Study could lead to new antibiotics to fight bacterial infections

One member of a large protein family that is known to stop the spread of bacterial infections by prompting infected human cells to self-destruct appears to kill the infectious bacteria instead, a new study led by UT Southwestern scientists shows. However, some bacteria have their own mechanism to thwart this attack, nullifying the deadly protein by tagging it for destruction.

The findings, published online today in Cell, could lead to new antibiotics to fight bacterial infections. And insight into this cellular conflict could shed light on a number of other conditions in which this protein is involved, including asthma, Type 1 diabetes, primary biliary cirrhosis, and Crohn's disease.

This is a wonderful example of an arms race between infectious bacteria and human cells."

Neal M. Alto, Ph.D., Study Leader and Professor of Microbiology at UT Southwestern and  Member of Harold C. Simmons Comprehensive Cancer Center

Previous research has shown that the protein, called gasdermin B (GSDMB), was different from other members of the mammalian gasdermin family. Related gasdermin proteins form pores in the membranes of infected cells, killing them while allowing inflammatory molecules to leak out and incite an immune response. However, GSDMB – found in humans but not in some other mammalian species, including rodents – doesn't form pores in the membranes of cultured mammalian cells, leaving its target a mystery.

Using a novel screening technology, Alto and colleagues discovered that a protein toxin called IpaH7.8 from shigella flexneri, a bacterium that causes diarrheal disease, directly inhibits GSDMB. Biochemical experiments show that IpaH7.8 places a chemical tag on GSDMB that marks it for cellular destruction.

To understand why shigella flexneri rids human cells of GSDMB, the researchers placed GSDMB within synthetic mammalian and bacterial cell membranes. While GSDMB left the synthetic mammalian membranes unharmed, it poked holes in the bacterial membranes. Further investigation showed that immune cells called natural killer cells stimulate this process.

Alto notes that inhibiting the ability of shigella IpaH7.8 to counteract GSDMB could lead to new types of antibiotics. And because genetic variants of GSDMB have been linked to a variety of inflammatory diseases and cancer, better understanding this protein could lead to new treatments for these conditions too.

Source:

UT Southwestern Medical Center

Journal reference:

Hansen, J. M., et al. (2021) Pathogenic ubiquitination of GSDMB inhibits NK cell bactericidal functions. Cell. doi.org/10.1016/j.cell.2021.04.036.

Posted in: Medical Research News | Disease/Infection News

Tags: Asthma, Bacteria, Cancer, Cell, Cirrhosis, Crohn's Disease, Diabetes, Education, Genetic, Immune Response, Mammalian Cells, Medical Research, Medicine, Microbiology, Natural Killer Cells, pH, Primary Biliary Cirrhosis, Protein, Research, Shigella, Toxin, Type 1 Diabetes

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Study highlights significant gap in evidence related to effectiveness of portable air filters in reducing COVID-19

Considerable gap in evidence around whether portable air filters reduce the incidence of COVID-19 and other respiratory infections.

There is an important absence of evidence regarding the effectiveness of a potentially cost-efficient intervention to prevent indoor transmission of respiratory infections, including COVID-19, warns a study by researchers at the University of Bristol.

Respiratory infections such as coughs, colds, and influenza, are common in all age groups, and can be either viral or bacterial. Bacteria and viruses can become airborne via talking, coughing or sneezing. The current global coronavirus (COVID-19) pandemic is also spread primarily by airborne droplets, and to date has led to over three million deaths worldwide.

Controlling how we acquire and transmit respiratory infections is of huge importance, particularly within indoor environments such as care homes, households, schools/day care, office buildings and hospitals where people are in close contact.

Several manufacturers of portable air filters have claimed their products remove potentially harmful bacteria and viruses from indoor air, including COVID-19 viral particles. However, there is often no detailed evidence provided on their websites to corroborate their claims for potential consumers to review before purchasing.

A team of UK researchers from the University of Bristol reviewed previous studies to investigate whether portable air filters used in any indoor setting can reduce incidence of respiratory infections and thus, whether there is any evidence to recommend their use in these settings to reduce the spread of COVID-19 and other respiratory infections. The team also explored whether portable air filters in indoor settings capture airborne bacteria and viruses within them, and if so, what specifically is captured.

The researchers found no studies investigating the effects of portable, commercially available air filters on the incidence of respiratory infections in any indoor community setting. Two studies reported removal or capture of airborne bacteria in indoor settings (an office and emergency room), demonstrating that the filters did capture airborne bacteria and reduced the amount of airborne bacteria in the air. Neither tested for the presence of viruses in the filters, nor a reduction in viral particles in the air.

The study, funded by Professor Alastair Hay's National Institute for Health Research Senior Investigator Award and published in PLoS One, was a systematic review of studies published after 2000 reporting (i) effects of portable air filters on incidence of respiratory infection, or (ii) whether filters capture and/or remove aerosolised bacteria and viruses from the air, including information of what is captured. Studies reporting non-portable air filters were excluded from this study.

Our study highlights the considerable gap in evidence related to the effectiveness of portable air filters in preventing respiratory infections, including COVID-19. Whilst we found some evidence suggesting use of air filters could theoretically contribute to reducing the spread of COVID-19 and other respiratory infections by capturing airborne particles, there is a complete absence of evidence as to whether they actually reduce the incidence of these infections."

Dr Ashley Hammond, Study Lead Author, Infectious Disease Epidemiologist, Centre for Academic Primary Care, University of Bristol

Professor Alastair Hay, a GP and Professor of Primary Care at the Centre for Academic Primary Care, University of Bristol, and the research group lead, said: "Randomised controlled trials are urgently needed to demonstrate the effects of portable air filters on incidence of respiratory infections, including COVID-19. The main research questions should focus primarily on whether use of portable air filters in any indoor environment can reduce respiratory infections compared to those environments without portable air filters."

Source:

University of Bristol

Journal reference:

Hammond, A., et al. (2021) Should homes and workplaces purchase portable air filters to reduce the transmission of SARS-CoV-2 and other respiratory infections? A systematic review. PLOS One. doi.org/10.1371/journal.pone.0251049.

Posted in: Device / Technology News | Medical Research News | Disease/Infection News

Tags: Bacteria, Coronavirus, Coronavirus Disease COVID-19, Coughing, Influenza, Pandemic, Primary Care, Research, Respiratory, SARS, SARS-CoV-2, Sneezing

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Novel platform has potential to detect many disease-related biomarkers in just one test

Most conventional molecular diagnostics usually detect only a single disease-related biomarker. Great examples are the PCR tests currently used to diagnose COVID-19 by detecting a specific sequence from SARS-CoV-2.

Such so-called singleplex methods provide reliable results because they are "calibrated" to a single biomarker. However, determining whether a patient is infected with a new SARS-CoV-2 variant or a completely different pathogen requires probing for many different biomarkers at one time.

Scientists from the Helmholtz Institute for RNA-based Infection Research (HIRI) and the Julius Maximilians University (JMU) in Würzburg have now paved the way for a completely new diagnostic platform with LEOPARD. It is a CRISPR-based method that is highly multiplexable, with the potential to detect a variety of disease-related biomarkers in just one test.

How LEOPARD works

LEOPARD, which stands for "Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection," is based on the finding that DNA cutting by Cas9 could be linked to the presence of a specific ribonucleic acid (RNA). This link allows LEOPARD to detect many RNAs at once, opening opportunities for the simultaneous detection of RNAs from viruses and other pathogens in a patient sample.

The study published today in "Science" was initiated by Chase Beisel, professor at JMU and research group leader at HIRI, and Professor Cynthia Sharma from JMU's Institute of Molecular Infection Biology (IMIB). "With LEOPARD, we succeeded in detecting RNA fragments from nine different viruses,' says Beisel. "We were also able to differentiate SARS-CoV-2 and one of its variants in a patient sample while confirming that each sample was correctly collected from the patient."

Background

CRISPR-Cas9 is principally known as a biomolecular tool for genome editing. Here, CRISPR-Cas9 function as molecular scissors that cut specific DNA sequences. These same scissors are naturally used by bacteria to cut DNA associated with invading viruses.

Whether editing genomes or eliminating viruses, Cas9 cutting is directed by guide RNAs. The guide RNAs found in bacteria must pair with a separate RNA called the tracrRNA. The RNA couple then can work with Cas9 to direct DNA cutting.

An unexpected discovery

The tracrRNA was thought to only pair with guide RNAs coming from the antiviral system. However, the Würzburg scientists discovered that the tracrRNA was pairing with other RNAs, turning them into guide RNAs.

When we searched for RNAs binding to Cas9 in our model organism Campylobacter, we surprisingly found that we detected not only guide RNAs, but also other RNA fragments in the cell that looked like guide RNAs. The tracrRNA was pairing with these RNAs, resulting in "non-canonical" guide RNAs that could direct DNA cutting by Cas9."

Cynthia Sharma, Chair, Molecular Infection Biology II, Institute of Molecular Infection Biology

Sharma is also a spokesperson of the Research Center for Infection Diseases (ZINF) at JMU. The LEOPARD diagnostic platform builds on this discovery. "We figured out how to reprogram the tracrRNAs to decide which RNAs become guide RNAs," says Beisel.

"By monitoring a set of matching DNAs, we can determine which RNAs were present in a sample based on which DNAs get cut. As part of the ongoing pandemic, LEOPARD could allow a doctor to figure out whether the patient is infected with SARS-CoV-2, if it's a unique variant, and whether the sample was correctly taken or needs to be repeated–all in one test."

In the future, LEOPARD's performance could dwarf even multiplexed PCR tests and other methods. "The technology has the potential to revolutionize medical diagnostics not only for infectious diseases and antibiotic resistances, but also for cancer and rare genetic diseases," says Oliver Kurzai, director of JMU's Institute of Hygiene and Microbiology, which provided patient samples for the study.

"The work highlights the excellent collaborative and interdisciplinary research taking place here in Würzburg," says Jörg Vogel, director of IMIB and HIRI, a joint facility of JMU with the Helmholtz Center for Infection Research in Braunschweig. "LEOPARD impressively demonstrates that we can cover the full spectrum of complementary cutting-edge research in Würzburg, from the fundamentals of RNA research to clinical applications."

Source:

University of Würzburg

Journal reference:

Jiao, C., et al. (2021) Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9. Science. doi.org/10.1126/science.abe7106.

Posted in: Molecular & Structural Biology | Cell Biology | Genomics

Tags: Antibiotic, Bacteria, Biomarker, Campylobacter, Cancer, Cas9, Cell, CRISPR, Diagnostic, Diagnostics, DNA, Doctor, Genetic, Genome, Genome Editing, Hygiene, Infectious Diseases, Microbiology, Molecular Diagnostics, Pandemic, Pathogen, Research, Ribonucleic Acid, RNA, SARS, SARS-CoV-2

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Nanobody cocktails neutralize SARS-CoV-2 and variants

Researchers report a collection of nanobodies derived from llamas, some of which can work synergistically and work against newer virus variants.

Although several strategies have been implemented to combat the COVID-19 pandemic, it continues unabated. Despite several vaccines being approved and used, vaccination rates have been slow, and there have been challenges in the equitable distribution of vaccines. These, along with decreasing immunity in persons already infected and the emergence of new virus variants, make it difficult to contain the pandemic.

Several therapeutic strategies have used convalescent sera and human monoclonal antibodies. However, new variants have emerged, with mutations that can evade these therapeutics.

An alternative to monoclonal antibodies is nanobodies. These are smaller proteins derived from animals like llamas and alpacas. Their small size allows them to bind to regions that are generally not accessible to the larger monoclonal or polyclonal antibodies. They are also simpler to manufacture as they can be easily cloned and expressed in bacteria. They can also be delivered directly to the lungs via nebulization.

However, the nanobodies also recognize regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain (RBD), which can cause escape mutations, reducing their potency.

Pre-screening protocol to select llamas with naturally strong immune responses, as determined by activity against standard animal vaccines

Testing nanobody neutralization capability

In a study published in the bioRxiv* preprint server, researchers report a collection of a large number of nanobodies that could potentially be resistant to escape mutations.

To build their suite of nanobodies, the team refined and optimized their previous method. They chose llamas with a strong immune response to SARS-CoV-2 and selected 113 nanobodies for further testing. A large proportion of the antibodies bind to the RBD, while the other bind to the non-RBD portion of the S1 subunit of the spike protein and the rest to the S2 subunit.

To identify nanobodies that will be resistant to virus mutations, the team selected a large number of high RBD binding nanobodies and tested them against the B.1.1.7 variant. Of the seven nanobodies they tested, they found six showed strong binding to the variants. In addition, they also chose nanobodies that bind to the non-RBD regions of the spike protein.

The authors then categorized the antibodies depending on what parts of the RBD they bind to. Their analysis revealed the RBD-binding nanobodies could be classified into three distinct groups. Within each group, the nanobodies could be grouped into bins so that some bind to distinct epitopes and partially overlap, binding to other separate epitopes.

This suggests two or more nanobodies can bind to the RBD at the same time. Further analysis revealed at least three different nanobodies could bind to the RBD simultaneously, which will be important in designing nanobody cocktails for therapies.

The nanobodies were potent in neutralizing SARS-CoV-2 pseudoviruses, with 16 nanobodies neutralizing the pseudovirus at less than 20 nM concentration. Nanobodies that target the non-RBD regions and the S2 subunit also neutralized the virus but at higher concentrations. The authors write this is the first evidence of nanobody neutralization targeting regions outside the RBD.

Nanobody cocktails more potent against escape variants

The team also tested the best nanobodies against pseudoviruses carrying the spike protein of the B.1.351 variant. Some nanobodies showed no neutralization activity against this variant, while one showed similar neutralization as the wild-type virus. Two nanobodies, however, showed increased neutralization activity against the variant. The nanobodies that neutralized the pseudovirus also neutralized the real SARS-CoV-2 virus and human airway epithelial cells. The team also found some nanobody combinations can work synergistically and increase potency dramatically.

Using nanobody cocktails mixed with the SARS-CoV-2 virus and allowing multiple replications, the authors also identified mutations that resist neutralization. Some potent nanobodies caused mutations at the same location as generated by antibodies in human convalescent sera, such as E484K, indicating the ACE2 binding site is a point of vulnerability for neutralization.

However, nanobody cocktails also increased the genetic barrier for escape. Mixing two nanobodies required the virus to undergo two different amino acid substitutions, making it more difficult for it to escape neutralization. Carefully choosing mixtures with more nanobodies may further decrease escape mutations.

Thus, the escape experiments show that currently used or newer therapeutics may yet lose their potency with newer virus variants emerging, which is very likely as some of the mutations seen in the experiments have not been seen in human isolates so far. However, using a combination of nanobodies, which can work synergistically to improve neutralization potency, the large collection of nanobodies generated by the team may help develop more potent treatments.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Mast, F. D. et al. (2021) Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2. bioRxiv, https://doi.org/10.1101/2021.04.08.438911, ​https://www.biorxiv.org/content/10.1101/2021.04.08.438911v1

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: ACE2, Amino Acid, Antibodies, Bacteria, Coronavirus, Coronavirus Disease COVID-19, Genetic, Immune Response, Lungs, Nanobodies, Pandemic, Protein, Pseudovirus, Receptor, Respiratory, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Therapeutics, Virus

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Written by

Lakshmi Supriya

Lakshmi Supriya got her BSc in Industrial Chemistry from IIT Kharagpur (India) and a Ph.D. in Polymer Science and Engineering from Virginia Tech (USA).

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To combat gum disease, help oral bacteria evolve

To combat gum disease, help oral bacteria evolve

Liver disease, from metabolic and bacterial causes, is a growing concern. What connects these dots? The gut, or more specifically, bacteria in the gut. Bacteria that cause inflammation in the mouth are transported through the digestive tract to the gut and liver, where they can cause liver inflammation. Lipopolysaccharides, important structural molecules in some bacteria, act as endotoxins, producing systemic effects that can manifest as non-alcoholic fatty liver disease (NAFLD). Now, a multidisciplinary team from the University of Tsukuba show that exercise could be used to improve the oral environment in people with NAFLD, potentially leading to a new treatment for the disease.

These researchers previously demonstrated that exercise benefits patients with NAFLD by reducing fat, inflammation, and scarring in the liver; improving the liver’s response to and clearance of the endotoxin; and reducing gum disease. With the latest study in their series, they add another signpost to uncharted territory:

“We know that exercise has innumerable benefits to health overall and for these specific conditions,” says corresponding author Professor Junichi Shoda. “With this study, we sought to characterize underlying mechanisms—that is, show how exercise alters physiology and how altered physiology induces changes in oral bacteria.”

The researchers carried out biochemical and genetic analyses on saliva from overweight men with NAFLD and gum disease before and after 12-week exercise or diet programs. Men in both groups were able to lose fat mass, but those following dietary restrictions also lost muscle mass, whereas those following the exercise program gained muscle mass. “More importantly, we found that reductions in lactoferrin, lipopolysaccharide, and IgA concentrations were only evident in the men who followed the exercise regimen,” Professor Shoda explains, “which suggested that the oral environment had been significantly altered by exercise.”

The samples from the exercise group also showed increased bacterial diversity and changes in the relative constituent bacterial populations. In the overall population, more bacteria expressed genes related to environmental information processing, and less bacteria expressed genes related to genetic information processing and metabolism. In fact, bacteria expressed fewer genes for producing lipopolysaccharides.

“Therefore, it seems that, in people with both non-alcoholic liver disease and gum disease, exercise causes a biochemical shift in the environment of the mouth that favors the survival of less harmful bacteria,” explains Professor Shoda.

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Study reveals molecular mechanisms of drug resistance in Mycobacterium tuberculosis

A consortium of researchers from Russia, Belarus, Japan, Germany and France led by a Skoltech scientist have uncovered the way in which Mycobacterium tuberculosis survives in iron-deficient conditions by utilizing rubredoxin B, a protein from a rubredoxin family that play an important role in adaptation to changing environmental conditions.

The new study is part of an effort to study the role of M. tuberculosis enzymes in developing resistance to the human immune system and medication. The paper was published in the journal Bioorganic Chemistry.

According to the World Health Organization, every year 10 million people fall ill with tuberculosis and about 1.5 million die from it, making it the world's top infectious killer. The bacterium that causes TB, Mycobacterium tuberculosis, is notorious for its ability to survive within macrophages, cells of the immune system that destroy harmful bacteria.

Continuing spread of drug resistance of M. tuberculosis to widely used therapeutics over recent decades became a substantial clinical problem. In this regard, the identification of novel molecular drug targets and deciphering the molecular mechanisms of drug resistance are of pivotal importance.

Natallia Strushkevich, Assistant Professor at the Skoltech Center for Computational and Data-Intensive Science and Engineering (CDISE), and her colleagues studied the crystal structure and function of rubredoxin B (RubB), a metalloprotein that ensures the proper functioning of cytochrome P450 (CYP) proteins essential to bacterial survival and pathogenicity.

The team hypothesizes that M. tuberculosis switched over to more iron-efficient RubB to survive iron starvation when granulomas are formed (these are largely unsuccessful attempts at defense against TB by the immune system).

During the long-term co-evolution with mammals, M. tuberculosis developed a variety of strategies to subvert or evade the host innate immune response, from recognition of the bacterium and phagosomal defenses within infected macrophages, to adaptive immune responses by antigen presenting cells. Iron assimilation, storage and utilization is essential for M. tuberculosis pathogenesis and also involved in emergence of multi- and extensively-drug resistant strains. Heme is the preferable iron source for M. tuberculosis and serves as a cofactor for various metabolic enzymes."

Natallia Strushkevich, Assistant Professor, Skoltech Center for Computational and Data-Intensive Science and Engineering (CDISE)

Based on our finding, we linked rubredoxin B to heme monoooxygenases important for metabolism of host immune oxysterols and anti tubercular drugs. Our findings indicate that M. tuberculosis has its own xenobiotics transformation system resembling human drug metabolizing system," explains Natallia Strushkevich.

According to Natallia: New targets for drug design efforts are in great demand and the cytochrome P450 enzymes have emerged as novel targets for the development of tuberculosis therapeutic agents.

The classic approaches to block these enzymes are not straightforward. Finding the alternative redox partner, such as RubB, enables further understanding of their function in different host microenvironments. This knowledge could be exploited to identify new ways to block their function in M. tuberculosis.

Earlier research by the consortium showed that one of the CYPs enabled by RubB can act against SQ109, a promising drug candidate against multidrug-resistant tuberculosis. Another study focused on how Mycobacterium tuberculosis protects itself by intercepting human immune signaling molecules — a hurdle that limits drug discovery.

Source:

Skolkovo Institute of Science and Technology (Skoltech)

Journal reference:

Sushko, T., et al. (2021) A new twist of rubredoxin function in M. tuberculosis. Bioorganic Chemistry. doi.org/10.1016/j.bioorg.2021.104721.

Posted in: Molecular & Structural Biology | Microbiology | Biochemistry

Tags: Aging, Antigen, Artificial Intelligence, Bacteria, Biochemistry, Cytochrome P450, Drug Discovery, Drugs, Evolution, Immune Response, Immune System, Metabolism, Photonics, Protein, Research, Structural Biology, Therapeutics, Tuberculosis

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Novel host-viral-microbiome interactions during COVID-19 may determine outcome

The current pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly for over a year. Though primarily a respiratory illness, its manifestations are often protean and may be life-threatening.

A new preprint on the medRxiv* server discusses how the underlying disease mechanisms are regulated such that the local or mucosal immune response is distinct from the systemic response.

Study: Distinct systemic and mucosal immune responses to SARS-CoV-2. Image Credit: Andrii Vodolazhskyi / Shutterstock

Viral suppression of innate but not adaptive immunity

The initial infection of epithelial cells in the upper respiratory tract, via the angiotensin-converting enzyme 2 (ACE2), triggers early innate defenses that prevent replicative infection and progressive disease.

These include immune and non-immune components, such as mucus and certain chemicals produced during the course of metabolism, as well as cell signaling proteins (cytokines) and interferons that are either produced during the normal cell cycle or in response to infection.

It has been established that the virus suppresses the activation of the innate immune system, beginning with the dendritic cells that present antigens to the immune effector and antibody-producing cells. It also reduces the intensity of type I and II interferon antiviral responses. The result is the hyperactivation of inflammatory macrophages.

Adaptive immune responses play a later role. These include antibodies such as the secretory immunoglobulin (Ig) A that guards the mucosal barriers and has been detected in COVID-19 patients, within blood, saliva and nasopharyngeal samples.

Lymphocyte counts in the peripheral blood are low in COVID-19 patients, but both B and T cells show efficient and specific antiviral memory responses. This includes high numbers of plasma cells which secrete specific neutralizing antibodies to the viral spike protein.

The specific T cell responses in the blood are associated with disease severity, which is therefore not the result of defective adaptive immunity, at least in the initial stages.

Cytokine storm in severe COVID-19

The cytokine storm characterized by systemic hyperinflammation, in proportion to viral ribonucleic acid (RNA) loads in the tissues, is a notable feature of severe and critical COVID-19.

The current study sought to identify the regulatory factors in local and systemic immunity to SARS-CoV-2 infection associated with the clinical phenotype.

The researchers found that nasal and systemic immunity were very different from each other in the same individual. The chief differences involve local cytokines in the nose and the nasal microbiome.

Strong local and systemic antibody responses

Following infection, the researchers found spike-specific IgG and IgA antibodies in plasma, with the titers and probability being proportional to disease severity. Plasma neutralizing activity was also proportional to disease severity, and to the frequency of anti-spike IgA and IgG.

Total IgM, IgG and IgA levels, and IgG subclasses, were similar in patients and healthy controls.

Anti-spike IgA, and IgG, responses, were higher in nasopharyngeal secretions as well, in proportion to the severity of the disease. Notably, critical patients showed an increase in total IgA in nasal secretions.

These findings show that they are mounted against the viral spike protein in acute COVID-19.

independent regulation of mucosal and systemic immunity

Making use of paired nasopharyngeal-plasma specimens, the researchers found that almost 90% of patients seroconverted, with both IgG and IgA anti-spike antibodies.

However, much fewer showed antibodies to the spike in their nasopharyngeal secretions. Those who did show such “nasoconversion,” however, developed anti-spike IgG and IgA.

Among the group of controls and infected individuals, both plasma and nasopharynx showed spike antibodies in about 30%. About 37% showed seroconversion but not nasoconversion. The latter occurred alone in 5%, and 30% of people showed neither.

All controls were in the last category, of course. However, two moderate COVID-19 patients were also seronegative and nasonegative at this point in time.

Two critically ill patients were seronegative but strongly nasopositive. The other patients were equally split between being both seropositive and nasopositive, or seropositive without nasoconversion.

About 12% failed to seroconvert altogether.

Strangely, there was no obvious relationship between the systemic and local spike antibodies in the same individual. Plasma levels of anti-spike IgA and IgG antibody levels vs antibody titers in the nasopharynx were not correlated, neither was IgA correlated with IgG responses.

This result suggests independent regulation of mucosal and systemic immune responses to SARS-CoV-2.”

Cytokines mediate inflammation and local immunity

The study also showed that ten cytokines in plasma were significantly different in critical COVID-19 patients compared to other patients, but in the nasopharynx, 13 cytokines were differentially regulated. Only two were common between the two groups.

Some of the nasal cytokines were higher in sicker patients. Thus, even cytokines appear to be differentially regulated in SARS-CoV-2 infection depending on the compartment of infection. The secretion of interferons was not associated with antiviral antibodies.

The higher levels of certain cytokines, but not interferons, in association with anti-spike antibodies in the nasopharyngeal secretions suggest that the former are involved in inflammation, and thus in the generation of local antibodies.

Viral load and immune response

The investigators found that the viral load is higher in both the local and the systemic compartments in COVID-19 patients, but they appeared to be independent of each other.

Plasma viral loads predicted the systemic inflammatory response and higher levels of specific regulatory cytokines, but a lower interferon response. This supports earlier findings of virus-induced hyper-inflammation. They also predicted high plasma anti-spike IgA and IgG, showing that they drive spike-specific antibody responses.

Nasopharyngeal viral loads showed inverse associations with inflammatory cytokines.

Microbiome regulation of immune responses

The researchers also found that SARS-CoV-2 infection is associated with disturbances of the nasopharyngeal microbiome, and that patients with critical COVID-19 patients show dysbiosis.

Moreover, following infection with this virus, the levels of certain cytokines fell, such as IL-33, IFNγ, IFNα/β and IFNλ3. These are associated with higher counts of ‘good’ bacteria that may increase resistance to SARS-CoV-2 and increased diversity overall.

Viral load, spike antibodies, neutralizing capacity and inflammatory cytokines were found to be associated with microbial community composition and growth. Thus, bacterial communities in the nose are closely associated with local and systemic inflammatory signaling and antibody responses during COVID-19.

Critical COVID-19 patients showed, in this study, a cluster of cytokines and growth factors at high levels in their blood which do not appear to be related to antiviral mechanisms. Instead, they are possibly linked to the regulation of the nasal microbiome, such that increases in these cytokines drive down cornerstone genera like Corynebacterium and Dolosigranulum.

Conversely, the inflammatory cytokine IL-6 is associated with higher levels of the pathobiontic Staphylococcus genus.

What are the implications?

The researchers suggest that protective immunity in SARS-CoV-2 infection hinges on several important regulatory points. One is the nasal microbiome, which is disrupted in this infection and results in a decrease in some cytokines that are important in controlling the virus.

The second is the local cytokine profile in the nasal mucosa, which determines the production of local antibodies in the nasopharynx. And thirdly, some bacterial genera increase in association with higher levels of inflammation, both mucosal and systemic. These appear to be the result of specific cytokine release patterns and are associated with poor outcomes.

This indicates the need to understand, through future studies, how the nasal microbiome is involved in the local and systemic reactions to the infection. Some researchers have shown a possible link between microbiota in the nose and the baseline production of type I and type III interferons.

Such individual differences in the amount of interferon secreted by the nasal bacteria could explain, in part, why different individuals respond so differently to the virus.

The range of mucosal immune responses (or the absence thereof) may also determine novel therapeutic modalities to enhance individual protection against the virus by increasing specific IgA production. Especially important may be the cytokine CCL2 and type I interferon.

The presence of SARS-CoV-2 infection could cause epithelial barrier breakdown and perturbations of nasal flora. These changes may, in turn, allow nasal pathobionts to enter the body, triggering systemic inflammation. A similar cycle has been reported to occur in the gut.

As a result, these patients may have a higher risk of severe COVID-19 disease. “Our study identifies novel host-viral-microbiome interactions during infection with SARS-CoV-2 which may help new strategies for identifying at risk individuals.”

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Smith, N. et al. (2021). Distinct systemic and mucosal immune responses to SARS-CoV-2. medRxiv preprint. doi: https://doi.org/10.1101/2021.03.01.21251633, https://www.medrxiv.org/content/10.1101/2021.03.01.21251633v1

Posted in: Medical Science News | Medical Research News | Miscellaneous News | Disease/Infection News | Healthcare News

Tags: ACE2, Angiotensin, Angiotensin-Converting Enzyme 2, Antibodies, Antibody, Bacteria, Blood, Cell, Cell Cycle, Cell Signaling, Coronavirus, Coronavirus Disease COVID-19, Cytokine, Cytokines, Dysbiosis, Enzyme, Frequency, Immune Response, Immune System, Immunoglobulin, Inflammation, Lymphocyte, Metabolism, Microbiome, Pandemic, Phenotype, Protein, Respiratory, Respiratory Illness, Ribonucleic Acid, RNA, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Virus

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Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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Technology Advances in Bacteria Detection

Bacterial infections are an increasing public health threat globally. The ability to rapidly detect infections in potentially life-threatening conditions such as sepsis is vital.

Researchers have presented novel detection techniques that can successfully identify and distinguish healthy and non-viable bacteria within minutes, potentially saving lives and money.  

Image Credit: nobeastsofierce/Shutterstock.com

The Important of Bacterial Testing

The modern medical practice relies heavily on bacterial testing. In progressive infections such as sepsis, the mortality rate increases by 8% per hour of delayed treatment.

While up to 30% of those presenting with urinary tract infections are not diagnosed using dipsticks, particularly in those with lower levels of infection.

In either instance, delays in diagnosis can lead to life-threatening consequences as the infection continues to become established. Similarly, delays in identifying cases of contamination in industrial samples can result in adverse economic outcomes.

Typical techniques of bacterial detection include enzyme-linked immunosorbent assay, plate culturing, and polymerase chain reaction. While the tests are credited for their sensitivity and accuracy, practitioners have to wait several days for the results.

Emerging research aimed to address these issues combining mathematical modeling, biology, and engineering principles to create a novel method to detect viable bacteria.

Ultra-fast Technology to Detect Bacteria

The investigation of a cell's electrical characteristic in response to being exposed to external electric fields, known as bioelectricity, is central to the current study.

As a topic, the investigation of bacterial bioelectricity is quite contemporary, but as a result, researchers have discovered that bacteria need a constant resting potential to replicate and use approximately half their energy to maintain this equilibrium.

Previously, fluorophore-mediated and time-lapse microscopy single-cell membrane studies have been used to identify proliferative capacity. One issue with such studies is that membrane potential changes can occur in several situations, creating generalized results if thorough calibrations are not conducted first.

The team of researchers from the University of Warwick, observed the changes in membrane potential and cell proliferation in response to electrical stimulation using a specially-developed device in two species of bacteria, Bacillus subtilis (B. subtilis) and Escherichia coli (E. coli).

A technique called phase-contrast time-lapse microscopy was used to observe when proliferative bacteria absorbed dyed fluorescent molecules used as membrane voltage indictors.

Following the administration of a 2.5-second electrical pulse, an intense fluorescence was observed indicating that the inside of the bacteria cell was more negatively charged compared to the outside.  

A proportion of the cells were exposed to ultraviolet light – a known inhibitor of bacterial growth. This impairment of growth was validated using phase-contrast time-lapse microscopy.

Control cells were also identified, and subject to the identical stimulation, irradiated cells became depolarized and the other cells hyperpolarized. This created a distinction between healthy and non-viable bacteria.

This shift was argued to be caused by an alteration in resting membrane potential in damaged cells and anticipated using the extended neuron model in the research.

The researchers treated a mixed culture of the two species of bacteria with an antibiotic, vancomycin, that inhibits B. subtilis proliferation only. Stimulation of both species resulted in hyperpolarization and depolarization of E. coil and B. subtilis, respectively.

The same was observed when the specimens were treated with protonophore or ethanol, resulting in cell damage. This method can be employed in addition to selective culture to identify antibiotic resistance.

Research Implications

James Stratford, the research's lead author, noted that "The system we have created can produce results which are similar to the plate counts used in medical and industrial testing but about 20x faster.

This could save many people's lives and also benefit the economy by detecting contamination in manufacturing processes."

As a result of the research, industrial devices are hoped to be available for clinical and commercial use to promptly identify live bacteria and investigate the effects on antibiotics on cultures.

Rapid Colorimetric Detection

Contemporary research within the field has employed detection techniques involving the use of chemical sensory, including colorimetric-derived detection methods. Such methods are credited for their ease of use and ability to identify the bacteria without the need for additional equipment.

Image Credit: Kallayanee Naloka/Shutterstock.com

A recent study reported on a novel technique called Bacterial Inhibition of GOX-catalyzed Reaction (BIGR) which can rapidly detect a broad spectrum of live bacterial species.

The researchers from Shanghai Jiao Tong University used the technique to identify Salmonella pullorum, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutants.

The assay utilized each bacteria's metabolism of glucose and enabled the detection of the species using the naked eye. Compared to traditional techniques, the authors noted that the test took 20 minutes to carry out and required only three microliters of sample.

The authors conclude that "the presented platform has great potential for rapid detection of bacteria in clinic and evaluation of bacteria viability. Future development of our study will focus on improving the sensitivity by utilizing a new colorimetric substrate."

Researchers at Gachon University have developed a similar colorimetric technique using nanoparticles. The technique was able to quantify both Escherichia coli and Staphylococcus aureus by monitoring a color change reduction using the naked eye and spectrophotometry within approximately 10 minutes.

The method relies on the activity of chitosan-coated magnetic nanoparticles, which when incubated with a bacteria-containing sample, results in a decrease in peroxidase-like activity.

Like the previous colorimetric technique, the assay presented is suggested by the authors to have great potential for rapidly diagnosing broad-spectrum bacterial infections in house, drastically reducing waiting times and saving lives.

References and Further Reading

  • Stratford, J. P., Edwards, C. L., Ghanshyam, M. J., Malyshev, D., Delise, M. A., Hayashi, Y., & Asally, M. (2019). Electrically induced bacterial membrane-potential dynamics correspond to cellular proliferation capacity. Proceedings of the National Academy of Sciences, 116(19), 9552-9557. https://doi.org/10.1073/pnas.1901788116
  • Alamer, S., Eissa, S., Chinnappan, R., Herron, P., & Zourob, M. (2018). Rapid colorimetric lactoferrin-based sandwich immunoassay on cotton swabs for the detection of foodborne pathogenic bacteria. Talanta, 185, 275-280. https://doi.org/10.1016/j.talanta.2018.03.072
  • Sun, J., Huang, J., Li, Y., Lv, J., & Ding, X. (2019). A simple and rapid colorimetric bacteria detection method based on bacterial inhibition of glucose oxidase-catalyzed reaction. Talanta, 197, 304-309. Doi: https://doi.org/10.1016/j.talanta.2019.01.039
  • Le, T. N., Tran, T. D., & Kim, M. I. (2020). A Convenient Colorimetric Bacteria Detection Method Utilizing Chitosan-Coated Magnetic Nanoparticles. Nanomaterials, 10(1), 92. Doi: 10.3390/nano10010092

Further Reading

  • All Bacteria Content
  • Does Vinegar Kill Bacteria?
  • What are Extensively Drug Resistant (XDR) Bacteria?

Last Updated: Apr 15, 2020

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Bacteria virus combo may be cause of neonatal brain infections in Uganda

A newly identified bacteria and a common virus may be the underlying cause of infection-induced hydrocephalus in Uganda, according to an international team of researchers.

“Thirteen years ago, while visiting Uganda and seeing a stream of kids with hydrocephalus after infection I asked the doctors, ‘What is the biggest problem you have that you can’t solve?'” said Steven J. Schiff, Brush Chair Professor of Engineering and professor of engineering science and mechanics, neurosurgery and physics, Penn State. “‘Why don’t you figure out what makes these kids sick?’ was the reply.”

By that time, the doctors at CURE Children’s Hospital of Uganda, had seen more than a thousand infants with infection-caused hydrocephalus and were unable to culture a single thing in the laboratory. They have now seen over 8,000 similar children in this one, small Ugandan hospital.

“Hydrocephalus is the most common childhood neurosurgical condition that we see in the population that we serve,” said Edith Mbabazi-Kabachelor, director of research, CURE Children’s Hospital of Uganda. “If hydrocephalus is left untreated in children less than two years old, the progressive increase in head size will lead to further brain damage, resulting in the majority of these children dying, and those that survive being left with severe cognitive and physical disability.”

Severe systemic bacterial infection during the first four weeks of life accounts for an estimated 680,000 to 750,000 yearly neonatal deaths worldwide. Hydrocephalus is the most common brain disorder in childhood and the largest single cause of childhood hydrocephalus is neonatal infection, accounting for an estimated 160,000 yearly cases, said Schiff.

Over a 5-year study in Uganda, supported by the U.S. National Institutes of Health, using advanced genomic techniques the team uncovered the major bacterial and viral underpinnings of these infections, the researchers report today (Sept. 30) in Science Translational Medicine.

Schiff and his team have studied this problem for more than 10 years, but in the last five years, they took a different approach, using DNA and RNA sequencing techniques to identify the causative agents. The researchers looked at blood and cerebrospinal fluid drawn from 100 cases of post-infectious hydrocephalus and control patients without infection in Uganda. There were 64 infants with post-infectious hydrocephalus and 36 with non-post-infectious hydrocephalus. All infants were under three months old. The researchers prepared the samples in two ways—fresh-frozen and preserved—and they sent samples to two different laboratories in the U.S., where samples were analyzed with different techniques. This was to ensure valid and reproducible results.

“We found this weird bacteria dominating,” said Schiff.

The bacteria was a previously unidentified strain of Paenibacillus thiaminolyticus, now named Mbale after the city where the CURE Children’s Hospital is located.

“The initial link between hydrocephalus and Paenibacillus was made through high-throughput sequencing and PCR analyses at the Center for Infection and Immunity in the Mailman School of Public Health at Columbia University, a renowned center led by W. Ian Lipkin,” said Schiff.

High-throughput sequencing allows sequencing of more than one DNA molecule at the same time, and PCR analysis multiplies existing DNA samples so that they are easier to analyze and identify.

“You build a field of dreams—in this case a platform for pathogen discovery—and wait for the right partner and the right project,” said W. Ian Lipkin, John Snow Professor of Epidemiology and director, Center for Infection and Immunity, Mailman School of Public Health, and professor of pathology and neurology, College of Physicians & Surgeons, Columbia University. “Steven Schiff is a remarkable investigator and this is such a project. It stands out for impact amongst hundreds we’ve done over a period of more than 30 years. Our team is delighted to have had an opportunity to help implicate an agent and contribute to control of this devastating disorder.”

The researchers managed to grow the difficult-to-culture new bacterial strain at Penn State, and tested it on mice. While the common variants of Paenibacillus are harmless, the Mbale strain was lethal to the mice.

The researchers found the new bacterial strain in the cerebrospinal fluid of the infection-induced hydrocephalic children and then only in the youngest patients.

“While we tested infants up to three months old, we mostly identified the cause of infections in those less than six weeks of age,” said Schiff. “If we didn’t study them really early in life, then the infection had already burned out. Between 6 and 12 weeks there were very few positive results.”

Schiff was not satisfied with finding the proposed bacterial cause of the problem, he said. He reasoned that other diseases had both a bacterial and viral component and so the team looked for viral, fungal and parasitic genetic material. They found cytomegalovirus (CMV) in the cerebrospinal fluid of the infection-caused hydrocephalic infants, but not in that of the other hydrocephalus patients.

CMV is a common virus found around the world. The virus causes minor symptoms, if any, in most adults, although babies may be born with congenital CMV or acquire it early in life and be significantly harmed by neurological damage. The researchers only found CMV in the cerebrospinal fluid of babies with post-infection hydrocephalus.

While the researchers believe they have found the source of the infections that cause the high prevalence of hydrocephalus, they do not know where the babies encounter the new bacteria. According to Schiff, the bacteria may be soil- or water-born and more work is necessary to find the bacterial source.

The researchers are creating predictive models that, coupled with data they are now analyzing from thousands of infants and satellite-acquired rainfall to predict optimal treatment for individual locations. The researchers said they do not know if this particular bacterial virus combination exists outside this area of Uganda. However, the same strategy of using DNA and RNA to diagnose previously unknown causes of similar infections can be used in many other regions in the developing world where similar cases are seen.

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Bacteria in the gut have a direct line to the brain

With its 100 million neurons, the gut has earned a reputation as the body’s “second brain”—corresponding with the real brain to manage things like intestinal muscle activity and enzyme secretions. A growing community of scientists are now seeking to understand how gut neurons interact with their brain counterparts, and how failures in this process may lead to disease.

Now, new research shows that gut bacteria play a direct role in these neuronal communications, determining the pace of intestinal motility. The research, conducted in mice and published in Nature, suggests a remarkable degree of communication between our nervous system and the microbiota. It may also have implications for treating gastrointestinal conditions.

“We describe how microbes can regulate a neuronal circuit that starts in the gut, goes to the brain, and comes back to the gut,” says Rockefeller’s Daniel Mucida, associate professor and head of the Laboratory of Mucosal Immunology. “Some of the neurons within this circuit are associated with irritable bowel syndrome, so it is possible that dysregulation of this circuit predisposes to IBS.”

The work was led by Paul A. Muller, a former graduate student in the Mucida lab.

How microbes control motility

To understand how the central nervous system senses microbes within the intestines, Mucida and his colleagues analyzed gut-connected neurons in mice that lacked microbes entirely, so-called germ-free mice that are raised from birth in an isolated environment, and given only food and water that has been thoroughly sterilized. They found that some gut-connected neurons are more active in the germ-free mice than in controls and express high levels of a gene called cFos, which is a marker for neuronal activity.

This increase in neuronal activity, in turn, causes food to move more slowly than usual through the digestive tract of the mice. When the researchers treated the germ-free mice with a drug that silences these gut neurons, they saw intestinal motility speed up.

It’s unclear how the neurons sense the presence of gut microbes, but Mucida and his colleagues found hints that the key may be a set of compounds known as short-chain fatty acids, which are made by gut bacteria. They found that lower levels of these fatty acids within the guts of mice were associated with greater activity of the gut-connected neurons. And when they boosted the animal’s gut levels of these compounds, the activity of their gut neurons decreased. Other microbial compounds and gut hormones that change with the microbiota were also found to regulate neuronal activity, suggesting additional players in this circuit.

Neurons in control

Further experiments revealed a conundrum, however. The scientists saw that the particular type of gut-connected neurons activated by the absence of microbes did not extend to the exposed surface of the intestines, suggesting that they cannot sense the fatty acid levels directly.

So Mucida and his colleagues decided to trace the circuit backwards and found a set of brainstem neurons that show increased activity in the germ-free mice. When the researchers manipulated control mice to specifically activate these same neurons, they saw an increase in the activity of the gut neurons and a decrease in intestinal motility.

The researchers continued to work backwards, next focusing their attention on the sensory neurons that send signals from the intestines to the brainstem. Their experiments revealed these sensory neurons extended to the interface of areas of the intestine that are exposed to high-levels of microbial compounds, including fatty acids. They turned off these neurons, to mimic what happens in germ-free mice that lack the fatty acids, or associated gut signals, and observed activated neurons in the brainstem, as well as activation of the gut neurons that control intestinal motility.

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