Unlocking the Antibiofilm Potential of Natural Compounds by Targeting the NADH:quinone Oxidoreductase WrbA.

Biofilm-dwelling cells endure adverse conditions, including oxidative imbalances. The NADH:quinone oxidoreductase enzyme WrbA has a crucial role in the mechanism of action of antibiofilm molecules such as ellagic and salicylic acids. This study aimed to exploit the potential of the WrbA scaffold as a valuable target for identifying antibiofilm compounds at non-lethal concentrations. A three-dimensional computational model, based on the published WrbA structure, was used to screen natural compounds from a virtual library of 800,000 compounds. Fisetin, morin, purpurogallin, NZ028, and NZ034, along with the reference compound ellagic acid, were selected. The antibiofilm effect of the molecules was tested at non-lethal concentrations evaluating the cell-adhesion of wild-type and WrbA-deprived Escherichia coli strains through fluorochrome-based microplate assays. It was shown that, except for NZ028, all of the selected molecules exhibited notable antibiofilm effects. Purpurogallin and NZ034 showed excellent antibiofilm performances at the lowest concentration of 0.5 µM, in line with ellagic acid. The observed loss of activity and the level of reactive oxygen species in the mutant strain, along with the correlation with terms contributing to the ligand-binding free energy on WrbA, strongly indicates the WrbA-dependency of purpurogallin and NZ034. Overall, the molecular target WrbA was successfully employed to identify active compounds at non-lethal concentrations, thus revealing, for the first time, the antibiofilm efficacy of purpurogallin and NZ034.

Mechanistic Insights into the Antibiofilm Mode of Action of Ellagic Acid.

Bacterial biofilm is a major contributor to the persistence of infection and the limited efficacy of antibiotics. Antibiofilm molecules that interfere with the biofilm lifestyle offer a valuable tool in fighting bacterial pathogens. Ellagic acid (EA) is a natural polyphenol that has shown attractive antibiofilm properties. However, its precise antibiofilm mode of action remains unknown. Experimental evidence links the NADH:quinone oxidoreductase enzyme WrbA to biofilm formation, stress response, and pathogen virulence. Moreover, WrbA has demonstrated interactions with antibiofilm molecules, suggesting its role in redox and biofilm modulation. This work aims to provide mechanistic insights into the antibiofilm mode of action of EA utilizing computational studies, biophysical measurements, enzyme inhibition studies on WrbA, and biofilm and reactive oxygen species assays exploiting a WrbA-deprived mutant strain of Escherichia coli. Our research efforts led us to propose that the antibiofilm mode of action of EA stems from its ability to perturb the bacterial redox homeostasis driven by WrbA. These findings shed new light on the antibiofilm properties of EA and could lead to the development of more effective treatments for biofilm-related infections.

Biofilm colonization of stone materials from an Australian outdoor sculpture: Importance of geometry and exposure.

The safeguarding of Australian outdoor stone heritage is currently limited by a lack of information concerning mechanisms responsible for the degradation of the built heritage. In this study, the bacterial community colonizing the stone surface of an outdoor sculpture located at the Church of St. John the Evangelist in Melbourne was analysed, providing an overview of the patterns of microbial composition associated with stone in an anthropogenic context. Illumina MiSeq 16S rRNA gene sequencing together with confocal laser microscope investigations highlighted the bacterial community was composed of both phototrophic and chemotrophic microorganisms characteristic of stone and soil, and typical of arid, salty and urban environments. Cardinal exposure, position and surface geometry were the most important factors in determining the structure of the microbial community. The North-West exposed areas on the top of the sculpture with high light exposure gave back the highest number of sequences and were dominated by Cyanobacteria. The South and West facing in middle and lower parts of the sculpture received significantly lower levels of radiation and were dominated by Actinobacteria. Proteobacteria were observed as widespread on the sculpture. This pioneer research provided an in-depth investigation of the microbial community structure on a deteriorated artistic stone in the Australian continent and provides information for the identification of deterioration-associated microorganisms and/or bacteria beneficial for stone preservation.

Corrigendum: In Living Color: Pigment-Based Microbial Ecology At the Mineral-Air Interface.

[This corrects the article DOI: 10.1093/biosci/biac091.].

In Living Color: Pigment-Based Microbial Ecology At the Mineral-Air Interface.

Pigment-based color is one of the most important phenotypic traits of biofilms at the mineral-air interface (subaerial biofilms, SABs), because it reflects the physiology of the microbial community. Because color is the hallmark of all SABs, we argue that pigment-based color could convey the mechanisms that drive microbial adaptation and coexistence across different terrestrial environments and link phenotypic traits to community fitness and ecological dynamics. Within this framework, we present the most relevant microbial pigments at the mineral-air interface and discuss some of the evolutionary landscapes that necessitate pigments as adaptive strategies for resource allocation and survivability. We report several pigment features that reflect SAB communities' structure and function, as well as pigment ecology in the context of microbial life-history strategies and coexistence theory. Finally, we conclude the study of pigment-based ecology by presenting its potential application and some of the key challenges in the research.

Correlation between Perturbation of Redox Homeostasis and Antibiofilm Capacity of Phytochemicals at Non-Lethal Concentrations.

Biofilms are the multicellular lifestyle of microorganisms and are present on potentially every type of biotic or abiotic surface. Detrimental biofilms are generally targeted with antimicrobial compounds. Phytochemicals at sub-lethal concentrations seem to be an exciting alternative strategy to control biofilms, as they are less likely to impose selective pressure leading to resistance. This overview gathers the literature on individual phytocompounds rather than on extracts of which the use is difficult to reproduce. To the best of our knowledge, this is the first review to target only individual phytochemicals below inhibitory concentrations against biofilm formation. We explored whether there is an overall mechanism that can explain the effects of individual phytochemicals at sub-lethal concentrations. Interestingly, in all experiments reported here in which oxidative stress was investigated, a modest increase in intracellular reactive oxygen species was reported in treated cells compared to untreated specimens. At sub-lethal concentrations, polyphenolic substances likely act as pro-oxidants by disturbing the healthy redox cycle and causing an accumulation of reactive oxygen species.

Metabolomic and Proteomic Changes in Candida albicans Biofilm in Response to Zosteric Acid Treatment.

Zosteric acid (ZA) is a secondary metabolite of the seagrass Zostera marina, with antibiofilm activity against fungi. Information concerning its mechanisms of action is lacking and this limits the development of more potent derivatives based on the same target and activity structure. The aim of this work was to investigate the ZA mode of action by analyzing the metabolic status of Candida albicans biofilm and its protein expression profile upon ZA treatment. Fourier-Transform Infrared Spectroscopy confirmed that ZA modified the metabolomic response of treated cells, showing changes in the spectral regions, mainly related to the protein compartment. Nano Liquid Chromatography-High-Resolution Mass Spectrometry highlighted that 10 proteins were differentially expressed in the C. albicans proteome upon ZA treatment. Proteins involved in the biogenesis, structure and integrity of cell walls as well as adhesion and stable attachment of hyphae were found downregulated, whereas some proteins involved in the stress response were found overexpressed. Additionally, ZA was involved in the modulation of non-DNA-based epigenetic regulatory mechanisms triggered by reactive oxygen species. These results partially clarified the ZA mechanism of action against fungi and provided insight into the major C. albicans pathways responsible for biofilm formation.

Understanding the Role of the Antioxidant Drug Erdosteine and Its Active Metabolite on Staphylococcus aureus Methicillin Resistant Biofilm Formation.

Increasing numbers of researches have suggested that some drugs with reactive oxygen species (ROS)-mediated mechanisms of action modulate biofilm formation of some pathogenic strains. However, the full contribution of ROS to biofilm development is still an open question. In this paper, the correlations between the antioxidant drug Erdosteine (Er) and its active Metabolite I (Met I), ROS and biofilm development of two strains of methicillin resistant Staphylococcus aureus are presented. Experiments revealed that Er and Met I at 2 and 5 mg/L increased up to three orders of magnitude the number of biofilm-dwelling cells, while the content of ROS within the biofilms was reduced above the 87%, with a major effect of Met I in comparison to Er. Comparative proteomics showed that, 5 mg/L Met I modified the expression of 30% and 65% of total proteins in the two strains respectively. Some proteins involved in cell replication were upregulated, and a nitric oxide-based mechanism is assumed to modulate the biofilm development by changing quorum sensitive pathways. Additionally, several proteins involved in virulence were downregulated in the presence of Met I, suggesting that treated cells, despite being greater in number, might have lost part of their virulence.

Biological risk assessment in the History and Historical Documentation Library of the University of Milan.

There are billions of books that in recent and in ancient times have been produced by the human race containing evidence of its intellectual and cultural efforts. Even when stored in libraries, not all these books survive over time undamaged, because in the biosphere their materials are potential nutrients. This is the unfortunate case of the History and Historical Documentation Library of the University of Milan, where biological agents have badly affected rare and valuable old books. An entomological monitoring was carried out using sticky traps and collecting insects during inspections. The beetle Gastrallus pubens Fairmaire, rarely identified in European libraries so far, was the main biological agent responsible for the book damage, since several tunnels due to larval activity and holes made by adults were observed. Using the Illumina MiSeq sequencing technology, Proteobacteria, Firmicutes and Actinobacteria were found to be the most abundant phyla. Ascomycota was the dominant phylum among three fungal phyla. As bacteria and fungi spread by the insects are primary indications of the insect presence in the library, in this paper a potential biomarker able to detect the G. pubens presence before visible infestation was searched for among the bacterial and fungal community peculiar in the insect frass and gut, but also found on books and the surfaces of shelves. Symbiotaphrina, an ascomycete fungus described as one of the symbiotic levuliform fungi, present in the anobiid beetles' gut, was the only one found in all samples analyzed and has therefore been proposed as a putative biomarker.

Bioremoval of graffiti using novel commercial strains of bacteria.

Previous studies have provided evidence that bioremediation deals a novel approach to graffiti removal, thereby overcoming well-known limitations of current cleaning methods. In the present study eight bacteria aerobic, mesophilic and culturable from the American ATCC and the German DSMZ collections of microorganisms, some isolated from car paint waste, colored deposits in a pulp dryer and wastewater from dye works, were tested in the removal of silver and black graffiti spray paints using immersion strategies with glass slides. Absorbance at 600 nm and live/dead assays were performed to estimate bacterial density and activity in all samples. Also, pH and dissolved organic carbon (DOC) and inorganic carbon (DIC) measurements in the liquid media were made, as well as, thickness, colorimetric and infrared (FTIR) spectroscopy measurements in graffiti paint layers were used to evaluate the presence of the selected bacteria in the samples and the graffiti bioremoval capacity of bacteria. Data demonstrated that of the eight bacteria studied, Enterobacter aerogenes, Comamonas sp. and a mixture of Bacillus sp., Delftia lacustris, Sphingobacterium caeni, and Ochrobactrum anthropi were the most promising for bioremoval of graffiti. According to significant changes in FTIR spectra, indicating an alteration of the paint polymeric structure, coupled with the presence of a consistent quantity of live bacteria in the medium as well as a significant increase of DIC (a measure of metabolic activity) and a change in paint color.

Dynamics of bacterial communities and substrate conversion during olive-mill waste dark fermentation: Prediction of the metabolic routes for hydrogen production.

The aim of this work was to study the biological catalysts and possible substrate conversion routes in mesophilic dark fermentation reactors aimed at producing H2 from olive mill wastewater. Bacillus and Clostridium were the most abundant phylotypes during the rapid stage of H2 production. Chemical analyses combined with predictive functional profiling of the bacterial communities indicated that the lactate fermentation was the main H2-producing route. In fact, during the fermentation process, lactate and acetate were consumed, while H2 and butyrate were being produced. The fermentation process was rich in genes that encode enzymes for lactate generation from pyruvate. Lactate conversion to butyrate through the generation of pyruvate produced H2 through the recycling of electron carriers via the pyruvate ferredoxin oxydoreductase pathway. Overall, these findings showed the synergy among lactate-, acetate- and H2-producing bacteria, which complex interactions determine the H2 production routes in the bioreactors.

The Control of Cultural Heritage Microbial Deterioration.

The microbial deterioration of cultural heritage includes physical and chemical damage as well as aesthetic alteration. With the technological advancement, a plethora of techniques for removing unwanted microorganisms have opened up new opportunities for microbiologists and conservators. This article reviews the most applied, up-to-date, and sustainable techniques developed for the control of cultural heritage microbial deterioration presenting noteworthy case studies. These techniques include chemical methods, i.e., traditional biocides and nanoparticles; physical methods, such as mechanical removal, UV irradiation, gamma radiation, laser cleaning, heat shocking, microwaves, and dry ice treatment; and biological methods, such as natural molecules with biocidal activity, enzymes, and microorganisms. The application of control systems requires the comprehension of their behavior toward the unwanted microorganisms and possible interactions with the heritage materials. This overview shows also the control methods drawbacks for the purpose of creating awareness in selecting the most suitable technique or combination of techniques.

Label-Free Proteomic Approach to Study the Non-lethal Effects of Silver Nanoparticles on a Gut Bacterium.

Among all the food-related nanoparticles consumed every day, silver nanoparticles (AgNPs) have become one of the most commonly utilized because of their antimicrobial properties. Despite their common use, the effects of sublethal concentrations of AgNPs, especially on gut biofilms, have been poorly investigated. To address this issue, we investigated in vitro the proteomic response of a monospecies Escherichia coli gut biofilm to chronic and acute exposures in sublethal concentrations of AgNPs. We used a new gel- and label-free proteomic approach based on shotgun nanoflow liquid chromatography-tandem mass spectrometry. This approach allows a quantification of the whole proteome at a dynamic range that is higher than the traditional proteomic investigation. To assess all different possible exposure scenarios, we compared the biofilm proteome of four treatments: (i) untreated cells for the control treatment, (ii) cells treated with 1 µg/ml AgNPs for 24 h for the acute treatment, (iii) cells grown with 1 µg/ml AgNPs for 96 h for the chronic treatment, and (iv) cells grown in the presence of 1 µg/ml AgNPs for 72 h and then further treated for 24 h with 10 µg/ml AgNPs for the chronic + acute treatment. Among the 1,917 proteins identified, 212 were significantly differentially expressed proteins. Several pathways were altered including biofilm formation, bacterial adhesion, stress response to reactive oxygen species, and glucose utilization.

Non-Lethal Effects of N-Acetylcysteine on Xylella fastidiosa Strain De Donno Biofilm Formation and Detachment.

This study investigated in-vitro the non-lethal effects of N-acetylcysteine (NAC) on Xylella fastidiosa subspecies pauca strain De Donno (Xf-DD) biofilm. This strain was isolated from the olive trees affected by the olive quick decline syndrome in southern Italy. Xf-DD was first exposed to non-lethal concentrations of NAC from 0.05 to 1000 µM. Cell surface adhesion was dramatically reduced at 500 µM NAC (-47%), hence, this concentration was selected for investigating the effects of pre-, post- and co-treatments on biofilm physiology and structural development, oxidative homeostasis, and biofilm detachment. Even though 500 µM NAC reduced bacterial attachment to surfaces, compared to the control samples, it promoted Xf-DD biofilm formation by increasing: (i) biofilm biomass by up to 78% in the co-treatment, (ii) matrix polysaccharides production by up to 72% in the pre-treatment, and (iii) reactive oxygen species levels by 3.5-fold in the co-treatment. Xf-DD biofilm detachment without and with NAC was also investigated. The NAC treatment did not increase biofilm detachment, compared to the control samples. All these findings suggested that, at 500 µM, NAC diversified the phenotypes in Xf-DD biofilm, promoting biofilm formation (hyper-biofilm-forming phenotype) and discouraging biofilm detachment (hyper-attachment phenotype), while increasing oxidative stress level in the biofilm.

The Ecology of Subaerial Biofilms in Dry and Inhospitable Terrestrial Environments.

The ecological relationship between minerals and microorganisms arguably represents one of the most important associations in dry terrestrial environments, since it strongly influences major biochemical cycles and regulates the productivity and stability of the Earth's food webs. Despite being inhospitable ecosystems, mineral substrata exposed to air harbor form complex and self-sustaining communities called subaerial biofilms (SABs). Using life on air-exposed minerals as a model and taking inspiration from the mechanisms of some microorganisms that have adapted to inhospitable conditions, we illustrate the ecology of SABs inhabiting natural and built environments. Finally, we advocate the need for the convergence between the experimental and theoretical approaches that might be used to characterize and simulate the development of SABs on mineral substrates and SABs' broader impacts on the dry terrestrial environment.

Testing Anti-Biofilm Polymeric Surfaces: Where to Start?

Present day awareness of biofilm colonization on polymeric surfaces has prompted the scientific community to develop an ever-increasing number of new materials with anti-biofilm features. However, compared to the large amount of work put into discovering potent biofilm inhibitors, only a small number of papers deal with their validation, a critical step in the translation of research into practical applications. This is due to the lack of standardized testing methods and/or of well-controlled in vivo studies that show biofilm prevention on polymeric surfaces; furthermore, there has been little correlation with the reduced incidence of material deterioration. Here an overview of the most common methods for studying biofilms and for testing the anti-biofilm properties of new surfaces is provided.

Promoting Beneficial and Inhibiting Undesirable Biofilm Formation with Mangrove Extracts.

The extracts of two mangrove species, Bruguiera cylindrica and Laguncularia racemosa, have been analyzed at sub-lethal concentrations for their potential to modulate biofilm cycles (i.e., adhesion, maturation, and detachment) on a bacterium, yeast, and filamentous fungus. Methanolic leaf extracts were also characterized, and MS/MS analysis has been used to identify the major compounds. In this study, we showed the following. (i) Adhesion was reduced up to 85.4% in all the models except for E. coli, where adhesion was promoted up to 5.10-fold. (ii) Both the sum and ratio of extracellular polysaccharides and proteins in mature biofilm were increased up to 2.5-fold and 2.6-fold in comparison to the negative control, respectively. Additionally, a shift toward a major production of exopolysaccharides was found coupled with a major production of both intracellular and extracellular reactive oxygen species. (iii) Lastly, detachment was generally promoted. In general, the L. racemosa extract had a higher bioactivity at lower concentrations than the B. cylindrica extract. Overall, our data showed a reduction in cells/conidia adhesion under B. cylindrica and L. racemosa exposure, followed by an increase of exopolysaccharides during biofilm maturation and a variable effect on biofilm dispersal. In conclusion, extracts either inhibited or enhanced biofilm development, and this effect depended on both the microbial taxon and biofilm formation step.

Sub-lethal concentrations of Perilla frutescens essential oils affect phytopathogenic fungal biofilms.

The lack of deep knowledge of plant pathogenic fungal biofilms is reflected in the few existing environmental-friendly options for controlling fungal plant disease. Indeed, chemical fungicides still dominate the market but present-day concerns about their real efficacy, increasing awareness of the risk they pose to human health and the environment, and the incidence of fungicide resistance have all led to the current trend of near zero-market-tolerance for pesticide residues in fruit and vegetables. Here, essential oils (PK and PK-IK) from the edible leaves of two cultivars of Perilla frutescens are proposed as new, effective, non-toxic, eco-friendly pesticide-free options suitable for a preventive or integrative approach for sustainable crop protection and product preservation. PK and PK-IK were extracted and characterized, and their ability to affect the biofilm formation of the phytopathogenic model fungi Colletotrichum musae, Fusarium dimerum and Fusarium oxysporum was studied at non-lethal doses. Both essential oils at 1000 and 2000 mg l-1 showed excellent anti-biofilm performance: i) reducing conidia adhesion up to 80.3 ±â€¯16.2%; ii) inhibiting conidia germination up to 100.0 ±â€¯0.0%; iii) affecting biofilm structural development, with a reduction in dry weight of up to 100.0 ±â€¯0.0% and extracellular polysaccharides and proteins up to 81.4 ±â€¯8.0% and 51.0 ±â€¯6.1% respectively. In all cases PK-IK showed better activity than PK.

Surface colour: An overlooked aspect in the study of cyanobacterial biofilm formation.

Cyanobacteria can grow as biofilms, communities that colonize surfaces and that play a fundamental role in the ecology of many diverse habitats and in the conversion of industrial production to green platforms. Although biofilm growth is known to be significantly affected by several characteristics, the effect of colour surface is an overlooked aspect that has not yet been investigated. In this study, we describe the effect of colour hues (white, red, blue and black) on the growth of cyanobacterial biofilms on air-exposed substrates. We measured growth, architecture, pigment production and levels of ATP and reactive oxygen species in cyanobacterial biofilms formed on different coloured substrates. The study findings demonstrate, for the first time, that the colour of a surface affects biofilm formation at the air-solid interface (with more biomass accumulating on white and red substrates than on blue and black substrates) and also alters the biofilm architecture. In addition, the roles of chromatic adaptation, phototrophic cells and reactive oxygen species as intermediates between colour sensing and biofilm response are discussed. Our results support the importance of colour as a new factor that favours surface colonization by cyanobacteria and its contribution to biofilm formation.

Impacts of dietary silver nanoparticles and probiotic administration on the microbiota of an in-vitro gut model.

Ingestion of silver nanoparticles (AgNPs) is inevitable linked to their widespread use in food, medicines and other consumer products. However, their effects on human microbiota at non-lethal concentrations remain poorly understood. In this study, the interactions among 1 µg mL-1 AgNPs, the intestinal microbiota, and the probiotic Bacillus subtilis (BS) were tested using in-vitro batch fermentation models inoculated with human fecal matter. Results from metagenomic investigations revealed that the core bacterial community was not affected by the exposure of AgNPs and BS at the later stage of fermentation, while the proportions of rare species changed drastically with the treatments. Furthermore, shifts in the Firmicutes/Bacteriodetes (F/B) ratios were observed after 24 h with an increase in the relative abundance of Firmicutes species and a decrease in Bacteroidetes in all fermentation cultures. The co-exposure to AgNPs and BS led to the lowest F/B ratio. Fluorescent in-situ hybridization analyses indicated that non-lethal concentration of AgNPs negatively affected the relative percentage of Faecalibacterium prausnitzii and Clostridium coccoides/Eubacterium rectales taxa in the fermentation cultures after 24 h. However, exposure to single and combined treatments of AgNPs and BS did not change the overall diversity of the fecal microflora. Functional differences in cell motility, translation, transport, and xenobiotics degradation occurred in AgNPs-treated fermentation cultures but not in AgNPs+BS-treated samples. Compared to the control samples, treated fecal cultures showed no significant statistical differences in terms of short-chain fatty acids profiles, cytotoxic and genotoxic effects on Caco-2 cell monolayers. Overall, AgNPs did not affect the composition and diversity of the core fecal microflora and its metabolic and toxic profiles. This work indicated a chemopreventive role of probiotic on fecal microflora against AgNPs, which were shown by the decrease of F/B ratio and the unaltered state of some key metabolic pathways.