What's Hot in the Microbial Sciences

What's Hot in the Microbial Sciences

Investigating Clove Oil (Eugenol) as an Adjuvant to Overcome Colistin Resistance

A recent study published in Microbiology Spectrum described an investigation of the combined activity of colistin, the “drug of last resort” used to treat infections by carbapenem-resistant members of the Enterobacteriaceae family, and eugenol, a naturally derived aromatic phenolic compound, against clinical isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae.

The misuse of antibiotics in human medicine and in agriculture has contributed to antimicrobial resistance (AMR), according to the World Health Organization (WHO). An urgent concern, in this regard, is that pathogenic bacteria are developing resistance to colistin, an antibiotic of great importance for patients lacking viable treatment alternatives, such as carbapenems and cephalosporins. The increasing frequency of AMR infections clearly demonstrates the need for new antimicrobials, yet few are in development in the pharmaceutical industry.

"" Clove oil traditionally has been used to bolster health. A compound called eugenol is one of its primary components.
Source: Wikimedia Commons.
As a result, researchers are casting the net far and wide for existing substances that can serve the purpose, including natural antimicrobial agents produced by plants, fungi and bacteria. Products from the natural world have served as useful therapeutic agents against pathogenic bacteria since the golden age of antibiotics in the mid-20th century.

Eugenol, also known as clove oil, is the major constituent (83%-95%) of the aromatic oil extracted from cloves (Syzygium aromaticum). It derives also from other plant products, such as nutmeg, cinnamon, ginger, turmeric, basil, bay laurel leaves and peppers (Solanaceae). Eugenol possesses known pharmacological properties and is used in antimicrobial, anticancer, antioxidant, anti-inflammatory and analgesic applications. Eugenol derivatives already have applications as local anesthetics and antiseptics.

In the study described in Microbiology Spectrum, 9 commonly used antibiotics were each tested in combination with eugenol against 14 colistin-resistant P. aeruginosa and K. pneumoniae clinical isolates. Cell viability assays indicated that eugenol at concentrations of up to 500 µg/mL was not toxic toward murine macrophage cells, and the colistin-eugenol combination displayed effectiveness against colistin-resistant bacteria in vivo in an infection model using greater wax moth larvae (Galleria mellonella). Treatments delayed larval death and increased their survival rates by 20%-30%.

Leakage of protein and DNA from bacteria significantly increased in the presence of eugenol, as measured by alkaline phosphatase (ALP) leakage assays, leading researchers to conclude that eugenol and colistin, in combination, increased bacterial membrane permeability. Furthermore, crystal violet staining and scanning electron microscopy revealed that, in concert, colistin and eugenol were found to disrupt biofilms, which act as bacterial shields from antibiotic pressure and are known to produce recalcitrant infections. Further studies will evaluate the safety of eugenol for clinical use.

The researchers concluded that colistin and eugenol in combination represent a formidable treatment option for colistin-resistant clinical infections involving P. aeruginosa and K. pneumoniae. While natural compounds alone may not demonstrate significant antibacterial activity, they can be used as adjuvants with conventional drugs to boost the drugs’ antimicrobial properties. Such use of adjuvants can circumvent antibiotic resistance, prevent its spread and reduce the adverse effects of drugs.

Could Engineered Living Material Being Used to Create Self-Healing Buildings?

Engineered living materials one day might be used to construct human habitations.
Source: Freepik.
Researchers at the University of California and the Scripps Institute of Oceanography in San Diego used synthetic biology to design an engineered living material consisting of a strain of cyanobacteria suspended in a gel matrix that can sense and decolorize a common textile dye pollutant, indigo carmine (IC). Such a system could be used for environmental remediation in the near term and, in the future, may even be expanded to produce biomimetic building materials that can sense and respond to their environment—such as walls that can sense structural damage or wear and automatically repair themselves (“self-healing”). The researchers reported their findings in Nature Communications.

IC, a coloring agent popular in the pharmaceutical, textile, leather and food industries, is widely used to dye denim fabric. Yet, it functions as an environmental contaminant, exerting toxic effects in aquatic ecosystems, and the discharge of textile wastewater into the environment can lead to adverse health conditions in humans, such as skin and eye irritation, corneal and conjunctiva lesions, dermatitis and even cancer with constant and prolonged contact. Effective methods to remove the dye from wastewater would prove useful.

To address this concern, researchers introduced a synthetic riboswitch to regulate expression of a yellow fluorescent protein reporter in the cyanobacterium, Synechococcus elongatus. Fluorescence of the reporter protein indicated successful introduction of the gene, as well as a gene encoding a laccase enzyme. Laccases are broad-spectrum enzymes that can degrade compounds in wastewater effluents from industries and hospitals. Then the researchers used 3D printing to fabricate a transparent and porous biocomposite material, an alginate hydrogel matrix, into which was “printed” a gel-based “ink” containing a strain of S. elongatus that can sense IC and respond by producing laccase.

The transparent and porous alginate hydrogel matrix surrounding the laccase-producing cyanobacteria allowed for photosynthesis and the transport of nutrients and gases, respectively. The gel-based “ink” protected the cells from mechanical shear stress during extrusion (“printing”) of the cyanobacterium-containing hydrogel matrix, minimizing cell membrane damage and enhancing cell viability in the printed pattern.

Upon completion of its bioengineering, subsections of the hydrogel (2 interconnected hollow squares) were suspended in medium containing IC. The responsive biomaterial produced laccase and decolorized the IC in the hydrogels. A “kill switch,” or inducible cell death, was engineered into the responsible cells so that they conveniently perished once the task was completed. Inducible cell death constitutes an important aspect of the process to contain the organism and reduce potential environmental impact.

This experiment constituted proof of concept that engineered living material can produce useful products and/or perform desired tasks in response to environmental cues. Such material has the potential to serve in environmental bioremediation of other pollutants and might one day be harnessed to produce self-healing components incorporated into built environments. Imagine a housing unit or laboratory facility constructed of engineered living material that can sense damage to itself and subsequently repair its own structure.

Engineered living materials that combine the structural properties of traditional building materials (such as concrete and cement) with living systems, which possess the ability to rapidly grow, sense their environment and even self-heal in response to environmental cues, have potential to solve numerous challenges associated with construction and maintenance of built environments. For example, development of advanced engineered living materials could improve methods for manufacture and maintenance of large physical systems, such as military bases and transport vehicles and, perhaps, even self-repairing scientific research and housing units far from Earth.

Do Vital Antibiotics Lurk Among Extinct Molecules?

"" Molecular de-extinction might allow us to reproduce useful antibiotic compounds from organisms that lived in the distant past.
Source: iStock.
Molecular de-extinction, a new field of science, involves the retrieval of organic molecules, such as nucleic acids, proteins and other compounds, from extinct organisms. In a recent study published in Cell Host & Microbe, University of Pennsylvania researchers investigated potentially beneficial applications of molecular de-extinction for drug discovery.

Unlike larger scale de-extinction of whole organisms, which has been discussed in the literature, these researchers concentrated on reintroducing bioactive molecules from nonextant organisms, hypothesizing that such molecules conferred benefits to extinct organisms and could, once again, be of use but in the modern world.

Recreating extinct molecules has the potential to reinforce human defenses against future challenges that resemble challenges from ancient environments, such as climate change and outbreaks of infectious diseases.

Previous researchers have defined methods for sequencing ancient DNA. However, with the advent of artificial intelligence (AI), the past 5 years have yielded an explosion of possibilities for drug discovery, including the rediscovery of antibiotics from remains that are hundreds of thousands of years old.

Utilizing an approach referred to as “paleoproteome mining,” the researchers employed the PanCleave Python pipeline, a protein informatics, open-source machine learning tool, to prospect for antimicrobial encrypted peptides in the secreted proteins of modern humans, as well as in the archaic proteomes of our closest extinct relatives, Neanderthals and Denisovans.

The team discovered small protein subsequences that displayed antibiotic qualities encrypted within the proteins of our long-gone relatives, and subsequently synthesized the corresponding molecules. They then tested the efficacy of these resurrected antibiotics. In both murine skin abscess infection models and thigh infection models, some of the protein fragments demonstrated efficacy against Acinetobacter baumannii, a known antimicrobial resistant pathogen that often functions as a hospital acquired infection (HAI) and causes blood, urinary tract and lung infections.

Molecular de-extinction shows promise in drug discovery by reintroducing unique antimicrobials from the distant past and opening the door to an entirely new pathway for future antibiotic discovery.

Can Phages Treat Acne?

"" Acne is a common scourge of adolescents. Scientists are exploring whether phages could be used to treat it.
Source: iStock.
Ah, adolescence. A time characterized by immense growth, a sprinkling of awkwardness and, for up to 80% of teens, acne. Though the development of acne is tied to multiple factors, from genetics to hormones, the skin-dwelling bacterium Cutibacterium acnes plays a key role in exacerbating inflammation. Antibiotics aimed at controlling C. acnes growth have been widely used to treat moderate-to-severe acne, yet the emergence of resistant strains has limited their efficacy. A study published in Nature Communications suggests that phages (viruses that infect bacteria) targeting C. acnes may offer a solution.

In the study, scientists screened skin swabs from patients with acne to uncover 8 phages with lytic activity against C. acnes. In vitro experiments with clinical C. acnes strains revealed that a majority (32/36, or 88%) were sensitive to the phages, including strains that were resistant to at least 1, and some that were resistant to all, antibiotics commonly used to treat acne (e.g., clindamycin, tetracycline and others). These findings highlighted the phages’ potential for combating C. acnes, including when antibiotics may be ineffective.

To test in vivo efficacy, researchers turned to a mouse model. Phages were suspended in a gel, which was then smeared onto C. acnes-induced, acne-like lesions on mice once a day for 5 days. Compared to animals treated with gel alone (control), phage treatment led to significant improvement in lesions, including a decrease in lesion bacterial load, diameter, elevation and presence of necrotic tissue. It also dampened C. acnes-triggered inflammation, as indicated via reduced neutrophil migration and expression of inflammatory molecules within the lesions. From these results, the scientists concluded that topical administration of phages could be a powerful method for treating acne—something that had never been shown before.

So, will doctors soon be prescribing phage-infused zit cream? Not quite. The study acknowledged several limitations, including differences in the characteristics and progression of acne-like lesions in the mouse model compared to human acne. Thus, clinical investigation of topical phage acne treatment in a patient population is warranted.

How Can Antimicrobial Peptides Lead to New Antivirals?

"" New antiviral compounds are needed to combat current and emerging viral threats.
Source: iStock.
The COVID-19 pandemic underscored the need for effective antivirals to combat existing and emerging viral threats. In a recent study published in ACS Infectious Disease, scientists describe antimicrobial peptide (AMP)-inspired molecules, called peptoids, that exhibit broad-spectrum antiviral activity by targeting conserved, host-derived lipids in the viral membrane. This mechanism may lower the potential for generation of resistant variants. The findings provide a basis for the development of new antiviral agents.

AMPs are released by diverse host cells to kill pathogens, including viruses. Though AMPs have been explored for clinical use, they have various drawbacks in their natural state, including poor bioavailability and the potential to trigger unwanted immune responses against the peptides themselves, which can reduce their efficacy. Yet, peptoids can be synthesized to mimic desirable aspects of AMPs and maintain structural differences that may make them more clinically applicable (e.g., greater stability and membrane permeability).

In this study, scientists examined the activity of 7 AMP-like peptoids against a diverse range of viruses, including Zika virus (ZIKV), Rift Valley Fever virus (RVFV), chikungunya virus (CHIKV) and coxsackie B3 virus (CVB3). Peptoids that had documented activity against SARS-CoV-2 and herpes simplex virus-1 were selected for the study.

While the efficacy of individual peptoids varied for each virus, 1 thing was consistent: the peptoids only inactivated enveloped viruses (ZIKV, RVFV, CHIKV). CVB3, a non-enveloped virus, was unaffected. Additional experimentation revealed that the peptoids showed high specificity for a host-derived lipid in the viral membrane, called phosphatidylserine (PS). In host cells, PS is normally maintained on the inner leaflet of the plasma membrane. During apoptosis, however, PS is flipped to the surface, where it serves as a signal for other cells to engulf their dying comrade.

Viruses can take advantage of this process—when PS is expressed on their membrane, they “look” like apoptotic cells, and are thus taken up by host cells. Once inside, the viruses have all the cellular machinery they need to reproduce. Notably, viruses do not have the enzymes needed to control where PS localizes. This means that, in contrast to host cells, PS is often present in high concentrations on the virus surface. With that in mind, the researchers proposed that the peptoids were not attracted solely to PS on the viruses, but how much of it was present. Indeed, CHIKV viruses expressing a higher proportion of PS on their surface were more sensitive to peptoid treatment than those expressing normal levels.

Besides highlighting the antiviral potential of antimicrobial peptoids, these findings are significant for several reasons. For 1, given viruses obtain membrane lipids from the host (i.e., they do not encode their own membrane proteins), designing antivirals that target conserved, host-derived membrane components may limit the development of resistant variants, as there is no selective pressure on the virus itself. Capitalizing on differences in localization and concentration of lipids, like PS, between host and virus may also be advantageous for promoting selectivity and limiting host cytotoxicity.

How Does Commensal Candida Stay Commensal?

"" C. albicans exhibits several morphologies, including a commensal yeast form and a pathogenic hyphal form.
Source: ASM Journals.
Candida albicans is a common fungal member of the gut microbiome, present in about 70% of people. While it can be pathogenic, C. albicans normally exists in a state of commensalism with its host. What underlies the maintenance of this commensal relationship? New research published in Science provides some clues, suggesting that a form of a hormone that controls appetite, called Peptide YY (PYY), plays a role.

PYY is secreted by enteroendocrine cells in the gut; upon its release, it is cleaved by a protease, and the resulting fragment helps regulate appetite satiety. In this study, scientists found that the full-length version of PYY is also secreted by a subset of gut cells, called Paneth cells (PC), which release a slew of antimicrobial peptides to control microbial growth in the gut. This led the researchers to wonder: does PC-secreted PYY (PC-PYY) have antimicrobial powers too?

While PC-PYY showed limited activity against various bacterial species, it was effective against C. albicans, indicating it could play a role in modulating growth of the fungus in the gut. Indeed, gut colonization by C. albicans in mice lacking PYY was 2-3 times higher than in mice expressing PYY.

However, there was a catch: in vitro experiments revealed that the peptide only worked against C. albicans hyphae (a branched, virulent form of the fungus), not the commensal yeast form of the microbe. Why? It appears the peptide is attracted to the negatively charged surface of the hyphal membrane—a characteristic not shared by the yeast. Once bound, the peptide wreaks havoc on the hyphal cell membrane. RNA-seq analyses showed that PC-PYY also interferes with hyphal expression of biofilm and virulence genes; in yeast, PC-PYY downregulates genes involved in the transition to the pathogenic hyphal form.

These data point to a mechanism whereby the host maintains C. albicans commensalism by specifically targeting the pathogenic version of the microbe, while leaving the non-pathogenic version unharmed. The authors found that PC-PYY exhibited similar activity against other fungal species, suggesting it could play a broader role in shaping the gut mycobiome.

Author: Madeline Barron

Madeline Barron
Madeline Barron, Ph.D., is the Science Communications Specialist at ASM. She obtained her Ph.D. from the University of Michigan in the Department of Microbiology and Immunology.

Author: John Bell

John Bell
John Bell is the Senior Communications Specialist at ASM. He moved to the ASM Science Communications team from ASM's Journals Department. He brings with him many years of experience in copyediting, proofreading and production.

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