3,3'-Diindolylmethane (DIM): A Potential Therapeutic Agent against Cariogenic Streptococcus mutans Biofilm.

Indole, a metabolite of the amino acid tryptophan, has been proven to act as a signal molecule in bacteria, acting in different aspects of biofilm formation. The oral biofilm is a type of biofilm that has consequences for human health. It is a complex, three-dimensional structure that develops on the surface of teeth via the attachment of primary microbial colonizers. Many oral infections are caused by an imbalance occurring in the microorganisms naturally found in oral biofilms and are considered major public health concerns. In this study, we test the effect of a natural bis-indole, 3,3'-Diindolylmethane (DIM), in mitigating the pathogenicity of the oral biofilm inhabiting bacterium Streptococcus mutans, a bacterium that is considered to be a principal etiological agent in dental caries. Our study found that DIM was able to attenuate S. mutans biofilm formation by 92%. Additionally, treatment with DIM lowered extracellular polymeric substance (EPS) production and decreased its durability significantly under acidic conditions. Therefore, the anti-biofilm and anti-virulence properties of DIM against S. mutans bacteria in an "oral setting" provides evidence for its usefulness in reducing biofilm formation and potentially for caries attenuation.

Conversations in the Gut: The Role of Quorum Sensing in Normobiosis.

An imbalance in gut microbiota, termed dysbiosis, has been shown to affect host health. Several factors, including dietary changes, have been reported to cause dysbiosis with its associated pathologies that include inflammatory bowel disease, cancer, obesity, depression, and autism. We recently demonstrated the inhibitory effects of artificial sweeteners on bacterial quorum sensing (QS) and proposed that QS inhibition may be one mechanism behind such dysbiosis. QS is a complex network of cell-cell communication that is mediated by small diffusible molecules known as autoinducers (AIs). Using AIs, bacteria interact with one another and coordinate their gene expression based on their population density for the benefit of the whole community or one group over another. Bacteria that cannot synthesize their own AIs secretly "listen" to the signals produced by other bacteria, a phenomenon known as "eavesdropping". AIs impact gut microbiota equilibrium by mediating intra- and interspecies interactions as well as interkingdom communication. In this review, we discuss the role of QS in normobiosis (the normal balance of bacteria in the gut) and how interference in QS causes gut microbial imbalance. First, we present a review of QS discovery and then highlight the various QS signaling molecules used by bacteria in the gut. We also explore strategies that promote gut bacterial activity via QS activation and provide prospects for the future.

Anti-Virulence Activity of 3,3'-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound Healing Benefits.

Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3'-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM.

Inhibitory Effects of Artificial Sweeteners on Bacterial Quorum Sensing.

Despite having been tagged as safe and beneficial, recent evidence remains inconclusive regarding the status of artificial sweeteners and their putative effects on gut microbiota. Gut microorganisms are essential for the normal metabolic functions of their host. These microorganisms communicate within their community and regulate group behaviors via a molecular system termed quorum sensing (QS). In the present study, we aimed to study the effects of artificial sweeteners on this bacterial communication system. Using biosensor assays, biophysical protein characterization methods, microscale thermophoresis, swarming motility assays, growth assays, as well as molecular docking, we show that aspartame, sucralose, and saccharin have significant inhibitory actions on the Gram-negative bacteria N-acyl homoserine lactone-based (AHL) communication system. Our studies indicate that these three artificial sweeteners are not bactericidal. Protein-ligand docking and interaction profiling, using LasR as a representative participating receptor for AHL, suggest that the artificial sweeteners bind to the ligand-binding pocket of the protein, possibly interfering with the proper housing of the native ligand and thus impeding protein folding. Our findings suggest that these artificial sweeteners may affect the balance of the gut microbial community via QS-inhibition. We, therefore, infer an effect of these artificial sweeteners on numerous molecular events that are at the core of intestinal microbial function, and by extension on the host metabolism.

Assessing the Molecular Targets and Mode of Action of Furanone C-30 on Pseudomonas aeruginosa Quorum Sensing.

Quorum sensing (QS), a sophisticated system of bacterial communication that depends on population density, is employed by many pathogenic bacteria to regulate virulence. In view of the current reality of antibiotic resistance, it is expected that interfering with QS can address bacterial pathogenicity without stimulating the incidence of resistance. Thus, harnessing QS inhibitors has been considered a promising approach to overriding bacterial infections and combating antibiotic resistance that has become a major threat to public healthcare around the globe. Pseudomonas aeruginosa is one of the most frequent multidrug-resistant bacteria that utilize QS to control virulence. Many natural compounds, including furanones, have demonstrated strong inhibitory effects on several pathogens via blocking or attenuating QS. While the natural furanones show no activity against P. aeruginosa, furanone C-30, a brominated derivative of natural furanone compounds, has been reported to be a potent inhibitor of the QS system of the notorious opportunistic pathogen. In the present study, we assess the molecular targets and mode of action of furanone C-30 on P. aeruginosa QS system. Our results suggest that furanone C-30 binds to LasR at the ligand-binding site but fails to establish interactions with the residues crucial for the protein's productive conformational changes and folding, thus rendering the protein dysfunctional. We also show that furanone C-30 inhibits RhlR, independent of LasR, suggesting a complex mechanism for the agent beyond what is known to date.

Anti-Quorum Sensing Activity of Stevia Extract, Stevioside, Rebaudioside A and Their Aglycon Steviol.

Governments are creating regulations for consumers to reduce their sugar intake, prompting companies to increase the ratio of artificial sweeteners in their products. However, there is evidence of some deleterious effects ascribed to the aforementioned synthetic agents and therefore consumers and food manufacturers have turned their attention to natural dietary sweeteners, such as stevia, to meet their sweetening needs. Stevia is generally considered safe; however, emerging scientific evidence has implicated the agent in gut microbial imbalance. In general, regulation of microbial behavior is known to depend highly on signaling molecules via quorum sensing (QS) pathways. This is also true for the gut microbial community. We, therefore, evaluated the possible role of these stevia-based natural sweeteners on this bacterial communication pathway. The use of a commercial stevia herbal supplement resulted in an inhibitory effect on bacterial communication, with no observable bactericidal effect. Purified stevia extracts, including stevioside, rebaudioside A (Reb A), and steviol revealed a molecular interaction, and possible interruption of Gram-negative bacterial communication, via either the LasR or RhlR receptor. Our in-silico analyses suggest a competitive-type inhibitory role for steviol, while Reb A and stevioside are likely to inhibit LasR-mediated QS in a non-competitive manner. These results suggest the need for further safety studies on the agents.

Functional marine metagenomic screening for anti-quorum sensing and anti-biofilm activity.

Quorum sensing (QS), a cell-to-cell communication process, entails the production of signaling molecules that enable synchronized gene expression in microbial communities to regulate myriad microbial functions, including biofilm formation. QS disruption may constitute an innovative approach to the design of novel antifouling and anti-biofilm agents. To identify novel quorum sensing inhibitors (QSI), 2,500 environmental bacterial artificial chromosomes (BAC) from uncultured marine planktonic bacteria were screened for QSI activity using soft agar overlaid with wild type Chromobacterium violaceum as an indicator. Of the BAC library clones, 7% showed high QSI activity (>40%) against the indicator bacterium, suggesting that QSI is common in the marine environment. The most active compound, eluted from BAC clone 14-A5, disrupted QS signaling pathways and reduced biofilm formation in both Pseudomonas aeruginosa and Acinetobacter baumannii. The mass spectra of the active BAC clone (14-A5) that had been visualized by thin layer chromatography was dominated by a m/z peak of 362.1.

Coral-associated bacteria, quorum sensing disrupters, and the regulation of biofouling.

Marine biofouling, the settlement of microorganisms and macroorganisms on structures submerged in seawater, although economically detrimental, is a successful strategy for survival in hostile environments, where coordinated bacterial communities establish biofilms via the regulation of quorum sensing (QS) communication systems. The inhibition of QS activity among bacteria isolated from different coral species was investigated to gain further insight into its potency in the attenuation, or even the prevention, of undesirable biofouling on marine organisms. It is hypothesized that coral mucus/microorganism interactions are competitive, suggesting that the dominant communities secrete QS disruptive compounds. One hundred and twenty bacterial isolates were collected from healthy coral species and screened for their ability to inhibit QS using three bioreporter strains. Approximately 12, 11, and 24% of the isolates exhibited anti-QS activity against Escherichia coli pSB1075, Chromobacterium violaceum CV026, and Agrobacterium tumefaciens KYC55 indicator strains, respectively. Isolates with positive activity against the bioluminescent monitor strains were scanned via a cytotoxic/genotoxic, E. coli TV1061 and DPD2794 antimicrobial panel. Isolates detected by C. violaceum CV026 and A. tumefaciens KYC55 reporter strains were tested for their ability to inhibit the growth of these reporter strains, which were found to be unaffected. Tests of the Favia sp. coral isolate Fav 2-50-7 (>98% similarity to Vibrio harveyi) for its ability to attenuate the formation of biofilm showed extensive inhibitory activity against biofilms of Pseudomonas aeruginosa and Acinetobacter baumannii. To ascertain the stability and general structure of the active compound, cell-free culture supernatants exposed to an increasing temperature gradient or to digestion by proteinase K, were shown to maintain potent QS attenuation and the ability to inhibit the growth of biofilms. Mass spectrometry confirmed the presence of a low molecular mass compound. The anti-QS strategy exemplified in the coral mucus is a model with potentially wide applications, including countering the ecological threat posed by biofilms. Manipulating synchronized bacterial behavior by detecting new QS inhibitors will facilitate the discovery of new antifouling compounds.

Characterization of quorum sensing signals in coral-associated bacteria.

Marine environment habitats, such as the coral mucus layer, are abundant in nutrients and rich with diverse populations of microorganisms. Since interactions among microorganisms found in coral mucus can be either mutualistic or competitive, understanding quorum sensing-based acyl homoserine lactone (AHL) language may shed light on the interaction between coral-associated microbial communities in the native host. More than 100 bacterial isolates obtained from different coral species were screened for their ability to produce AHL. When screening the isolated coral bacteria for AHL induction activity using the reporter strains Escherichia coli K802NR-pSB1075 and Agrobacterium tumefaciens KYC55, we found that approximately 30% of the isolates tested positive. Thin layer chromatography separation of supernatant extracts revealed different AHL profiles, with detection of at least one active compound in the supernatant of those bacterial extracts being able to induce AHL activity in the two different bioreporter strains. The active extract of bacterial isolate 3AT 1-10-4 was subjected to further analysis by preparative thin layer chromatography and liquid chromatography tandem mass spectrometry. One of the compounds was found to correspond with N-(3-hydroxydecanoyl)-L-homoserine lactone. 16S rRNA gene sequencing of the isolates with positive AHL activity affiliated them with the Vibrio genus. Understanding the ecological role of AHL in the coral environment and its regulatory circuits in the coral holobiont-associated microbial community will further expand our knowledge of such interactions.

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis.