Tag Archives: Laboratory

Microbiology

Study provides evidence for a strong role of autophagy in controlling intracellular infections

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Reviewed by Emily Henderson, B.Sc.Mar 23 2023

Researchers at the Francis Crick Institute have found that the body’s process of removing old and damaged cell parts, is also an essential part of tackling infections that take hold within our cells, like TB.

If this natural process can be harnessed with new treatments, it could present an alternative to, or improve use of antibiotics, especially where bacteria have become resistant to existing drugs.

In their study, published in Nature Microbiology today, ahead of World TB Day on the 24th March, the team studied genes key to bacteria’s ability to evade autophagy, a pathway that cells use to destroy themselves when they are under stress or infected.

They engineered human immune cells called macrophages from specialist stem cells called induced pluripotent stem cells, which have the ability to become any cell type in the body. They then used genome editing tools to manipulate the macrophages ability to perform autophagy. When genes key to autophagy were removed and the cells were infected with Mycobacterium tuberculosis (bacilli that cause TB), the bacterial infection took hold, replicating more within the engineered cells and causing mass host cell death.

These results are evidence for a strong role of autophagy in controlling intracellular infections like TB. If this pathway can be boosted or strengthened, it could be a new avenue for tackling antibiotic resistance, by making existing antibiotic drugs more effective or presenting an alternative to drugs in cases where bacteria have evolved resistance.

I first studied the role of autophagy in infection during my PhD, so it’s incredible to see renewed interest in this field. Using the latest technologies, we’ve been able to show a key role for this pathway in controlling infection.

As immunotherapies have harnessed the immune system to fight cancer, boosting this immune defense with a host-directed therapy, could be a valuable new tool in the fight against infections, particularly those becoming resistant to antibiotics.”

Max Gutierrez, Head of the Host-Pathogen Interactions in Tuberculosis Laboratory at Francis Crick Institute

The team also validated their results using macrophages isolated from blood samples, confirming the importance of autophagy in human defenses.

Beren Aylan, joint first author and PhD student at the Crick together with Elliott Bernard and Enrica Pellegrino, said: “Antibiotic resistance is a huge threat to our health so it’s incredibly important to understand how our bodies fight infection and where there might be room for improvement.

“TB is a great example of where targeting our own immune defenses could be really effective, because it takes a very long course of different antibiotic treatments to effectively remove the infection. Anything that can be done to more effectively remove bacteria, could also make a huge difference to the cost and accessibility of treatments.”

The team are now planning to screen for drug compounds that could be used to boost autophagy in a targeted way.

“Boosting the autophagy pathway isn’t as simple as it might seem,” adds Max. This is because all parts of the body use autophagy as a way to recycle old and damaged cells. In order to safely increase autophagy in the location of infections, we need to target the pathway in macrophages alone.”

Source:

Francis Crick Institute

Journal reference:

Aylan, B., et al. (2023). ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages. Nature Microbiology. doi.org/10.1038/s41564-023-01335-9

Microbiology

High-resolution mass spectrometric rapid identification of Candida auris

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Dr. Priyom Bose, Ph.D.By Dr. Priyom Bose, Ph.D.Mar 23 2023Reviewed by Danielle Ellis, B.Sc.

A recent study published in the Journal of Fungi used a novel OrbitrapTM high-resolution mass spectrometric technology coupled with liquid chromatography to identify geographically different clades of Candida auris (C. auris) isolates. This proof-of-concept methodology could accurately detect C. auris in the microbiology laboratory.

Study: Fast and Accurate Identification of Candida auris by High Resolution Mass Spectrometry. Image Credit: Jens Goepfert / ShutterstockStudy: Fast and Accurate Identification of Candida auris by High Resolution Mass Spectrometry. Image Credit: Jens Goepfert / Shutterstock

Background

Over a decade ago, C. auris was first found in East Asia, causing bloodstream infections. Although this fungal infection was initially found in India, South America, South Africa, and the Middle East, it soon prevailed globally. 

C. auris soon became a common nosocomial fungal pathogen, particularly among intensive care unit (ICU) patients. As a result, the Centers for Disease Control and Prevention (CDC) has classified C. auris as an urgent threat pathogen.

An important factor that allows C. auris outbreaks worldwide is the improper identification of yeast pathogens in hospital laboratories. Hence, there is an urgent need for accurate and rapid identification of C. auris in hospital laboratories, which can reduce their transmission in healthcare facilities.

Genomic analysis of worldwide C. auris isolates has indicated that around five clades have emerged in the last 20 years, independently and simultaneously. These five distinct geographically restricted clades are clade I: South Asia, clade II: East Asia, clade III: Africa, clade IV: South America, and clade V: Iran. Each clade differs from the other by around ten thousand single-nucleotide polymorphisms. 

Each clade has differential resistance to antifungal agents; for example, clade I is more resistant to fluconazole, while clade II exhibits susceptibility. Currently, C. auris isolates belonging to these clades have been introduced to many countries worldwide. Scientists have highlighted the importance of quickly identifying and monitoring these clades to restrict further spread. 

C. auris possesses several structurally unique sphingolipids and mannoproteins, enabling it to adhere to medical devices and hospital environments persistently. These proteins also aid in biofilm formation and prevent elimination by common disinfectants.

Several studies have indicated that molecular techniques fail to identify C. auris, whereas matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) technology can accurately identify this fungus at the species level.

The Study and its Findings

102 clinical C. auris strains were selected, representing all five clades. These clades were determined based on a short tandem repeat (STR) typing assay, which was subsequently compared to whole-genome sequencing results.

The current study applied OrbitrapTM high-resolution mass spectrometric technology to identify C. auris based on protein analysis methods. This technique was combined with liquid chromatography (LC) for initial separation. In this method, electrospray ionization (ESI) transfers proteins into the gas phase for ionization and is subsequently introduced to the mass spectrometer (LC-MS).

Mass analysis is conducted by either fragment ions or intact mass (MS) through tandem mass spectrometry (MS/MS). Some of the key features of the OrbitrapTM mass analyzer are a high resolution of up to 200,000, a high mass-to-charge ratio of 6,000, high mass accuracy between 2 and 5 ppm, and a dynamic range greater than 104.

C. auris clade differentiation using monoisotopic mass measurements depicted as heat map. Color scale ranges from blue (max signal) to dark red (no signal), representing abundance of measured monoisotopic masses in each strain. Clade specific differential protein masses are visible from the rectangular vertical boxes indicating the geographic affiliation and clade assignment and its vertically associated dendrogram indicating observed protein masses (columns vs. rows). X-axis indicating clade assignment and y-axis indicating observed MS1 protein masses.

In addition, this method is highly sensitive and can measure the exact mass of a compound. It can also identify minor structural changes due to a translated single nucleotide polymorphism into an amino acid change.

Importantly, this newly developed technology could identify all C. auris isolates with high confidence. Furthermore, it could differentiate C. auris across clades. Even though a limited number of isolates were present from each clade, this spectrometric technology identified C. auris clades with 99.6% identification accuracy.

Based on a principal component analysis (PCA) and a subsequent affinity clustering study, the South Asian, East Asian, and Iranian C. auris clades were more proteomically closely related. Long branches in the affinity clustering analysis suggested that the C. auris strains were present as outliers that required more attention, regardless of the detection technique.

Proteomic typing results indicated the capacity to track strains of the same origin isolated from diverse geographical locations. In the future, more precise matching and alignment of typing schemes (based on next-generation sequencing) is required to build on these results. This would significantly reduce false identifications and classifications of unknown strains associated with new clades or lineage.

Conclusions

Although the workflow linked to mass spectrometry and next-generation sequencing are not directly comparable, their results are similar, i.e., identifying unknown clinical microbes. The standard next-generation sequencing method is a highly time-consuming process that requires many delicate time-intensive quality-control steps, particularly during multiplexed sample runs.

In contrast, the newly developed methodology can provide results within 60 minutes. Therefore, applying the high-resolution OrbitrapTM mass spectrometer to accurately and rapidly identify C. auris clades is an attractive alternative to conventional platforms.

Journal reference:
  • Jamalian, A. et al. (2023) “Fast and Accurate Identification of Candida auris by High Resolution Mass Spectrometry”, Journal of Fungi, 9(2), p. 267. doi: 10.3390/jof9020267, https://www.mdpi.com/2309-608X/9/2/267
Microbiology

Co-infection with MRSA ‘superbug’ could make COVID-19 outcomes even more deadly

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Reviewed by Emily Henderson, B.Sc.Mar 21 2023

Global data shows nearly 10 per cent of severe COVID-19 cases involve a secondary bacterial co-infection – with Staphylococcus aureus, also known as Staph A., being the most common organism responsible for co-existing infections with SARS-CoV-2. Researchers at Western have found if you add a ‘superbug’ – methicillin-resistant Staphylococcus aureus (MRSA) – into the mix, the COVID-19 outcome could be even more deadly.

The mystery of how and why these two pathogens, when combined, contribute to the severity of the disease remains unsolved. However, a team of Western researchers has made significant progress toward solving this “whodunit”.

New research by Mariya Goncheva, Richard M. Gibson, Ainslie C. Shouldice, Jimmy D. Dikeakos and David E. Heinrichs, has revealed that IsdA, a protein found in all strains of Staph A., enhanced SARS-CoV-2 replication by 10- to 15-fold. The findings of this study are significant and could help inform the development of new therapeutic approaches for COVID-19 patients with bacterial co-infections.

Interestingly, the study, which was recently published in iScience, also showed that SARS-CoV-2 did not affect the bacteria’s growth. This was contrary to what the researchers had initially expected.

We started with an assumption that SARS-CoV-2 and hospitalization due to COVID-19 possibly caused patients to be more susceptible to bacterial infections which eventually resulted in worse outcomes.”

Mariya Goncheva

Goncheva is a former postdoctoral associate, previously with the department of microbiology and immunology at Schulich School of Medicine & Dentistry.

Goncheva said bacterial infections are most commonly acquired in hospital settings and hospitalization increases the risk of co-infection. “Bacterial infections are one of the most significant complications of respiratory viral infections such as COVID-19 and Influenza A. Despite the use of antibiotics, 25 per cent of patients co-infected with SARS-CoV-2 and bacteria, die as a result. This is especially true for patients who are hospitalized, and even more so for those in intensive care units. We were interested in finding why this happens,” said Goncheva, lead investigator of the study.

Goncheva, currently Canada Research Chair in virology and professor of biochemistry and microbiology at the University of Victoria, studied the pathogenesis of multi-drug resistant bacteria (such as MRSA) supervised by Heinrichs, professor of microbiology and immunology at Schulich Medicine & Dentistry.

When the COVID-19 pandemic hit, she pivoted to study interactions between MRSA and SARS-CoV-2.

For this study, conducted at Western’s level 3 biocontainment lab, Imaging Pathogens for Knowledge Translation (ImPaKT), Goncheva’s work created an out-of-organism laboratory model to study the interactions between SARS-CoV-2 and MRSA, a difficult-to-treat multi-drug resistant bacteria.

“At the beginning of the pandemic, the then newly opened ImPaKT facility made it possible for us to study the interactions between live SARS-CoV-2 virus and MRSA. We were able to get these insights into molecular-level interactions due to the technology at ImPaKT,” said Heinrichs, whose lab focuses on MRSA and finding drugs to treat MRSA infections. “The next step would be to replicate this study in relevant animal models.”

Source:

University of Western Ontario

Journal reference:

Goncheva, M. I., et al. (2023). The Staphylococcus aureus protein IsdA increases SARS CoV-2 replication by modulating JAK-STAT signaling. IScience. doi.org/10.1016/j.isci.2023.105975.

Microbiology

Avanced genome editing technology could be used as a one-time treatment for CD3 delta SCID

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Reviewed by Emily Henderson, B.Sc.Mar 21 2023

A new UCLA-led study suggests that advanced genome editing technology could be used as a one-time treatment for the rare and deadly genetic disease CD3 delta severe combined immunodeficiency.

The condition, also known as CD3 delta SCID, is caused by a mutation in the CD3D gene, which prevents the production of the CD3 delta protein that is needed for the normal development of T cells from blood stem cells.

Without T cells, babies born with CD3 delta SCID are unable to fight off infections and, if untreated, often die within the first two years of life. Currently, bone marrow transplant is the only available treatment, but the procedure carries significant risks.

In a study published in Cell, the researchers showed that a new genome editing technique called base editing can correct the mutation that causes CD3 delta SCID in blood stem cells and restore their ability to produce T cells.

The potential therapy is the result of a collaboration between the laboratories of Dr. Donald Kohn and Dr. Gay Crooks, both members of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA and senior authors of the study.

Kohn’s lab has previously developed successful gene therapies for several immune system deficiencies, including other forms of SCID. He and his colleagues turned their attention to CD3 delta SCID at the request of Dr. Nicola Wright, a pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute in Canada, who reached out in search of a better treatment option for her patients.

CD3 delta SCID is prevalent in the Mennonite community that migrates between Canada and Mexico.

Because newborns are not screened for SCID in Mexico, I often see babies who have been diagnosed late and are returning to Canada quite sick.”

Dr. Nicola Wright, pediatric hematologist and immunologist at the Alberta Children’s Hospital Research Institute

When Kohn presented Wright’s request to his lab, Grace McAuley, then a research associate who joined the lab at the end of her senior year at UCLA, stepped up with a daring idea.

“Grace proposed we try base editing, a very new technology my lab had never attempted before,” said Kohn, a distinguished professor of microbiology, immunology and molecular genetics, and of pediatrics.

Base editing is an ultraprecise form of genome editing that enables scientists to correct single-letter mutations in DNA. DNA is made up of four chemical bases that are referred to as A, T, C and G; those bases pair together to form the “rungs” in DNA’s double-helix ladder structure.

While other gene editing platforms, like CRISPR-Cas9, cut both strands of the chromosome to make changes to DNA, base editing chemically changes one DNA base letter into another -; an A to a G, for example -; leaving the chromosome intact.

“I had a very steep learning curve in the beginning, when base editing just wasn’t working,” said McAuley, who is now pursuing an M.D.-Ph.D. at UC San Diego and is the study’s co-first author. “But I kept pushing forward. My goal was help get this therapy to the clinic as fast as was safely possible.”

McAuley reached out to the Broad Institute’s David Liu, the inventor of base editing, for advice on how to evaluate the technique’s safety for this particular use. Eventually, McAuley identified a base editor that was highly efficient at correcting the disease-causing genetic mutation.

Because the disease is extremely rare, obtaining patient stem cells for the UCLA study was a significant challenge. The project got a boost when Wright provided the researchers with blood stem cells donated by a CD3 delta SCID patient who was undergoing a bone marrow transplant.

The base editor corrected an average of almost 71% of the patient’s stem cells across three laboratory experiments.

Next, McAuley worked with Dr. Gloria Yiu, a UCLA clinical instructor in rheumatology, to test whether the corrected cells could give rise to T cells. Yiu used artificial thymic organoids, which are stem cell-derived tissue models developed by Crooks’ lab that mimic the environment of the human thymus -; the organ where blood stem cells become T cells.

When the corrected blood stem cells were introduced into the artificial thymic organoids, they produced fully functional and mature T cells.

“Because the artificial thymic organoid supports the development of mature T cells so efficiently, it was the ideal system to show that base editing of patients’ stem cells could fix the defect seen in this disease,” said Yiu, who is also a co-first author of the study.

As a final step, McAuley studied the longevity of the corrected stem cells by transplanting them into a mouse. The corrected cells remained four months after transplant, indicating that base editing had corrected the mutation in true, self-renewing blood stem cells. The findings suggest that corrected blood stem cells could persist long-term and produce the T cells patients would need to live healthy lives.

“This project was a beautiful picture of team science, with clinical need and scientific expertise aligned,” said Crooks, a professor of pathology and laboratory medicine. “Every team member played a vital role in making this work successful.”

The research team is now working with Wright on how to bring the new approach to a clinical trial for infants with CD3 delta SCID from Canada, Mexico and the U.S.

This research was funded by the Jeffrey Modell Foundation, the National Institutes of Health, the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute, the V Foundation and the A.P. Giannini Foundation.

The therapeutic approach described in this article has been used in preclinical tests only and has not been tested in humans or approved by the Food and Drug Administration as safe and effective for use in humans. The technique is covered by a patent application filed by the UCLA Technology Development Group on behalf of the Regents of the University of California, with Kohn and McAuley listed as co-inventors.

Source:

University of California – Los Angeles Health Sciences

Journal reference:

McAuley, G.E., et al. (2023) Human T cell generation is restored in CD3δ severe combined immunodeficiency through adenine base editing. Cell. doi.org/10.1016/j.cell.2023.02.027.

Microbiology

Multiplex PCR panels associated with reduced administration of antibiotics to hospitalized GI patients

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Reviewed by Emily Henderson, B.Sc.Mar 16 2023

Acute gastroenteritis afflicts adults of all ages, causing significant suffering and inflicting significant costs on the American healthcare system. A new study encompassing nearly 40,000 hospital visits from a geographically diverse healthcare database shows that sampling a single stool, using multiple polymerase chain reaction (PCR) panels, can identify more pathogens, notably diarrhea-causing E. coli and enteric viruses, and do so more rapidly than a conventional workup. The research is published in Journal of Clinical Microbiology, a publication of the American Society for Microbiology.

Using multiple PCR panels, “Fewer patients received antibiotics, required additional visits or diagnostic tests, or were hospitalized for gastroenteritis within 30 days [of index visit],” said Rena C. Moon, M.D., M.P.H., Principal Research Scientist, PINC AI Applied Sciences, Charlotte, NC. Additionally, healthcare costs were lower than with a conventional workup. Conventional workups may include testing a stool culture for a single suspect species of pathogen, use of a single pathogen PCR test, or identifying a pathogen using microscopy, immunology or an ova and parasites test.

Earlier studies showed that large multiplex PCR panels improve the speed and accuracy of diagnostic testing in patients with acute gastroenteritis, but their impact on costs and clinical outcomes had been uncertain. Our study shows that the benefits of multiplex panels can be achieved without increasing overall healthcare costs, and also facilitates more appropriate use of antibiotics.”

Ferric C. Fang, M.D., Professor of Laboratory Medicine, Pathology, and Microbiology at the University of Washington School of Medicine, Seattle

“This study illustrates the power of big data to analyze the healthcare impacts of diagnostic testing, and help laboratories select testing approaches that improve meaningful clinical outcomes,” Fang said.

Using multiplex PCR, more patients could be discharged and did not require hospitalization during the following month. That resulted in similar healthcare costs to patients undergoing the traditional stool work-up plus follow-up visits over the following month. Furthermore, multiplex PCR (using 12 or more) panels were associated with reduced administration of antibiotics to hospitalized patients.

The overall result: improved care with lower costs.

Source:

American Society for Microbiology

Microbiology

Host immune system forms small lesions in the intestines in response to bacterial infection

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Reviewed by Emily Henderson, B.Sc.Mar 14 2023

Yersinia bacteria cause a variety of human and animal diseases, the most notorious being the plague, caused by Yersinia pestis. A relative, Yersinia pseudotuberculosis, causes gastrointestinal illness and is less deadly but naturally infects both mice and humans, making it a useful model for studying its interactions with the immune system.

These two pathogens, as well as a third close cousin, Y. enterocolitica, which affects swine and can cause food-borne illness if people consume infected meat, have many traits in common, particularly their knack for interfering with the immune system’s ability to respond to infection.

The plague pathogen is blood-borne and transmitted by infected fleas. Infection with the other two depends on ingestion. Yet the focus of much of the work in the field had been on interactions of Yersinia with lymphoid tissues, rather than the intestine. A new study of Y. pseudotuberculosis led by a team from Penn’s School of Veterinary Medicine and published in Nature Microbiology demonstrates that, in response to infection, the host immune system forms small, walled-off lesions in the intestines called granulomas. It’s the first time these organized collections of immune cells have been found in the intestines in response to Yersinia infections.

The team went on to show that monocytes, a type of immune cell, sustain these granulomas. Without them, the granulomas deteriorated, allowing the mice to be overtaken by Yersinia.

“Our data reveal a previously unappreciated site where Yersinia can colonize and the immune system is engaged,” says Igor Brodsky, senior author on the work and a professor and chair of pathobiology at Penn Vet. “These granulomas form in order to control the bacterial infection in the intestines. And we show that if they don’t form or fail to be maintained, the bacteria are able to overcome the control of the immune system and cause greater systemic infection.”

The findings have implications for developing new therapies that leverage the host immune system, Brodsky says. A drug that harnessed the power of immune cells to not only keep Yersinia in check but to overcome its defenses, they say, could potentially eliminate the pathogen altogether.

A novel battlefield

Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica share a keen ability to evade immune detection.

“In all three Yersinia infections, a hallmark is that they colonize lymphoid tissues and are able to escape immune control and replicate, cause disease, and spread,” Brodsky says.

Earlier studies had shown that Yersinia prompted the formation of granulomas in the lymph nodes and spleen but had never observed them in the intestines until Daniel Sorobetea, a research fellow in Brodsky’s group, took a closer look at the intestines of mice infected with Y. pseudotuberculosis.

“Because it’s an orally acquired pathogen, we were interested in how the bacteria behaved in the intestines,” Brodsky says. “Daniel made this initial observation that, following Yersinia pseudotuberculosis infection, there were macroscopically visible lesions all along the length of the gut that had never been described before.”

The research team, including Sorobetea and later Rina Matsuda, a doctoral student in the lab, saw that these same lesions were present when mice were infected with Y. enterocolitica, forming within five days after an infection.

A biopsy of the intestinal tissues confirmed that the lesions were a type of granuloma, known as a pyogranuloma, composed of a variety of immune cells, including monocytes and neutrophils, another type of white blood cell that is part of the body’s front line in fighting bacteria and viruses.

Granulomas form in other diseases that involve chronic infection, including tuberculosis, for which Y. pseudotuberculosis is named. Somewhat paradoxically, these granulomas-;while key in controlling infection by walling off the infectious agent-;also sustain a population of the pathogen within those walls.

The team wanted to understand how these granulomas were both formed and maintained, working with mice lacking monocytes as well as animals treated with an antibody that depletes monocytes. In the animals lacking monocytes “these granulomas, with their distinct architecture, wouldn’t form,” Brodsky says.

Instead, a more disorganized and necrotic abscess developed, neutrophils failed to be activated, and the mice were less able to control the invading bacteria. These animals experienced higher levels of bacteria in their intestines and succumbed to their infections.

Groundwork for the future

The researchers believe the monocytes are responsible for recruiting neutrophils to the site of infection and thus launching the formation of the granuloma, helping to control the bacteria. This leading role for monocytes may exist beyond the intestines, the researchers believe.

We hypothesize that it’s a general role for the monocytes in other tissues as well.”

Igor Brodsky, senior author

But the discoveries also point to the intestines as a key site of engagement between the immune system and Yersinia.

“Previous to this study we knew of Peyer’s patches to be the primary site where the body interacts with the outside environment through the mucosal tissue of the intestines,” says Brodsky. Peyer’s patches are small areas of lymphoid tissue present in the intestines that serve to regulate the microbiome and fend off infection.

In future work, Brodsky and colleagues hope to continue to piece together the mechanism by which monocytes and neutrophils contain the bacteria, an effort they’re pursing in collaboration with Sunny Shin’s lab in the Perelman School of Medicine’s microbiology department.

A deeper understanding of the molecular pathways that regulate this immune response could one day offer inroads into host-directed immune therapies, by which a drug could tip the scales in favor of the host immune system, unleashing its might to fully eradicate the bacteria rather than simply corralling them in granulomas.

“These therapies have caused an explosion of excitement in the cancer field,” Brodsky says, “the idea of reinvigorating the immune system. Conceptually we can also think about how to coax the immune system to be reinvigorated to attack pathogens in these settings of chronic infection as well.”

Source:

University of Pennsylvania

Journal reference:

Sorobetea, D., et al. (2023). Inflammatory monocytes promote granuloma control of Yersinia infection. Nature Microbiology. doi.org/10.1038/s41564-023-01338-6.

Microbiology

New study focuses on genetic diversity of E. coli bacteria in hospitalized patients

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Reviewed by Emily Henderson, B.Sc.Mar 13 2023

The human intestine is an environment inhabited by many bacteria and other microorganisms collectively known as the gut microbiome, gut microbiota or intestinal flora. In most people, it contributes to wellness. A healthy gut indicates a stronger immune system, improved metabolism, and a healthy brain and heart, among other functions.

Escherichia coli is one of the bacteria found in practically everyone’s gut microbiota, where it performs important functions, such as producing certain vitamins.

But there’s a vast amount of genetic diversity in the species. Some of its members are pathogenic and can cause diseases such as urinary tract infections. E. coli is the main agent of this type of infection among both healthy people and hospitalized patients or users of healthcare services.”

Tânia Gomes do Amaral, Head of the Experimental Enterobacterial Pathogenicity Laboratory (LEPE), Federal University of São Paulo’s Medical School (EPM-UNIFESP), Brazil

Amaral is first author of an article published in the journal Pathogens on the virulence of these bacteria and their resistance to antibiotics in hospitalized patients.

“Our study focused on hospitalized patients because patients who stay in hospital for a long period are more likely to undergo various procedures, such as urine catheter insertion or venous access. Although these procedures are performed to assure life support, they may facilitate the entry of bacteria into the organism and cause an infection,” Amaral explained.

She earned a PhD in microbiology from EPM-UNIFESP in 1988, conducting part of her research at New York University Medical School and the Center for Vaccine Development at the University of Maryland, Baltimore (UMB) in the United States.

The article reports the findings of a broader study led by Amaral, with 12 co-authors who are researchers and graduate students, on the virulence and drug resistance of E. coli strains associated with urinary tract infections. The study was supported by FAPESP via three projects (18/17353-7, 19/21685-8 and 17/14821-7).

The main aim of this part of the study, described in the master’s dissertation of José Francisco Santos Neto, was to evaluate the diversity and drug resistance of pathogenic E. coli strains isolated from the gut microbiota of inpatients, and to analyze the frequency of endogenous infection (caused by bacteria from the patient’s own microbiota).

The UNIFESP group first investigated the genetic diversity and drug resistance of E. coli strains isolated from the gut microbiota of hospitalized patients, sequencing these strains as well as others isolated from their urine and comparing the results in order to evaluate dissemination of the bacteria in the hospital environment.

“We also compared the genomes of these strains with those of E. coli strains isolated in different parts of the world in order to see if any globally disseminated pathogenic bacteria were present in the study sample,” said Ana Carolina de Mello Santos, a postdoctoral researcher working on the LEPE team.

Urinary tract infections proved to be endogenous for the vast majority of the patients in the study (more than 70%). The results also showed that the patients’ gut microbiota contained at least two genetically different populations of E. coli and that about 30% were colonized by non-lactose-fermenting E. coli strains, which are less common, with some of the patients studied having only such strains in their gut microbiota.

“This finding is most interesting because previous research conducted in other countries to analyze the composition of human gut microbiota didn’t investigate non-lactose-fermenting E. coli,” Santos said.

The authors also note the presence of bacteria with all the genetic markers required for classification as pathogenic and the detection of pathogenic bacteria in the gut microbiota of all patients that had not yet developed an infection. “Hospitalized patients are more susceptible to infection because by definition they are already unwell. Colonization by pathogens is the first step in the spread of hospital-acquired infections now so frequent worldwide,” Santos said.

With regard to antibiotics and other antimicrobials, the authors stress that drug resistance is also a growing global problem, and enterobacterial resistance to third-generation cephalosporins as well as colistin is critical. In all patients whose gut microbiota was colonized by drug-resistant bacteria, the same bacteria also caused endogenous urinary tract infections. In other words, the multidrug-resistant bacteria colonized the gut and traveled to the urinary tract, where they caused an infection.

“In light of these findings, early assessment of gut microbiota in hospitalized patients, at least in cases of E. coli infection, can facilitate and guide their treatment, while also identifying patients who risk progressing to extra-intestinal diseases such as urinary tract infections, which were part of the focus for our study,” Amaral said. “We don’t yet know whether the findings also apply to other bacteria found in gut microbiota, such as the genera Klebsiella, Enterobacter, Pseudomonas and others that can cause infections when they travel to extra-intestinal sites.”

These bacterial genera tend to be even more drug-resistant than E. coli, representing a major public health problem in the hospital environment. As the researchers noted, the World Health Organization (WHO) considers E. coli strains resistant to cephalosporin and colistin to be a critical global health threat. “The presence in human gut microbiota of drug-resistant bacteria associated with severe infectious disease is a matter of great concern, not least because they could spread to people outside the hospital environment,” Amaral said.

Another point raised by the study is the importance of finding out when colonization of the patient’s gut by drug-resistant virulent bacteria occurred. The authors of the article were unable to determine whether the bacteria resistant to cephalosporins and colistin colonized the patients before or after they were hospitalized.

By analyzing the genomes of the strains, however, the researchers were able to identify global risk clones that can cause severe disease and are associated with antimicrobial resistance. “One such clone found in the gut microbiota of two patients was identical to others isolated from urinary tract infections in Londrina, Paraná [a state in South Brazil], and in the United States, as well as European and Asian countries. This shows that some strains found in the study are clones generally associated with infections in all regions of the world,” Amaral said.

This type of information is important when patients are hospitalized. Knowledge of bacterial virulence and drug resistance can be used to prevent infection in parts of the organism outside the intestine and stop the bacteria from spreading to other patients in the same hospital.

Source:

São Paulo Research Foundation (FAPESP)

Journal reference:

Santos-Neto, J.F., et al. (2023) Virulence Profile, Antibiotic Resistance, and Phylogenetic Relationships among Escherichia coli Strains Isolated from the Feces and Urine of Hospitalized Patients. Pathogens. doi.org/10.3390/pathogens11121528.

Microbiology

New discoveries made regarding autism onset in mouse models

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Reviewed by Emily Henderson, B.Sc.Mar 10 2023

Although autism is a common neurodevelopmental disorder, the multiple factors behind its onset are still not fully understood. Animal models of idiopathic autism, especially mice, are often used to help researchers understand the complicated mechanisms behind the disorder, with BTBR/J being the most commonly used mouse model in the world.

Now, an international research collaboration including Kobe University’s Professor TAKUMI Toru and Researcher Chia-wen Lin et al. have made new discoveries regarding autism onset in mouse models.

In their detailed series of experiments and analyses of BTBR/J mice and the other subspecies BTBR/R, they revealed that endogenous retrovirus activation increases a fetus’s susceptibility to autism. They also discovered that BTBR/R exhibits autistic-like behaviors without reduced learning ability, making it a more accurate model of autism than the widely-used BTBR/J model.

It is hoped that further research will contribute towards better classification of autism types, as well as the creation of new treatment strategies for neurodevelopmental disorders.

These research results were published in Molecular Psychiatry on March 7, 2023

Main points

  • The researchers analyzed BTBR/J, a widely used mouse model of autism, and its subspecies BTBR/Rusing MRI. This revealed that the corpus callosum, which connects the left and right hemispheres of the brain, was impaired in BTBR/J mice but not in BTBR/R mice.
  • Genome and transcription analysis showed that BTBR mice have increased levels of endogenous retrovirus genes.
  • Furthermore, single-cell RNA analysis of BTBR/R mice revealed changes in the expression of various genes (including stress response genes) that are indicative of endogenous retrovirus activation.
  • Even though BTBR/J and BTBR/R mice have the same ancestry, the results of various behavioral analysis experiments revealed differences in spatial learning ability and other behaviors between the two types of model mice.

Research background

Autism (autism spectrum disorder) is a neurodevelopmental disorder that remains largely unexplored despite the rapidly increasing number of patients. Reasons for this continuing increase in people diagnosed with autism include changes to diagnostic criteria and older fathers becoming more common. Autism is strongly related to genetic factors and can be caused by abnormalities in DNA structure, such as copy number variations. Animal models, especially mice, are often used in research to illuminate the pathology of autism. Among these models, BTBR/J is a mouse model of the natural onset of autism that is commonly used. Studies have reported various abnormalities in BTBR/J mice including impairment of the corpus callosum (which connects the left and right hemispheres of the brain) and excessive immune system signaling. However, it is not fully understood why this particular lineage displays autistic-like behavioral abnormalities.

The aim of the current study was to shed light on the onset mechanism of these autistic-like behavioral abnormalities by conducting comparative analysis on BTBR/J and its subspecies BTBR/R.

Research findings

First of all, the researchers conducted MRI scans on BTBR/J and BTBR/R mice to investigate structural differences in each region of the brain. The results revealed that there were differences between BTBR/J and BTBR/R mice in 33 regions including the amygdala. A particularly prominent difference discovered was that even though BTBR/J’s corpus callosum is impaired, BTBR/R’s is normal.

Next, the research group used the array CGH method to compare BTBR/R’s copy number variations with that of a normal mouse model (B6). They revealed that BTBR/R mice had significantly increased levels of endogenous retroviruses (ERV) in comparison to B6 mice. Furthermore, qRT-PCR tests revealed that these retroviruses were activated in BTBR/R mice. On the other hand, in B6 mice there was no change in the expression of LINE ERV (which is classified in the same repetitive sequence), indicating that this retroviral activation is specific to BTBR.

Subsequently, the researchers carried out single-cell RNA analysis on the tissue of embryonic BTBR mice (on the AGM and yolk sac). The results provide evidence of ERV activation in BTBR mice, as expression changes were observed in a group of genes downstream of ERV.

Lastly, the researchers comprehensively investigated the differences between BTBR/J and BTBR/R on a behavioral level. BTBR/R mice were less anxious than BTBR/J and showed qualitative changes in ultrasound vocalizations, which are measured as a way to assess communicative ability in mice. BTBR/R mice also exhibited more self-grooming behaviors and buried more marbles in the marble burying test. These two tests were designed to detect repetitive behavioral abnormalities in autistic individuals. From the results, it was clear that BTBR/R exhibits more repetitive behaviors (i.e. it is more symptomatic) than BTBR/J. The 3-chamber social interaction test, which measures how closely a mouse will approach another mouse, also revealed more pronounced social deficits in BTBR/R than BTBR/J mice (Figure 4i). In addition, a Barnes maze was used to conduct a spatial learning test, in which BTBR/J mice exhibited reduced learning ability compared to B6 (normal mice). BTBR/R mice, on the other hand, exhibited similar ability to B6.

Overall, the study revealed that retrovirus activation causes the copy number variants in BTBR mice to increase, which leads to the differences in behavior and brain structure seen in BTBR/J and BTBR/R mice (Figure 5).

Further developments

BTBR/J mice are widely used by researchers as a mouse model of autism. However, the results of this study highlight the usefulness of the other lineage of BTBR/R mice because they exhibit autistic-like behavior without compromised spatial learning ability. The results also suggest that it may be possible to develop new treatments for autism that suppress ERV activation. Furthermore, it is necessary to classify autism subtypes according to their onset mechanism, which is a vital first step towards opening up new avenues of treatment for autism.

Source:

Kobe University

Journal reference:

Lin, C-W., et al. (2023) An old model with new insights: endogenous retroviruses drive the evolvement toward ASD susceptibility and hijack transcription machinery during development. Molecular Psychiatry. doi.org/10.1038/s41380-023-01999-z.

Microbiology

Study finds community-onset bacterial coinfection in children with critical COVID-19 is infrequent but empiric antibiotics are commonly prescribed

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Pooja Toshniwal PahariaBy Pooja Toshniwal PahariaMar 8 2023Reviewed by Danielle Ellis, B.Sc.

In a recent study published in Open Forum Infectious Diseases, researchers evaluated the use of empiric antibiotics to determine the prevalence rates of community-acquired bacterial coinfections among hospitalized pediatric critical coronavirus disease 2019 (COVID-19) patients and to identify opportunities for de-escalating antibiotic usage in case of no bacteria-caused sepsis among high-risk individuals, and those presenting with shock.

Study: Community-onset bacterial coinfection in children critically ill with SARS-CoV-2 infection. Image Credit: nokwalai/Shutterstock
Study: Community-onset bacterial coinfection in children critically ill with SARS-CoV-2 infection. Image Credit: nokwalai/Shutterstock

Background

Community-acquired bacterial coinfections among hospitalized adult coronavirus disease 2019 (COVID-19) patients are uncommon; however, empiric antibiotic usage is reportedly high. Data on empiric antibiotic usage and bacterial coinfections among pediatric individuals with critical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are limited.

The clinical manifestations of severe SARS-CoV-2 infections often include pulmonary distress and fever, findings that could be difficult to discriminate from serious bacterial infections, which might prompt the use of empiric antibiotics in the initial days of hospitalization, particularly among high-risk individuals.

About the study

In the present study, researchers investigated whether any radiographic, laboratory, or clinical features ascertainable during hospitalization were related to empiric antibiotic usage or were estimative of bacterial coinfections acquired in community settings.

The team evaluated individuals below 19.0 years and admitted to pediatric high-acuity units (HAU) or intensive care units (ICU) due to SARS-CoV-2 infections from March 2020 to December 2020. On the basis of microbiology reports from the initial 72 hours of hospitalization, the team adjudicated if patients had community-acquired bacterial coinfections.

Clinical and demographic variables of individuals with and without antibiotic prescriptions and bacterial coinfections in the initial days of hospitalization were compared. Poisson regression modeling was performed to assess factors related to the outcome, and the adjusted relative risk (aRR) values were calculated.

Data were obtained from patient electronic medical records and data from the nationwide overcoming COVID-19 population health active surveillance registry of patients hospitalized due to COVID-19-associated complications between 15 March 2020 and 31 December 2020 across >70.0 pediatric hospitals in 25 states.

COVID-19 diagnosis was confirmed using polymerase chain reaction (PCR). The team excluded multisystem inflammatory syndrome among children (MIS-C) patients diagnosed using the Centers for Disease Control and Prevention (CDC) criteria. Data were obtained on demographic parameters, clinical symptoms and signs, comorbidities, radiographical and laboratory investigations, and data on antibiotics prescribed at admission and the course of critical COVID-19, including clinical outcomes and hemodynamic and respiratory support needed.

The primary study outcome assessed was the prescriptions of empirical antimicrobials, for which enteral or intravenous antimicrobials administered in the initial two days of hospital admission were assessed. The second outcome evaluated community-acquired bacterial infection presence, for which relevant case report form (CRF) information from individuals with SARS-CoV-2-positive microbiological cultures, and PCR, were analyzed in the initial 72 hours of hospital admission.

Results

Out of 532 individuals, 63.0% were administered empiric antibiotics; however, only seven percent developed bacterial coinfections, of which only three percent were respiratory-type. Empirical antibiotics had a greater likelihood of being prescribed to immunosuppressed individuals (aRR of 1.3), requiring non-mechanical ventilator-type respiratory aid (aRR of 1.4), or requiring invasive-type mechanical ventilators (aRR of 1.8), than no respiratory aid.

The most frequently prescribed antimicrobials were ceftriaxone (41%) and vancomycin (28%), followed by cefepime (20%). Most individuals were prescribed multiple antimicrobials, with 21%, 10%, and 18% receiving 2.0, 3.0, and ≥4.0 antibiotics in the initial two days of hospital admission. More than 33% of individuals received antibiotics for ≥5.0 days, despite no evidence of bacterial coinfections. The median social vulnerability index (SVI) values were significantly greater among those who received antibiotics than those who did not.

The median C-reactive protein (CRP) levels were greater among those who received antibiotics versus those who did not (4.6 mg per dL vs. 2.2 mg per dL), as were the median procalcitonin levels (0.4 ng per mL vs. 0.1 ng per mL). The median leukocyte counts showed no significant differences between the two groups. Antibiotic usage was related to COVID-19 severity, indicated by greater median values for PEdiatric Logistic Organ Dysfunction-2 (PELOD-2) scores at hospitalization among individuals who received antibiotics than those who did not.

Seven percent (n=38) of individuals had true community-onset bacterial coinfections, of which 13, 16, 8.0, and 4.0 were bloodstream infections, respiratory infections, urinary tract infections, and bacterial infections at other sites (peritonitis, colitis, meningitis, and pharyngitis), respectively.

No particular pathogenic organism predominantly caused bacterial coinfections, although most pulmonary coinfections were caused by Staphylococcus aureus and/or Pseudomonas aeruginosa. Greater PELOD-2 scores at admission were associated with bacterial coinfections (aRR of 1.2), in addition to age, sex, and pulmonary conditions other than asthma (aRR 2.3).

Conclusion

Overall, the study findings showed that community-onset bacterial coinfections among children with critical COVID-19 are not frequent; however, empiric antibiotics are usually prescribed. The study findings inform antibiotic use and underpin swift de-escalation in case assessments indicating that coinfections are not likely.

Journal reference:
Microbiology

Antibiotics can destroy many types of bacteria, but increasingly, bacterial pathogens are gaining resistance to many commonly used …

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Antibiotics can destroy many types of bacteria, but increasingly, bacterial pathogens are gaining resistance to many commonly used types. As the threat of antibiotic resistance looms large, researchers have sought to find new antibiotics and other ways to destroy dangerous bacteria. But new antibiotics can be extremely difficult to identify and test. Bacteriophages, which are viruses that only infect bacterial cells, might offer an alternative. Bacteriophages (phages) were studied many years ago, before the development of antibiotic drugs, and they could help us once again.

Image credit: Pixabay

If we are going to use bacteriophages in the clinic to treat humans, we should understand how they work, and how bacteria can also become resistant to them. Microbes are in an arms race with each other, so while phages can infect bacteria, some bacterial cells have found ways to thwart the effects of those phages. New research reported in Nature Microbiology has shown that when certain bacteria carry a specific genetic mutation, phages don’t work against them anymore.

In this study, the researchers used a new technique so they could actually see a phage attacking bacteria. Mycobacteriophages infect Mycobacterial species, including the pathogens Mycobacterium tuberculosis and Mycobacterium abscessus, as well as the harmless Mycobacterium smegmatis, which was used in this research.

The scientists determined that Mycobacterial gene called lsr2 is essential for many mycobacteriophages to successfully infect Mycobacteria. Mycobacteria that carry a mutation that renders the Lsr2 protein non-functional are resistant to these phages.

Normally, Lsr2 aids in DNA replication in bacterial cells. Bacteriophages can harness this protein, however, and use it to reproduce the phage’s DNA. Thus, when Lsr2 stops working, the phage cannot replicate and it cannot manipulate bacterial cells.

In the video above, by first study author Charles Dulberger, a genetically engineered mutant phage infects Mycobacterium smegmatis. First, one phage particle (red dot at 0.42 seconds) binds to a bacterium. The phage DNA (green fluorescence) is injected into the bacterial cell (2-second mark). The bright green dots at the cells’ ends are not relevant. For a few seconds, the DNA forms a zone of phage replication, and fills the cell. Finally, the cell explodes at 6:25 seconds. (About three hours have been compressed to make this video.)

The approach used in this study can also be used to investigate other links between bacteriophages and the bacteria they infect.

“This paper focuses on just one bacterial protein,” noted co-corresponding study author Graham Hatfull, a Professor at the University of Pittsburgh. But there are many more opportunities to use this technique. “There are lots of different phages and lots of other proteins.”

Sources: University of Pittsburgh, Nature Microbiology


Carmen Leitch