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Lactic acid bacteria (LAB) are known to exhibit various beneficial roles in fermentation, serving as probiotics, and producing a plethora of valuable compounds including antimicrobial activity such as bacteriocin-like inhibitory substance (BLIS) that can be used as biopreservative to improve food safety and quality. However, the yield of BLIS is often limited, which poses a challenge to be commercially competitive with the current preservation practice. Therefore, the present work aimed to establish an optimised two-plasmid CRISPR/Cas9 system to redirect the carbon flux away from lactate towards compounds with antimicrobial activity by disrupting lactate dehydrogenase gene (ldh) on various strains of LAB. The lactic acid-deficient (ldhΔ) strains caused a metabolic shift resulting in increased inhibitory activity against selected foodborne pathogens up to 78 % than the wild-type (WT) strain. The most significant effect was depicted by Enterococcus faecalis-ldh∆ which displayed prominent bactericidal effects against all foodborne pathogens as compared to the WT that showed no antimicrobial activity. The present work provided a framework model for economically important LAB and other beneficial bacteria to synthesise and increase the yield of valuable food and industrial compounds. The present work reported for the first time that the metabolism of selected LAB can be manipulated by modifying ldh to attain metabolites with higher antimicrobial activity.

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Clostridium perfringens is an important opportunistic microorganism in commercial poultry production that is implicated in necrotic enteritis (NE) outbreaks. This disease poses a severe financial burden on the global poultry industry, causing estimated annual losses of $6 billion globally. The ban on in-feed antibiotic growth promoters has spurred investigations into approaches of alternatives to antibiotics, among which Bacillus probiotics have demonstrated varying degrees of effectiveness against NE. However, the precise mechanisms underlying Bacillus-mediated beneficial effects on host responses in NE remain to be further elucidated. In this manuscript, we conducted in vitro and genomic mining analysis to investigate anti-C. perfringens activity observed in the supernatants derived from 2 Bacillus amyloliquefaciens strains (FS1092 and BaD747). Both strains demonstrated potent anti-C. perfringens activities in in vitro studies. An analysis of genomes from 15 B. amyloliquefaciens, 11 B. velezensis, and 2 B. subtilis strains has revealed an intriguing clustering pattern among strains known to possess anti-C. perfringens activities. Furthermore, our investigation has identified 7 potential antimicrobial compounds, predicted as secondary metabolites through antiSMASH genomic mining within the published genomes of B. amyloliquefaciens species. Based on in vitro analysis, BaD747 may have the potential as a probiotic in the control of NE. These findings not only enhance our understanding of B. amyloliquefaciens's action against C. perfringens but also provide a scientific rationale for the development of novel antimicrobial therapeutic agents against NE.

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In mammary glands, the formation of less-permeable tight junctions (TJs) and the production of antimicrobial compounds like lactoferrin and defensins are important for preventing mastitis. Resveratrol, a polyphenol contained in red grapes, is known to protect mammary epithelial cells (MECs) from oxidative stress; however, oral administration of resveratrol causes a decrease in certain biological processes through conjugation and metabolic conversion. In this study, we determined the beneficial effects of resveratrol on TJs and antimicrobial compounds in cultured goat MECs by adding it to the medium, and in lactating goat mammary glands by topical application for percutaneous absorption. TJ barrier function was evaluated by transepithelial resistance and expression or localization pattern of claudins for culture model in vitro and by somatic cell count, Na+, albumin, and IgG in milk for topical application in vivo. Concentrations of antimicrobial compounds and cytokines were measured using ELISA. Activation of STAT3 was evaluated by Western blotting. Resveratrol strengthened TJ barrier function by upregulating claudin-3 in cultured MECs and topical application to udders reduced somatic cell count, Na+, albumin, and IgG in milk. Resveratrol increased ß-defensin and S100A7 levels in cultured MECs and milk. In addition, resveratrol down-regulated cytokine production and STAT3 pathway. These findings suggest that the topical application of resveratrol to udders may be effective in preventing mastitis.

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OBJECTIVES: This study aimed to isolate antibacterial compounds active against periodontopathic bacteria from mulberry (Morus alba) leaves. METHODS: The acetone-soluble fraction of mulberry leaves was extracted from the oil layer by oil/water separation. The extract was purified using silica gel open-column chromatography. The minimum inhibitory concentration (MIC) of the crude extract or purified fractions against Porphyromonas gingivalis was measured at each step. RESULTS: The MIC of the crude extract against P. gingivalis was 62.5-125 µg/mL. The fractions showing activity against P. gingivalis were designated Cf K and Cf P. The MICs of Cf K against P. gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus mutans were 6.25 µg/mL, 25 µg/mL, 12.5 µg/mL, and 12.5 µg/mL, respectively. In contrast, the MICs of Cf P against P. gingivalis, F. nucleatum, P. intermedia, and S. mutans were 25.0 µg/mL, >50 µg/mL, 50 µg/mL, and 12.5-25.0 µg/mL, respectively. CONCLUSIONS: Mulberry leaves contain antibacterial components against periodontopathic bacteria such as P. gingivalis, F. nucleatum, and P. intermedia.

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This study proposes an innovative strategy to enhance the pharmacophore model of antimicrobial bismuth thiolato complex drugs by substituting hydrocarbon ligand structures with boron clusters, particularly icosahedral closo-dicarbadodecaborane (C2B10H12, carboranes). The hetero- and homoleptic mercaptocarborane complexes BiPh2L (1) and BiL3 (2) (L=9-S-1,2-C2B10H11) were prepared from 9-mercaptocarborane (HL) and triphenylbismuth. Comprehensive characterization using NMR, IR, MS, and XRD techniques confirmed their successful synthesis. Evaluation of antimicrobial activity in a liquid broth microdilution assay demonstrated micromolar to submicromolar minimum inhibitory concentrations (MIC) suggesting high effectiveness against S. aureus and limited efficacy against E. coli. This study highlights the potential of boron-containing bismuth complexes as promising antimicrobial agents, especially targeting Gram-positive bacteria, thus contributing to the advancement of novel therapeutic approaches.

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Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its ß-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

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Pathogenic bacteria in food are a public health problem worldwide. Polyphenolic bioactive compounds with antimicrobial activity and antioxidant capacity represent a tangible alternative to overcome this problem. To preserve the biological functions of phenolic compounds such as tannic acid, which has been described to possess antioxidant and antimicrobial activity, this study describes the synthesis of a zinc nanohydroxide to stabilize its properties. Characterization by XRD, FT-IR, SEM, DLS, and UV-vis evidenced the presence of tannic acid in the nanohybrid TA-Zn-LHS which was further confirmed by DPPH, ABTS and FRAP antioxidant activity techniques. Bacterial growth inhibition of Escherichia coli ATCC 8739, Salmonella Enteritidis, and Staphylococcus aureus ATCC 25923 was over 80% at 50 mg/mL of the TA-Zn-LHS and over 90% with Zn-LHS. Antibiofilm evaluation of these same strains showed biofilm formation inhibition > 90% and > 80% for Zn-LHS and TA-Zn-LHS, respectively. The toxicity evaluation of the materials in Artemia salina showed a classification of the materials as non-toxic to slightly toxic in concentrations up to 1 mg/mL. These results allow us to introduce a new nanohybrid useful for food safety with safe biological functions.

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Copper (Cu)-based antimicrobial compounds (CBACs) have been widely used to control phytopathogens for nearly fourteen decades. Since the first commercialized Bordeaux mixture was introduced, CBACs have been gradually developed from highly to slightly soluble reagents and from inorganic to synthetic organic, with nanomaterials being a recent development. Traditionally, slightly soluble CBACs form a physical film on the surface of plant tissues, separating the micro-organisms from the host, then release divalent or monovalent copper ions (Cu2+ or Cu+) to construct a secondary layer of protection which inhibits the growth of pathogens. Recent progress has demonstrated that the release of a low concentration of Cu2+ may elicit immune responses in plants. This supports a triple-tiered protection role of CBACs: break contact, inhibit microorganisms, and stimulate host immunity. This spatial defense system, which is integrated both inside and outside the plant cell, provides long-lasting and broad-spectrum protection, even against emergent copper-resistant strains. Here, we review recent findings and highlight the perspectives underlying mitigation strategies for the sustainable utilization of CBACs.

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Owing to its antimicrobial activity, electrochemically activated water (ECAW) is a potential alternative to chemical disinfectants for eliminating foodborne pathogens, including Salmonella Heidelberg, from food processing facilities. However, their antibiofilm activity remains unclear. This study aimed to evaluate the antibiofilm activity of ECAW against S. Heidelberg biofilms formed on stainless steel and polyethylene and to determine its corrosive capacity. ECAW (200 ppm) and a broad-spectrum disinfectant (0.2%) were tested for their antibiofilm activity against S. Heidelberg at 25 °C and 37 °C after 10 and 20 min of contact with stainless steel and polyethylene. Potentiostatic polarization tests were performed to compare the corrosive capacity of both compounds. Both compounds were effective in removing S. Heidelberg biofilms. Bacterial counts were significantly lower with ECAW than with disinfectant in polyethylene, regardless the time of contact. The time of contact and the surface significantly influenced the bacterial counts of S. Heidelberg. Temperature was not an important factor affecting the antibiofilm activities of the compounds. ECAW was less corrosive than the disinfectant. ECAW demonstrated a similar or even superior effect in the control of S. Heidelberg biofilms, when compared to disinfectants, reducing bacterial counts by up to 5 log10 CFU cm-2. The corrosion of stainless steel with ECAW was similar to that of commercial disinfectants. This technology is a possible alternative for controlling S. Heidelberg in the food production chain.

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Marine environments and salty inland ecosystems encompass various environmental conditions, such as extremes of temperature, salinity, pH, pressure, altitude, dry conditions, and nutrient scarcity. The extremely halophilic archaea (also called haloarchaea) are a group of microorganisms requiring high salt concentrations (2-6 M NaCl) for optimal growth. Haloarchaea have different metabolic adaptations to withstand these extreme conditions. Among the adaptations, several vesicles, granules, primary and secondary metabolites are produced that are highly significant in biotechnology, such as carotenoids, halocins, enzymes, and granules of polyhydroxyalkanoates (PHAs). Among halophilic enzymes, reductases play a significant role in the textile industry and the degradation of hydrocarbon compounds. Enzymes like dehydrogenases, glycosyl hydrolases, lipases, esterases, and proteases can also be used in several industrial procedures. More recently, several studies stated that carotenoids, gas vacuoles, and liposomes produced by haloarchaea have specific applications in medicine and pharmacy. Additionally, the production of biodegradable and biocompatible polymers by haloarchaea to store carbon makes them potent candidates to be used as cell factories in the industrial production of bioplastics. Furthermore, some haloarchaeal species can synthesize nanoparticles during heavy metal detoxification, thus shedding light on a new approach to producing nanoparticles on a large scale. Recent studies also highlight that exopolysaccharides from haloarchaea can bind the SARS-CoV-2 spike protein. This review explores the potential of haloarchaea in the industry and biotechnology as cellular factories to upscale the production of diverse bioactive compounds.

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Soil-derived microorganisms have been sampled intensively throughout the last decades in order to discover bacterial strains that produce new antibiotics. The increasing emergence of multidrug-resistant bacteria and the constant high demand for new antibiotic classes are leading to the sampling and investigation of new microbiomes that contain antimicrobial producers. Human-associated microbiomes are therefore gaining more and more attention. This chapter presents a detailed description of how human microbiomes can be sampled and how microbiota members from skin and nasal samples can be isolated. Different methods for antimicrobial compound screening are presented.

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Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

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Background: Microbial secondary metabolites have shown promise as a source of novel antimicrobial agents. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activity of compound from a novel Streptomyces albofaciens strain MS38. The objective was to identify a potential bioactive compound with broad-spectrum antimicrobial properties. Methods: The isolated strain MS38 on starch casein agar was characterized using morphological, physiological, and molecular identification techniques. The compound was obtained from the fermented broth through extraction with n-butanol and further purification using silica gel column chromatography and high-performance liquid chromatography (HPLC). Structural elucidation was conducted using Ultraviolet (UV), Infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) techniques. The antimicrobial activity was evaluated using the agar well diffusion method and the microplate Alamar blue assay (MABA). Results: The isolated strain MS38 was identified as novel S. albofaciens based on morphological characteristics and confirmed by 16S sequences analysis and MALDI-TOF MS. The compound obtained from the fermented broth exhibited substantial antimicrobial activity against a variety of pathogenic bacteria and fungi. Structural analysis revealed a complex chemical structure with characteristic functional groups indicative of potential antimicrobial properties. The compound demonstrated strong activity against both Gram-positive (Staphylococcus Spp.) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria, as well as fungi, including Candida albicans and Trichophyton rubrum. Conclusion: This study successfully isolated and characterized a bioactive compound from a novel S. albofaciens MS38. The compound exhibited significant antimicrobial activity against a range of pathogenic microorganisms. These findings underscore the importance of exploring microbial biodiversity for the discovery of novel antimicrobial agents. This study contributes to the growing knowledge of microbial secondary metabolites with potential therapeutic value.

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AIMS: The gastro-intestinal tract is a major reservoir of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli. Bacillus spores may be used as probiotics to decrease digestive colonization by ESBL-E. coli. Our aim was to assess the in vitro and in vivo activity of new Bacillus strains against ESBL-E. coli. METHODS AND RESULTS: We screened the in vitro activity of 50 Bacillus strains against clinical isolates of ESBL-E. coli and selected B. subtilis strains CH311 and S3B. Both strains decreased ESBL-E. coli titers by 4 log10 CFU L-1 in an in vitro model of gut content, whereas the B. subtilis CU1 strain did not. In a murine model of intestinal colonization by ESBL-E. coli, CH311 and S3B did not decrease fecal titers of ESBL-E. coli. Ten sequences of putative antimicrobial peptides were identified in the genomes of CH311 and S3B, but not in CU1. CONCLUSIONS: Two new B. subtilis strains showed strong in vitro activity against ESBL-E. coli. SIGNIFICANCE AND IMPACT OF STUDY: Despite strong in vitro activities of new B. subtilis strains against ESBL-E. coli, intestinal colonisation was not altered by curative Bacillus treatment even if their spores proved to germinate in the gut. Thus, this work underlines the importance of in vivo experiments to identify efficient probiotics. The use of potential antimicrobial compounds identified by genome sequencing remains an attractive alternative to explore.

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ABSTRACT@#Phenolic compounds are secondary metabolites of plants metabolism and can be found in olive oil. They exhibit antimicrobial activity towards both gram-positive and gram-negative bacteria. However, little is known about the antibacterial activity of the compounds towards periodontopathogens. The study aimed to investigate the potential of these compounds as antibacterial agents towards pathogens, specifically Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. Phenolic compounds were extracted from extra virgin olive oil (EVOO) through liquid-liquid separation using methanol:water (70:30), and hexane. It was then prepared in various concentrations to determine its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against the periodontopathogens. The anti-adhesion activity was quantified using crystal violet staining while the effects on the morphology were examined through scanning electron microscopy (SEM). The MICs of the phenolic compounds on A. actinomycetemcomitans, P. gingivalis and F. nucleatum were 31.25 mg/mL, 62.5 mg/mL and 125 mg/mL, respectively. The MBCs of the phenolic compounds on A. actinomycetemcomitans and F. nucleatum were 62.5 mg/mL and 125 mg/mL, respectively suggesting this compound can eradicate these bacteria. There was no bactericidal effect on P. gingivalis. The adhesion of all the bacteria was interrupted by the compounds at the lowest concentration (1.95 mg/mL). SEM findings showed disruption of bacterial cell surfaces such as blebs and disintegration of cells after exposure to this extract. Phenolic compounds of olive oil exhibited antibacterial activity against the tested pathogens, with bactericidal effects on A. actinomycetemcomitans and F. nucleatum and bacteriostatic effects on P. gingivalis.

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Antimicrobial peptides (AMPs) are a promising class of compounds being developed against multi-drug resistant bacteria. Hybridization has been reported to increase antimicrobial activity. Here, two proline-rich peptides (consP1: VRKPPYLPRPRPRPL-CONH2 and Bac5-v291: RWRRPIRRRPIRPPFWR-CONH2) were combined with two arginine-isoleucine-rich peptides (optP1: KIILRIRWR-CONH2 and optP7: KRRVRWIIW-CONH2). Proline-rich antimicrobial peptides (PrAMPs) are known to inhibit the bacterial ribosome, shown also for Bac5-v291, whereas it is hypothesized a "dirty drug" model for the arginine-isoleucine-rich peptides. That hypothesis was underpinned by transmission electron microscopy and biological small-angle X-ray scattering (BioSAXS). The strength of BioSAXS is the power to detect ultrastructural changes in millions of cells in a short time (seconds) in a high-throughput manner. This information can be used to classify antimicrobial compounds into groups according to the ultrastructural changes they inflict on bacteria and how the bacteria react towards that assault. Based on previous studies, this correlates very well with different modes of action. Due to the novelty of this approach direct identification of the target of the antimicrobial compound is not yet fully established, more research is needed. More research is needed to address this limitation. The hybrid peptides showed a stronger antimicrobial activity compared to the proline-rich peptides, except when compared to Bac5-v291 against E. coli. The increase in activity compared to the arginine-isoleucine-rich peptides was up to 6-fold, however, it was not a general increase but was dependent on the combination of peptides and bacteria. BioSAXS experiments revealed that proline-rich peptides and arginine-isoleucine-rich peptides induce very different ultrastructural changes in E. coli, whereas a hybrid peptide (hyP7B5GK) shows changes, different to both parental peptides and the untreated control. These different ultrastructural changes indicated that the mode of action of the parental peptides might be different from each other as well as from the hybrid peptide hyP7B5GK. All peptides showed very low haemolytic activity, some of them showed a 100-fold or larger therapeutic window, demonstrating the potential for further drug development.

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We hypothesized that environmental microbiomes contain a wide range of bacteria that produce yet uncharacterized antimicrobial compounds (AMCs) that can potentially be used to control pathogens. Over 600 bacterial strains were isolated from soil and food compost samples, and 68 biocontrol bacteria with antimicrobial activity were chosen for further studies based on inhibition assays against a wide range of food and plant pathogens. For further characterization of the bioactive compounds, a new method was established that used living pathogens in a liquid culture to stimulate bacteria to produce high amounts of AMCs in bacterial supernatants. A peptide gel electrophoresis microbial inhibition assay was used to concurrently achieve size separation of the antimicrobial peptides. Fifteen potential bioactive peptides were then further characterized by tandem MS, revealing cold-shock proteins and 50S ribosomal proteins. To identify non-peptidic AMCs, bacterial supernatants were analyzed by HPLC followed by GC/MS. Among the 14 identified bioactive compounds, 3-isobutylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione and 2-acetyl-3-methyl-octahydropyrrolo[1,2-a]piperazine-1,4-dione were identified as new AMCs. Our work suggests that antimicrobial compound production in microbes is enhanced when faced with a threat from other microorganisms, and that this approach can rapidly lead to the development of new antimicrobials with the potential for upscaling.

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This review summarizes the theory of zeta potential (ZP) and the most relevant data about how it has been used for studying bacteria. We have especially focused on the discovery and characterization of novel antimicrobial compounds. The ZP technique may be considered an indirect tool to estimate the surface potential of bacteria, a physical characteristic that is key to maintaining optimal cell function. For this reason, targeting the bacterial surface is of paramount interest in the development of new antimicrobials. Surface-acting agents have been found to display a remarkable bactericidal effect and have simultaneously revealed a low tendency to trigger resistance. Changes in the bacterial surface as a result of various processes can also be followed by ZP measurements. However, due to the complexity of the bacterial surface, some considerations regarding the assessment of ZP must first be taken into account. Evidence on the application of ZP measurements to the characterization of bacteria and biofilm formation is presented next. We finally discuss the feasibility of using the ZP technique to assess antimicrobial-induced changes in the bacterial surface. Among these changes are those related to the interaction of the agent with different components of the cell envelope, membrane permeabilization, and loss of viability.

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Antibiotics from few culturable microorganisms have saved millions of lives since the 20th century. But with resistance formation, these compounds become increasingly ineffective, while the majority of microbial and with that chemical compound diversity remains inaccessible for cultivation and exploration. Culturing recalcitrant bacteria is a stochastic process. But conventional methods are limited to low throughput. By increasing (i) throughput and (ii) sensitivity by miniaturization, we innovate microbiological cultivation to comply with biological stochasticity. Here, we introduce a droplet-based microscale cultivation system, which is directly coupled to a high-throughput screening for antimicrobial activity prior to strain isolation. We demonstrate that highly parallelized in-droplet cultivation starting from single cells results in the cultivation of yet uncultured species and a significantly higher bacterial diversity than standard agar plate cultivation. Strains able to inhibit intact reporter strains were isolated from the system. A variety of antimicrobial compounds were detected for a selected potent antibiotic producer.

Antibiotics are chemicals derived from microorganisms that can kill the bacteria that harm human health. In the 20^th and 21^st centuries antibiotics saved millions of lives, but new strains of dangerous bacteria that cannot be killed by antibiotics, known as antibiotic resistant strains, are becoming more frequent. Most antibiotics are produced by only a small group of microorganisms, but many more microorganisms exist in nature. So it is possible that microorganisms outside this small group can produce different antibiotics that are effective against antibiotic resistant strains. Unfortunately, finding the microorganisms that produce these alternative antibiotics is challenging because researchers do not know which bacteria are producing these molecules and how to grow these microorganisms in the laboratory. To solve this problem, Mahler et al. developed a new method for growing a new subset of microorganisms in the laboratory. This would allow researchers to study the new microorganisms under controlled conditions, and determine whether any of the substances they produce have antibiotic properties. Mahler et al. generated tiny droplets that could only contain a single cell of a microorganism, so each microbe could grow alone in its own protected environment. Using this approach, it was possible to grow completely different types of microorganisms than with traditional techniques, and keep them isolated from each other. This allowed each different species of microbe to be screened for antimicrobial activity, allowing the identification of chemicals that could potentially be developed into new antibiotics. This new method is automated and miniaturized, paving the way for growing many more cells in few hours, with very low material and space requirements. These results showcase a way of growing new types of microorganisms in the laboratory, making it easier and faster to study them and determine what chemicals they produce. Understanding a greater variety of microorganisms in detail can help identify new chemicals for industrial applications, including new ways of combating infections.

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Microbial metabolites have been recognized as an important source for the discovery of new antifungal agents because of their diverse chemical structures with novel modes of action. In the course of our screening for new antifungal agents from microbes, we found that culture filtrates of two fungal species Aspergillus candidus SFC20200425-M11 and Aspergillus montenegroi SFC20200425-M27 have the potentials to reduce the development of fungal plant diseases such as tomato late blight and wheat leaf rust. From these two Aspergillus spp., we isolated a total of seven active compounds, including two new compounds (4 and 6), and identified their chemical structures based on the NMR spectral analyses: sphaeropsidin A (1), (R)-formosusin A (2), (R)-variotin (3), candidusin (4), asperlin (5), montenegrol (6), and protulactone A (7). Based on the results of the in vitro bioassays of 11 plant pathogenic fungi and bacteria, sphaeropsidin A (1), (R)-formosusin A (2), (R)-variotin (3), and asperlin (5) exhibited a wide range of antimicrobial activity. Furthermore, when plants were treated with sphaeropsidin A (1) and (R)-formosusin A (2) at a concentration of 500 µg/ml, sphaeropsidin A (1) exhibited an efficacy disease control value of 96 and 90% compared to non-treated control against tomato late blight and wheat leaf rust, and (R)-formosusin A (2) strongly reduced the development of tomato gray mold by 82%. Asperlin (5) at a concentration of 500 µg/ml effectively controlled the development of tomato late blight and wheat leaf rust with a disease control value of 95%. Given that culture filtrates and active compounds derived from two Aspergillus spp. exhibited disease control efficacies, our results suggest that the Aspergillus-produced antifungal compounds could be useful for the development of new natural fungicides.