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

Recent research advances on Chromobacterium violaceum

Chromobacterium violaceum is a gram-negative bacterium, which has been used widely in microbiology labs involved in quorum sensing (QS) research. Among the QS-regulated traits of this bacterium, violacein production has received the maximum attention. Violacein production in this organism, however is not under sole control of QS machinery, and other QS-regulated traits of this bacterium also need to be investigated in better detail. Though not often involved in human infections, this bacterium is being viewed as an emerging pathogen. This review attempts to highlight the recent research advances on C. violaceum, with respect to violacein biosynthesis, development of various applications of this bacterium and its bioactive metabolite violacein, and its pathogenicity.

Recent research advances on Chromobacterium violaceum

Chromobacterium violaceum is a gram-negative bacterium, which has been used widely in microbiology labs involved in quorum sensing (QS) research. Among the QS-regulated traits of this bacterium, violacein production has received the maximum attention. Violacein production in this organism, however is not under sole control of QS machinery, and other QS-regulated traits of this bacterium also need to be investigated in better detail. Though not often involved in human infections, this bacterium is being viewed as an emerging pathogen. This review attempts to highlight the recent research advances on C. violaceum, with respect to violacein biosynthesis, development of various applications of this bacterium and its bioactive metabolite violacein, and its pathogenicity.

Efeito da violaceína sobre formas evolutivas de trypanosoma cruzi

A Organização Mundial de Saúde estima que aproximadamente 7 a 8 milhões de pessoas encontram-se infectadas pelo Trypanosoma cruzi no mundo. O tratamento da doença de Chagas apresenta eficácia limitada e efeitos colaterais que limitam a tolerabilidade e a adesão dos pacientes. A busca de novas alternativas terapêuticas a partir de substâncias bioativas cresceu bastante nos últimos anos. A violaceína (VIO), um pigmento bacteriano produzido por Chromobacterium violace um tem mostrado diversas ações biológicas, dentre elas, ações antiulcerogênica, antitumoral, antiviral e antiparasitária. No presente trabalho, estudamos os efeitos da VIO sobre as formas evolutivas doTrypanosoma cruzi. As formas epimastigotas foram cultivadas em meio LIT, a 28°C, na presença deVIO (0,97; 1,9; 3,9; 7,8; 15,62; 31,25; 62,5; 125; 250; 500; 1000μM) por 24, 48 e 72h. As formas tripomastigotas, foram obtidas após infecção em células LLC-MK2, ressuspensas em meio DMEM2% de SBF e incubadas com VIO (0,97; 1,9; 3,9; 7,8; 15,62; 31,25μM) por 24h. Formas amastigotas foram cultivadas em lamínulas circulares no interior de placas de cultura contendo células LLC-MK2e tratadas com violaceína (4,97 e 9,94 μM)...

The World Health Organization estimates that approximately 7 to 8 million individuals areinfected with Trypanosoma cruzi worldwide. The treatment of Chagas disease has limited efficacy andside effects that limit patient tolerability and compliance. The search for new therapeutic alternativesbased on bioactive substances has increased significantly in recent years. Violacein (VIO), a bacterialpigment produced by Chromobacterium violaceum, has shown several biological actions and amongthem, antiulcer, antitumor, antiviral, and antiparasitic action. In this study, we assessed the effects ofVIO on the evolutionary forms of Trypanosoma cruzi. Epimastigotes were cultured in liver infusiontryptose (LIT) medium at 28 °C in the presence of VIO (0.97; 1.9; 3.9; 7.8; 15.62; 31.25; 62.5; 125;250 ; 500; 1000μM) for 24, 48 and 72 hours. Trypomastigotes were obtained after infection in LLCMK2cells resuspended in 2% DMEM medium of FBS and incubated with VIO (0.97; 1.9; 3.9; 7.8;15.62; 31.25μM ) for 24h. Amastigotes were cultured on circular glass slides within culture platescontaining LLC-MK2 cells and treated with violacein (4.97 and 9.94 μM)...

Composition, anti-quorum sensing and antimicrobial activity of essential oils from Lippia alba

Many Gram-negative pathogens have the ability to produce N-acylhomoserine lactones (AHLs) as signal molecules for quorum sensing (QS). This cell-cell communication system allows them to coordinate gene expression and regulate virulence. Strategies to inhibit QS are promising for the control of infectious diseases or antibiotic resistant bacterial pathogens. The aim of the present study was to evaluate the anti-quorum sensing (anti-QS) and antibacterial potential of five essential oils isolated from Lippia alba on the Tn-5 mutant of Chromobacterium violaceum CV026, and on the growth of the gram-positive bacteria S. aureus ATCC 25923. The anti-QS activity was detected through the inhibition of the QS-controlled violacein pigment production by the sensor bacteria. Results showed that two essential oils from L. alba, one containing the greatest geranial:neral and the other the highest limonene:carvone concentrations, were the most effective QS inhibitors. Both oils also had small effects on cell growth. Moreover, the geranial/neral chemotype oil also produced the maximum zone of growth inhibition against S. aureus ATCC 25923. These data suggest essential oils from L. alba have promising properties as QS modulators, and present antibacterial activity on S. aureus.

Effect of certain natural products and organic solvents on quorum sensing in Chromobacterium violaceum

Objective: To investigate the effect of seed extracts of Pongamia pinnata, Pyrus pyrifolia, and Manilkara hexandra, bacterial pigment prodigiosin, and three organic solvents (ethanol, methanol, and dimethylsulfoxide), on quorum sensing (QS) in Chromobacterium violaceum (C. violaceum). Methods: C. violaceum was challenged with plant extracts prepared by microwave assisted extraction method, prodigiosin, and organic solvents. Effect of these test substances on C. violaceum growth, and quorum sensing regulated pigment (violacein) production was studied by broth dilution assay. High performance liquid chromatography was also applied to generate chromatographic fingerprint of the active extracts. Effect of sub-minimum inhibitory concentration level of the antibiotic streptomycin on quorum sensing regulated pigment production was also studied. Results: Pongamia pinnata seed extracts and prodigiosin were found to possess anti-QS, and Manilkara hexandra and Pyrus pyrifolia seed extracts to possess QS-enhancing effect in C. violaceum. Dimethylsulfoxide was found to enhance violacein production, whereas ethanol and methanol reduced violacein production in C. violaceum. Streptomycin at sub-minimum inhibitory concentration level was able to significantly arrest QS-regulated pigment production in C. violaceum and Serratia marcescens. Conclusions: Prodigiosin and the seed extracts used in this study could affect quorum sensing in C. violaceum to a notable extent. Results of this study also emphasize the importance of inclusion of appropriate solvent controls (negative controls) in bioassays designed for screening of antimicrobial and/or anti-QS compounds. Antipathogenic potential of low concentrations of streptomycin was also demonstrated.

Effect of certain natural products and organic solvents on quorum sensing in Chromobacterium violaceum

OBJECTIVE@#To investigate the effect of seed extracts of Pongamia pinnata, Pyrus pyrifolia, and Manilkara hexandra, bacterial pigment prodigiosin, and three organic solvents (ethanol, methanol, and dimethylsulfoxide), on quorum sensing (QS) in Chromobacterium violaceum (C. violaceum).@*METHODS@#C. violaceum was challenged with plant extracts prepared by microwave assisted extraction method, prodigiosin, and organic solvents. Effect of these test substances on C. violaceum growth, and quorum sensing regulated pigment (violacein) production was studied by broth dilution assay. High performance liquid chromatography was also applied to generate chromatographic fingerprint of the active extracts. Effect of sub-minimum inhibitory concentration level of the antibiotic streptomycin on quorum sensing regulated pigment production was also studied.@*RESULTS@#Pongamia pinnata seed extracts and prodigiosin were found to possess anti-QS, and Manilkara hexandra and Pyrus pyrifolia seed extracts to possess QS-enhancing effect in C. violaceum. Dimethylsulfoxide was found to enhance violacein production, whereas ethanol and methanol reduced violacein production in C. violaceum. Streptomycin at sub-minimum inhibitory concentration level was able to significantly arrest QS-regulated pigment production in C. violaceum and Serratia marcescens.@*CONCLUSIONS@#Prodigiosin and the seed extracts used in this study could affect quorum sensing in C. violaceum to a notable extent. Results of this study also emphasize the importance of inclusion of appropriate solvent controls (negative controls) in bioassays designed for screening of antimicrobial and/or anti-QS compounds. Antipathogenic potential of low concentrations of streptomycin was also demonstrated.

In vitro and in vivo antitumor activity of crude extracts obtained from Brazilian Chromobacterium sp isolates

Natural products produced by microorganisms have been an important source of new substances and lead compounds for the pharmaceutical industry. Chromobacterium violaceum is a Gram-negative β-proteobacterium, abundant in water and soil in tropical and subtropical regions and it produces violacein, a pigment that has shown great pharmaceutical potential. Crude extracts of five Brazilian isolates of Chromobacterium sp (0.25, 2.5, 25, and 250 µg/mL) were evaluated in an in vitro antitumor activity assay with nine human tumor cells. Secondary metabolic profiles were analyzed by liquid chromatography and electrospray ionization mass spectrometry resulting in the identification of violacein in all extracts, whereas FK228 was detected only in EtCE 308 and EtCE 592 extracts. AcCE and EtCE 310 extracts showed selectivity for NCI/ADR-RES cells in the in vitro assay and were evaluated in vivo in the solid Ehrlich tumor model, resulting in 50.3 and 54.6% growth inhibition, respectively. The crude extracts of Chromobacterium sp isolates showed potential and selective antitumor activities for certain human tumor cells, making them a potential source of lead compounds. Furthermore, the results suggest that other compounds, in addition to violacein, deoxyviolacein and FK228, may be involved in the antitumor effect observed.

Isolation and Identification of Marine Actinomycetes with Quorum Sensing Inhibitory Activity / 微生物学通报

Quorum sensing systems of pathogens are central regulators for the expression of virulence factors. Increasing evidence implies that targeting the quorum sensing system of many pathogenic bacteria is a promising therapeutic approach to control infections. In this work,we isolated 47 strains of actinomycetes from the mud sample of Jiaozhou Bay. Quorum sensing inhibitory activity was monitored by Chromobacterium violaceum CV026. As a result,the culture broth extract of actinomycetes WA-7 was found to have significant quorum sensing inhibitory activity. This strain was assigned to the genus Streptomyces based on its 16S rDNA sequence. Further investigation revealed that the extract could inhibit the quorum sensing-controlled violacein and proteases production of C. violaceum in a concentration-dependent manner.