Violacein improves vemurafenib response in melanoma spheroids.

Chemotherapy resistance is one of the main challenges in melanoma treatment. Violacein, a natural pigment produced by Chromobacterium violaceum, induces apoptosis in a variety of tumours, including melanoma. Here, we used BRAF-mutated melanoma spheroids to test the potential of violacein as a sensitizer of cellular viability and levels of the proteins p62 and fatty acid synthase (FASN). Importantly, violacein in combination with vemurafenib (ViVe) was able to interfere with spheroid survival at subtoxic concentrations. The results demonstrated that the ViVe protocol triggered cell death assessed by calcein and ethidium homodimer dyes. Accordingly, melanoma cells in 2D systems also showed a higher apoptosis rate when treated with ViVe. In the current study, we show evidence that ViVe downregulates crucial mediators like FASN, which partially explains how it acts as a sensitizer and ultimately improves the effectiveness of vemurafenib against melanoma cells.

Metabolomic Analysis of the Effect of Lippia origanoides Essential Oil on the Inhibition of Quorum Sensing in Chromobacterium violaceum.

Bacteria can communicate through quorum sensing, allowing them to develop different survival or virulence traits that lead to increased bacterial resistance against conventional antibiotic therapy. Here, fifteen essential oils (EOs) were investigated for their antimicrobial and anti-quorum-sensing activities using Chromobacterium violaceum CV026 as a model. All EOs were isolated from plant material via hydrodistillation and analyzed using GC/MS. In vitro antimicrobial activity was determined using the microdilution technique. Subinhibitory concentrations were used to determine anti-quorum-sensing activity by inhibition of violacein production. Finally, a possible mechanism of action for most bioactive EOs was determined using a metabolomic approach. Among the EOs evaluated, the EO from Lippia origanoides exhibited antimicrobial and anti-quorum activities at 0.37 and 0.15 mg/mL, respectively. Based on the experimental results, the antibiofilm activity of EO can be attributed to the blockage of tryptophan metabolism in the metabolic pathway of violacein synthesis. The metabolomic analyses made it possible to see effects mainly at the levels of tryptophan metabolism, nucleotide biosynthesis, arginine metabolism and vitamin biosynthesis. This allows us to highlight the EO of L. origanoides as a promising candidate for further studies in the design of antimicrobial compounds against bacterial resistance.

Enzymatic Active Release of Violacein Present in Nanostructured Lipid Carrier by Lipase Encapsulated in 3D-Bioprinted Chitosan-Hydroxypropyl Methylcellulose Matrix With Anticancer Activity.

Violacein (Viol) is a bacterial purple water-insoluble pigment synthesized by Chromobacterium violaceum and other microorganisms that display many beneficial therapeutic properties including anticancer activity. Viol was produced, purified in our laboratory, and encapsulated in a nanostructured lipid carrier (NLC). The NLC is composed of the solid lipid myristyl myristate, an oily lipid mixture composed of capric and caprylic acids, and the surfactant poloxamer P188. Dormant lipase from Rhizomucor miehei was incorporated into the NLC-Viol to develop an active release system. The NLC particle size determined by dynamic light scattering brings around 150 nm particle size and ζ≈ -9.0 mV with or without lipase, but the incorporation of lipase increase the PdI from 0.241 to 0.319 (≈32%). For scaffold development, a 2.5 hydroxypropyl methylcellulose/chitosan ratio was obtained after optimization of a composite for extrusion in a 3D-bioprinter developed and constructed in our laboratory. Final Viol encapsulation efficiency in the printings was over 90%. Kinetic release of the biodye at pH = 7.4 from the mesh containing NLC-lipase showed roughly 20% Viol fast release than without the enzyme. However, both Viol kinetic releases displayed similar profiles at pH = 5.0, where the lipase is inactive. The kinetic release of Viol from the NLC-matrices was modeled and the best correlation was found with the Korsmeyer-Peppas model (R2 = 0.95) with n < 0.5 suggesting a Fickian release of Viol from the matrices. Scanning Electron Microscope (SEM) images of the NLC-meshes showed significant differences before and after Viol's release. Also, the presence of lipase dramatically increased the gaps in the interchain mesh. XRD and Fourier Transform Infrared (FTIR) analyses of the NLC-meshes showed a decrease in the crystalline structure of the composites with the incorporation of the NLC, and the decrease of myristyl myristate in the mesh can be attributed to the lipase activity. TGA profiles of the NLC-meshes showed high thermal stability than the individual components. Cytotoxic studies in A549 and HCT-116 cancer cell lines revealed high anticancer activity of the matrix mediated by mucoadhesive chitosan, plus the biological synergistic activities of violacein and lipase.

Violacein switches off low molecular weight tyrosine phosphatase and rewires mitochondria in colorectal cancer cells.

In the last decade, emerging evidence has shown that low molecular weight protein tyrosine phosphatase (LMWPTP) not only contributes to the progression of cancer but is associated with prostate low survival rate and colorectal cancer metastasis. We report that LMWPTP favors the glycolytic profile in some tumors. Therefore, the focus of the present study was to identify metabolic enzymes that correlate with LMWPTP expression in patient samples. Exploratory data analysis from RNA-seq, proteomics, and histology staining, confirmed the higher expression of LMWPTP in CRC. Our descriptive statistical analyses indicate a positive expression correlation between LMWPTP and energy metabolism enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In addition, we examine the potential of violacein to reprogram energetic metabolism and LMWPTP activity. Violacein treatment induced a shift of glycolytic to oxidative metabolism associated with alteration in mitochondrial efficiency, as indicated by higher oxygen consumption rate. Particularly, violacein treated cells displayed higher proton leak and ATP-linked oxygen consumption rate (OCR) as an indicator of the OXPHOS preference. Notably, violacein is able to bind and inhibit LMWPTP. Since the LMWPTP acts as a hub of signaling pathways that offer tumor cells invasive advantages, such as survival and the ability to migrate, our findings highlight an unexplored potential of violacein in circumventing the metabolic plasticity of tumor cells.

Violacein negatively modulates the colorectal cancer survival and epithelial-mesenchymal transition.

Violacein is a secondary metabolite produced by several microorganisms including Chromobacterium violaceum, and it is already used in food and cosmetics. However, due to its potent anticancer and low side effects, its molecular action needs to be deeply scrutinized. Therefore, the main objective of this study was to evaluate the violacein's ability to interfere with three cancer hallmarks: growth factors receptor-dependent signaling, proliferation, and epithelial-mesenchymal transition (EMT). Violacein has been associated with the induction of apoptosis in colorectal cancer (CRC) cells. Here, we demonstrate that this molecule is also active in CRC spheroids and inhibits cell migration. Violacein treatment reduced the amount of EGFR and AXL receptors in the HT29 cell line. Accordingly, the inhibition of the AKT, ERK, and PKCδ kinases, which are downstream mediators of the signaling pathways triggered by EGFR and AXL, is detected. Another interesting finding was that even when the cells were stimulated with transforming growth factor-ß, the EMT marker (N-cadherin) decreased. Therefore, this study provides further evidence that reinforces the potential of violacein as an antitumor agent, once this biomolecule can "switch off" properties associated with cancer plasticity.

Violacein and its antifungal activity: comments and potentialities.

Violacein is an important natural antimicrobial pigment that is mainly produced by Chromobacterium violaceum and Janthinobacterium lividum. It presents a significant range of effects against phytopathogenic and human fungi, besides being featured as having low toxicity, and by its important ecological role in protecting amphibian species and applications in dyed medical fabric. The hypothesis about violacein's action mechanisms against mucormycosis (Rhizopus arrhizus) and candidiasis (Candida auris) is herein discussed based on data available in the scientific literature.

Molecular signatures of Janthinobacterium lividum from Trinidad support high potential for crude oil metabolism.

BACKGROUND: Janthinobacterium lividum is considered to be a psychrotrophic bacterial species. For the first time in the literature, J. lividum strains were isolated from Trinidad presenting with atypical features - hydrocarbonoclastic and able to survive in a tropical environment. METHODS: Identification of the Trinidad strains was carried out through 16S rRNA phylogenetic analysis. Gene-specific primers were designed to target the VioA which encodes violacein pigment and the EstA/B gene which encodes secreted extracellular lipase. Bioinformatics analyses were carried out on the nucleotide and amino acid sequences of VioA and EstA/B genes of the Trinidad Janthinobacterium strains to assess functionality and phylogenetic relatedness to other Janthinobacterium sequences specifically and more broadly, to other members of the Oxalobacteraceae family of betaproteobacteria. RESULTS: 16S rRNA confirmed the identity of the Trinidad strains as J. lividum and resolved three of the Trinidad strains at the intra-specific level. Typical motility patterns of this species were recorded. VioAp sequences were highly conserved, however, synonymous substitutions located outside of the critical sites for enzyme function were detected for the Trinidad strains. Comparisons with PDB 6g2p model from aa231 to aa406 further indicated no functional disruption of the VioA gene of the Trinidad strains. Phylogeny of the VioA protein sequences inferred placement of all J. lividum taxa into a highly supported species-specific clade (bs = 98%). EstA/Bp sequences were highly conserved, however, synonymous substitutions were detected that were unique to the Trinidad strains. Phylogenetic inference positioned the Trinidad consensus VioA and EstA protein sequences in a clearly distinct branch. CONCLUSIONS: The findings showed that the primary sequence of VioAp and EstA/Bp were unique to the Trinidad strains and these molecular signatures were reflected in phylogenetic inference. Our results supported chemotaxis, possible elective inactivation of VioA gene expression and secreted lipase activity as survival mechanisms of the Trinidad strains in petrogenic conditions.

Multi-target drug with potential applications: violacein in the spotlight.

The aim of the current review is to address updated research on a natural pigment called violacein, with emphasis on its production, biological activity and applications. New information about violacein's action mechanisms as antitumor agent and about its synergistic action in drug delivery systems has brought new alternatives for anticancer therapy. Thus, violacein is introduced as reliable drug capable of overcoming at least three cancer hallmarks, namely: proliferative signaling, cell death resistance and metastasis. In addition, antimicrobial effects on several microorganisms affecting humans and other animals turn violacein into an attractive drug to combat resistant pathogens. Emphasis is given to effects of violacein combined with different agents, such as antibiotics, anticancer agents and nanoparticles. Although violacein is well-known for many decades, it remains an attractive compound. Thus, research groups have been making continuous effort to help improving its production in recent years, which can surely enable its pharmaceutical and chemical application as multi-task compound, even in the cosmetics and food industries.

Plant compounds and nonsteroidal anti-inflammatory drugs interfere with quorum sensing in Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative, saprophytic bacterium that can infect humans and its virulence may be regulated by quorum sensing via N-acyl homoserine lactones. A virtual screening study with plant compounds and nonsteroidal anti-inflammatory drugs for inhibition of C. violaceum quorum sensing system has been performed. In vitro evaluation was done to validate the in silico results. Molecular docking showed that phytol, margaric acid, palmitic acid, dipyrone, ketoprofen, and phenylbutazone bound to structures of CviR proteins of different C. violaceum strains. Phytol presented higher binding affinities than AHLs and furanones, recognized inducers, and inhibitors of quorum sensing, respectively. When tested in vitro, phytol at a non-inhibitory concentration was the most efficient tested compound to reduce phenotypes regulated by quorum sensing. The results indicate that in silico compound prospection to inhibit quorum sensing may be a good tool for finding alternative lead molecules.

Violacein-Induced Chaperone System Collapse Underlies Multistage Antiplasmodial Activity.

Antimalarial drugs with novel modes of action and wide therapeutic potential are needed to pave the way for malaria eradication. Violacein is a natural compound known for its biological activity against cancer cells and several pathogens, including the malaria parasite, Plasmodium falciparum (Pf). Herein, using chemical genomic profiling (CGP), we found that violacein affects protein homeostasis. Mechanistically, violacein binds Pf chaperones, PfHsp90 and PfHsp70-1, compromising the latter's ATPase and chaperone activities. Additionally, violacein-treated parasites exhibited increased protein unfolding and proteasomal degradation. The uncoupling of the parasite stress response reflects the multistage growth inhibitory effect promoted by violacein. Despite evidence of proteotoxic stress, violacein did not inhibit global protein synthesis via UPR activation-a process that is highly dependent on chaperones, in agreement with the notion of a violacein-induced proteostasis collapse. Our data highlight the importance of a functioning chaperone-proteasome system for parasite development and differentiation. Thus, a violacein-like small molecule might provide a good scaffold for development of a novel probe for examining the molecular chaperone network and/or antiplasmodial drug design.

Pigments from Antarctic bacteria and their biotechnological applications.

Pigments from microorganisms have triggered great interest in the market, mostly by their "natural" appeal, their favorable production conditions, in addition to the potential new chemical structures or naturally overproducing strains. They have been used in: food, feed, dairy, textile, pharmaceutical, and cosmetic industries. The high rate of pigment production in microorganisms recovered from Antarctica in response to selective pressures such as: high UV radiation, low temperatures, and freezing and thawing cycles makes this a unique biome which means that much of its biological heritage cannot be found elsewhere on the planet. This vast arsenal of pigmented molecules has different functions in bacteria and may exhibit different biotechnological activities, such as: extracellular sunscreens, photoprotective function, antimicrobial activity, biodegradability, etc. However, many challenges for the commercial use of these compounds have yet to be overcome, such as: the low stability of natural pigments in cosmetic formulations, the change in color when subjected to pH variations, the low yield and the high costs in their production. This review surveys the different types of natural pigments found in Antarctic bacteria, classifying them according to their chemical structure. Finally, we give an overview of the main pigments that are used commercially today.

Patents on Violacein: A Compound with Great Diversity of Biological Activities and Industrial Potential.

BACKGROUND: This review outlines the current impact of violacein-derivative materials in several technological areas through patents. METHODS: A comprehensive examination of patent databases on violacein demonstrated the relevance of this pigment, as well as the pertinent topics related to its technological development in order to obtain adaptable new pharmaceuticals, cosmetics, and new quality fiber materials, together with other applications of violacein in different areas. RESULTS: At present, there is no efficient and economical technique for violacein preparation at the industrial scale. Many attempts have been made, but none have overcome the challenge of being an effective and inexpensive process. However, some potential applications of violacein in fields such as biomedicine make the pigment worthy of continuous investigation. In particular, violacein patents covering biosynthesis for different applications have been reported recently. CONCLUSION: Violacein has been used as a unique pigment in distinct specialty areas, such as in medical and industrial fields. This review of patents provides an update on violacein innovations that are useful for researchers working in the expanding and interesting field of biotechnology with natural pigments.

Production and antiproliferative effect of violacein, a purple pigment produced by an Antarctic bacterial isolate.

We studied the production and the potential use of a purple-pigment produced by an Antarctic bacterial isolate. This pigment was identified as violacein, a metabolite produced by many bacterial strains and reported that it has antiproliferative activity in many cell lines. We analyzed the effect of temperature and the composition of the growth medium on pigment production, achieving the highest yield at 20 °C in Tryptic Soy Broth medium supplemented with 3.6 g/L glucose. We doubled the yield of the pigment production when the process was scaled up in a 5 L bioreactor (77 mg/L of crude pigment). The pigment was purified and identified by mass spectrometry (DI-EI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy as violacein. We performed survival assays that showed that the pure pigment has antiproliferative activity and sensitize HeLa cells (cervix cell carcinoma) to cisplatin. Besides, the pigment did not show genotoxic activity in HeLa cells as found performing micronucleus assays. These results suggest that this pigment may be used as anticancer or sensitizer to cisplatin drug in cervix cancer.

Isolation and characterization of violacein from an Antarctic Iodobacter: a non-pathogenic psychrotolerant microorganism.

Violacein is an intensely purple pigment synthesized by various genera of bacteria that has been discovered to have a wide range of interesting biological activities which range from anticarcinogenic to antibacterial. One of the hindrances for its real-life application is that the first microorganisms found to produce the compound may act as opportunistic pathogens. Here, we report the isolation and characterization of violacein from a non-pathogenic Antarctic Iodobacter strain. Its anti-microbial properties were also tested. The method proposed here for the purification of violacein shows high yields, indicating that this Antarctic microorganism could be a valuable source for this important pigment. This is the first characterization of violacein from an Antarctic Iodobacter strain and here we also present a viable method to obtain this pigment for potential biotechnological applications.

Violacein antimicrobial activity on Staphylococcus epidermidis biofilm.

The aim of this study was to evaluate the antimicrobial potential of violacein (VIO) on Staphylococcus epidermidis biofilm. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined, as well as the effect of VIO exposure time on microbial viability in mature biofilm. Violacein showed good antibiofilm action, inhibiting biofilm formation and eradicating mature biofilm of S. epidermidis at concentrations of 20 µg.mL-1 and 160 µg.mL-1, respectively. At concentrations equal to MBEC and 2x MBEC, the biofilm was eradicated in 3 h and 2h30min of incubation, respectively.When evaluating VIO modulating effect on the action of clinically-used drugs (vancomycin, cefepime, ciprofloxacin and meropenem), especial synergism was observed in the violacein-ciprofloxacin association, it can completely erradicated the mature biofilm at the concentration of 1/2xMBEC and 1/4xMBEC, respectively. VIO shows good antimicrobial action on S. epidermidis biofilm and has the potential to synergistically modulate the activity of clinically-used antimicrobials.

Violacein@Biogenic Ag system: synergistic antibacterial activity against Staphylococcus aureus.

OBJECTIVES: To examine the synergistic antibacterial activity of violacein and silver nanoparticles (AgNPs) against ATCC bacteria, Staphylococcus aureus, Escherichia coli and two bacteria isolated from bovine mastitis. METHODS: Violacein from Chromobacterium violaceum and biogenic AgNPs from Fusarium oxysporum were evaluated in antimicrobial tests. RESULTS: E. coli isolates were not inhibited by violacein at concentrations up to 400 µM and they showed sensitivity for AgNPs between 62.5 and 250 µM. Staphylococcus aureus showed sensitivity to violacein with MIC of 200 µM, and the MIC with AgNPs between 250 µM and 125 µM. It was also tested the association between the two compounds through a concentration gradient and was observed the reduction of the MIC in the combination for both strains. CONCLUSION: The bactericidal effect of violacein against S. aureus was better when combined with AgNPs (synergistic).

Violacein Targets the Cytoplasmic Membrane of Bacteria.

Violacein is a tryptophan-derived purple pigment produced by environmental bacteria, which displays multiple biological activities, including strong inhibition of Gram-positive pathogens. Here, we applied a combination of experimental approaches to identify the mechanism by which violacein kills Gram-positive bacteria. Fluorescence microscopy showed that violacein quickly and dramatically permeabilizes B. subtilis and S. aureus cells. Cell permeabilization was accompanied by the appearance of visible discontinuities or rips in the cytoplasmic membrane, but it did not affect the cell wall. Using in vitro experiments, we showed that violacein binds directly to liposomes made with commercial and bacterial phospholipids and perturbs their structure and permeability. Furthermore, molecular dynamics simulations were employed to reveal how violacein inserts itself into lipid bilayers. Thus, our combined results demonstrate that the cytoplasmic membrane is the primary target of violacein in bacteria. The implications of this finding for the development of violacein as a therapeutic agent are discussed.

EmrR-Dependent Upregulation of the Efflux Pump EmrCAB Contributes to Antibiotic Resistance in Chromobacterium violaceum.

Chromobacterium violaceum is an environmental Gram-negative bacterium that causes infections in humans. Treatment of C. violaceum infections is difficult and little is known about the mechanisms of antibiotic resistance in this bacterium. In this work, we identified mutations in the MarR family transcription factor EmrR and in the protein GyrA as key determinants of quinolone resistance in C. violaceum, and we defined EmrR as a repressor of the MFS-type efflux pump EmrCAB. Null deletion of emrR caused increased resistance to nalidixic acid, but not to other quinolones or antibiotics of different classes. Moreover, the ΔemrR mutant showed decreased production of the purple pigment violacein. Importantly, we isolated C. violaceum spontaneous nalidixic acid-resistant mutants with a point mutation in the DNA-binding domain of EmrR (R92H), with antibiotic resistance profile similar to that of the ΔemrR mutant. Other spontaneous mutants with high MIC values for nalidixic acid and increased resistance to fluoroquinolones presented point mutations in the gene gyrA. Using DNA microarray, Northern blot and EMSA assays, we demonstrated that EmrR represses directly a few dozen genes, including the emrCAB operon and other genes related to transport, oxidative stress and virulence. This EmrR repression on emrCAB was relieved by salicylate. Although mutation of the C. violaceum emrCAB operon had no effect in antibiotic susceptibility or violacein production, deletion of emrCAB in an emrR mutant background restored antibiotic susceptibility and violacein production in the ΔemrR mutant. Using a biosensor reporter strain, we demonstrated that the lack of pigment production in ΔemrR correlates with the accumulation of quorum-sensing molecules in the cell supernatant of this mutant strain. Therefore, our data revealed that overexpression of the efflux pump EmrCAB via mutation and/or derepression of EmrR confers quinolone resistance and alters quorum-sensing signaling in C. violaceum, and that point mutation in emrR can contribute to emergence of antibiotic resistance in bacteria.

Inibição do sistema quorum sensing AI-1 por Capsicum frutescens e Capsicum annuum em bactérias Gram-negativas / Inhibition of AI-1 quorum-sensing system by Capsicum frutescens and Capsicum annuum in Gram-negative bacteria

A inibição do quorum sensing (QS) altera a comunicação bacteriana, reduzindo a expressão de fatores de virulência e a formação de biofilmes, o que pode conferir menor pressão seletiva em comparação aos antibióticos tradicionais. As frutas e hortaliças constituem uma fonte rica em compostos com propriedades potenciais de inibição do QS. Entretanto, há pouca referência sobre o potencial de pimentas do gênero Capsicum e de seus compostos isolados como inibidores do QS. Esse trabalho teve como objetivo avaliar o efeito de extratos orgânicos obtidos das variedades de pimenta-malagueta e pimentão vermelho sobre o sistema QS dependente do sinalizador AI-1 (acil homoserina lactona - AHL) em bactérias Gram-negativas. Os extratos foram obtidos por extração em fase sólida e separados em uma fração metanólica e outra amônica; sendo os compostos característicos identificados e quantificados por cromatografia líquida de alta eficiência (CLAE). A atividade antimicrobiana dos extratos foi avaliada pela determinação da concentração inibitória mínima (MIC) e pela curva de crescimento de Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 e Pseudomonas aeruginosa PAO1. O efeito anti-QS dos extratos foi avaliado pelos testes de difusão em ágar e quantificação da produção de violaceína em meio líquido por C. violaceum e sobre a formação de biofilme, avaliado pelo ensaio de cristal violeta e microscopia confocal, em S. liquefaciens e P. aeruginosa nas temperaturas 30 ºC e 37 ºC. Os resultados obtidos pela CLAE indicaram que o extrato metanólico de pimenta-malagueta (EMPM) continha capsaicinoides como a capsaicina e dihidrocapsaicina, luteolina e outros compostos não identificados; já o extrato amônico desta não continha os compostos capsaicinoides. Ambos os extratos de pimentão vermelho continham luteolina e compostos não identificados, mas não apresentaram capsaicinoides. Como o EMPM era representativo dos demais extratos, por conter tanto capsaicinóides quanto luteolina, o foco deste trabalho foi avaliar os efeitos do EMPM sobre fenótipos microbianos nas concentrações 5; 2,5; 1,25 e 0,625 mg/ml, além de utilizar a capsaicina como controle comparativo em concentrações equivalentes às do extrato (25, 50 e 100 µg/ml). Os resultados da atividade antimicrobiana mostraram inibição parcial do crescimento das bactérias nas concentrações sub-MIC (MIC >5 mg/ml) de 5 e 2,5 mg/ml de EMPM. A capsaicina também inibiu parcialmente o crescimento das bactérias a 100 µg/ml, com exceção de S. liquefaciens a 37 ºC, cujo crescimento foi induzido em 50 e 25 µg/ml. A produção de violaceína foi reduzida pelo EMPM a 1,25 e 0,625 mg/ml, sem afetar o crescimento de C. violaceum. Ensaios com C. violaceum CV026, estirpe biosensora capaz de produzir o pigmento na presença de AI-1 exógeno, sugerem que o possível mecanismo de atuação do extrato sobre o sistema QS em C. violaceum 12472 é sobre a síntese do sinalizador, já que não foi observada inibição da produção de violaceína em CV026 pelo extrato. Contrariamente, a capsaicina incrementou a produção do pigmento na estirpe 12472, mas ensaios com a estirpe CV026 indicaram que a capsaicina não atua como sinalizador do QS, uma vez que esta não induziu a produção de violaceína nesta estirpe. Já a formação de biofilme foi incrementada na presença do EMPM, sendo consideravelmente maior em P. aeruginosa a 30 ºC. Igualmente, observou-se indução da formação de biofilme por capsaicina em S. liquefaciens (37 ºC) e P. aeruginosa (30 ºC). Porém, a capsaicina não teve efeito sobre a formação de biofilme de S. liquefaciens quando cultivada a 30 ºC, nem P. aeruginosa a 37 ºC. Os resultados revelam que a produção de violaceína em C. violaceum ATCC 12472 é inibida pelo EMPM, mas não pela capsaicina. Já, o EMPM e a capsaicina, de forma geral, não inibem a formação de biofilme de S. liquefaciens MG1 nem P. aeruginosa PAO1. Outros estudos são necessários para elucidar os mecanismos pelos quais o EMPM e a capsaicina agem sobre os fenótipos avaliados neste trabalho

Quorum sensing inhibition alters bacterial communication by reducing virulence factors expression and biofilm formation, exerting less selective pressure compared to antibiotics. Fruits and vegetables are rich sources of compounds with potential QS-inhibition properties. However, there are few references about the potential of peppers belonging to the genus Capsicum and its isolated compounds as QS inhibitors. This study aimed to assess the effect of organic extracts obtained from Capsicum varieties, pimenta-malagueta (red chili) and pimentão vermelho (red bell pepper), on the AI-1 dependent QS system. The extracts were obtained by solid phase extraction and split into a methanolic and an ammonic fraction. Characteristic compounds were identified and quantified by high performance liquid chromatography (HPLC). The antimicrobial activity of the extracts was assessed by determining the minimal inhibitory concentration (MIC) and the growth curve of Chromobacterium violaceum ATCC 12472, Serratia liquefaciens MG1 and Pseudomonas aeruginosa PAO1. The anti-QS effect of the extracts was evaluated by the agar diffusion assay and the quantification of violacein production was assessed in liquid medium by C. violaceum, as well as in the biofilm formation test determined by the crystal violet assay and confocal microscopy with S. liquefaciens and P. aeruginosa at 30 ºC and 37 ºC. HPLC results showed that the methanolic extract of pimenta-malagueta (EMPM) contained capsaicinoids such as capsaicin and dihidrocapsaicin, luteolin and other unidentified compounds in lower concentrations; while its ammonic extract did not have capsaicinoids. Both pimentão vermelho extracts contained luteolin and other unidentified compounds in low concentrations, but they did not contain capsaicinoids. As EMPM was representative among the extracts because it contained capsaicinoids and luteolin, the focus of this work was to assess the effect of EMPM over microbial phenotypes at concentrations of 5, 2.5, 1.25 and 0.625 mg/ml, using capsaicin as a comparative control at equivalent concentrations to those in EMPM (25, 50 and 100 µg/ml). Antimicrobial activity assays showed a partial inhibition growth of bacteria at sub-MIC concentrations (MIC >5 mg/ml) of EMPM at 5 and 2.5 mg/ml. Similarly, capsaicin partially inhibited bacterial growth at 100 µg/ml, except for S. liquefaciens at 37 ºC in which growth was induced at 50 and 25 µg/ml. Violacein production was reduced by EMPM at 1,25 and 0,625 mg/ml without affecting C. violaceum growth. Assays with C. violaceum CV026, a biosensor strain that produces violacein in the presence of exogenous AI-1, suggest that EMPM reduced violacein production in C. violaceum 12472 by interfering with the AI-1 synthesis. In contrast, capsaicin incremented violacein synthesis in strain 12472, but experiments with strain CV026 revealed that capsaicin does not function as an analog of AI-1. Biofilm formation was increased in EMPM presence, being remarkably superior in P. aeruginosa cultivated at 30 ºC, as opposed to cultivation at 37 ºC. Similarly, capsaicin induced biofilm formation in S. liquefaciens (37 ºC) and P. aeruginosa (30 ºC). However, capsaicin did not affect biofilm formation on S. liquefaciens cultured at 30 ºC, neither on P. aeruginosa at 37 ºC. These results show that violacein production in C. violaceum ATCC 12472 is inhibited by EMPM, but not by capsaicin. In general, EMPM and capsaicin did not inhibit biofilm formation in S. liquefaciens MG1 neither in P. aeruginosa PAO1. More studies are necessary to elucidate the mechanisms by which EMPM and capsaicin affect the studied phenotypes in this work