Research opens possibility of reducing risk of gut bacterial infections with next-generation probiotic

A team of researchers is exploring the possibility that next-generation probiotics – live bacteria that are good for your health – would reduce the risk of infection with the bacterium Clostridium difficile. In laboratory-grown bacterial communities, the researchers determined that, when supplied with glycerol, the probiotic Lactobacillus reuteri produced reuterin, an antibacterial compound that selectively killed C. difficile. The study appears in Infection and Immunity.

C. difficile causes thousands of deaths and billions of dollars in healthcare expenses in the U.S. each year. Although most patients respond to antibiotic treatment, up to 35 percent will relapse and require extended antibiotic treatments,” said first and corresponding author Dr. Jennifer K. Spinler, instructor of pathology & immunology at Baylor College of Medicine, who oversees microbial genetics and genomics efforts at the Texas Children’s Microbiome Center at Texas Children’s Hospital.

C. difficile infections are the most common cause of diarrhea associated with the use of antibiotics. If these bacteria attempt to invade the human gut, the ‘good bacteria,’ which outnumber C. difficile, usually prevent them from growing. However, when a person takes antibiotics, for example to treat pneumonia, the antibiotic also can kill the good bacteria in the gut, opening an opportunity for C. difficile to thrive into a potentially life-threatening infection.

“When repeated antibiotic treatments fail to eliminate C. difficile infections, some patients are resorting to fecal microbiome transplant – the transfer of fecal matter from a healthy donor – which treats the disease but also could have negative side effects,” Spinler said. “We wanted to find an alternative treatment, a prophylactic strategy based on probiotics that could help prevent C. difficile from thriving in the first place.”

“Probiotics are commonly used to treat a range of human diseases, yet clinical studies are generally fraught by variable clinical outcomes and protective mechanisms are poorly understood in patients. This study provides important clues on why clinical efficacy may be seen in some patients treated with one probiotic bacterium but not with others,” said senior author Dr. Tor Savidge, associate professor of pathology & immunology and of pediatrics at Baylor and the Texas Children’s Microbiome Center.

Working in the Texas Children’s Microbiome Center, Spinler and her colleagues tested the possibility that probiotic L. reuteri, which is known to produce antibacterial compounds, could help prevent C. difficile from establishing a microbial community in lab cultures.

An unexpected result with major implications for a preventative strategy

Spinler and Savidge established a collaboration with co-author Dr. Robert A. Britton, professor of molecular virology and microbiology at Baylor and member of the Dan L Duncan Comprehensive Cancer Center.

The Britton lab uses mini-bioreactor arrays – multiple small culture chambers – that provide a platform in which researchers could recreate the invasion of an antibiotic-treated human intestinal community by C. difficile.

“Using the mini-bioreactors model we showed that L. reuteri reduced the burden of C. difficile infection in a complex gut community,” Britton said. “To achieve its beneficial effect, L. reuteri requires glycerol and converts it into the antimicrobial reuterin.”

The literature reports reuterin as a broad-spectrum antibiotic; it affects the growth of a wide variety of bacteria when they are tested individually in the lab. What was intriguing in this study is that reuterin didn’t have a broad-spectrum effect in the mini-bioreactor bacterial community setting.

“I expected reuterin to have an antibacterial effect on several different types of bacteria in the community, but it only affected C. difficile and not the good bacteria, which was exciting because it has major implications for a preventative strategy,” Spinler said.

“Although these results are too preliminary to be translated directly into human therapy, they provide a foundation upon which to further develop treatments based on co-administration of L. reuteri and glycerol to prevent C. difficile infection,” said co-author Dr. Jennifer Auchtung, director of the Cultivation Core at Baylor’s Alkek Center for Metagenomics and Microbiome Research and assistant professor of molecular virology and microbiology at Baylor.

In the future, this potential treatment could be administered prophylactically to patients before they take antibiotics known to disrupt normal gut microbes. The L. reuteri/glycerol formulation would help maintain the healthy gut microbial community and also help prevent the growth of C. difficile, which would result in decreased hospital stay and costs and reduced long-term health consequences of C. difficile recurrent infections.

Penn study details impact of antibiotics, antiseptics on skin microbiomes

The use of topical antibiotics can dramatically alter communities of bacteria that live on the skin, while the use of antiseptics has a much smaller, less durable impact. The study, conducted in mice in the laboratory of Elizabeth Grice, PhD, an assistant professor of Dermatology in the Perelman School of Medicine at the University of Pennsylvania, is the first to show the long-term effects of antimicrobial drugs on the skin microbiome. Researchers published their findings today in the journal Antimicrobial Agents and Chemotherapy.

The skin, much like the gut, is colonized by a diverse multitude of microorganisms which generally coexist as a stable ecosystem — many of which are harmless or even beneficial to the host. However, when that ecosystem is disturbed or destabilized, colonization and/or infection by more dangerous microbes can occur. Antiseptics, such as ethanol or iodine, are commonly used to disinfect the skin prior to surgical procedures or following exposure to contaminated surfaces or objects. Topical antibiotics may be used to decolonize skin of specific types of bacteria or for rashes, wounds, or other common conditions.

In the gut, research shows medication that alters microbial communities can lead to complications like Clostridium difficile, or C. diff — which causes diarrhea and is the most common hospital-acquired infection. But when it comes to the skin, the impact of these medications on bacteria strains like Staphylococcus aureus, or S. aureus — the most common cause of skin infections — is still largely unstudied.

“We know antibiotics and antiseptics can be effective in stopping the growth of certain bacteria, but we wanted to know about the larger impact these treatments can have on the resident microbial communities on the skin,” said the study’s lead author, Adam J. SanMiguel, PhD, a researcher in the Grice Laboratory at Penn.

Researchers treated the skin of hairless mice with a variety of antibiotics, including a narrowly targeted mupirocin ointment and a broadly applicable triple-antibiotic ointment (TAO) containing bacitracin, neomycin, and polymyxin B. All of the antibiotics changed the makeup of the microbial communities, and, in a key finding of the study, the impact of that change lasted for days after treatment stopped.

“The problem in this case isn’t antibiotic resistance, but instead, how long the disruption of the skin microbiomes continues,” SanMiguel said. “That disruption opens the door for colonization by an unwanted strain.”

The researchers similarly evaluated antiseptics, using alcohol or povidone-iodine and comparing those treatments with two control groups – mice treated with water and mice entirely untreated. They found neither antiseptic caused responses similar enough to cluster the mice together into groups based on their microbiomes. They also found no clear difference between the treatment groups and the control groups when comparing the relative number of individual bacteria strains.

“We thought antiseptics would be even more disruptive to microbial communities than antibiotics since they are less targeted, but it turns out the opposite is true,” SanMiguel said. “It shows how stable the skin microbiome can be in the face of stress.”

However, both antibiotic and antiseptic treatments removed skin resident bacteria that compete against the pathogenic S. aureus to colonize the skin. Colonization with S. aureus is a risk factor for developing a skin infection.

“This gives us a better understanding of how topical antimicrobials affect the skin microbiome and what kind of impact their disturbance can have in the context of pathogenic colonization,” said Grice, the study’s senior author. “This helps us anticipate their potential effects.”

The researchers say this work can provide the foundation for greater understanding of how the skin defends against infection. They have already begun similar testing in humans.

Gut bacteria could protect cancer patients and pregnant women from Listeria, study suggests

Researchers at Memorial Sloan Kettering Cancer Center in New York have discovered that bacteria living in the gut provide a first line of defense against severe Listeria infections. The study, which will be published June 6 in The Journal of Experimental Medicine, suggests that providing these bacteria in the form of probiotics could protect individuals who are particularly susceptible to Listeria, including pregnant women and cancer patients undergoing chemotherapy.

Listeria monocytogenes is a major pathogen acquired by eating contaminated food, but healthy adults can generally fend off an infection after suffering, at worst, a few days of gastroenteritis. However, some individuals, including infants, pregnant women, and immunocompromised cancer patients, are susceptible to more severe forms of listeriosis, in which the bacterium escapes the gastrointestinal tract and disseminates throughout the body, causing septicemia, meningitis, and, in many cases, death.

Patients with some forms of cancer are as much as 1,000 times more likely to develop listeriosis, possibly because chemotherapy drugs can suppress a patient’s immune system. But a team of researchers led by Simone Becattini and Eric G. Pamer wondered whether the gut microbiome–the community of bacteria that naturally lives in the gastrointestinal tract–might also play a role in limiting L. monocytogenes infection. Chemotherapy disrupts the microbiome, and gut bacteria are known to prevent other food-borne pathogens from colonizing the gastrointestinal tract by, for example, secreting antibacterial toxins.

The researchers found that disrupting the microbiome with antibiotics made laboratory mice more susceptible to L. monocytogenes infection, increasing the pathogen’s ability to colonize the gastrointestinal tract and spread into the circulatory system to cause the animals’ death. The effect of antibiotics was even more noticeable in immunocompromised mice lacking key immune cells; these animals succumbed to even small doses of L. monocytogenes if their microbiomes were disrupted by antibiotic treatment.

Mice treated with the common chemotherapy drugs doxorubicin and cyclophosphamide were vulnerable to Listeria infection, and they became even more susceptible when they were also treated with antibiotics.

The researchers identified four species of gut bacteria–all members of the Clostridiales order–that together were able to limit L. monocytogenes growth in laboratory cultures. Transferring these probiotic bacteria into germ-free mice protected the rodents from Listeria infection by limiting the pathogen’s ability to colonize the gastrointestinal tract and disseminate into other tissues. “Thus, augmenting colonization resistance functions in immunocompromised patients by introducing these protective bacterial species might represent a novel clinical approach to prevent L. monocytogenes infection,” says Becattini.

“Our results also raise the possibility that in other at-risk categories for listeriosis, such as infants or pregnant women, disruptions to the gut microbiome could be a contributing factor to susceptibility,” Becattini continues. “Pregnant women in their third trimester, the phase in which susceptibility to Listeria is known to be highest, show an altered microbiome, with a marked reduction in Clostridiales species.”

Engineers design a new weapon against bacteria

Over the past few decades, many bacteria have become resistant to existing antibiotics, and few new drugs have emerged. A recent study from a U.K. commission on antimicrobial resistance estimated that by 2050, antibiotic-resistant bacterial infections will kill 10 million people per year, if no new drugs are developed.

To help rebuild the arsenal against infectious diseases, many scientists are turning toward naturally occurring proteins known as antimicrobial peptides, which can kill not only bacteria but other microbes such as viruses and fungi. A team of researchers at MIT, the University of Brasilia, and the University of British Columbia has now engineered an antimicrobial peptide that can destroy many types of bacteria, including some that are resistant to most antibiotics.

“One of our main goals is to provide solutions to try to combat antibiotic resistance,” says MIT postdoc Cesar de la Fuente. “This peptide is exciting in the sense that it provides a new alternative for treating these infections, which are predicted to kill more people annually than any other cause of death in our society, including cancer.”

De la Fuente is the corresponding author of the new study, and one of its lead authors along with Osmar Silva, a postdoc at the University of Brasilia, and Evan Haney, a postdoc at the University of British Columbia. Timothy Lu, an MIT associate professor of electrical engineering and computer science, and of biological engineering, is also an author of the paper, which appears in the Nov. 2 issue of Scientific Reports.

Improving on nature

Antimicrobial peptides, produced by all living organisms as part of their immune defenses, kill microbes in several different ways. First, they poke holes in the invaders’ cell membranes. Once inside, they can disrupt several cellular targets, including DNA, RNA, and proteins.

These peptides also have another critical ability that sets them apart from traditional antibiotics: They can recruit the host’s immune system, summoning cells called leukocytes that secrete chemicals that help kill the invading microbes.

Scientists have been working for several years to try to adapt these peptides as alternatives to antibiotics, as bacteria become resistant to existing drugs. Naturally occurring peptides can be composed of 20 different amino acids, so there is a great deal of possible variation in their sequences.

“You can tailor their sequences in such a way that you can tune them for specific functions,” de la Fuente says. “We have the computational power to try to generate therapeutics that can make it to the clinic and have an impact on society.”

In this study, the researchers began with a naturally occurring antimicrobial peptide called clavanin-A, which was originally isolated from a marine animal known as a tunicate. The original form of the peptide kills many types of bacteria, but the researchers decided to try to engineer it to make it even more effective.

Antimicrobial peptides have a positively charged region that allows them to poke through bacterial cell membranes, and a hydrophobic stretch that enables interaction with and translocation into membranes. The researchers decided to add a sequence of five amino acids that would make the peptides even more hydrophobic, in hopes that it would improve their killing ability.

This new peptide, which they called clavanin-MO, was very potent against many bacterial strains. In tests in mice, the researchers found that it could kill strains of Escherichia coli and Staphylococcus aureus that are resistant to most antibiotics.

Suppressing sepsis

Another key advantage of these peptides is that while they recruit immune cells to combat the infection, they also suppress the overactive inflammatory response that can cause sepsis, a life threatening condition.

“In this single molecule, you have a synthetic peptide that can kill microbes — both susceptible and drug-resistant — and at the same time can act as an anti-inflammatory mediator and enhance protective immunity,” de la Fuente says.

The researchers also found that these peptides can destroy certain biofilms, which are thin layers of bacterial cells that form on surfaces. That raises the possibility of using them to treat infections caused by biofilms, such as the Pseudomonas aeruginosa infections that often affect the lungs of cystic fibrosis patients. Or, they could be embedded into surfaces such as tabletops to make them resistant to microbial growth.

Other possible applications for these peptides include antimicrobial coatings for catheters, or ointments that could be used to treat skin infections caused by Staphylococcus aureus or other bacteria.

If these peptides are developed for therapeutic use, the researchers anticipate that they could be used either in stand-alone therapy or together with traditional antibiotics, which would make it more difficult for bacteria to evolve drug resistance. The researchers are now investigating what makes the engineered peptides more effective than the naturally occurring ones, with hopes of making them even better.

Flesh-Eating Infections In Rheumatoid Arthritis Patients Spur New Discovery

Rheumatoid arthritis patients taking medications that inhibit interleukin-1beta (IL-1beta), a molecule that stimulates the immune system, are 300 times more likely to experience invasive Group A Streptococcal infections than patients not on the drug, according to University of California San Diego School of Medicine researchers. Their study, published August 19 in Science Immunology, also uncovers a critical new role for IL-1beta as the body’s independent early warning system for bacterial infections.

“The more we know about each step in the body’s immune response to bacterial infections, the better equipped we are to design more personalized, targeted therapies for autoimmune diseases — therapies that are effective, but minimize risk of infection,” said senior author Victor Nizet, MD, professor of pediatrics and pharmacy at UC San Diego School of Medicine and Skaggs School of Pharmacy and Pharmaceutical Sciences.

IL-1beta is a molecule that stimulates an immune response, calling white blood cells to the site of an infection so they can engulf and clear away invading pathogens. The body first produces the molecule in a longer, inactive form that must be cleaved to be activated. The scientific community long believed that only the body itself could cleave and activate IL-1beta, by employing a cellular structure known as the inflammasome. But in experiments using cell cultures and mouse models of infection, Nizet and team found that SpeB, an enzyme secreted by strep bacteria, also cleaves and activates IL-1beta, triggering a protective immune response.

“This finding may explain why some of the more invasive, flesh-eating strep strains have a genetic mutation that blocks SpeB production — it helps them avoid tripping the alarm and setting off an immune response,” said first author Christopher LaRock, PhD, a postdoctoral researcher in Nizet’s lab.

The researchers hypothesize that for less invasive strains, like those that cause strep throat, producing SpeB and activating IL-1beta might be advantageous — the resulting immune response may wipe out competing bacteria and help strep establish a foothold in the body.

While the human immune system can quickly recognize and respond to bacterial infections, sometimes this reaction can go overboard, leading to autoimmune diseases such as rheumatoid arthritis. In this case, a person’s own immune system attacks “self” proteins instead of just foreign invaders.

In their efforts to investigate IL-1beta function, Nizet, LaRock and team analyzed a U.S. Food and Drug Administration (FDA) database on adverse events in rheumatoid patients who took anakinra, a drug that dampens autoimmunity by inhibiting IL-1beta. They found that patients receiving anakinra were more than 300 times more likely to experience invasive, flesh-eating strep infections than patients not taking the drug.

“A likely explanation for this increased risk is that with IL-1beta out of the picture, as is the case with patients taking anakinra, strep strains can progress to invasive infection even while producing SpeB, which goes unnoticed by the immune system,” LaRock said.

This finding underscores IL-1beta’s importance as an early warning system that’s triggered not only by the host, but also directly by bacterial enzymes, essentially “taking out the middle man,” Nizet said. The UC San Diego researchers believe this capacity for direct pathogen detection represents IL-1beta’s original role in immunity, going all the way back in evolution to simpler animals, such as fish.

“Inhibiting the body’s bacterial sensor can put a person at risk for invasive infection,” Nizet said, “but just the fact that we now know that this patient population is at higher risk and why means we can take simple steps — such as close monitoring and prophylactic antibiotics — to prevent it from happening. ”