Brominated Furanones Inhibit Biofilm Formation by Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is a main cause of bacterial food-borne diseases. As Salmonella can form biofilms in which it is better protected against antimicrobial agents on a wide diversity of surfaces, it is of interest to explore ways to inhibit biofilm formation. Brominated furanones, originally extracted from the marine alga Delisea pulchra, are known to interfere with biofilm formation in several pathogens. In this study, we have synthesized a small focused library of brominated furanones and tested their activity against S. enterica serovar Typhimurium biofilm formation. We show that several furanones inhibit Salmonella biofilm formation at non-growth-inhibiting concentrations. The most interesting compounds are (Z)-4-bromo-5-(bromomethylene)-3-alkyl-2(5H)-furanones with chain lengths of two to six carbon atoms. A microarray study was performed to analyze the gene expression profiles of Salmonella in the presence of (Z)-4-bromo-5-(bromomethylene)-3-ethyl-2(5H)-furanone. The induced genes include genes that are involved in metabolism, stress response, and drug sensitivity. Most of the repressed genes are involved in metabolism, the type III secretion system, and flagellar biosynthesis. Follow-up experiments confirmed that this furanone interferes with the synthesis of flagella by Salmonella. No evidence was found that furanones act on the currently known quorum-sensing systems in Salmonella. Interestingly, pretreatment with furanones rendered Salmonella biofilms more susceptible to antibiotic treatment. Conclusively, this work demonstrates that particular brominated furanones have potential in the prevention of biofilm formation by Salmonella serovar Typhimurium.     

Identifying the important structural elements of brominated furanones for inhibiting biofilm formation by Escherichia coli

A collection of structurally closely related furanones was synthesized to identify the most important structural elements in brominated furanones for inhibiting the formation of bacterial biofilms. The results suggest that a conjugated exocyclic vinyl bromide on the furanone ring is the most important structural element for the non-toxic but inhibition activity for Escherichia coli biofilm formation. Furanones bearing monosubstituted bromide groups on saturated carbons were found to have a toxic effect that attenuates the bacterial growth.     

Effects of natural and chemically synthesized furanones on quorum sensing in <Emphasis Type="Italic">Chromobacterium violaceum</Emphasis>

Background  

Cell to cell signaling systems in Gram-negative bacteria rely on small diffusible molecules such as the N-acylhomoserine lactones (AHL). These compounds are involved in the production of antibiotics, exoenzymes, virulence factors and biofilm formation. They belong to the class of furanone derivatives which are frequently found in nature as pheromones, flavor compounds or secondary metabolites. To obtain more information on the relation between molecular structure and quorum sensing, we tested a variety of natural and chemically synthesized furanones for their ability to interfere with the quorum sensing mechanism using a quantitative bioassay with Chromobacterium violaceum CV026 for antagonistic and agonistic action. We were looking at the following questions:     

A quorum sensing-disrupting brominated thiophenone with a promising therapeutic potential to treat luminescent vibriosis

Vibrio harveyi is amongst the most important bacterial pathogens in aquaculture. Novel methods to control this pathogen are needed since many strains have acquired resistance to antibiotics. We previously showed that quorum sensing-disrupting furanones are able to protect brine shrimp larvae against vibriosis. However, a major problem of these compounds is that they are toxic toward higher organisms and therefore, they are not safe to be used in aquaculture. The synthesis of brominated thiophenones, sulphur analogues of the quorum sensing-disrupting furanones, has recently been reported. In the present study, we report that these compounds block quorum sensing in V. harveyi at concentrations in the low micromolar range. Bioluminescence experiments with V. harveyi quorum sensing mutants and a fluorescence anisotropy assay indicated that the compounds disrupt quorum sensing in this bacterium by decreasing the ability of the quorum sensing master regulator LuxR to bind to its target promoter DNA. In vivo challenge tests with gnotobiotic brine shrimp larvae showed that thiophenone compound TF310, (Z)-4-((5-(bromomethylene)-2-oxo-2,5-dihydrothiophen-3-yl)methoxy)-4-oxobutanoic acid, completely protected the larvae from V. harveyi BB120 when dosed to the culture water at 2.5 μM or more, whereas severe toxicity was only observed at 250 μM. This makes TF310 showing the highest therapeutic index of all quorum sensing-disrupting compounds tested thus far in our brine shrimp model system.     

A synthetic furanone potentiates the effect of disinfectants on Salmonella in biofilm

Aims: To study a possible effect of a synthetic brominated furanone on biofilm formation and biofilm resistance to disinfectants in Salmonella enterica. Methods and Results: The effect of a synthetic furanone on biofilm formation of Salm. enterica serovar Agona and Salm. enterica serovar Typhimurium (11 strains of different origins) was evaluated in a microtiterplate assay. A significant reduction in biofilm build‐up in microtiterplates by the furanone was observed for seven of the strains tested. Biofilms by two Salm. Agona feed factory strains and the effects on survival after exposures to disinfectants (hypochlorite and benzalkonium chloride) were assessed for both strains. Pretreatment with furanone significantly potentiated the effect of the two disinfectants for both strains. Conclusions: The effect of disinfectants on Salmonella in biofilm was significantly enhanced when the biofilm was grown in the presence of furanone. This was probably because of an effect on biofilm architecture, composition and in some cases also biofilm build‐up. Significance and Impact of the Study: The present study gives valuable new knowledge in the fight against Salmonella biofilm in the environment because of the potentiated effect of conventional disinfectants.     

A naturally occurring brominated furanone covalently modifies and inactivates LuxS

Halogenated furanones, a group of natural products initially isolated from marine red algae, are known to inhibit bacterial biofilm formation, swarming, and quorum sensing. However, their molecular targets and the precise mode of action remain elusive. Herein, we show that a naturally occurring brominated furanone covalently modifies and inactivates LuxS (S-ribosylhomocysteine lyase, EC 4.4.1.21), the enzyme which produces autoinducer-2 (AI-2).     

Cost of chemical defence in the red alga Delisea pulchra

The concept of a cost of defence is fundamental to theories for the evolution of defences against consumers. However, the evidence for a cost of plant chemical defences is mixed, and often indirect. This is particularly true for marine macroalgae (seaweeds), for which inferences of cost to date rely almost exclusively on phenotypic correlations between one class of secondary metabolites (brown algal phlorotannins) and growth (or, in one instance, fecundity). No studies of the cost of seaweed chemical defense have experimentally manipulated the presence of secondary metabolites in a controlled fashion and only one previous study has considered genetic background as a factor. Here we measured the cost of halogenated furanones to the red seaweed Delisea pulchra in three ways: a) phenotypic correlations between concentrations of furanones and fecundity in field collected thalli; b) genetic correlations between concentrations of furanones and growth for clones of thalli grown from tetraspores, and c) by comparing growth rates of thalli for which furanone production was experimentally inhibited (furanone -) vs thalli which produced furanones (furanone +). Two of our three tests-correlations between furanones and fecundity, and the growth of furanone (+) vs furanone (−) thalli-indicated a cost of furanones to D. pulchra but genetic correlations between furanones and growth did not. We suggest that these apparently conflicting results are consistent with the consequences of apical growth in this alga, and may further result from a cost of furanones only being manifested at critical developmental stages or times of tissue differentiation.     

Differential gene expression to investigate the effect of (5Z)-4-bromo- 5-(bromomethylene)-3-butyl-2(5H)-furanone on Bacillus subtilis

(5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming, and biofilm formation of gram-positive bacteria. Using the gram-positive bacterium Bacillus subtilis as a test organism, we observed cell killing by 20 microg of furanone per ml, while 5 microg of furanone per ml inhibited growth approximately twofold without killing the cells. To discover the mechanism of this inhibition on a genetic level and to investigate furanone as a novel antibiotic, full-genome DNA microarrays were used to analyze the gene expression profiles of B. subtilis grown with and without 5 microg of furanone per ml. This agent induced 92 genes more than fivefold (P < 0.05) and repressed 15 genes more than fivefold (P < 0.05). The induced genes include genes involved in stress responses (such as the class III heat shock genes clpC, clpE, and ctsR and the class I heat shock genes groES, but no class II or IV heat shock genes), fatty acid biosynthesis, lichenan degradation, transport, and metabolism, as well as 59 genes with unknown functions. The microarray results for four genes were confirmed by RNA dot blotting. Mutation of a stress response gene, clpC, caused B. subtilis to be much more sensitive to 5 microg of furanone per ml (there was no growth in 8 h, while the wild-type strain grew to the stationary phase in 8 h) and confirmed the importance of the induction of this gene as identified by the microarray analysis.     

Bicyclic brominated furanones: A new class of quorum sensing modulators that inhibit bacterial biofilm formation

Both natural and synthetic brominated furanones are known to inhibit biofilm formation by bacteria, but their toxicity to mammalian cells is often not reported. Here, we designed and synthesized a new class of brominated furanones (BBFs) that contained a bicyclic structure having one bromide group with well-defined regiochemistry. This class of molecules exhibited reduction in the toxicity to mammalian cells (human neuroblastoma SK-N-SH) and did not inhibit bacteria (Pseudomonas aeruginosa and Escherichia coli) growth, but retained the inhibitory activity towards biofilm formation of bacteria. In addition, all the BBFs inhibited the production of virulence factor elastase B in P. aeruginosa. To explore the effect of BBFs on quorum sensing, we used a reporter gene assay and found that 6-BBF and 7-BBF exhibited antagonistic activities for LasR protein in the lasI quorum sensing circuit, while 5-BBF showed agonistic activity for the rhlI quorum sensing circuit. This study suggests that structural variation of brominated furanones can be designed for targeted functions to control biofilm formation.     

γ-Alkylidene-γ-lactones and isobutylpyrrol-2(5H)-ones analogues to rubrolides as inhibitors of biofilm formation by Gram-positive and Gram-negative bacteria

Several molecules have been discovered that interfere with formation of bacterial biofilms, opening a new strategy for the development of more efficient treatments in case of antibiotic resistant bacteria. Amongst the most active compounds are some natural brominated furanones from marine algae Delisea pulchra that have proven to be able to control pathogenic biofilms. We have recently reported that some rubrolide analogues are able to inhibit biofilm formation of Enterococcus faecalis. In the present Letter we describe results of the biological evaluation of a small library of 28 compounds including brominated furanones and the corresponding lactams against biofilm formation of Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis and Streptococcus mutans. Our results showed that in general these compounds were more active against biofilms of S. epidermidis and P. aeruginosa, with little or no inhibition of planktonic bacterial growth. In some cases they were able to prevent biofilm formation of P. aeruginosa at concentrations as low as 0.6 μg/mL (1.3 μM, compound 3d) and 0.7 μg/mL (1.3 μM, 3f). Results also indicate that, in general, lactams are more active against biofilms than their precursors, thus designating this class of molecules as good candidates for the development of a new generation of antimicrobial drugs targeted to biofilm inhibition.     

The Effect of Brominated Furanones on The Formation of Staphylococcus aureus Biofilm on PVC

To study the influence of brominated furanones on the formation of Staphylococcus aureus (SA) biofilm on PVC thus providing new avenues of research on the surface modification of materials and clinical treatment of biomaterial-centered infection. Three brominated furanones (furanone-1, furanone-2, and furanone-3) were coated on the surface of PVC material. Both the modified PVC materials and SA were co-cultivated together. To assess the thickness of bacterial biofilm and bacterium colony unit area on PVC materials, confocal laser scanning microscopy and scanning electron microscopy (SEM) were used to observe the surface structure of SA biofilm formation. All treatments were compared with the control group which was not coated with furanones. PVC materials coated with furanone-1 had an increase in bacterial biofilm as well as SA colony area when compared with control. However, there was no significant difference between treating with furanone-1 and furanone-3 (P > 0.05). The impact of different brominated furanones on SA biofilm formation on the surface of PVC materials is different, furanone-1 can promote the SA biofilm formation on the surface of PVC material.     

Inhibition of biofilm formation and swarming of Bacillus subtilis by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone

AIMS: (5Z)-4-Bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone(furanone) of the marine alga Delisea pulchra was synthesized, and its inhibition of swarming motility and biofilm formation of Bacillus subtilis was investigated. METHODS AND RESULTS: Furanone was found to inhibit both the growth of B. subtilis and its swarming motility in a concentration-dependent way. In addition, as shown by confocal scanning laser microscopy, furanone inhibited the biofilm formation of B. subtilis. At 40 microg ml(-1), furanone decreased the biofilm thickness by 25%, decreased the number of water channels, and reduced the percentage of live cells by 63%. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Natural furanone has potential for controlling the multicellular behaviour of Gram-positive bacteria.     

DNA binding-uptake system: a link between cell-to-cell communication and biofilm formation

DNA has recently been described as a major structural component of the extracellular matrix in biofilms. In streptococci, the competence-stimulating peptide (CSP) cell-to-cell signal is involved in competence for genetic transformation, biofilm formation, and autolysis. Among the genes regulated in response to the CSP are those involved in binding and uptake of extracellular DNA. We show in this study that a functional DNA binding-uptake system is involved in biofilm formation. A comGB mutant of Streptococcus mutans deficient in DNA binding and uptake, but unaffected in signaling, showed reduced biofilm formation. During growth in the presence of DNase I, biofilm was reduced in the wild type to levels similar to those found with the comGB mutant, suggesting that DNA plays an important role in the wild-type biofilm formation. We also showed that growth in the presence of synthetic CSP promoted significant release of DNA, with similar levels in the wild type and in the comGB mutant. The importance of the DNA binding-uptake system in biofilm formation points to possible novel targets to fight infections.     

Transition from reversible to irreversible attachment during biofilm formation by Pseudomonas fluorescens WCS365 requires an ABC transporter and a large secreted protein

We report the identification of an ATP-binding cassette (ABC) transporter and an associated large cell-surface protein that are required for biofilm formation by Pseudomonas fluorescens WCS365. The genes coding for these proteins are designated lap for large adhesion protein. The LapA protein, with a predicted molecular weight of approximately 900 kDa, is found to be loosely associated with the cell surface and present in the culture supernatant. The LapB, LapC and LapE proteins are predicted to be the cytoplasmic membrane-localized ATPase, membrane fusion protein and outer membrane protein component, respectively, of an ABC transporter. Consistent with this prediction, LapE, like other members of this family, is localized to the outer membrane. We propose that the lapEBC-encoded ABC transporter participates in the secretion of LapA, as strains with mutations in the lapEBC genes do not have detectable LapA associated with the cell surface or in the supernatant. The lap genes are conserved among environmental pseudomonads such as P. putida KT2440, P. fluorescens PfO1 and P. fluorescens WCS365, but are absent from pathogenic pseudomonads such as P. aeruginosa and P. syringae. The wild-type strain of P. fluorescens WCS365 and its lap mutant derivatives were assessed for their biofilm forming ability in static and flow systems. The lap mutant strains are impaired in an early step in biofilm formation and are unable to develop the mature biofilm structure seen for the wild-type bacterium. Time-lapse microscopy studies determined that the lap mutants are unable to progress from reversible (or transient) attachment to the irreversible attachment stage of biofilm development. The lap mutants were also found to be defective in attachment to quartz sand, an abiotic surface these organisms likely encounter in the environment.     

Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.     

Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora

The plant pathogen Erwinia carotovora regulates expression of virulence factors and antibiotic production via an N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL) dependent quorum sensing mechanism. The marine alga Delisea pulchra produces halogenated furanones known to antagonise 3-oxo-C6-HSL activity. We have tested the effects of a halogenated furanone on the production of carbapenem, cellulase and protease in E. carotovora. Despite differences in the regulatory mechanisms controlling carbapenem and exoenzyme production each was inhibited by the algal metabolite. We present evidence to suggest that the furanone dependent inhibition of carbapenem production is a result of the disruption of the 3-oxo-C6-HSL dependent expression of the carABCDEFGH operon.     

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone reduces corrosion from Desulfotomaculum orientis

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming and biofilm formation of Gram-positive bacteria (Ren et al., 2002, Lett Appl Microbiol 34: 293-299). In the present study, the Gram-positive sulphate-reducing bacterium (SRB), Desulfotomaculum orientis, was used to study the inhibition of mild steel corrosion due to the addition of furanone. The weight loss from batch coupon experiments incubated with 40 microg x ml(-1) furanone was reduced fivefold compared with samples that lacked furanone. Analysis of the metal surface with environmental scanning electron microscopy further confirmed the protection afforded by the addition of furanone. In agreement with the corrosion inhibition, most probable number (MPN) analysis showed that 20 and 40 microg x ml(-1) furanone inhibited 58% and 96% of the D. orientis growth respectively. Hence, furanone has the potential to inhibit microbial-induced corrosion related to Gram-positive bacteria.