Reversing the biofilm-inducing effect of two xanthones from Garcinia mangostana by 3-methyl-2(5H)-furanone and N-butyryl-D-L homoserine lactone.

BACKGROUND: According to the WHO, 12 bacteria cause numerous human infections, including Enterobacteriaceae Klebsiella pneumoniae, and thus represent a public health problem. Microbial resistance is associated with biofilm formation; therefore, it is critical to know the biofilm-inducing potential of various compounds of everyday life. Likewise, the reversibility of biofilms and the modulation of persister cells are important for controlling microbial pathogens. In this work, we investigated the biofilm-inducing effects of xanthones from Garcinia mangostana on Klebsiella pneumoniae. Furthermore, we investigated the reversal effect of 3-methyl-2(5H)-furanone and the formation of persister cells induced by xanthones and their role in modulating the biofilm to the antibiotic gentamicin. METHODS: To analyze the biofilm-inducing role of xanthones from Garcinia mangostana, cultures of K. pneumoniae containing duodenal probe pieces were treated with 0.1-0.001 µM α- and γ-mangostin, and the biofilm levels were measured using spectrophotometry. To determine biofilm reversion, cultures treated with xanthones, or gentamicin were mixed with 3-methyl-2(5H)-furanone or N-butyryl-DL-homoserine lactone. The presence of K. pneumoniae persister cells was determined by applying the compounds to the mature biofilm, and the number of colony-forming units was counted. RESULTS: The xanthones α- and γ-mangostin increased K. pneumoniae biofilm production by 40% with duodenal probes. However, 3-methyl-2(5H)-furanone at 0.001 µΜ reversed biofilm formation by up to 60%. Moreover, adding the same to a culture treated with gentamicin reduced the biofilm by 80.5%. This effect was highlighted when 3-methyl-2(5H)-furanone was administered 6 h later than xanthones. At high concentrations of α-mangostin, persister K. pneumoniae cells in the biofilm were about 5 - 10 times more abundant than cells, whereas, with γ-mangostin, they were about 100 times more. CONCLUSION: Two xanthones, α- and γ-mangostin from G. mangostana, induced biofilm formation in K. pneumoniae and promoted persister cells. However, the biofilm formation was reversed by adding 3-methyl-2(5H)-furanone, and even this effect was achieved with gentamicin. In addition, this compound controlled the persister K. pneumoniae cells promoted by α-mangostin. Thus, synthetic, and natural biofilm-inducing compounds could harm human health. Therefore, avoiding these substances and looking for biofilm inhibitors would be a strategy to overcome microbial resistance and recover antibiotics that are no longer used.

Atmospheric oxidation of furanones by •OH and •Cl radicals: In situ FTIR rate coefficient determinations, SAR and theoretical studies.

Gas-phase kinetics of the overall reactions of •OH and •Cl radicals with dihydrofuran-3(2H)-one (oxolan-3-one) and dihydro-2-methyl-3(2H)-furanone (2MTHF-3-one) were studied at 298 K and atmospheric pressure. The rate coefficients were determined using the relative method in a 480 L multipass glass reactor coupled to an FT-IR detection system. The rate coefficients found for oxolan 3-one and 2MTHF-one with •OH radicals (k1 and k2) and with •Cl atoms (k3 and k4) at 298 K and atmospheric pressure (in cm3 molecule-1 s-1) were: k1 = (1.86 ± 0.29) × 10-11, k2 = (2.64 ± 0.47) × 10-11, k3= (1.15 ± 0.28) × 10 -10, and k4 = (1.33 ± 0.32) × 10-10, respectively. Reactivity trends were developed by comparison with other similar structures and Fukui indices employed to determine the reactivity of different sites on the ring. The singularity of the reaction with •OH was assessed by computational studies which showed the formation of several stable hydrogen bonded complexes, explaining the difference with the reaction with the •Cl atom. SAR estimations of the rate coefficients were calculated and compared to the experimental values.

Furanone and phytol influence metabolic phenotypes regulated by acyl-homoserine lactone in Salmonella.

Salmonella is an important foodborne pathogen, and it is unable to produce the quorum sensing signaling molecules called acyl-homoserine lactones (AHLs). However, it synthesizes the SdiA protein, detecting AHL molecules, also known as autoinducer-1 (AI-1), in the external environment. Exogenous AHLs can regulate specific genes related to virulence and stress response in Salmonella. Thus, interfering with quorum sensing can be a strategy to reduce virulence and help elucidate the cell-to-cell communication role in the pathogens' response to extracellular signals. This study aimed to evaluate the influence of the quorum sensing inhibitors furanone and phytol on phenotypes regulated by N-dodecanoyl homoserine lactone (C12-HSL) in Salmonella enterica serovar Enteritidis. The furanone C30 at 50 nM and phytol at 2 mM canceled the alterations promoted by C12-HSL on glucose consumption and the levels of free cellular thiol in Salmonella Enteritidis PT4 578 under anaerobic conditions. In silico analysis suggests that these compounds can bind to the SdiA protein of Salmonella Enteritidis and accommodate in the AHL binding pocket. Thus, furanone C30 and phytol act as antagonists of AI-1 and are likely inhibitors of the quorum sensing mechanism mediated by AHL in Salmonella.

A Brominated Furanone Inhibits Pseudomonas aeruginosa Quorum Sensing and Type III Secretion, Attenuating Its Virulence in a Murine Cutaneous Abscess Model.

Quorum sensing (QS) and type III secretion systems (T3SSs) are among the most attractive anti-virulence targets for combating multidrug-resistant pathogenic bacteria. Some halogenated furanones reduce QS-associated virulence, but their role in T3SS inhibition remains unclear. This study aimed to assess the inhibition of these two systems on Pseudomonas aeruginosa virulence. The halogenated furanones (Z)-4-bromo-5-(bromomethylene)-2(5H) (C-30) and 5-(dibromomethylene)-2(5H) (named hereafter GBr) were synthesized, and their ability to inhibit the secretion of type III exoenzymes and QS-controlled virulence factors was analyzed in P. aeruginosa PA14 and two clinical isolates. Furthermore, their ability to prevent bacterial establishment was determined in a murine cutaneous abscess model. The GBr furanone reduced pyocyanin production, biofilm formation, and swarming motility in the same manner or more effectively than C-30. Moreover, both furanones inhibited the secretion of ExoS, ExoT, or ExoU effectors in all tested strains. The administration of GBr (25 and 50 µM) to CD1 mice infected with the PA14 strain significantly decreased necrosis formation in the inoculation zone and the systemic spread of bacteria more efficiently than C-30 (50 µM). Molecular docking analysis suggested that the gem position of bromine in GBr increases its affinity for the active site of the QS LasR regulator. Overall, our findings showed that the GBr furanone displayed efficient multi-target properties that may favor the development of more effective anti-virulence therapies.

Synthesis and cytotoxic evaluation of protoanemonin and three brominated derivatives / Síntesis y evaluación citotóxica de la protoanemonina y tres derivados bromados / Síntese e avaliação citotóxica de protoanemonina e três derivados bromados

Abstract The protoanemonin, a natural furanone, was synthesized from (Z)-4-bromo-5-(bromomethylene)-furan-2(5H)-one by a reductive dehalogenation reaction with zinc. The 5-(dibromomethylene)-2(5H)-furanone and (E)-5-(bromomethylene)-2(5H)-furanone were also synthetized from levulinic acid bromination and acid promoted cyclization. The antiproliferative activity of all synthesized compounds against the human cancer cell lines PC-3 (prostate) and U-251 (glioblastoma) was investigated. The results showed that all the obtained furanones are more active than the reference drug cisplatin, with iC50 values in the range of 0.31 ± 0.02 to 7.30 ± 0.08 µM. However, (E)-5-(bromomethylene)-2(5H)-furanone, with a bromine atom in the double bond, was the most active, and demonstrated to be about 25-fold more active than the reference drug cisplatin.

Resumen La protoanemonina, una furanona de origen natural, fue sintetizada a partir de la (Z)-4-bromo-5-(bromometilen)-furan-2(5H)-ona mediante una deshalogenación reductiva con zinc. La 5-(dibromometilen)-2(5H)-furanona y la (E)-5-(bromometilen)-2(5H)-furanona también fueron sintetizadas a partir del ácido levulínico mediante bromación y posterior ciclación promovida por ácido. Se investigó la actividad antiproliferativa de todos los compuestos sintetizados en las líneas celulares de cáncer humano PC-3 (próstata) y U-251 (glioblastoma). Los resultados demostraron que todas las furanonas obtenidas son más activas que el fármaco de referencia cisplatino, con un intervalo de Ci50 de 0,31 ± 0,02 hasta 7,30 ± 0,08 µM. Sin embargo, la (E)-5-(bromometilen)-2(5H)-furanona, con un átomo de bromo en el doble enlace, demostró ser 25 veces más activa que el fármaco de referencia cisplatino.

Resumo Protoanemonina, uma furanona natural, foi sintetizada de (Z)-4-bromo-5-(bromometileno)-furan-2(5H)-ona por desalógenação redutora do zinco. Também foram sintetizados 5-(Dibromometileno)-2(5H)-furanona e (E)-5-(Bromometileno)-2(5H)-furanona a partir da brominação com ácido levulínico e ciclização promovida por ácido. Foi investigada a atividade antiproliferativa de todos os compostos sintetizados nas linhas celulares humanas de câncer PC-3 (próstata) e U-251 (glioblastoma). Os resultados mostram que todos os furanonas obtidos são mais ativos que a cisplatina do medicamento de referência, com intervalo Ci50 de 0,31 ± 0,02 até 7,30 ± 0,08 µM. Entretanto, a mais ativa foi a (E)-5-(Bromometileno)-2(5H)-furanona, que com um átomo de bromo na dupla ligação exocíclica, provou ser 25 vezes mais ativa que a medicamento de referência cisplatina.

Simultaneous determination of acrylamide and 4-hydroxy-2,5-dimethyl-3(2H)-furanone in baby food by liquid chromatography-tandem mass spectrometry.

A liquid chromatography triple quadrupole mass spectrometry method was developed and validated for the simultaneous determination of acrylamide and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) in baby food. The sample preparation involves acetonitrile-based extraction combined with dispersive primary secondary amine (PSA) cleanup and cation-exchange solid-phase extraction (SPE), which promotes efficient removal of matrix interferences. Analytical selectivity and sensitivity were achieved for the quantification of acrylamide and HDMF in complex matrices such as fruit, cereal and milk-based baby foods; furthermore, adequate linearity (range 10-300µgkg-1) in solvent and matrix-matched calibration curves, and appropriate recoveries (94-110%) and precision (RSD≤10%), under repeatability and within-laboratory reproducibility conditions, were also obtained. Expanded measurement uncertainty was estimated at the 20µgkg-1 level (limit of quantification) on the basis of data obtained from in-house validation, with values of 25.5 and 16.5% for acrylamide and HDMF, respectively. The fitness for purpose of developed method was verified by analyzing 15 commercial baby foods available in the Brazilian market. Acrylamide was detected in one plum-based baby food (35µgkg-1) while HDMF in 67% of the samples analyzed (levels between 25 and 262µgkg-1).

Acyl homoserine lactone-based quorum sensing stimulates biofilm formation by Salmonella Enteritidis in anaerobic conditions.

Quorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12-HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37 °C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50 nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36 h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.

Novel insights from molecular docking of SdiA from Salmonella Enteritidis and Escherichia coli with quorum sensing and quorum quenching molecules.

Quorum sensing is a cell-to-cell communication mechanism leading to differential gene expression in response to high population density. The autoinducer-1 (AI-1) type quorum sensing system is incomplete in Escherichia coli and Salmonella due to the lack of the AI-1 synthase (LuxI homolog) responsible for acyl homoserine lactone (AHL) synthesis. However, these bacteria encode the AHL receptor SdiA (a LuxR homolog) leading to gene regulation in response to AI-1 produced by other bacteria. This study aimed to model the SdiA protein of Salmonella enterica serovar Enteritidis PT4 578 based on three crystallized SdiA structures from Enterohemorrhagic E. coli (EHEC) with different ligands. Molecular docking of these predicted structures with AHLs, furanones and 1-octanoyl-rac-glycerol were also performed. The available EHEC SdiA structures provided good prototypes for modeling SdiA from Salmonella. The molecular docking of these proteins showed that residues Y63, W67, Y71, D80 and S134 are common binding sites for different quorum modulating signals, besides being conserved among other LuxR type proteins. We also show that AHLs with twelve carbons presented better binding affinity to SdiA than AHLs with smaller side chains in our docking analysis, regardless of the protein structures used. Interestingly, the conformational changes provided by AHL binding resulted in structural models with increased affinities to brominated furanones. These results suggest that the use of brominated furanones to inhibit phenotypes controlled by quorum sensing in Salmonella and EHEC may present a good strategy since these inhibitors seem to specifically compete with AHLs for binding to SdiA in both pathogens.

2(5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition

Biofouling of membranes demands costly periodic cleaning and membrane replacement. A sustainable and environmentally friendly solution for maintenance is not available and would be of great interest for many purposes including economical. As complex biofilm formation by environmental strains is the major cause of biofouling and biofilm formation in most cases are controlled by N-Acylhomoserine lactone (AHL)mediated Quorum Sensing (QS). An effort was made to understand the appropriateness of 2(5H)-furanone, to use against biofouling of membranes. QS inhibition activity by 2(5H)-furanone was studied using bioindicator strains and known AHLs of different acyl chain lengths. The biofilm inhibition was studied by growth analysis on polystyrene plate of Aeromonas hyrdrophila, an environmental biofilm strain isolated from a bio-fouled reverse osmosis (RO) membrane. Results showed a QS inhibition activity against a wide range of AHLs and also biofilm formation by 2(5H)-furanone, which is believed to act as a potential quorum inhibition agent in a bacterial biofilm community.

2(5H)-Furanone: A Prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition.

Biofouling of membranes demands costly periodic cleaning and membrane replacement. A sustainable and environmentally friendly solution for maintenance is not available and would be of great interest for many purposes including economical. As complex biofilm formation by environmental strains is the major cause of biofouling and biofilm formation in most cases are controlled by N-Acylhomoserine lactone (AHL)-mediated Quorum Sensing (QS). An effort was made to understand the appropriateness of 2(5H)-furanone, to use against biofouling of membranes. QS inhibition activity by 2(5H)-furanone was studied using bioindicator strains and known AHL of different acyl chain lengths. The biofilm inhibition was studied by growth analysis on polystyrene plate of Aeromonas hyrdrophila, an environmental biofilm strain isolated from a bio-fouled reverse osmosis (RO) membrane. Results showed a QS inhibition activity against a wide range of AHLs and also biofilm formation by 2(5H)-furanone, which is believed to act as a potential quorum inhibition agent in a bacterial biofilm community.

2(5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensing inhibition

Biofouling of membranes demands costly periodic cleaning and membrane replacement. A sustainable and environmentally friendly solution for maintenance is not available and would be of great interest for many purposes including economical. As complex biofilm formation by environmental strains is the major cause of biofouling and biofilm formation in most cases are controlled by N-Acylhomoserine lactone (AHL)mediated Quorum Sensing (QS). An effort was made to understand the appropriateness of 2(5H)-furanone, to use against biofouling of membranes. QS inhibition activity by 2(5H)-furanone was studied using bioindicator strains and known AHLs of different acyl chain lengths. The biofilm inhibition was studied by growth analysis on polystyrene plate of Aeromonas hyrdrophila, an environmental biofilm strain isolated from a bio-fouled reverse osmosis (RO) membrane. Results showed a QS inhibition activity against a wide range of AHLs and also biofilm formation by 2(5H)-furanone, which is believed to act as a potential quorum inhibition agent in a bacterial biofilm community.