During the 2022-2023 winter season Denmark experienced a surge in infections caused by group A streptococci (GAS), including the more dangerous, invasive types of infections (iGAS). Incidence of iGAS is highest among the elderly, but the largest relative increase from previous seasons was seen among children. The study is being presented to the European Congress of Clinical Microbiology & Infectious Diseases (ECCMID 2023, Copenhagen, 15-18 April), by Thor Bech Johannesen and Steen Hoffmann, Statens Serum Institut, Copenhagen, Denmark, and colleagues.
Following the implementation of lockdown measures to prevent spread of COVID-19 in March 2020, the number of invasive infections caused by GAS, including more dangerous invasive types (iGAS), decreased. However, during November 2022, an increasing number of these infections occurred in all regions of Denmark, with incidence rates reaching three times the pre-lockdown levels in January-March 2023. While there is no policy on mandatory reporting of GAS infection in Denmark, clinical microbiology laboratories nationwide submit isolates of iGAS to Statens Serum Institut (SSI) for further characterization on a voluntary basis.
Since 2018 approximately 90% of all iGAS cases in Denmark have been submitted to SSI for whole genome sequencing (WGS). For the period 2018 through March 2023, the authors extracted these WGS data and all records from the Danish Microbiology Database (MiBa) with culture-proven GAS and iGAS (invasive GAS being defined as GAS isolated from an anatomical region that should be sterile). Repeated specimens from the same patient of either GAS or iGAS within a 30-day-period were excluded. Potential date of death was collected from the Danish Civil Registration System.
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Incidence of GAS as well as iGAS decreased notably following the restrictions in March 2020. The incidence of both remained at low levels until October 2022 and then dramatically increased in December 2022, peaking between January and March 2023 (Figure 1). The incidence of iGAS was highest in the age groups 65-84 years (4.0/100,000) and 85+ years (5.2/100,000) (Figure 2). Fatalities from iGAS in absolute numbers have also increased, but the case fatality rates for all age groups were similar to previous seasons (approximately 15% overall, and 30% in those aged 85 years and older – rates in children are low and vary due to low absolute numbers).
The strains ST28 emm1 (also known as M1) and ST36 emm12, which have both been virtually absent since April 2020, accounted for 53% and 28%, respectively, of iGAS infections in 2023. A new subvariant of M1 emerged in 2022 and has become the dominant subvariant in 2023, accounting for 30% of all iGAS cases (Figure 3). In addition to a distinct core genome, this variant is characterised by its acquisition of a bacteriophage carrying the virulence factor SpeC, a known key exotoxin. From initial analyses, the novel M1 subvariant does not appear to be significantly more virulent than other M1 variants circulating in Denmark, however, M1 variants in general are more likely to cause invasive disease, and iGAS patients infected with M1 variants are more often in need of intensive care. No significant difference was found in mortality rates for individual variants.
The authors conclude: “Since December 2022, the incidence of iGAS-cases in Denmark has been unusually high, partly driven by the emergence of a new M1 subvariant, which has been responsible for 30% of iGAS cases in 2023. Although a large proportion of the variants currently circulating in Denmark have a high capacity for virulence, we estimate that the current surge is largely due to extensive community spread, possibly combined with a low level of immunity in the general population following two years of extraordinarily low incidence rates.“
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Viruses are usually associated with illness. But our bodies are full of both bacteria and viruses that constantly proliferate and interact with each other in our gastrointestinal tract. While we have known for decades that gut bacteria in young children are vital to protect them from chronic diseases later on in life, our knowledge about the many viruses found there is minimal.
A few years back, this gave University of Copenhagen professor Dennis Sandris Nielsen the idea to delve more deeply into this question. As a result, a team of researchers from COPSAC (Copenhagen Prospective Studies on Asthma in Childhood) and the Department of Food Science at UCPH, among others, spent five years studying and mapping the diaper contents of 647 healthy Danish one-year-olds.
“We found an exceptional number of unknown viruses in the feces of these babies. Not just thousands of new virus species – but to our surprise, the viruses represented more than 200 families of yet to be described viruses. This means that, from early on in life, healthy children are tumbling about with an extreme diversity of gut viruses, which probably have a major impact on whether they develop various diseases later on in life,” says Professor Dennis Sandris Nielsen of the Department of Food Science, senior author of the research paper about the study, now published in Nature Microbiology.
The researchers found and mapped a total of 10,000 viral species in the children’s feces – a number ten times larger than the number of bacterial species in the same children. These viral species are distributed across 248 different viral families, of which only 16 were previously known. The researchers named the remaining 232 unknown viral families after the children whose diapers made the study possible. As a result, new viral families include names like Sylvesterviridae, Rigmorviridae and Tristanviridae.
Bacterial viruses are our allies
This is the first time that such a systematic an overview of gut viral diversity has been compiled. It provides an entirely new basis for discovering the importance of viruses for our microbiome and immune system development. Our hypothesis is that, because the immune system has not yet learned to separate the wheat from the chaff at the age of one, an extraordinarily high species richness of gut viruses emerges, and is likely needed to protect against chronic diseases like asthma and diabetes later on in life.”
Shiraz Shah, first author and senior researcher at COPSAC
Ninety percent of the viruses found by the researchers are bacterial viruses – known as bacteriophages. These viruses have bacteria as their hosts and do not attack the children’s own cells, meaning that they do not cause disease. The hypothesis is that bacteriophages primarily serve as allies:
“We work from the assumption that bacteriophages are largely responsible for shaping bacterial communities and their function in our intestinal system. Some bacteriophages can provide their host bacterium with properties that make it more competitive by integrating its own genome into the genome of the bacterium. When this occurs, a bacteriophage can then increase a bacterium’s ability to absorb e.g. various carbohydrates, thereby allowing the bacterium to metabolize more things,” explains Dennis Sandris Nielsen, who continues:
“It also seems like bacteriophages help keep the gut microbiome balanced by keeping individual bacterial populations in check, which ensures that there are not too many of a single bacterial species in the ecosystem. It’s a bit like lion and gazelle populations on the savannah.”
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Shiraz Shah adds:
“Previously, the research community mostly focused on the role of bacteria in relation to health and disease. But viruses are the third leg of the stool and we need to learn more about them. Viruses, bacteria and the immune system most likely interact and affect each other in some type of balance. Any imbalance in this relationship most likely increases the risk of chronic disease.”
The remaining ten percent of viruses found in the children are eukaryotic – that is, they use human cells as hosts. These can be both friends and foes for us:
“It is thought-provoking that all children run around with 10-20 of these virus types that infect human cells. So, there is a constant viral infection taking place, which apparently doesn’t make them sick. We just know very little about what’s really at play. My guess is that they’re important for training our immune system to recognise infections later. But it may also be that they are a risk factor for diseases that we have yet to discover,” says Dennis Sandris Nielsen.
Could play an important role in inflammatory diseases
The researchers have yet to discover where the many viruses in the one-year-olds come from. Their best answer thus far is the environment:
“Our gut is sterile until we are born. During birth, we are exposed to bacteria from the mother and environment. It is likely that some of the first viruses come along with these initial bacteria, while many others are introduced later via dirty fingers, pets, dirt that kids put in their mouths and other things in the environment,” says Dennis Sandris Nielsen.
As Shiraz Shah points out, the entire field of research speaks to a huge global health problem:
“A lot of research suggests that the majority of chronic diseases that we’re familiar with – from arthritis to depression – have an inflammatory component. That is, the immune system is not working as it ought to – which might be because it wasn’t trained properly. So, if we learn more about the role that bacteria and viruses play in a well-trained immune system, it can hopefully lead us to being able to avoid many of the chronic diseases that afflict so many people today.”
The research groups have begun investigating the role of gut viruses in relation to a number of different diseases that occur in childhood, such as asthma and ADHD.
Extremely long tail provides a window into how bacteria-infecting viruses assemble.
A recent study in the Journal of Biological Chemistry has revealed the secret behind an evolutionary marvel: a bacteriophage with an extremely long tail. This extraordinary tail is part of a bacteriophage that lives in inhospitable hot springs and preys on some of the toughest bacteria on the planet.
Bacteriophages are a group of viruses that infect and replicate in bacteria and are the most common and diverse things on Earth.
“Bacteriophages, or phages for short, are everywhere that bacteria are, including the dirt and water around you and in your own body’s microbial ecosystem as well,” said Emily Agnello, a graduate student at the University of Massachusetts Chan Medical School and the lead author on the study.
Unlike many of the viruses that infect humans and animals that contain only one compartment, phages consist of a tail attached to a spiky, prismlike protein shell that contains their DNA.
Phage tails, like hairstyles, vary in length and style; some are long and bouncy while others are short and stiff. While most phages have short, microscopic tails, the “Rapunzel bacteriophage” P74-26 has a tail 10 times longer than most and is nearly 1 micrometer long, about the width of some spider’s silk. The “Rapunzel” moniker is derived from the fairy tale in which a girl with extremely long hair was locked in a tower by an evil witch.
Brian Kelch, an associate professor of biochemistry and molecular biotechnology at UMass Chan who supervised the work, described P74-26 as having a “monster of a tail.”
Phage tails are important for puncturing bacteria, which are coated in a dense, viscous substance. P74-26’s long tail allows it to invade and infect the toughest bacteria. Not only does P74-26 have an extremely long tail, but it is also the most stable phage, allowing it to exist in and infect bacteria that live in hot springs that can reach over 170° F. Researchers have been studying P74-26 to find out why and how it can exist in such extreme environments.
To work with a phage that thrives in such high temperatures, Agnello had to adjust the conditions of her experiments to coax the phage tail to assemble itself in a test tube. Kelch said Agnello created a system with which she could induce rapid tail self-assembly.
“Each phage tail is made up of many small building blocks that come together to form a long tube. Our research finds that these building blocks can change shape, or conformation, as they come together,” Agnello said. “This shape-changing behavior is important in allowing the building blocks to fit together and form the correct structure of the tail tube.”
The researchers used high-power imaging techniques as well as computer simulations and found that the building blocks of the tail lean on each other to stabilize themselves.
“We used a technique called cryo-electron microscopy, which is a huge microscope that allows us to take thousands of images and short movies at a very high magnification,” Agnello explained. “By taking lots of pictures of the phage’s tail tubes and stacking them together, we were able to figure out exactly how the building blocks fit together.”
They found P74-26 uses a “ball and socket” mechanism to sturdy itself. In addition, the tail is formed from vertically stacking rings of molecules that make a hollow canal.
“I like to think about these phage building blocks as kind of like Legos,” Kelch said. “The Lego has studs on one side and the holes or sockets on the other.”
He added: “Imagine a Lego where the sockets start off closed. But as you start to build with the Legos, the sockets begin to open up to allow the studs on other Legos to build a larger assembly. This movement is an important way that these phage building blocks self-regulate their assembly.”
Kelch pointed out that, compared with most phages, P74-26 uses half the number of building blocks to form stacking rings that make up the tail.
“We think what has happened is that some ancient virus fused its building blocks into one protein. Imagine two small Lego bricks are fused into one large brick with no seams. This long tail is built with larger, sturdier building blocks,” Kelch explained. “We think that could be stabilizing the tail at high temperatures.”
The researchers now plan to use genetic manipulation to alter the length of the phage tail and see how that changes its behavior.
Phages occupy almost every corner of the globe and are important to a variety of industries like healthcare, environmental conservation and food safety. In fact, long-tailed phages like P74-26 have been used in preliminary clinical trials to treat certain bacterial infections.
“Bacteriophages are gaining ever-growing interest as an alternative to antibiotics for treating bacterial infections,” Agnello said. “By studying phage assembly, we can better understand how these viruses interact with bacteria, which could lead to the development of more effective phage-based therapies. … I believe that studying unique, interesting things can lead to findings and applications that we can’t even yet imagine.”
Reference: “Conformational dynamics control assembly of an extremely long bacteriophage tail tube” by Emily Agnello, Joshua Pajak, Xingchen Liu and Brian A. Kelch, 14 March 2023, Journal of Biological Chemistry. DOI: 10.1016/j.jbc.2023.103021
American Society for Biochemistry and Molecular Biology
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.
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.”
Even though humans are complex organisms and bacteria are single cells, and each are made of completely different cell types (eukaryotic and prokaryotic cells, respectively), there are some similar immune mechanisms at work in both of them. Scientists have now learned more about how a complex found in both human and bacterial cells, a group of enzymes called ubiquitin transferases, works to regulate immune pathways. The findings, which have been reported in Nature, may provide new insights into treatments for a wide range of human diseases, suggested the researchers.
“This study demonstrates that we’re not all that different from bacteria,” said senior study author Aaron Whiteley, an assistant professor at the University of Colorado Boulder. “We can learn a lot about how the human body works by studying these bacterial processes.”
Some research has suggested that the immune system found in humans has its origins in bacterial cells. Bacteria have to fight their own infections from other microbes like bacteriophages, viruses that infect bacterial cells. The CRISPR gene editing tool is derived from a bacterial immune defense.
An enzyme called cGAS (cyclic GMP-AMP synthase) can be found in humans, and a simpler version of it is also carried by bacteria; cGAS works to activate an immune defense when viral pathogens are detected.
Researchers have now analyzed the structure of bacterial cGAS, and revealed other proteins that are involved in the response to a viral infection. This study has shown that in bacteria, cGAS is modified by a simplified form of ubiquitin transferase, a crucial enzyme also found in human cells.
Bacteria are far easier to manipulate genetically compared to human cells, so this opens up a world of new research opportunities, said co-first study author Hannah Ledvina, PhD, a postdoctoral researcher. “The ubiquitin transferases in bacteria are a missing link in our understanding of the evolutionary history of these proteins.”
In this research, the scientists have also revealed two critical parts of ubiquitin transferase: Cap2 and Cap3 (CD-NTase-associated protein 2 and 3) that activate and deactivate the cGAS response, respectively.
In humans cells, ubiquitin tags also work to mark cellular garbage, like dysfunctional or unnecessary proteins that have to be degraded and disposed. Problems with this system can lead to a buildup of cellular trash, which may lead to some disorders, such as neurodegeneration.
Thus, while more research is needed, the study authors are hopeful that this work will enable us to learn more about many diseases, including autoimmune disorders like arthritis or neurodegenerative diseases such as Parkinson’s disease
Parts of the bacterial ubiquitin transferase complex, like Cap3 – the off switch, could be harnessed to eliminate some pathologies related to human disease, suggested Whiteley.
CRISPR, the revolutionary gene-editing tool, is making waves in the scientific community once more with its potential to edit the genomes of viruses that infect bacteria.
Led by CRISPR pioneers Jennifer Doudna and Jill Banfield, a team has used a rare form of CRISPR to engineer custom bacteriophages, a development that could aid in the treatment of drug-resistant infections and allow researchers to control microbiomes without the use of antibiotics. The research, published in Nature Microbiology, represents a significant achievement as the engineering of bacteriophages has long been a challenge for the scientific community.
“Bacteriophages are some of the most abundant and diverse biological entities on Earth. Unlike prior approaches, this editing strategy works against the tremendous genetic diversity of bacteriophages,” said first author Benjamin Adler, a postdoctoral fellow in Doudna’s lab. “There are so many exciting directions here – discovery is literally at our fingertips!”
Bacteriophages, also simply called phages, insert their genetic material into bacterial cells using a syringe-like apparatus, then hijack the protein-building machinery of their hosts in order to reproduce themselves – usually killing the bacteria in the process. (They’re harmless to other organisms, including us humans, even though electron microscopy images have revealed that they look like sinister alien spaceships.)
CRISPR-Cas is a type of immune defense mechanism that many bacteria and archaea use against phages. A CRISPR-Cas system consists of short snippets of RNA that are complementary to sequences in phage genes, allowing the microbe to recognize when invasive genetic material has been inserted, and scissor-like enzymes that neutralize the phage genes by cutting them into harmless pieces, after being guided into place by the RNA.
Over millennia, the perpetual evolutionary battle between phage offense and bacterial defense forced phages to specialize. There are a lot of microbes, so there are also a lot of phages, each with unique adaptations. This astounding diversity has made phage editing difficult, including making them resistant to many forms of CRISPR, which is why the most commonly used system – CRISPR-Cas9 – doesn’t work for this application.
“Phages have many ways to evade defenses, ranging from anti-CRISPRs to just being good at repairing their own DNA,” said Adler. “So, in a sense, the adaptations encoded in phage genomes that make them so good at manipulating microbes are the exact same reason why it has been so difficult to develop a general-purpose tool for editing their genomes.”
Project leaders Doudna and Banfield have developed numerous CRISPR-based tools together since they first collaborated on an early investigation of CRISPR in 2008. That work – performed at Lawrence Berkeley National Laboratory (Berkeley Lab) – was cited by the Nobel Prize committee when Doudna and her other collaborator, Emmanuelle Charpentier, received the prize in 2020. Doudna and Banfield’s team of Berkeley Lab and UC Berkeley researchers were studying the properties of a rare form of CRISPR called CRISPR-Cas13 (derived from a bacterium commonly found in the human mouth) when they discovered that this version of the defense system works against a huge range of phages.
The phage-fighting potency of CRISPR-Cas13 was unexpected given how few microbes use it, explained Adler. The scientists were doubly surprised because the phages it defeated in testing all infect using double-stranded DNA, but the CRISPR-Cas13 system only targets and chops single-stranded viral RNA. Like other types of viruses, some phages have DNA-based genomes and some have RNA-based genomes. However, all known viruses use RNA to express their genes. The CRISPR-Cas13 system effectively neutralized nine different DNA phages that all infect strains of E. coli, yet have almost no similarity across their genomes.
According to co-author and phage expert Vivek Mutalik, a staff scientist in Berkeley Lab’s Biosciences Area, these findings indicate that the CRISPR system can defend against diverse DNA-based phages by targeting their RNA after it has been converted from DNA by the bacteria’s own enzymes prior to protein translation.
Next, the team demonstrated that the system can be used to edit phage genomes rather than just chop them up defensively.
First, they made segments of DNA composed of the phage sequence they wanted to create flanked by native phage sequences and put them into the phage’s target bacteria. When the phages infected the DNA-laden microbes, a small percentage of the phages reproducing inside the microbes took up the altered DNA and incorporated it into their genomes in place of the original sequence. This step is a longstanding DNA editing technique called homologous recombination. The decades-old problem in phage research is that although this step, the actual phage genome editing, works just fine, isolating and replicating the phages with the edited sequence from the larger pool of normal phages is very tricky.
This is where the CRISPR-Cas13 comes in. In step two, the scientists engineered another strain of host-microbe to contain a CRISPR-Cas13 system that senses and defends against the normal phage genome sequence. When the phages made in step one were exposed to the second-round hosts, the phages with the original sequence were defeated by the CRISPR defense system, but the small number of edited phages were able to evade it. They survived and replicated themselves.
Experiments with three unrelated E. coli phages showed a staggering success rate: more than 99% of the phages produced in the two-step processes contained the edits, which ranged from enormous multi-gene deletions all the way down to precise replacements of a single amino acid.
“In my opinion, this work on phage engineering is one of the top milestones in phage biology,” said Mutalik. “As phages impact microbial ecology, evolution, population dynamics, and virulence, seamless engineering of bacteria and their phages has profound implications for foundational science but also has the potential to make a real difference in all aspects of the bioeconomy. In addition to human health, this phage engineering capability will impact everything from biomanufacturing and agriculture to food production.”
Buoyed by their initial results, the scientists are currently working to expand the CRISPR system to use it on more types of phages, starting with ones that impact microbial soil communities. They are also using it as a tool to explore the genetic mysteries within phage genomes. Who knows what other amazing tools and technologies can be inspired by the spoils of microscopic war between bacteria and viruses?
Reference: “Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing” by Benjamin A. Adler, Tomas Hessler, Brady F. Cress, Arushi Lahiri, Vivek K. Mutalik, Rodolphe Barrangou, Jillian Banfield and Jennifer A. Doudna, 31 October 2022, Nature Microbiology. DOI: 10.1038/s41564-022-01258-x
The study was was funded by the Department of Energy Microbial Community Analysis & Functional Evaluation in Soils (m-CAFES) Scientific Focus Area.
Microbes can easily share genes. Not only can different types of bacteria do this, there is also evidence that entirely different branches of life – archaea and bacteria can also share genes. Some microbial genes can be found on small bits of DNA called mobile genetic elements, which are not a part of a microbe’s genome, but can still be expressed when they’re a microbial cell. These mobile genetic elements can move from one cell to another in a process known as horizontal gene transfer. Researchers have now found that bacteria in the maternal microbiome can share genes with bacteria in the infant microbiome, in the period just before birth until a few weeks after delivery – the perinatal period. Horizontal gene transfer enables maternal microbes to influence how bacteria in the infant microbiome are functioning, without actually moving the maternal microbes themselves. These findings have been reported in Cell.
“This is the first study to describe the transfer of mobile genetic elements between maternal and infant microbiomes,” said senior study author Ramnik Xavier of the Broad Institute of MIT and Harvard. “Our study also, for the first time, integrated gut microbiome and metabolomic profiles from both mothers and infants and discovered links between gut metabolites, bacteria and breastmilk substrates. This investigation represents a unique perspective into the codevelopment of infant gut microbiomes and metabolomes under the influence of known maternal and dietary factors.”
The gut microbiome produces metabolites that can affect various aspects of infant development, such as immune system maturation and cognitive development during the perinatal period, a critical window. At birth, microbes move from the maternal microbiome to the infant microbiome, but we still have a lot to learn about how microbes are affecting development, and how they are developing into a microbiome themselves.
In this study, the researchers tracked the microbiomes and metabolites of 70 infant-mother pairs, from late pregnancy until the babies were one year old. This research showed that mobile genetic elements moved from microbes carried by moms and into microbes carried by infants. The mobile genetic elements that were transferred were often related to diet.
Infants were also found to have less diversity in their metabolomes compared to moms, however, there were metabolites, and links between microbes and metabolites that were identified exclusively by infants. Infants that got regular formula (that was not excessively hydrolyzed) also had metabolomes and cytokine signatures that were different from infants that were exclusively breastfed.
“The infant gut harbored thousands of unique metabolites, many of which were likely modified from breastmilk substrates by gut bacteria,” noted co-first study author Tommi Vatanen of the Broad Institute of MIT and Harvard. “Many of these metabolites likely impact immune system and cognitive development.”
This process seems to be a way for the maternal microbiome to exert an influence on the infant microbiome withouth transmitting specific species of bacteria.
Prophages, which are dormant bacteriophages, also seem to be involved in the movement of mobile genetic elements between the maternal and infant microbiomes, added Xavier.
For decades, humans have relied on antibiotics to eliminate bacterial infections, and for a long time, those antibiotics worked reliably. But microbes, like other forms of life, can evolve to find ways around the things that impede their growth and survival. Pathogenic bacteria are doing that with antibiotics, and while scientists are searching for new antibiotic compounds that can be used therapeutically, creating new medicines presents many challenges and can take a long time. Bacteriophages, which are viruses that only infect bacterial cells, might be a solution to the problem of antibiotic resistance. But if they are going to work against infections in humans, we need to know more about them first.
Scientists have now used a variety of methods including cryo-electron microscopy, machine learning, and simulations to characterize the structure and function of a bacteriophage that normally lives in the human gastrointestinal tract. The phage, called ϕKp24, could be useful against multidrug resistant strains of Klebsiella pneumoniae. The work has been reported in Nature Communications.
The World Health Organization has designated K. pneumoniae a priority 1 pathogen, meaning that it is critical to create new antibiotics that can eliminate these infections. There are over one hundred genetically distinct types of K. pneumoniae, which can cause bacteremia, urinary tract infection, pneumonia, and other diseases. Immunocompromised individuals are at particular risk.
Although bacteriophages can destroy multi-drug resistant K. pneumoniae, most of these phages are very individualized, and will only attack specific strains of the pathogen. This study aimed to reveal more about the ϕKp24 phage, which may be able to attack multiple strains of K. pneumoniae.
The study authors suggested that ϕKp24 may be a good candidate for the development of phage therapy for humans.
“The problem with phage therapy is that bacteriophages don’t work on all bacteria, even from the same species. In contrast to many known bacteriophages, this one is special because it works on many different subtypes. This makes it a good candidate for phage therapy,” explained corresponding study author Professor Ariane Briegel of Institute Biology Leiden. “By learning more about how the bacteriophage works, we can hopefully treat people with it in the future.”
Cryo-electron microscopy by University of Alabama at Birmingham researchers has exposed the structure of a bacterial virus with unprecedented detail. This is the first structure of a virus able to infect Staphylococcus epidermidis, and high-resolution knowledge of structure is a key link between viral biology and potential therapeutic use of the virus to quell bacterial infections.
Bacteriophages or “phages” is the terms used for viruses that infect bacteria. The UAB researchers, led by Terje Dokland, Ph.D., in collaboration with Asma Hatoum-Aslan, Ph.D., at the University of Illinois Urbana-Champaign, have described atomic models for all or part of 11 different structural proteins in phage Andhra. The study is published in Science Advances.
Andhra is a member of the picovirus group. Its host range is limited to S. epidermidis. This skin bacterium is mostly benign but also is a leading cause of infections of indwelling medical devices. “Picoviruses are rarely found in phage collections and remain understudied and underused for therapeutic applications,” said Hatoum-Aslan, a phage biologist at the University of Illinois.
With emergence of antibiotic resistance in S. epidermidis and the related pathogen Staphylococcus aureus, researchers have renewed interest in potentially using bacteriophages to treat bacterial infections. Picoviruses always kill the cells they infect, after binding to the bacterial cell wall, enzymatically breaking through that wall, penetrating the cell membrane and injecting viral DNA into the cell. They also have other traits that make them attractive candidates for therapeutic use, including a small genome and an inability to transfer bacterial genes between bacteria.
Knowledge of protein structure in Andhra and understanding of how those structures allow the virus to infect a bacterium will make it possible to produce custom-made phages tailored to a specific purpose, using genetic manipulation.
The structural basis for host specificity between phages that infect S. aureus and S. epidermidis is still poorly understood. With the present study, we have gained a better understanding of the structures and functions of the Andhra gene products and the determinants of host specificity, paving the way for a more rational design of custom phages for therapeutic applications. Our findings elucidate critical features for virion assembly, host recognition and penetration.”
Terje Dokland, professor of microbiology at UAB and director of the UAB Cryo-Electron Microscopy Core
Staphylococcal phages typically have a narrow range of bacteria they can infect, depending on the variable polymers of wall teichoic acid on the surface of different bacterial strains. “This narrow host range is a double-edged sword: On one hand, it allows the phages to target only the specific pathogen causing the disease; on the other hand, it means that the phage may need to be tailored to the patient in each specific case,” Dokland said.
The general structure of Andhra is a 20-faced, roundish icosahedral capsid head that contains the viral genome. The capsid is attached to a short tail. The tail is largely responsible for binding to S. epidermidis and enzymatically breaking the cell wall. The viral DNA is injected into the bacterium through the tail. Segments of the tail include the portal from the capsid to the tail, and the stem, appendages, knob and tail tip.
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The 11 different proteins that make up each virus particle are found in multiple copies that assemble together. For instance, the capsid is made of 235 copies each of two proteins, and the other nine virion proteins have copy numbers from two to 72. In total, the virion is made up of 645 protein pieces that include two copies of a 12th protein, whose structure was predicted using the protein structure prediction program AlphaFold.
The atomic models described by Dokland, Hatoum-Aslan, and co-first authors N’Toia C. Hawkins, Ph.D., and James L. Kizziah, Ph.D., UAB Department of Microbiology, show the structures for each protein -; as described in molecular language like alpha-helix, beta-helix, beta-strand, beta-barrel or beta-prism. The researchers have described how each protein binds to other copies of that same protein type, such as to make up the hexameric and pentameric faces of the capsid, as well as how each protein interacts with adjacent different protein types.
Electron microscopes use a beam of accelerated electrons to illuminate an object, providing much higher resolution than a light microscope. Cryo-electron microscopy adds the element of super-cold temperatures, making it particularly useful for near-atomic structure resolution of larger proteins, membrane proteins or lipid-containing samples like membrane-bound receptors, and complexes of several biomolecules together.
In the past eight years, new electron detectors have created a tremendous jump in resolution for cryo-electron microscopy over normal electron microscopy. Key elements of this so-called “resolution revolution” for cryo-electron microscopy are:
Flash-freezing aqueous samples in liquid ethane cooled to below -256 degrees F. Instead of ice crystals that disrupt samples and scatter the electron beam, the water freezes to a window-like “vitreous ice.”
The sample is kept at super-cold temperatures in the microscope, and a low dose of electrons is used to avoid damage to the proteins.
Extremely fast direct electron detectors are able to count individual atoms at hundreds of frames per second, allowing sample movement to be corrected on the fly.
Advanced computing merges thousands of images to generate three-dimensional structures at high resolution. Graphics processing units are used to churn through terabytes of data.
The microscope stage that holds the sample can also be tilted as images are taken, allowing construction of a three-dimensional tomographic image, similar to a CT scan at the hospital.
The analysis of Andhra virion structure by the UAB researchers started with 230,714 particle images. Molecular reconstruction of the capsid, tail, distal tail and tail tip started with 186,542, 159,489, 159,489 and 159,489 images, respectively. Resolution ranged from 3.50 to 4.90 angstroms.
Thought LeadersMatthew DunneDirector for Drug DiscoveryMicreos Pharamceuticals
For World Antimicrobial Awareness Week 2022, we speak to Matthew Dunne, Director for Drug Discovery at Micreos Pharmaceuticals, about the importance of creating new targeted antibacterial products.
Please can you introduce yourself and tell us about your role at Micreos?
My name is Matthew Dunne, and I am a Director for Drug Discovery at Micreos Pharmaceuticals in Switzerland. I provide strategic and technical leadership for R&D and preclinical activities within our newly established Division of Antimicrobial Vector Innovation. I joined Micreos in May of 2022 from the Swiss Federal Institute of Technology Zurich (ETH Zurich) at the same time as Dr. Samuel Kilcher, who sits alongside me as co-Director within the new division, which is developing a new class of medicines we have coined Antimicrobial Vectors.
In my capacity as Director, I work from Micreos’ state-of-the-art research facility in Switzerland, where I analyze data together with our growing team of genetic engineers and biologists. In addition to providing leadership of this new, highly innovative drug discovery division, I provide assistance with developing our regulatory affairs strategy, the management of external innovation development projects with industry partners and academia, as well as dealing with a variety of diverse tasks that are typical for a fast-growing biotech company.
Image Credit: Inspiring/Shutterstock.com
You are currently a Director of Drug Discovery at Micreos, a company working to develop the world’s first targeted antibacterial products. Can you tell us more about Micreos’ vision and the importance of finding alternatives to antibiotics?
Micreos is working towards providing innovative therapeutic solutions that deliver a profound and transformational impact to improve the standard of care for people living with devastating illnesses.
Antimicrobial resistance, or AMR is a naturally occurring process that cannot be eliminated; it can only be controlled. Unfortunately, decades of overprescribing antibiotics in combination with the use of antibiotics in agriculture and farming, such as growth factors for livestock (that has been banned in the EU since 2006), has driven the spread of antimicrobial resistance genes among bacterial pathogens. AMR is estimated to have caused 1.27 million deaths in 2019, with this number expected to keep on growing. Nevertheless, we are fighting back.
At Micreos, we are developing two classes of antimicrobials: Endolysins and Antimicrobial Vectors. Both have different modes of action compared to antibiotics, making them capable of killing all AMR bacteria. Both technologies provide other important advantages, too, such as their ability to precisely kill a specific pathogenic species while leaving commensal or “good” bacteria unaffected. Also, due to their alternative mechanisms of action, they are able to circumvent some of the harmful side effects of antibiotic use.
The drug discovery sector has seen considerable advances in the last decade, thanks largely to technology and increased collaboration. How do you feel this sector has changed in recent years and what has personally been the most exciting development that you have seen?
Global healthcare is rapidly transitioning towards precision medicine. Personally, I think the most impressive advancements over the last decade have been realized with nucleic acids. For example, antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) that modulate gene expression are being designed for large indications, rare diseases, and even patients with ultrarare, “n-of-1” diseases.
In the last two years, we all witnessed another form of nucleic acid therapy, mRNA. In less than a year, scientists went from sequencing the SARS-CoV-2 virus to designing different mRNA vaccines for global distribution. I am sure there are going to be many more exciting developments within this space in the near future. I am especially interested to see how the mRNA field progresses with regard to gene therapy, where mRNA can be administered to compensate for a faulty gene or protein.
Image Credit: paulista/Shutterstock.com
Micreos has recently launched a new world-class antimicrobial vector engineering team to ramp up the fight against antimicrobial resistance (AMR). Can you tell us more about why this team was created and the work you are carrying out?
Bacteriophages are natural predators of bacteria that, for over 100 years, have sat on the sidelines of modern medicine. They have mostly been applied as an experimental treatment, reserved for patients suffering from chronic infections that are untreatable with conventional antibiotics. As the threat of AMR intensifies, there is a significant demand for developing and enhancing the capabilities of alternative therapeutics to treat bacterial infections, among many other chronic and rare diseases.
At Micreos Pharmaceuticals, we are heavily invested in harnessing the power of genetic information. In the Division of Antimicrobial Vectors, we use the genomes of bacterial viruses or bacteriophages as “blueprints” for engineering using CRISPR-Cas technology as well as various synthetic approaches. First, we isolate and sequence bacteriophages from different environments that are predisposed to target and kill certain pathogenic species. Next, the fun starts, as the team and I get to apply our knowledge and expertise in bacteriophages, biochemistry, and structural biology to reprogram these genetic “blueprints” to generate Antimicrobial Vector libraries.
We can engineer structural genes for improved stability, introduce heterologous payloads for improved potency, remove unneeded elements for better efficiency and safety, and reprogram their targeting capabilities to reach bacteria in niche locations, such as intracellular reservoirs or biofilms. The resulting libraries of Antimicrobial Vectors provide unique and therapeutically important functions when used against bacterial infections.
This new team combines individuals with a variety of knowledge across various sectors, including molecular microbiology, genetic engineering, and phage therapy. Why is having a multidisciplinary team vital when developing new ways to tackle infectious diseases?
We are fortunate to have assembled a multidisciplinary team of experts proficient in all aspects of the Antimicrobial Vector R&D process, from selecting and testing environmental bacteriophages, to designing genetic scaffolds for reprogramming, to early-stage production, efficacy assessment, manufacturing optimization, and preclinical testing.
Our team also works very closely with experts in clinical trial design and regulatory affairs. This not only makes for interesting coffee breaks, where ideas and alternative perspectives are thrown around, but it ensures that we have a drug development pipeline that runs as efficiently as possible. It is important to have frequent input regarding aspects of safety, translatability, and efficacy to ensure our medicines will translate as quickly as possible from bench to bedside.
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How can the technology developed at Micreos help to tackle AMR through the creation of precision antimicrobials?
We are seeing more and more biologics and recombinant protein-based therapies in development to accompany small-molecule antibiotics in the fight against AMR. Micreos has already established itself as the global experts in engineering of endolysins, which has led to an array of precision protein-based antimicrobials capable of targeted killing of harmful S. aureus pathogen while leaving beneficial bacteria intact and without triggering resistance development.
During my PhD studies at EMBL Hamburg, I solved the atomic structures of these cell wall degrading enzymes and witnessed firsthand how miniscule amounts of endolysin could eradicate entire monocultures of specific bacteria in minutes with no off-target effects against “good” bacteria, such as those found on our skin or in our guts. Unlike the development of AMR against antibiotics, scientists do not expect to see similar resistance mechanisms emerge for endolysins due to their targeting of essential cell wall components that are extremely difficult for bacteria to modify.
Currently, our pharmaceutical grade endolysins are being developed for atopic dermatitis, diabetic foot ulcers, cutaneous T-cell lymphoma (based on excessive skin colonization by S. aureus) and bloodstream infections.
At the right time, and following extensive preclinical testing, we are all excited to witness our Antimicrobial Vector technology follow in the footsteps of our endolysins as it translates from discovery to clinical trials and onto improving the standard of care for people suffering from infections and many other devastating disorders.
Every year, the world celebrates World Antimicrobial Awareness Week (WAAW), dedicated to spreading awareness about AMR. The theme for 2022 is ‘Preventing Antimicrobial Resistance Together‘. What does this message mean to you, and how can international collaboration help to tackle this global health threat?
In 2019, nearly 5 million people died from illnesses involving AMR bacteria. Based on the current trajectory, these numbers are only going to keep rising – and at quicker and quicker rates – with predictions estimating AMR will cause 10 million deaths by the year 2050. The solution to controlling antimicrobial resistance is to work together internationally to implement more effective governance surrounding antimicrobials, improve public awareness surrounding antibiotics, and fund the development of new classes of antimicrobials to bolster our arsenal of available medicines.
It is important that drug developers, researchers, health authorities, and academics all play a part, no matter how big (e.g., establishing initiatives and investment funds) or small (e.g., tweets, chats among friends in the pub), to help raise public awareness surrounding AMR. Events such as those taking place during WAAW and their ability to disseminate information about AMR and its threat to our everyday lives are incredibly important. The public needs to know that AMR could impact our normal way of life. We risk reversing nearly a century of progress in public health if we allow normally innocuous infections to again become untreatable.
In addition to WAAW, we are seeing an expansion in other AMR initiatives, the introduction of innovation funds, and a growing number of collaborative organizations providing much-needed platforms for engagement and collaboration between industry, researchers, non-profit organizations, charities, and governments around the world.
Micreos has always focused on forging strong collaborations with other industry partners, clinicians, and academia to help advance the development of our precision antimicrobials. For instance, our proprietary endolysin technology was created together with ETH Zurich, which remains an important partner to us moving forwards with our Antimicrobial Vector technology.
Image Credit: The World Health Organization
Despite AMR being described as one of the top 10 threats to humanity, many people still do not understand its wide-reaching effects. Why is this, and why is it therefore so critical to continue to raise awareness?
I believe this is due to poor public communication and education regarding what antibiotics are, how they work, and what AMR really means. In 2015, when the WHO asked 10,000 people from 12 different countries about antibiotics, 76% of respondents believed that antibiotic resistance happens when the body becomes resistant to antibiotics – rather than bacteria becoming resistant to the antibiotics. Moreover, 44% of people believed they are not at risk of antibiotic-resistant infections if they simply take antibiotics as prescribed and of course, that is not correct.
Governments, academics, drug developers, and health professionals must do better at communicating a clearer message about what antibiotics are and – most importantly – why they are a precious resource that we cannot continue to take for granted.
What do you believe the future of antimicrobials to look like? Is it possible to one day see a world without resistance?
Another imminent threat to human existence is climate change, which shares many similarities, such as urgency, severity, and global effects, as we are seeing with the spread of AMR. What gives me hope for the future of antimicrobials and tackling AMR is witnessing the growth in public conversation and awareness surrounding climate change; the same will happen with AMR.
Improving awareness for AMR is about educating and mobilizing audiences so they are driven to take their own actions and make their own decisions toward confronting this growing crisis. I am hopeful that everyone will play a part through communication, the sharing of novel solutions, and advocating for change that will be shaped by our different experiences, cultures, and underlying values.
Originally from Macclesfield in the Northwest of England, Matthew studied Biochemistry at the University of Birmingham before obtaining a Ph.D. in Biochemistry and Structural Biology from the European Molecular Biology Laboratory (EMBL) in Hamburg and the University College Cork, Ireland, where he characterized the atomic structure and function of endolysins.
For the last eight years, Matthew has worked as a Postdoc and then Senior Scientist at the Swiss Institute of Technology in Zurich (ETH Zurich), where he investigated the molecular-level interactions of bacteriophages against a wide variety of foodborne and clinical pathogens, produced novel bacterial diagnostics, and developed genetic engineering tools that have been used to produce different types of bacteriophage-based therapeutics and diagnostic elements. Matthew maintains a research group within the lab of Prof. Martin Loessner at ETH Zurich, where he is actively involved in using genetic engineering to further explore how bacteriophages interact with their hosts, as well as lead a team of researchers developing bacteriophages to treat urinary tract infections for assessment in future clinical trials.
Matthew lives in Zurich with his wife, Alyssa Hill, also a Senior Scientist in Pharmaceutical Chemistry at ETH Zurich. In his free time, you will find Matthew swimming in the lakes and rivers dotted around the city, coaching and playing field hockey for the Red Sox HC, or skiing, hiking, and exploring Switzerland with Alyssa.
Eichenseher F, Herpers BL, Badoux P, Leyva-Castillo JM, Geha RS, van der Zwart M, McKellar J, Janssen F, de Rooij B, Selvakumar L, Röhrig C, Frieling J, Offerhaus M, Loessner MJ, Schmelcher M. Linker-Improved Chimeric Endolysin Selectively Kills Staphylococcus aureus In Vitro, on Reconstituted Human Epidermis, and in a Murine Model of Skin Infection. Antimicrob Agents Chemother. 2022 May 17;66(5):e0227321. doi: 10.1128/aac.02273-21. Epub 2022 Apr 13. PMID: 35416713; PMCID: PMC9112974.
Microbiome: From Research and Innovation to Market
