Schisandrin A enhances pathogens resistance by targeting a conserved p38 MAPK pathway.

Schizandrin A (SA), also known as deoxyschizandrin, is one of the most biologically active lignans isolated from the traditional Chinese medicine Fructus schisandrae chinensis. Schisandrin A has proven benefits for anti-cancer, anti-inflammation, hepatoprotection, anti-oxidation, neuroprotection, anti-diabetes. But the influence of Schisandrin A to the innate immune response and its molecular mechanisms remain obscure. In this study, we found that Schisandrin A increased resistance to not only the Gram-negative pathogens Pseudomonas aeruginosa and Salmonella enterica but also the Gram-positive pathogen Listeria monocytogenes. Meanwhile, Schisandrin A protected the animals from the infection by enhancing the tolerance to the pathogens infection rather than by reducing the bacterial burden. Through the screening of the conserved immune pathways in Caenorhabditis elegans, we found that Schisandrin A enhanced innate immunity via p38 MAPK pathway. Furthermore, Schisandrin A increased the expression of antibacterial peptide genes, such as K08D8.5, lys-2, F35E12.5, T24B8.5, and C32H11.12 by activation PMK-1/p38 MAPK. Importantly, Schisandrin A-treated mice also enhanced resistance to P. aeruginosa PA14 infection and significantly increased the levels of active PMK-1. Thus, promoted PMK-1/p38 MAPK-mediated innate immunity by Schisandrin A is conserved from worms to mammals. Our work provides a conserved mechanism by which Schisandrin A enhances innate immune response and boosts its therapeutic application in the treatment of infectious diseases.

Exploration of the inhibitory effect of Cassia fistula on quorum sensing mediated virulence factor production and biofilm activity in Pseudomonas aeruginosa: an in vivo study in model organism Caenorhabditis elegans.

Introduction. Resistance to antibiotics is leading to challenges in the treatment of microbial diseases. One amongst the various approaches to control these pathogens is quorum sensing (QS), which is used to rectify resistance issues. Blocking the bacterial QS circuit is the most reliable anti-virulence therapy to control pathogenicity-associated genes. Pseudomonas aeruginosa is a contagious bacterium that proliferates in the host by using signalling molecules like acyl-homoserine lactones; these molecules generate and disseminate toxins and virulence factors for increasing host infection.Hypothesis. The herb Cassia fistula is known to have antimicrobial, antidiabetic, anti-inflammatory, antitumor medicinal properties amongst others. We hypothesize that its crude extracts will inhibit the QS circuit of Pseudomonas aeruginosa (P. aeruginosa).Aim. The research work was aimed at evaluating anti-quorum sensing and anti-biofilm activity of various crude extracts from Cassia fistula against P. aeruginosa.Methodology. Various extraction methods and solvents were availed for maximum separation, and the extracts were screened for anti-quorum sensing activity. The most potent Fruit Ethyl acetate (FEE) extract at non-inhibitory concentrations was found to interrupt both short-chain (RhlI/R) and long-chain (LasI/R) QS circuits and other virulence factors (P<0.05) such as elastase, protease, rhamnolipids and pyocyanin levels in P. aeruginosa. Biofilm inhibitory properties of FEE were demonstrated using atomic force microscopy, scanning electron microscope and confocal laser microscope. Caenorhabditis elegans infection model (Paralytic assay) was developed to determine the protective role of FEE by reducing the pathogenicity of P. aeruginosa.Results. The study results suggest that hot crude FEE extract interfered in the QS circuit, leading to comprehensive debilitation of QS-controlled virulence factors. The extract reduced virulence factor production in P. aeruginosa at 4 mg ml-1 concentration whilst paradoxically promoting biofilm formation. Possibly, higher sugar content in the extract promoted clump formation of biofilm architecture by increasing exopolysaccharide production. Moreover, in vivo analysis of bacterial pathogenesis on Caenorhabditis elegans reveals a drastic increase in survival rates in FEE treated worms compared to untreated control.Conclusions. FEE showed promising QS inhibitory activity against P. aeruginosa. In the future, additional purification of crude FEE is required to remove carbohydrates, and pure isolated phytochemicals from FEE could be used as therapeutic agents to control QS-mediated infections in P. aeruginosa.

Whole transcriptome analysis of schinifoline treatment in Caenorhabditis elegans infected with Candida albicans.

Candida albicans is an opportunistic fungal human pathogen that has been causing an increasing number of deaths each year. Due to the widespread use of broad-spectrum antibiotics and immunosuppressants, C. albicans resistance to these therapies has increased. Thus, natural plant inhibitors are being investigated for treating C. albicans infections. Schinifoline is a 4-quinolinone alkaloid with antibacterial, insecticidal, antitumor, and other biological activities. Here, we explored the effects of schinifoline on C. albicans in C. elegans and extracted RNA from uninfected C. elegans, C. elegans infected with C. albicans, and C. elegans infected with C. albicans and treated with 100 mg/l schinifoline. Our results showed that there were significant differences among the three groups. The GO and KEGG pathway analysis suggested that the pathogenicity of C. albicans to C. elegans was caused by abnormal protein function. Schinifoline regulates lysosomal pathway related genes that accelerate the metabolism and degradation of abnormal proteins, thereby inhibiting the negative effects of C. albicans in vivo. These findings advance our understanding of the molecular mechanisms underlying schinifoline inhibition of C. albicans.

Physicochemical characterization and antioxidant effects of green microalga Chlorella pyrenoidosa polysaccharide by regulation of microRNAs and gut microbiota in Caenorhabditis elegans.

A novel polysaccharide from Chlorella pyrenoidosa (CPP) was separated and purified with the average molecular weight 15.8 kDa. It was composed of seven monosaccharides including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. FT-IR and NMR spectra analysis further revealed that CPP was an acidic polysaccharide consisting of ß-L-Arap-(1→, →2)-α-L-Rhap-(1→, ß-D-GlcpA-(1→, →4)-α-D-GalpA-(1→, →6)-ß-D-Glcp-(1→, →3)-ß-D-Manp-(1→, and →3, 6)-ß-D-Galp-(1→. The CPP treatment could effectively prolong lifespan of Caenorhabditis elegans under the oxidative stress conditions and inhibit the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) as well as enhancing the level of superoxide dismutase (SOD). It could up-regulate the expressions of Daf-16 and Skn-1 genes via declining miR-48-3p, miR-48-5p, and miR-51-5p translocation. Moreover, 16S rRNA sequencing revealed that the CPP-enriched Faecalibacterium, Haemophilus, Vibrio, and Shewanella were strongly correlated with SOD, MDA, apoptosis, and ROS. These results indicated that CPP may be considered as a desired ingredient on regulating the aging and oxidative diseases.

Mitochondrial Signature in Human Monocytes and Resistance to Infection in C. elegans During Fumarate-Induced Innate Immune Training.

Monocytes can develop immunological memory, a functional characteristic widely recognized as innate immune training, to distinguish it from memory in adaptive immune cells. Upon a secondary immune challenge, either homologous or heterologous, trained monocytes/macrophages exhibit a more robust production of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, than untrained monocytes. Candida albicans, ß-glucan, and BCG are all inducers of monocyte training and recent metabolic profiling analyses have revealed that training induction is dependent on glycolysis, glutaminolysis, and the cholesterol synthesis pathway, along with fumarate accumulation; interestingly, fumarate itself can induce training. Since fumarate is produced by the tricarboxylic acid (TCA) cycle within mitochondria, we asked whether extra-mitochondrial fumarate has an effect on mitochondrial function. Results showed that the addition of fumarate to monocytes induces mitochondrial Ca2+ uptake, fusion, and increased membrane potential (Δψm), while mitochondrial cristae became closer to each other, suggesting that immediate (from minutes to hours) mitochondrial activation plays a role in the induction phase of innate immune training of monocytes. To establish whether fumarate induces similar mitochondrial changes in vivo in a multicellular organism, effects of fumarate supplementation were tested in the nematode worm Caenorhabditis elegans. This induced mitochondrial fusion in both muscle and intestinal cells and also increased resistance to infection of the pharynx with E. coli. Together, these findings contribute to defining a mitochondrial signature associated with the induction of innate immune training by fumarate treatment, and to the understanding of whole organism infection resistance.

Anti-MRSA agent discovery using Caenorhabditis elegans-based high-throughput screening.

Staphylococcus aureus is a leading cause of hospital- and community-acquired infections. Despite current advances in antimicrobial chemotherapy, the infections caused by S. aureus remain challenging due to their ability to readily develop resistance. Indeed, antibiotic resistance, exemplified by methicillin-resistant S. aureus (MRSA) is a top threat to global health security. Furthermore, the current rate of antibiotic discovery is much slower than the rate of antibiotic-resistance development. It seems evident that the conventional in vitro bacterial growth-based screening strategies can no longer effectively supply new antibiotics at the rate needed to combat bacterial antibiotic-resistance. To overcome this antibiotic resistance crisis, screening assays based on host-pathogen interactions have been developed. In particular, the free-living nematode Caenorhabditis elegans has been used for drug screening against MRSA. In this review, we will discuss the general principles of the C. elegans-based screening platform and will highlight its unique strengths by comparing it with conventional antibiotic screening platforms. We will outline major hits from high-throughput screens of more than 100,000 small molecules using the C. elegans-MRSA infection assay and will review the mode-of-action of the identified hit compounds. Lastly, we will discuss the potential of a C. elegans-based screening strategy as a paradigm shift screening platform.

Dietary serine-microbiota interaction enhances chemotherapeutic toxicity without altering drug conversion.

The gut microbiota metabolizes drugs and alters their efficacy and toxicity. Diet alters drugs, the metabolism of the microbiota, and the host. However, whether diet-triggered metabolic changes in the microbiota can alter drug responses in the host has been largely unexplored. Here we show that dietary thymidine and serine enhance 5-fluoro 2'deoxyuridine (FUdR) toxicity in C. elegans through different microbial mechanisms. Thymidine promotes microbial conversion of the prodrug FUdR into toxic 5-fluorouridine-5'-monophosphate (FUMP), leading to enhanced host death associated with mitochondrial RNA and DNA depletion, and lethal activation of autophagy. By contrast, serine does not alter FUdR metabolism. Instead, serine alters E. coli's 1C-metabolism, reduces the provision of nucleotides to the host, and exacerbates DNA toxicity and host death without mitochondrial RNA or DNA depletion; moreover, autophagy promotes survival in this condition. This work implies that diet-microbe interactions can alter the host response to drugs without altering the drug or the host.

Bacterial vitamin B12 production enhances nematode predatory behavior.

Although the microbiota is known to affect host development, metabolism, and immunity, its impact on host behavior is only beginning to be understood. In order to better characterize behavior modulation by host-associated microorganisms, we investigated how bacteria modulate complex behaviors in the nematode model organism Pristionchus pacificus. This nematode is a predator that feeds on the larvae of other nematodes, including Caenorhabditis elegans. By growing P. pacificus on different bacteria and testing their ability to kill C. elegans, we reveal large differences in killing efficiencies, with a Novosphingobium species showing the strongest enhancement. This enhanced killing was not accompanied by an increase in feeding, which is a phenomenon known as surplus killing, whereby predators kill more prey than necessary for sustenance. Our RNA-seq data demonstrate widespread metabolic rewiring upon exposure to Novosphingobium, which facilitated screening of bacterial mutants with altered transcriptional responses. We identified bacterial production of vitamin B12 as an important cause of such enhanced predatory behavior. Although vitamin B12 is an essential cofactor for detoxification and metabolite biosynthesis, shown previously to accelerate development in C. elegans, supplementation with this enzyme cofactor amplified surplus killing in P. pacificus, whereas mutants in vitamin B12-dependent pathways reduced surplus killing. By demonstrating that production of vitamin B12 by host-associated microbiota can affect complex host behaviors, we reveal new connections between animal diet, microbiota, and nervous system.

Repurposing bioactive compounds for treating multidrug-resistant pathogens.

Introduction. Antimicrobial development is being outpaced by the rising rate of antimicrobial resistance in the developing and industrialized world. Drug repurposing, where novel antibacterial functions can be found for known molecular entities, reduces drug development costs, reduces regulatory hurdles, and increases rate of success.Aim. We sought to characterize the antimicrobial properties of five known bioactives (DMAQ-B1, carboplatin, oxaliplatin, CD437 and PSB-069) that were discovered in a high-throughput phenotypic screen for hits that extend Caenorhabditis elegans survival during exposure to Pseudomonas aeruginosa PA14.Methodology. c.f.u. assays, biofilm staining and fluorescence microscopy were used to assay the compounds' effect on various virulence determinants. Checkerboard assays were used to assess synergy between compounds and conventional antimicrobials. C. elegans-based assays were used to test whether the compounds could also rescue against Enterococcus faecalis and Staphyloccus aureus. Finally, toxicity was assessed in C. elegans and mammalian cells.Results. Four of the compounds rescued C. elegans from a second bacterial pathogen and two of them (DMAQ-B1, a naturally occurring insulin mimetic, and CD437, an agonist of the retinoic acid receptor) rescued against all three. The platinum complexes displayed increased antimicrobial activity against P. aeruginosa. Of the molecules tested, only CD437 showed slight synergy with ampicillin. The two most effective compounds, DMAQ-B1 and CD437, showed toxicity to mammalian cells.Conclusion. Although these compounds' potential for repurposing is limited by their toxicity, our results contribute to this growing field and provide a simple road map for using C. elegans for preliminary testing of known bioactive compounds with predicted antimicrobial activity.

A Combined Proteomics, Metabolomics and In Vivo Analysis Approach for the Characterization of Probiotics in Large-Scale Production.

The manufacturing processes of commercial probiotic strains may be affected in different ways in the attempt to optimize yield, costs, functionality, or stability, influencing gene expression, protein patterns, or metabolic output. Aim of this work is to compare different samples of a high concentration (450 billion bacteria) multispecies (8 strains) formulation produced at two different manufacturing sites, United States of America (US) and Italy (IT), by applying a combination of functional proteomics, metabolomics, and in vivo analyses. Several protein-profile differences were detected between IT- and US-made products, with Lactobacillus paracasei, Streptococcus thermophilus, and Bifidobacteria being the main affected probiotics/microorganisms. Performing proton nuclear magnetic spectroscopy (1H-NMR), some discrepancies in amino acid, lactate, betaine and sucrose concentrations were also reported between the two products. Finally, we investigated the health-promoting and antiaging effects of both products in the model organism Caenorhabditis elegans. The integration of omics platforms with in vivo analysis has emerged as a powerful tool to assess manufacturing procedures.

Occurrence of Salmonella typhimurium resistance under sublethal/repeated exposure to cauliflower infusion and infection effects on Caernohabditis elegans host test organism.

Resistant bacteria to antimicrobials are increasingly emerging in medical, food industry and livestock environments. The present research work assesses the capability of Salmonella enterica var Typhimurium to become adapted under the exposure to a natural cauliflower antimicrobial by-product infusion in consecutive repeated exposure cycles. Caenorhabditis elegans was proposed as in vivo host-test organism to compare possible changes in the virulent pattern of the different rounds treated S. enterica var Typhimurium and untreated bacterial cells. According to the obtained results, S. enterica var Typhimurium was able to generate resistance against a repeated exposure to cauliflower by-product infusion 5% (w/v), increasing the resistance with the number of exposed repetitions. Meanwhile, at the first exposure, cauliflower by-product infusion was effective in reducing S. enterica var Typhimurium (≈1 log10 cycle), and S. enterica var Typhimurium became resistant to this natural antimicrobial after the second and third treatment-round and was able to grow (≈1 log10 cycle). In spite of the increased resistance observed for repeatedly treated bacteria, the present study reveals no changes on C. elegans infection effects between resistant and untreated S. enterica var Typhimurium, according to phenotypic parameters evaluation (lifespan duration and egg-laying).

Lactobacillus rhamnosus Lcr35 as an effective treatment for preventing Candida albicans infection in the invertebrate model Caenorhabditis elegans: First mechanistic insights.

The increased recurrence of Candida albicans infections is associated with greater resistance to antifungal drugs. This involves the establishment of alternative therapeutic protocols, such as probiotic microorganisms whose antifungal potential has already been demonstrated using preclinical models (cell cultures, laboratory animals). Understanding the mechanisms of action of probiotic microorganisms has become a strategic need for the development of new therapeutics for humans. In this study, we investigated the prophylactic anti-C. albicans properties of Lactobacillus rhamnosus Lcr35® using the in vitro Caco-2 cell model and the in vivo Caenorhabditis elegans model. In Caco-2 cells, we showed that the strain Lcr35® significantly inhibited the growth (~2 log CFU.mL-1) and adhesion (150 to 6,300 times less) of the pathogen. Moreover, in addition to having a pro-longevity activity in the nematode (+42.9%, p = 3.56.10-6), Lcr35® protects the animal from the fungal infection (+267% of survival, p < 2.10-16) even if the yeast is still detectable in its intestine. At the mechanistic level, we noticed the repression of genes of the p38 MAPK signalling pathway and genes involved in the antifungal response induced by Lcr35®, suggesting that the pathogen no longer appears to be detected by the worm immune system. However, the DAF-16/FOXO transcription factor, implicated in the longevity and antipathogenic response of C. elegans, is activated by Lcr35®. These results suggest that the probiotic strain acts by stimulating its host via DAF-16 but also by suppressing the virulence of the pathogen.

Host-Microbe-Drug-Nutrient Screen Identifies Bacterial Effectors of Metformin Therapy.

Metformin is the first-line therapy for treating type 2 diabetes and a promising anti-aging drug. We set out to address the fundamental question of how gut microbes and nutrition, key regulators of host physiology, affect the effects of metformin. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we developed a high-throughput four-way screen to define the underlying host-microbe-drug-nutrient interactions. We show that microbes integrate cues from metformin and the diet through the phosphotransferase signaling pathway that converges on the transcriptional regulator Crp. A detailed experimental characterization of metformin effects downstream of Crp in combination with metabolic modeling of the microbiota in metformin-treated type 2 diabetic patients predicts the production of microbial agmatine, a regulator of metformin effects on host lipid metabolism and lifespan. Our high-throughput screening platform paves the way for identifying exploitable drug-nutrient-microbiome interactions to improve host health and longevity through targeted microbiome therapies. VIDEO ABSTRACT.

A Delicate Balance between Bacterial Iron and Reactive Oxygen Species Supports Optimal C. elegans Development.

Iron is an essential micronutrient for all forms of life; low levels of iron cause human disease, while too much iron is toxic. Low iron levels induce reactive oxygen species (ROS) by disruption of the heme and iron-sulfur cluster-dependent electron transport chain (ETC). To identify bacterial metabolites that affect development, we screened the Keio Escherichia coli collection and uncovered 244 gene deletion mutants that slow Caenorhabditis elegans development. Several of these genes encode members of the ETC cytochrome bo oxidase complex, as well as iron importers. Surprisingly, either iron or anti-oxidant supplementation reversed the developmental delay. This suggests that low bacterial iron results in high bacterial ROS and vice versa, which causes oxidative stress in C. elegans that subsequently impairs mitochondrial function and delays development. Our data indicate that the bacterial diets of C. elegans provide precisely tailored amounts of iron to support proper development.

Whole-genome analysis of the colonization-resistant bacterium Phytobacter sp. SCO41T isolated from Bacillus nematocida B16-fed adult Caenorhabditis elegans.

Colonization resistance is an important attribute for bacterial interactions with hosts, but the mechanism is still not completely clear. In this study, we found that Phytobacter sp. SCO41T can effectively inhibit the in vivo colonization of Bacillus nematocida B16 in Caenorhabditis elegans, and we revealed the colonization resistance mechanism. Three strains of colonization-resistant bacteria, SCO41T, BX15, and BC7, were isolated from the intestines of the free-living nematode C. elegans derived from rotten fruit and soil. The primary characteristics and genome map of one of the three isolates was investigated to explore the underlying mechanism of colonization resistance in C. elegans. In addition, we performed exogenous iron supplementation and gene cluster knockout experiments to validate the sequencing results. The results showed that relationship was close among the three strains, which was identified as belonging to the genus Phytobacter. The type strain is SCO41T (= CICC 24103T = KCTC 52362T). Whole genome analysis showed that csgA, csgB, csgC, csgE, csgF, and csgG were involved in the curli adhesive process and that fepA, fepB, fepC, fepD, and fepG played important roles in SCO41T against the colonization of B. nematocida B16 in C. elegans by competing for iron. Exogenous iron supplementation showed that exogenous iron can increase the colonization of B. nematocida B16, which was additionally confirmed by a deletion mutant strain. The csg gene family contributes to the colonization of SCO41T in C. elegans. Curli potentially contribute to the colonization of SCO41T in C. elegans, and enterobactin has a key role in SCO41T to resist the colonization of B. nematocida B16 by competing for iron.

Validation of the anti-infective potential of a polyherbal 'Panchvalkal' preparation, and elucidation of the molecular basis underlining its efficacy against Pseudomonas aeruginosa.

BACKGROUND: A Panchvalkal formulation (Pentaphyte P-5®) mentioned in ancient texts of Indian traditional medicine was investigated for its anti-infective potential against Pseudomonas aeruginosa. METHODS: Effect of the test formulation on bacterial growth and pigment production was evaluated by broth dilution assay. In vivo efficacy was evaluated using Caenorhabditis elegans as the model host. Whole transcriptome approach was taken to study the effect of test formulation on bacterial gene expression. RESULTS: This formulation in vitro was found to be capable of affecting quorum sensing (QS)-regulated traits (pyocyanin, pyoverdine, biofilm) of Pseudomonas aeruginosa. In combination with antibiotics, it enhanced susceptibility of the test bacterium to antibiotics like cephalexin and tetracycline. Effect of Panchvalkal formulation (PF) on QS-regulated traits of P. aeruginosa was not reversed even after repeated exposure of the bacterium to PF. In vivo efficacy of PF was demonstrated employing Caenorhabditis elegans as the model host, wherein PF-treated bacteria were able to kill lesser worms than their extract-unexposed counterparts. Whole transcriptome study revealed that approximately 14% of the P. aeruginosa genome was expressed differently under the influence of PF. CONCLUSIONS: Major mechanisms through which Panchvalkal seems to exert its anti-virulence effect are generation of nitrosative and oxidative stress, and disturbing iron and molybdenum homeostasis, besides interfering with QS machinery. This study is a good demonstration of the therapeutic utility of the 'polyherbalism' concept, so common in ayurved. It also demonstrates utility of the modern 'omics' tools for validating the traditional medicine i.e. ayuromics.

Anti-pathogenic potential of a classical ayurvedic Triphala formulation.

A classical ayurvedic polyherbal formulation namely Triphala was assessed for its anti-pathogenic potential against five different pathogenic bacteria. Virulence of four of them towards the model host Caenorhabditis elegans was attenuated (by 18-45%) owing to pre-treatment with Triphala (≤20 µg/ml). Triphala could also exert significant therapeutic effect on worms already infected with Chromobacterium violaceum, Serratia marcescens or Staphylococcus aureus. Prophylactic use of Triphala allowed worms to score 14-41% better survival in face of subsequent pathogen challenge. Repeated exposure to this formulation induced resistance in S. marcescens, but not in P. aeruginosa. It also exerted a post-extract effect (PEE) on three of the test pathogens. Triphala was able to modulate production of quorum sensing (QS)-regulated pigments in three of the multidrug-resistant gram-negative test bacteria. Haemolytic activity of S. aureus was heavily inhibited under the influence of this formulation. P. aeruginosa's lysozyme-susceptibility was found to increase by ~25-43% upon Triphala-pretreatment. These results validate therapeutic potential of one of the most widely used polyherbal ayurvedic formulations called Triphala.

Seed Extract of Psoralea corylifolia and Its Constituent Bakuchiol Impairs AHL-Based Quorum Sensing and Biofilm Formation in Food- and Human-Related Pathogens.

The emergence of multi-drug resistance in pathogenic bacteria in clinical settings as well as food-borne infections has become a serious health concern. The problem of drug resistance necessitates the need for alternative novel therapeutic strategies to combat this menace. One such approach is targeting the quorum-sensing (QS) controlled virulence and biofilm formation. In this study, we first screened different fractions of Psoralea corylifolia (seed) for their anti-QS property in the Chromobacterium violaceum 12472 strain. The methanol fraction was found to be the most active fraction and was selected for further bioassays. At sub-inhibitory concentrations, the P. corylifolia methanol fraction (PCMF) reduced QS-regulated virulence functions in C. violaceum CVO26 (violacein); Pseudomonas aeruginosa (elastase, protease, pyocyanin, chitinase, exopolysaccharides (EPS), and swarming motility), A. hydrophila (protease, EPS), and Serratia marcescens (prodigiosin). Biofilm formation in all the test pathogens was reduced significantly (p ≤ 0.005) in a concentration-dependent manner. The ß-galactosidase assay showed that the PCMF at 1,000 µg/ml downregulated las-controlled transcription in PAO1. In vivo studies with C. elegans demonstrated increased survival of the nematodes after treatment with the PCMF. Bakuchiol, a phytoconstituent of the extract, demonstrated significant inhibition of QS-regulated violacein production in C. violaceum and impaired biofilm formation in the test pathogens. The molecular docking results suggested that bakuchiol efficiently binds to the active pockets of LasR and RhlR, and the complexes were stabilized by several hydrophobic interactions. Additionally, the molecular dynamics simulation of LasR, LasR-bakuchiol, RhlR, and RhlR-bakuchiol complexes for 50 ns revealed that the binding of bakuchiol to LasR and RhlR was fairly stable. The study highlights the anti-infective potential of the PCMF and bakuchiol instead of bactericidal or bacteriostatic action, as the extract targets QS-controlled virulence and the biofilm.

Prophylactic potential of a Panchgavya formulation against certain pathogenic bacteria.

A Panchgavya preparation was evaluated for its prophylactic efficacy against bacterial infection, employing the nematode worm Caenorhabditis elegans as a model host. Worms fed with the Panchgavya preparation prior to being challenged with pathogenic bacteria had a better survival rate against four out of five test bacterial pathogens, as compared to the control worms. Panchgavya feeding prior to bacterial challenge was found to be most effective against Staphylococcus aureus, resulting in 27% (p=0.0001) better worm survival. To the best of our awareness, this is the first report demonstrating in vivo prophylactic efficacy of Panchgavya mixture against pathogenic bacteria.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.