 25 2023

A new tool which could help reduce the spread of antimicrobial resistance is showing early promise, through exploiting a bacterial immune system as a gene editing tool.

Antimicrobial resistance is a major global threat, with nearly five million deaths annually resulting from antibiotics failing to treat infection, according to the World Health Organisation.

Bacteria often develop resistance when resistant genes are transported between hosts. One way that this occurs is via plasmids – circular strands of DNA, which can spread easily between bacteria, and swiftly replicate. This can occur in our bodies, and in environmental settings, such as waterways.

The Exeter team harnessed the CRISPR-Cas gene editing system, which can target specific sequences of DNA, and cuts through them when they are encountered. The researchers engineered a plasmid which can specifically target the resistance gene for Gentamicin – a commonly used antibiotic.

In laboratory experiments, the new research, published in Microbiology, found that the plasmid protected its host cell from developing resistance. Furthermore, researchers found that the plasmid effectively targeted antimicrobial-resistant genes in hosts to which it transferred, reversing their resistance.

Antimicrobial resistance threatens to outstrip covid in terms of the number of global deaths. We urgently need new ways to stop resistance spreading between hosts. Our technology is showing early promise to eliminate resistance in a wide range of different bacteria. Our next step is to conduct experiments in more complex microbial communities. We hope one day it could be a way to reduce the spread of antimicrobial resistance in environments such as sewage treatment plants, which we know are breeding grounds for resistance.”

David Walker-Sünderhauf, Lead Author, University of Exeter

The research is supported by GW4, the Medical Research Council, the Lister Institute, and JPI-AMR.

Source:

University of Exeter

Journal reference:

Walker-Sünderhauf, D., et al. (2023) Removal of AMR plasmids using a mobile, broad host-range, CRISPR-Cas9 delivery tool. Microbiology. doi.org/10.1099/mic.0.001334.

Microbiology

Exploring nutritional factors during pregnancy and in infancy to find clues for childhood tooth decay

23 May, 2023 Admin Leave a comment

Reviewed by Danielle Ellis, B.Sc.May 23 2023

Researchers from Rochester Institute of Technology and the University of Rochester Medical Center are taking a closer look at nutritional factors during pregnancy and in infancy associated with severe tooth decay in young children.

Brenda Abu, assistant professor in RIT’s Wegmans School of Health and Nutrition and a researcher in maternal and child health, is collaborating on a study to investigate the Oral Microbiome in Early Infancy (OMEI) and Nutrition. Perinatal oral health expert Dr. Jin Xiao, associate professor at the Eastman Institute for Oral Health, is leading a large project funded by the National Institutes of Health’s Dental and Craniofacial Research.

The researchers will examine relationships between perinatal nutritive behavior-;such as dietary iron intake-;and nonnutritive behavior-;such as pica-;and the oral microbiome during pregnancy and early life. Abu and Xiao will assess the impact on infants’ early-life oral yeast colonization and infection and explore microbial compositions of pica substances. A two-year $380,000 award from the NIH supports Abu’s collaboration.

Pica is the compulsive eating of items lacking nutritional value. The behavior occurs most often in women and children, and substances consumed include seemingly harmless items, such as ice, or dangerous materials, such as dried paint, clay, soil, or metal. Pica may cause infections and deplete iron stores in pregnant women. The results can be devastating on maternal health and fetal development and carry long-lasting consequences, according to Abu.

People who have iron deficiency crave the taste and smell of non-food substances that make iron deficiency worse. Pregnant women who develop iron deficiency anemia have increased risk of miscarriages, low-birthweight babies, and other poor-birth outcomes.”

Brenda Abu, Assistant Professor, RIT’s Wegmans School of Health and Nutrition

Other risk factors revealed from this study could inform prenatal counseling for underserved women and predict and prevent “Early Childhood Caries,” or severe tooth decay in young children. Xiao’s research among underserved racial and ethnic minority groups has shown that the presence of certain bacteria and yeast in the mother’s mouth increases the child’s likelihood of developing the condition.

“The OMEI + Nutrition is the first study that examines the relationship between nutritive and nonnutritive factors on perinatal oral microbiome among underserved U.S. pregnant women and their children,” said Dr. Xiao. “The data generated will strengthen the understanding of children’s oral microbiome development and their association to tooth decay.”

Abu’s collaboration with Xiao and other URMC researchers began with an earlier study assessing pica practice, oral health, and oral microbiome during pregnancy. The NIH award supports Abu’s career development and complements her international research focused on micronutrient nutrition and consequences among women and children living in Ghana. Findings from the current study exploring maternal nutrition and the oral microbiome in early infancy will influence the scope of Abu’s international research.

“With my training and expertise in nutrition, my long-term career goal is to bridge gaps in nutritional and oral research and generate groundbreaking interventions for early warning, early detections, and prevention of oral disease and iron deficiency among underserved mothers and young children,” Abu said.

Dr. Eli Eliav, professor and director of the UR’s Eastman Institute for Oral Health, is the adviser for the OMEI + Nutrition research. UR team members who will play key roles on the project include Steven Gill, professor of microbiology and immunology; Tong Tong Wu, associate professor of biostatistics and computational biology; and Dr. Kevin Fiscella, professor of family medicine. The entire team is listed online.

Source:

Rochester Institute of Technology

Microbiology

Novel gene-editing strategy harnesses an unusual protective ability to eliminate HIV-1 infection

19 May, 2023 Admin Leave a comment

Reviewed by Danielle Ellis, B.Sc.May 19 2023

Genetic alterations that give rise to a rare, fatal disorder known as MOGS-CDG paradoxically also protect cells against infection by viruses. Now, scientists at the Lewis Katz School of Medicine at Temple University have harnessed this unusual protective ability in a novel gene-editing strategy aimed at eliminating HIV-1 infection with no adverse effects on cell mortality.

The new approach, described online April 28 in the journal Molecular Therapy – Nucleic Acids, is based on a combination of two gene-editing constructs, one that targets HIV-1 DNA and one that targets a gene called MOGS – defects in which cause MOGS-CDG. In cells from persons infected with HIV-1, the Temple researchers show that disrupting the virus’s DNA while also deliberately altering MOGS blocks the production of infectious HIV-1 particles. The discovery opens up new avenues in the development of a cure for HIV/AIDS.

Proper MOGS function is essential for glycosylation, a process by which some cellular proteins synthesized in the body are modified to make them stable and functional. Glycosylation, however, is leveraged by certain kinds of infectious viruses. In particular, viruses like HIV, influenza, SARS-CoV-2, and hepatitis C, which are surrounded by a viral envelope, rely on glycosylated proteins to enter host cells.

In the new study, lead investigators Kamel Khalili, PhD, Laura H. Carnell Professor and Chair of the Department of Microbiology, Immunology, and Inflammation, Director of the Center for Neurovirology and Gene Editing, and Director of the Comprehensive NeuroAIDS Center at the Lewis Katz School of Medicine, and Rafal Kaminski, PhD, Assistant Professor at the Center for Neurovirology and Gene Editing at the Lewis Katz School of Medicine designed a genetic approach to exclusively turn on CRISPR to impede MOGS gene expression through DNA editing within immune cells that harbor replication competent, HIV-1. Their novel approach is expected to avoid any impact on the health of uninfected cells that retain normal MOGS gene function. Stimulation of the apparatus in HIV-1 infected cells disrupted the glycan structure of the HIV-1 envelope protein, culminating in the production of non-infectious virus particles.

“This approach is conceptually very interesting,” said Dr. Khalili, who is also senior investigator on the new study. “By mitigating the ability of the virus to enter cells, which requires glycosylation, MOGS may offer another target, in addition to the integrated viral DNA for developing the next generation of CRISPR gene-editing technology for HIV elimination.”

Dr. Kaminski, Dr. Khalili, and Tricia H. Burdo, PhD, Professor and Vice Chair in the Department of Microbiology, Immunology, and Inflammation and the Center for Neurovirology and Gene Editing at Temple and an expert in the use of non-human primate models for HIV-1, have been working together to further assess the efficacy and safety of CRISPR-MOGS strategy in preclinical studies. In previous work, the team demonstrated that CRISPR-based technology can successfully remove viral DNA from the cells of infected non-human primates.

Other researchers who contributed to the study include Hong Liu, Chen Chen, Shuren Liao, and Shohreh Amini, Department of Microbiology, Immunology, and Inflammation, Center for Neurovirology and Gene Editing, Lewis Katz School of Medicine at Temple University; Danielle K. Sohaii, Conrad R.Y. Cruz, and Catherine M. Bollard, Center for Cancer and Immunology Research, Children’s National Health System, The George Washington University; Thomas J. Cradick and Jennifer Gordon, Excision Biotherapeutics, San Francisco, CA; Anand Mehta, Stephane Grauzam, and James Dressman, Department of Cell and Molecular Pharmacology, Medical University of South Carolina; and Carlos Barrero and Magda Florez, Department of Pharmaceutical Sciences, School of Pharmacy, Temple University.

The research was supported in part by grants from the National Institutes of Health and the W.W. Smith Charitable Trust.

Source:

Temple University Health System

Journal reference:

Liu, H., et al. (2023) Strategic Self-Limiting Production of Infectious HIV Particles by CRISPR in Permissive Cells. Molecular Therapy — Nucleic Acids. doi.org/10.1016/j.omtn.2023.04.027.

Microbiology

Transforming antibiotic resistance testing: a novel, rapid and affordable technique

19 May, 2023 Admin Leave a comment

Interview conducted by Lily Ramsey, LLMMay 19 2023

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Thought LeadersDr. Sandor KasasResearch LeadEcole Polytechnique Fédérale de Lausanne

News Medical speaks with Dr. Sandor Kasas, a lead researcher at Ecole Polytechnique Fédérale de Lausanne in Switzerland. Here we discuss his recent development of a novel and highly efficient method for rapid antibiotic susceptibility testing using optical microscopy.

The new technique, known as Optical Nanomotion Detection (ONMD), is an extremely rapid, label-free, and single-cell sensitive method to test for antibiotic sensitivity. ONMD requires only a traditional optical microscope equipped with a camera or mobile phone. The simplicity and efficiency of the technique could prove to be a game changer in the field of antibiotic resistance.

Please can you introduce yourself, tell us about your career background, and what inspired your career in biology and medicine?

I graduated in medicine but never practiced in hospitals or medical centers. After my studies, I started working as an assistant in histology at the University of Fribourg in Switzerland. My first research projects included image processing, scanning tunneling, and atomic force microscopy.

Later, and for most of the rest of my scientific carrier, I focused primarily on the biological applications of AFM. For the past ten years, my research interest is about nanomotion, i.e., the study of oscillations at a nanometric scale of living organisms.

Image Credit: dominikazara/Shutterstock.com

You started working on biological applications of the atomic force microscope (AFM) in 1992. From your perspective, how has the antibiotic resistance landscape changed over the last two decades? What role has the advancement in technology played in furthering our understanding?

In the early ’90s, the AFM was mainly used for imaging. Later, AFM microscopists noticed that the instrument could also be used to explore the mechanical properties of living organisms. More recently, many “exotic” applications of the AFM have emerged, such as its use to weigh single cells or study their oscillations at the nanometric scale. In the 1990s, antibiotic resistance was not as serious a problem as today, but several teams were already using AFM to assess the effects of antibiotics on bacterial morphology.

The first investigations were limited to structural changes, but later, as the fields of application of AFM expanded, the instrument made it possible to monitor the mechanical properties of the bacterial cell wall upon exposure to antibiotics. In the 2010s, with G. Longo and G. Dietler, we demonstrated that AFM could also track nanoscale oscillations of living organisms. The very first application we had in mind was using the instrument to perform rapid antibiotic susceptibility testing.

We have therefore developed devices based on dedicated AFM technology to perform fast AST (i.e., in 2-4h). AFM-based nanomotion detection instruments are already implemented in medical centers in Switzerland, Spain, and Austria. However, this type of device has some drawbacks, including the need to fix the organism of interest on a cantilever. To overcome this limitation, we have developed with R. Willaert a nanomotion detector based on an optical microscope.

Your most recent research has led to the development of a novel and highly efficient technique for rapid antibiotic susceptibility testing using optical microscopy. Please could you tell us why the development of rapid, affordable, and efficient testing methods is so important in the world of antimicrobial resistance?

Rapid antibiotic susceptibility testing could reduce the use of broad-spectrum antibiotics. Traditional ASTs based on replication rate require 24 hours (but up to 1 month in the case of tuberculosis) to identify the most effective antibiotic. Doctors prescribe broad-spectrum antibiotics between the patient’s admission to a medical center and the results of the AST.

These drugs quickly improve patients’ conditions but, unfortunately, promote resistance. A rapid AST that could identify the most suitable antibiotic within 2-4 hours would eliminate broad-spectrum antibiotics and increase treatment efficiency and reduce the development of resistant bacterial strains. Since bacterial resistance is a global problem, rapid ASTs should also be implemented in developing countries. Therefore, affordable and simple-to-use tests are needed.

Image Credit: Fahroni/Shutterstock.com

Were there any limitations and obstacles you faced in the research process? If so, how did you overcome them?

Antibiotic sensitivity detection with ONMD is very similar to the AFM-based technique. As long as the bacterium is alive, it oscillates; if the antibiotic is effective, it kills the micro-organism, and its oscillations stop. The first limitation we faced when developing the ONMD was our microscopes’ depth of field of view. To prevent the bacteria from leaving the focal plane of the optical microscope during the measurement, we had to constrain the microbes into microfluidic channels a few micrometers high.

Microfabrication of such devices is relatively straightforward in an academic environment, but we were looking for simpler solutions. One option for constructing such a device is to use 10-micron double-sided rubber tape. It allows you to “build” a microfluidic chamber in 5 minutes with two glass coverslips and a puncher.

Another challenge was nanoscale motion detection. For this purpose, we used freely available cross-correlation algorithms that achieve sub-pixel resolution. (i.e., a few nanometers). We first developed the ONMD for larger organisms, such as yeast cells, and expanded the method to bacteria. This further development took us around two years.

You worked alongside Dr. Ronnie Willaert, a professor of structural biology at Vrije Universiteit Brussel, on developing this new rapid AST technique. How did your areas of expertise and research backgrounds complement each other in developing ONMD?

R. Willaert is an expert in yeast microbiology and microfluidics, while our team in Lausanne is primarily involved in AFM-based nanomotion detection and applying AFM to clinically relevant problems. The two teams were supported by a joint grant from the Swiss National Science Foundation and the Research Foundation Flanders (FWO) which enabled the development of the method.

The field of antimicrobial resistance requires a high level of international collaboration, with everyone working together to achieve a common goal. With antimicrobial resistance rising to dangerously high levels in all parts of the world, how important is collaboration in this field?

Our project required expertise in various fields, such as microbiology, microscopy, microfluidics, programming, and data processing. In the development of rapid AST instruments and many others, only a multidisciplinary approach and close collaboration between teams with complementary expertise is today the only path to success.

You and Dr. Willaert have said, ‘The simplicity and efficiency of the method make it a game-changer in the field of AST.’ Can you please expand on what makes ONMD a game changer in the AST field and what implications this research could have in clinical and research settings?

As mentioned earlier, bacterial resistance is a global health problem. Rapid AST should also be easily implemented in developing countries to limit the spread of resistant strains. The cheaper and simpler the technique, the more likely it is to be used on a large scale. We are convinced that the ONMD approach can meet these requirements. ONMD could also be used for drug discovery or basic research.

While we recognize the importance of rapid AST, what next steps must be taken before this technique can be used globally in research and clinical landscapes?

For fundamental research, there are no other important developments to be made. Any reasonably equipped research center can implement the technique and use it. Regarding implementing the technique in developing countries or extreme environments, stand-alone devices have to be used, which have yet to be manufactured.

There is a rapidly expanding need for efficient AST globally; however, the need for affordable, accessible, and simple techniques are of grave importance in developing countries disproportionately affected by antibiotic resistance due to existing global health disparities. Could this rapid AST technique be utilized in low-middle-income countries to slow the growing spread of multi-resistant bacteria? What would this mean for global health?

We are confident that ONMD-based AST testing can soon be implemented in research centers in both developed and developing countries. However, accreditation by the health authorities is necessary to use it as a standard diagnostic tool; this process can take several years, depending on the government health policy.

What’s next for you and your research? Are you involved in any exciting upcoming projects?

We want to develop a self-contained device for extreme environments. It would consist of a small microscope equipped with a camera and a data processing unit. The microfluidic part of the device could contain different antibiotics ready to be tested.

The ONMD technique could also monitor contamination levels in enclosed environments such as submarines, spacecraft, and space stations. One of our recent projects is funded by the European Space Agency (ESA) to develop a rapid antifungal susceptibility test that could work in microgravity. Additionally, ONMD could be used for even more exciting projects, such as chemistry-independent life detection in the search for extraterrestrial life.

Where can readers find more information?

Villalba MI, Rossetti E, Bonvallat A, Yvanoff C, Radonicic V, Willaert RG*, Kasas S.*.Simple optical nanomotion method for single-bacterium viability and antibiotic response testing. PNAS 2023, May 2;120(18):e2221284120. doi: 10.1073/pnas.2221284120. Epub 2023 Apr 24. PMID: 37094120. * Contributed equally. https://doi.org/10.1073/pnas.2221284120
Radonicic, V.; Yvanoff, C.; Villalba, M.I.; Devreese, B.; Kasas, S.; Willaert, R.G. Single-Cell Optical Nanomotion of Candida albicans in Microwells for Rapid Antifungal Susceptibility Testing. Fermentation 2023, 9:365. https://doi.org/10.3390/fermentation9040365
Parmar P, Villalba MI, Horii Huber AS, Kalauzi A, Bartolić D, Radotić K, Willaert RG, MacFabe DF and Kasas S. Mitochondrial nanomotion measured by optical microscopy. Front. Microbiol. 2023, 14:1133773. https://doi.org/10.3389/fmicb.2023.1133773
Starodubtseva MN, Irina A. Chelnokova IA, Shkliarava NM, Villalba MI, Tapalski DV, Kasas S, Willaert RG. Modulation of the nanoscale motion rate of Candida albicans by X-rays. Front. Microbiol. 2023, 14:1133027. https://doi.org/10.3389/fmicb.2023.1133027
Radonicic V, Yvanoff C, Villalba MI, Kasas S, Willaert RG. The Dynamics of Single-Cell Nanomotion Behaviour of Saccharomyces cerevisiae in a Microfluidic Chip for Rapid Antifungal Susceptibility Testing. Fermentation. 2022; 8(5):195. https://doi.org/10.3390/fermentation8050195

About Dr. Sandor Kasas

Nanomotion is a fascinating and novel approach to observing living organisms.

Our team focuses almost exclusively on recording the nanomotion of bacterial mitochondria and mammalian cells with optical and AFM-based devices.

Recently, we demonstrated that the technique could be used not only for fast antimicrobial sensitivity testing but also to explore the metabolism of unicellular organisms. We hope our efforts will permit us to expand the application domains of ONMD.

Microbiology

Mouse study offers clues to developing an effective vaccine for Klebsiella bacteria

19 May, 2023 Admin Leave a comment

Reviewed by Danielle Ellis, B.Sc.May 18 2023

A mouse study at Washington University School of Medicine in St. Louis points to data that could be key to developing an effective vaccine for the bacterium Klebsiella pneumoniae. The bug is often resistant to antibiotics, making it difficult to treat in some.

In the U.S., the bacterium Klebsiella pneumoniae is a common cause of urinary tract infection, bloodstream infection and pneumonia. While infections with the bacterium can be easily treated in some, Klebsiella has a dangerous flip side: It also is frequently resistant to antibiotics, making it extraordinarily difficult to treat in others. About half of people infected with a hypervirulent, drug-resistant strain of the bacterium die.

Scientists are working on vaccines for Klebsiella, but the optimal vaccine design is still unknown. However, a new study in mice by scientists at Washington University School of Medicine in St. Louis and Omniose, a St. Louis startup company specializing in vaccine production, provides critical data that could be key to developing an effective vaccine for Klebsiella. The findings, published in PLoS Pathogens, are a step toward taming the superbug.

When you think about the bugs that can be resistant to almost all antibiotics — the scary superbugs in the news — a lot of them are strains of Klebsiella. For a long time, the bacterium wasn’t even a pressing issue. But now it is, due to an explosion in antibiotic-resistant Klebsiella. Our goal is to diminish Klebsiella’s superbug status by developing a vaccine before hypervirulent or resistant strains sicken and kill even more people.”

David A. Rosen, MD, PhD, study’s senior author, assistant professor of pediatrics and of molecular microbiology at Washington University

Hypervirulent Klebsiella strains have spread globally, often causing community-acquired infections.

In the U.S., Klebsiella infections primarily occur in health-care facilities where medically vulnerable patients are immunocompromised, require long courses of antibiotics to treat other conditions, have chronic diseases, or are elderly people or newborns. “But now we’re seeing the emergence of hypervirulent strains dangerous enough to cause serious disease or death among healthy people in the community,” Rosen said.

Most concerning among scientists are the strains of Klebsiella impervious to carbapenems, a class of broad-spectrum antibiotics used to treat the most severe bacterial infections. For this reason, the World Health Organization and the U.S. Centers for Disease Control and Prevention have identified carbapenem-resistant Klebsiella as an urgent threat to public health.

The rod-shaped bacterium is immobile and, like chocolate-covered candies, encapsulated in sugar coatings. In the new study, researchers created two experimental vaccines based on two different sugars, or polysaccharides, on Klebsiella’s surface: the terminal sugars on lipopolysaccharide, called O-antigen, and a capsular polysaccharide, or K-antigen. Since sugars by themselves tend to produce weak immune responses, the researchers linked each of the sugars to a protein to boost the immune response, creating so-called conjugate vaccines. Sugar-protein conjugate vaccines have proven successful in combating several bacteria including Streptococcus pneumoniae, the most common cause of pneumonia. Historically, this connection between the sugar and protein carrier has been achieved using synthetic chemistry in a test tube; however, the vaccines created for this study are called bioconjugate vaccines, because the researchers connected the sugar to the protein all within an engineered bacteria system.

Once the vaccines were created, the researchers tested the experimental bioconjugate vaccines’ ability to protect mice from disease caused by Klebsiella.

“It turned out that the capsule vaccine was far superior to the O-antigen vaccine,” said the study’s first author, Paeton Wantuch, PhD, a postdoctoral associate in Rosen’s lab. “Mice that received the capsule vaccine were significantly more likely to survive Klebsiella infection in their lungs or their bloodstream than mice that received the O-antigen vaccine.”

Both vaccines elicited high levels of antibodies against their respective targets. But the antibodies against the O-antigen just weren’t as effective as the ones against the capsule. In some strains of Klebsiella, the O-antigen may be obscured by other sugars, so the antibodies that target the O-antigen cannot make contact with their target.

“Our findings suggest that we may also need to include the capsule-based antigens in vaccine formulations developed against Klebsiella,” Rosen said. “This is why it’s so important for us to continue studying antibody-antigen interactions in the different strains, with the goal of identifying the ideal vaccine composition for clinical trials soon. The need has never been more imperative, especially as Klebsiella’s drug-resistant, hypervirulent strains become stronger, bolder and more dangerous to human health.”

Source:

Washington University School of Medicine

Journal reference:

Wantuch, P. L., et al. (2023) Area-deprivation, social care spending and the rates of children in care proceedings in local authorities in Engl Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae. PLOS Pathogens. doi.org/10.1371/journal.ppat.1011367.

Microbiology

UK’s first Centre for Phage Research highlights Actiphage in the KTN UK Phage Innovation Showcase

18 May, 2023 Admin Leave a comment

May 18 2023Reviewed by Danielle Ellis, B.Sc.

Tuberculosis is caused by a bacterium that has evolved to evade the body’s immune system; now, its natural enemy, a phage, is being used to detect the infection at an early stage. The UK’s first Centre for Phage Research is being officially launched on 16^th May and featured in the Innovate UK KTN’s UK Phage Innovation Showcase will be Actiphage^®, a phage-based molecular diagnostic for tuberculosis.

UK’s first Centre for Phage Research highlights Actiphage in the KTN UK Phage Innovation Showcase — *Actiphage finds live mycobacterium tuberculosis in the blood and releases its DNA for identification with qPCR. Image Credit: PBD Biotech.*

Catherine Rees, Professor of Microbiology at University of Nottingham, is a co-founder of PBD Biotech, developers of Actiphage.

I am delighted to be supporting the launch of the University of Leicester’s Centre for Phage Research and to be given an opportunity to highlight our phage-based technology in the KTN UK Phage Innovation Showcase. These developments emphasise the UK’s recognised strength in phage research and I look forward to the exciting new collaborations and technology developments that will arise from these important networking events.”

Catherine Rees, Professor of Microbiology, University of Nottingham | Co-founder, PBD Biotech | Developers of Actiphage

A quarter of the world’s population are carriers of tuberculosis but show no symptoms. It can remain hidden in the body and only progress to full disease when the immune system is weakened, perhaps by other disease or malnutrition. Tuberculosis disease is preventable but can be fatal unless treated. The challenge is how to detect the people that have active disease and treat them before they become infective – thereby breaking the cycle of infection.

The bacterium, Mycobacterium tuberculosis (Mtb) has proven difficult to detect as it has a hard cell wall that prevents the release of its DNA, and it is slow to culture. Actiphage uses a phage, a virus specific to Mtb, to find live cells of Mtb in the blood.

Research at the NIHR Respiratory Biomedical Research Centre in Leicester by Clinical Senior Lecturer and Honorary Consultant Physician, Dr Pranabashis Haldar, has shown that Mtb detected in the blood using Actiphage is an indicator of active TB disease; this is a potential breakthrough in the fight to End TB.

As we see with the Actiphage test, phages can enhance the scope and sensitivity of molecular diagnostics for bacterial infections by providing a mechanism to identify and release target genetic material for PCR based amplification.

There are specific phages known to infect the vast majority of bacteria that cause infections. Phages are also versatile and may be modified to enable different approaches to the development of clinical diagnostics.”

Dr Pranabashis Haldar, NIHR Respiratory Biomedical Research Centre in Leicester by Clinical Senior Lecturer and Honorary Consultant Physician

Dr Haldar has been invited to join a panel group at the event and he thinks that phages will have multiple roles in the prevention and treatment of bacterial infections.

For more information about the Centre for Phage Research visit le.ac.uk/research/centres/phage-research.

To find out more about Actiphage visit pbdbio.com.

Source:

PBD Biotech

Microbiology

A novel approach to quantify personal information contained within gut metagenome data

17 May, 2023 Admin Leave a comment

By Neha MathurMay 17 2023Reviewed by Lily Ramsey, LLM

In a recent study published in Nature Microbiology, researchers used shotgun sequencing to extract human reads from deoxyribonucleic acid (DNA) in fecal samples of 343 Japanese individuals comprising the main dataset of this study.

They used this gut metagenome data to reconstruct personal information. Some study participants also provided whole genome sequencing (WGS) data for ultra-deep metagenome shotgun sequencing analysis.

Study: Reconstruction of the personal information from human genome reads in gut metagenome sequencing data. Image Credit: KaterynaKon/Shutterstock.com

Background

The knowledge regarding the human microbiome, microorganisms inhabiting the human body, has expanded considerably in the last ten years, thanks to rapid advancements in technologies like metagenome shotgun sequencing.

This technology allows the sequencing of the non-bacterial component of the microbiome samples, including host DNA. For instance, in fecal samples, the amount of host DNA is less than 10% but is removed to protect the privacy of donors.

Human germline genotype in metagenome data is substantial to enable the re-identification of individuals. However, researchers and donors should recognize that it is highly confidential, so sharing it with the community requires careful consideration.

Apart from ethical concerns related to sharing this data, it is necessary to understand that if human reads in metagenome data are not removed before deposition, what kind of personal information (e.g., sex and ancestry) could this data help recover?

In addition, human reads in gut metagenome data could be a good resource for stool-based forensics, robust variant calling, and polygenic risk scores based estimates of disease risks (e.g., type 2 diabetes).

Since this data could help quantitatively and precisely reconstruct genotype information, it could complement human WGS data.

About the study

In the present study, researchers applied a few humans reads in the gut metagenome data of the main study dataset to reconstruct personal information, including genetic sex and ancestry. For predicting genetic sex and the ancestries of these 343 individuals, they used sequencing depth of the sex chromosomes and modified likelihood score-based method, respectively.

In addition, the researchers developed methods to re-identify a person from a genotype dataset. Furthermore, they combined two harmonized genotype-calling approaches, the direct calling of rare variants and the two-step imputation of common variants, to reconstruct genotypes.

The main dataset of the study included 343 Japanese participants, whereas the validation dataset for the genetic sex prediction analysis comprised 113 Japanese individuals.

The multi-ancestry dataset, which helped the researchers validate ancestry prediction analysis, comprised 73 individuals of various nationalities, including samples from individuals in New Delhi, India.

The female and male participants in each dataset were 196 & 147, 65 & 48, and 25 & 48, respectively. Likewise, the age range for these three datasets was 20 to 88, 20 to 81, and 20 to 61 years, respectively.

Results and conclusion

Given that human reads in the gut metagenome data were derived consistently from all chromosomes, the read depth of the X chromosome was nearly double in females and that of the Y chromosome in males.

So, in a logistic regression analysis, when the researchers applied a 0.43 Y:X chromosome read-depth ratio to the validation dataset, which correctly predicted the genetic sex of 97.3% of the study samples.

In human microbiome and genetic research, the feasibility of sex prediction using human gut metagenome data could help remove mislabelled samples.

The study analysis also helped researchers remarkably predict ancestry in 98.3% of individuals using 1,000 Genomes Project (1KG) data as a reference.

However, the likelihood score-based method often misclassified South Asian (SAS) samples as American (AMR) and European (EUR), especially when the number of human reads was small. It is understandable because the genetic diversity of the SAS population is complex.

The likelihood score-based method also efficiently utilized the data from genomic areas with low coverage demonstrating the quantitative power of gut metagenome data to re-identify individuals and successfully re-identified 93.3% of individuals.

Despite ethical concerns, the re-identification method used in this study could help in the quality control of multi-omics datasets comprising gut metagenome and human germline genotype data.

In addition, the authors successfully reconstructed genome-wide common variants using genomic approaches. Historically researchers used stool samples as a source of germline genomes for wild and domestic animals but not humans.

Thus, further development of suitable methodologies could help efficiently utilize the human genome in gut metagenome data and benefit animal research.

Nonetheless, the study remarkably demonstrated that optimized methods could help reconstruct personal information from the human reads in gut metagenome data.

Moreover, the findings of this study could serve as a guiding resource to devise best practices for using the already accumulated gut metagenome data of humans.

Journal reference:

Tomofuji, Y. et al. (2023) “Reconstruction of the personal information from human genome reads in gut metagenome sequencing data”, Nature Microbiology. doi: 10.1038/s41564-023-01381-3. https://www.nature.com/articles/s41564-023-01381-3

Microbiology

Long-ignored antibiotic could help fight against multi-drug resistant bacteria

17 May, 2023 Admin Leave a comment

Reviewed by Megan Craig, M.Sc.May 16 2023

“The end of modern medicine as we know it.” That’s how the then-director general of the World Health Organization characterized the creeping problem of antimicrobial resistance in 2012. Antimicrobial resistance is the tendency of bacteria, fungus and other disease-causing microbes to evolve strategies to evade the medications humans have discovered and developed to fight them. The evolution of these so-called “super bugs” is an inevitable natural phenomenon, accelerated by misuse of existing drugs and intensified by the lack of new ones in the development pipeline.

Without antibiotics to manage common bacterial infections, small injuries and minor infections become potentially fatal encounters. In 2019, more than 2.8 million antimicrobial-resistant infections occurred in the United States, and more than 35,000 people died as a result, according to the Centers for Disease Control and Prevention (CDC). In the same year, about 1.25 million people died globally. A report from the United Nations issued earlier this year warned that number could rise to ten million global deaths annually if nothing is done to combat antimicrobial resistance.

For nearly 25 years, James Kirby, MD, director of the Clinical Microbiology Laboratory at Beth Israel Deaconess Medical Center (BIDMC), has worked to advance the fight against infectious diseases by finding and developing new, potent antimicrobials, and by better understanding how disease-causing bacteria make us sick. In a recent paper published in PLOS Biology, Kirby and colleagues investigated a naturally occurring antimicrobial agent discovered more than 80 years ago.

Using leading-edge technology, Kirby’s team demonstrated that chemical variants of the antibiotic, called streptothricins, showed potency against several contemporary drug-resistant strains of bacteria. The researchers also revealed the unique mechanism by which streptothricin fights off bacterial infections. What’s more, they showed the antibiotic had a therapeutic effect in an animal model at non-toxic concentrations. Taken together, the findings suggest streptothricin deserves further pre-clinical exploration as a potential therapy for the treatment of multi-drug resistant bacteria.

We asked Dr. Kirby to tell us more about this long-ignored antibiotic and how it could help humans stave off the problems of antimicrobial resistance a little longer.

Q: Why is it important to look for new antimicrobials? Can’t we preserve the drugs we have through more judicious use of antibiotics?

Stewardship is extremely important, but once you’re infected with one of these drug-resistant organisms, you need the tools to address it.

Much of modern medicine is predicated on making patients temporarily — and sometimes for long periods of time — immunosuppressed. When these patients get colonized with these multidrug-resistant organisms, it’s very problematic. We need better antibiotics and more choices to address multidrug resistance.

We have to realize that this is a worldwide problem, and organisms know no borders. So, a management approach for using these therapies may work well in Boston but may not in other areas of the world where the resources aren’t available to do appropriate stewardship.

Q: Your team investigated an antimicrobial discovered more than 80 years ago. Why was so little still known about it?

The first antibiotic, penicillin, was discovered in 1928 and mass produced for the market by the early 1940s. While a game-changing drug, it worked on only one of the two major classes of bacteria that infect people, what we call gram-positive bacteria. The gram-positive bacteria include staphylococcal infections and streptococcal infections which cause strep throat, skin infections and toxic shock. There still was not an antibiotic for the other half of bacteria that can cause human infections, known as gram-negative organisms.

In 1942, scientists discovered this antibiotic that they isolated from a soil bacterium called streptothricin, possibly addressing gram-negative organisms. A pharmaceutical company immediately licensed the rights to it, but the development program was dropped soon after when some patients developed renal or kidney toxicity. Part of the reason for not pursuing further research was that several additional antibiotics were identified soon thereafter which were also active against gram-negatives. So, streptothricin got shelved.

Q: What prompted you to look at streptothricin specifically now?

It was partly serendipity. My research laboratory is interested in finding new, or old and forgotten, solutions to treat highly drug-resistant gram-negative pathogens like E. coli or Klebsiella or Acinetobacter that we commonly see in hospitalized, immunocompromised patients. The problem is that they’re increasingly resistant to many if not all of the antibiotics that we have available.

Part of our research is to understand how these superbugs cause disease. To do that, we need a way to manipulate the genomes of these organisms. Commonly, the way that’s done is to create a change in the organism linked with the ability to resist a particular antibiotic that’s known as a selection agent. But for these super resistant gram-negative pathogens, there was really nothing we could use. These bugs were already resistant to everything.

We started searching around for drugs that we could use, and it turns out these super resistant bugs were highly susceptible to streptothricin, so we were able to use it as a selection agent to do these experiments.

As I read the literature on streptothricin and its history, I had the realization that it was not sufficiently explored. Here was this antibiotic with outstanding activity against gram-negative bacteria – and we confirmed that by testing it against a lot of different pathogens that we see in hospitals. That raised the question of whether we could get really good antibiotic activity at concentrations that are not going to cause damage to the animal or person in treatment.

Q: But it did cause kidney toxicity in people in 1942. What would be different now?

What scientists were isolating in 1942 was not as pure as what we are working with today. In fact, what was then called streptothricin is actually a mixture of several streptothricin variants. The natural mixture of different types of streptothricins is now referred to as nourseothricin.

In animal models, we tested whether we could kill the harmful microorganism without harming the host using a highly purified single streptothricin variant. We used a very famous strain of Klebsiella pneumoniae called the Nevada strain which was the first pan-drug-resistant, gram-negative organism isolated in the United States, an organism for which there was no treatment. A single dose cleared this organism from an infected animal model while avoiding any toxicity. It was really remarkable. We’re still in the very early stages of development, but I think we’ve validated that this is a compound that’s worth investing in further studies to find even better variants that eventually will meet the properties of a human therapeutic.

Q. How does nourseothricin work to kill gram-negative bacteria?

That’s another really important part of our study. The mechanism hadn’t been figured out before and we showed that nourseothricin acts in a completely new way compared to any other type of antibiotic.

It works by inhibiting the ability of the organism to produce proteins in a very sneaky way. When a cell makes proteins, they make them off a blueprint or message that tells the cell what amino acids to link together to build the protein. Our studies help explain how this antibiotic confuses the machinery so that the message is read incorrectly, and it starts to put together gibberish. Essentially the cell gets poisoned because it’s producing all this junk.

In the absence of new classes of antibiotics, we’ve been good at taking existing drugs like penicillin for example and modifying them; we’ve been making variations on the same theme. The problem with that is that the resistance mechanisms against penicillin and other drugs already exist. There’s a huge environmental reservoir of resistance out there. Those existing mechanisms of resistance might not work perfectly well against your new variant of penicillin, but they will evolve very quickly to be able to conquer it.

So, there’s recognition that what we really want is new classes of antibiotics that act in a novel way. That’s why streptothricin’s action uncovered by our studies is so exciting. It works in a very unique way not seen with any other antibiotic, and that is very powerful because it means there’s not this huge environmental reservoir of potential resistance.

Q. You emphasize these are early steps in development. What are the next steps?

My lab is working very closely with colleagues at Northeastern University who figured out a way to synthesize streptothricin from scratch in a way that will allow us to cast many different variants. Then we can look for ones that have the ideal properties of high potency and reduced toxicity.

We are also continuing our collaboration with scientists at Case Western Reserve University Medical Center, diving more deeply to understand exactly how this antibiotic works. Then we can use that fundamental knowledge in our designs of future variants and be smarter about how we try to make the best antibiotic.

We have great collaborators that have allowed us to pursue a project that crosses multiple fields. This work is an example of collaborative science really at its best.

Co-authors included first author Christopher E. Morgan and Edward W. Yuof Case Western Reserve; Yoon-Suk Kang,Alex B. Green, Kenneth P. Smith, Lucius Chiaraviglio, Katherine A. Truelson, Katelyn E. Zulauf, Shade Rodriguez, and Anthony D. Kang of BIDMC; Matthew G. Dowgiallo,Brandon C. Miller, and Roman Manetsch of Northeastern University.

Source:

Beth Israel Deaconess Medical Center

Microbiology

Study identifies key genetic mechanism of drug resistance in the deadliest malaria parasites

13 May, 2023 Admin Leave a comment

Reviewed by Emily Henderson, B.Sc.May 11 2023

An important genetic mechanism of drug resistance in one of the deadliest human malaria parasites has been identified in a new study published in Nature Microbiology.

A second key gene, pfaat1, responsible for encoding a protein that transports amino acids in the membrane of Plasmodium falciparum, is involved in its resistance to the major anti-malaria drug, chloroquine.

The findings may have implications for the ongoing battle against malaria, which infects an estimated 247 million people worldwide and kills more than 619,000 each year, most of which are young children.

Chloroquine is a major antimalaria drug, however in recent years, resistance has emerged in malaria parasites, first spreading through Southeast Asia and then through Africa in the 1970s and 1980s. Although alternative antimalarial drugs have been developed, resistance to chloroquine remains a big challenge.

Since its discovery in 2000, only one gene has been believed to have been responsible for resistance to chloroquine – the resistance transporter pfcrt which helps the malaria parasite transport the drug out of a key region in their cells, subsequently rendering it ineffective.

In this study, researchers from the Medical Research Council (MRC) Unit The Gambia at the London School of Hygiene & Tropical Medicine (LSHTM) analysed more than 600 genomes of P. falciparum that were collected in The Gambia over a period of 30 years. The team found that mutant variants of a second gene, pfaat1, which encodes an amino acid transporter, increased in frequency from undetectable to very high levels between 1984 and 2014. Importantly, their genome-wide population analyses also indicated long term co-selection on this gene alongside the previously-known resistance gene pfcrt.

In the laboratory, a further team of researchers including from Texas Biomed, University of Notre Dame and Seattle Children’s Research Institute found that replacing these mutations in parasite genomes using CRISPR gene-editing technology impacted drug resistance. A team from Nottingham University also found that these mutations could impact the function of pfaat1 in yeast, resulting in drug resistance.

Complementary analysis of malaria genome datasets additionally suggested that parasites from Africa and Asia may carry different mutations in pfaat1 which could help explain differences in the evolution of drug resistance across these continents.

Alfred Amambua-Ngwa, Professor of Genetic Epidemiology at MRC Unit The Gambia at LSHTM said: “This is a very clear example of natural selection in action – these mutations were preferred and passed on with extremely high frequency in a very short amount of time, suggesting they provide a significant survival advantage.

“The mutations in pfaat1 very closely mirror the increase of pfcrt mutations. This, and other genetic analyses in the paper demonstrate that the transporter AAT1 has a major role in chloroquine resistance.”

Grappling with drug resistance, for malaria and other pathogens, requires taking a holistic approach to both drug development and pathogen surveillance. We must be aware that different genes and molecules will be working together to survive treatments. That is why looking at whole genomes and whole populations is so critical.”

David Conway, Professor of Biology, LSHTM

Source:

London School of Hygiene & Tropical Medicine (LSHTM)

Journal reference:

Amambua-Ngwa, A., et al. (2023). Chloroquine resistance evolution in Plasmodium falciparum is mediated by the putative amino acid transporter AAT1. Nature Microbiology. doi.org/10.1038/s41564-023-01377-z.

The grants

Please can you introduce yourself, tell us about your career background, and what inspired your career in biology and medicine?

You started working on biological applications of the atomic force microscope (AFM) in 1992. From your perspective, how has the antibiotic resistance landscape changed over the last two decades? What role has the advancement in technology played in furthering our understanding?

Were there any limitations and obstacles you faced in the research process? If so, how did you overcome them?

You worked alongside Dr. Ronnie Willaert, a professor of structural biology at Vrije Universiteit Brussel, on developing this new rapid AST technique. How did your areas of expertise and research backgrounds complement each other in developing ONMD?

The field of antimicrobial resistance requires a high level of international collaboration, with everyone working together to achieve a common goal. With antimicrobial resistance rising to dangerously high levels in all parts of the world, how important is collaboration in this field?

You and Dr. Willaert have said, ‘The simplicity and efficiency of the method make it a game-changer in the field of AST.’ Can you please expand on what makes ONMD a game changer in the AST field and what implications this research could have in clinical and research settings?

While we recognize the importance of rapid AST, what next steps must be taken before this technique can be used globally in research and clinical landscapes?

What’s next for you and your research? Are you involved in any exciting upcoming projects?

Where can readers find more information?

About Dr. Sandor Kasas

Background

About the study

Results and conclusion

Microbiome: From Research and Innovation to Market