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

The quorum-sensing disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) of the alga Delisea pulchra was found to inhibit the swarming motility of Escherichia coli completely at 13 microg cm-2 (also at 20 microg ml-1) but did not inhibit its growth rate at 13-52 microg cm-2 or from 20 to 100 microg ml-1. Swimming was not inhibited by the furanone at 20-40 microg ml-1. In addition, confocal scanning laser microscopy revealed that this furanone at 60 microg ml-1 inhibited the biofilm formation of E. coli, as it decreased its thickness by 55%, reduced the number of water channels and decreased the percentage of live cells by 87%. This suggests that natural furanone may be used as a new method to control bacterial biofilms that does not involve toxicity. Furanone at 10 microg ml-1 also inhibited by 3300-fold the quorum sensing of Vibrio harveyi via autoinducer 1 (AI-1) and inhibited by 5500-fold that of V. harveyi via of autoinducer 2 (AI-2) as well as inhibited by 26-600-fold the quorum sensing of E. coli via AI-2; hence, this furanone is a non-specific intercellular signal antagonist.     

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.     

(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.     

MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis

The stator-force generator that drives Na+-dependent motility in alkaliphilic Bacillus pseudofirmus OF4 is identified here as MotPS, MotAB-like proteins with genes that are downstream of the ccpA gene, which encodes a major regulator of carbon metabolism. B. pseudofirmus OF4 was only motile at pH values above 8. Disruption of motPS resulted in a non-motile phenotype, and motility was restored by transformation with a multicopy plasmid containing the motPS genes. Purified and reconstituted MotPS from B. pseudofirmus OF4 catalysed amiloride analogue-sensitive Na+ translocation. In contrast to B. pseudofirmus, Bacillus subtilis contains both MotAB and MotPS systems. The role of the motPS genes from B. subtilis in several motility-based behaviours was tested in isogenic strains with intact motAB and motPS loci, only one of the two mot systems or neither mot system. B. subtilis MotPS (BsMotPS) supported Na+-stimulated motility, chemotaxis on soft agar surfaces and biofilm formation, especially after selection of an up-motile variant. BsMotPS also supported motility in agar soft plugs immersed in liquid; motility was completely inhibited by an amiloride analogue. BsMotPS did not support surfactin-dependent swarming on higher concentration agar surfaces. These results indicate that BsMotPS contributes to biofilm formation and motility on soft agar, but not to swarming, in laboratory strains of B. subtilis in which MotAB is the dominant stator-force generator. BsMotPS could potentially be dominant for motility in B. subtilis variants that arise in particular niches.     

Synthesis of (R)-norbgugaine and its potential as quorum sensing inhibitor against Pseudomonas aeruginosa

(R)-Bgugaine is a natural pyrrolidine alkaloid from Arisarum vulgare, which shows antifungal and antibacterial activity. In this Letter, we have accomplished the simple synthesis of norbgugaine (demethylated form of natural bgugaine) employing Wittig olefination and cat. hydrogenation as the key steps and its biological studies are reported for the first time. The synthesized norbgugaine was evaluated for inhibition of quorum sensing mediated virulence factors (motility, biofilm formation, pyocyanin pigmentation, rhamnolipid production and LasA protease) in Pseudomonas aeruginosa wherein swarming motility is reduced by 95%, and biofilm formation by 83%.     

Extracellular proteolytic activity plays a central role in swarming motility in Bacillus subtilis

Natural isolates of Bacillus subtilis exhibit a robust multicellular behavior known as swarming. A form of motility, swarming is characterized by a rapid, coordinated progression of a bacterial population across a surface. As a collective bacterial process, swarming is often associated with biofilm formation and has been linked to virulence factor expression in pathogenic bacteria. While the swarming phenotype has been well documented for Bacillus species, an understanding of the molecular mechanisms responsible remains largely isolated to gram-negative bacteria. To better understand how swarming is controlled in members of the genus Bacillus, we investigated the effect of a series of gene deletions on swarm motility. Our analysis revealed that a strain deficient for the production of surfactin and extracellular proteolytic activity did not swarm or form biofilm. While it is known that surfactin, a lipoprotein surfactant, functions in swarming motility by reducing surface tension, this is the first report demonstrating that general extracellular protease activity also has an important function. These results not only help to define the factors involved in eliciting swarm migration but support the idea that swarming and biofilm formation may have overlapping control mechanisms.     

Inhibition of quorum sensing in Serratia marcescens AS-1 by synthetic analogs of N-acylhomoserine lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C(6)-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C(9)-CPA), had a strong inhibitory effect on prodigiosin production. C(9)-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C(9)-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C(6)-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C(9)-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用*

摘要目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用 （尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus Mirabilis ）为研究对象,考 察它的发酵滤液对一种机会致病菌———铜绿假单胞菌（ Pseudomonas aeruginose）多细胞行为的作用,以期得到一株多细胞行为抑 制菌：在不影响 P.aeruginosa 生长的前提下,抑制生物膜形成、EPS 产生以及定向丛集运动,解除保护,减缓扩散,为降低P.aeruginosa 耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮-硫酸法、平板检测法,探究P.aeruginosa 发酵滤 液对P.aeruginosa 生物膜、胞外多聚物、定向丛集运动和生长的影响。结果： P.aeruginosa 发酵滤液能显著抑制生物膜 量,在体积百分比浓度为1 %时,抑制率可达60.9 %。该菌的发酵滤液还能阻碍的定向丛集运动,减弱它的吸附和扩 散运动;同时,也减少了P.aeruginosa 胞外多聚物的产量,在滤液体积百分比浓度为1 %时,抑制率达到45.9%。更重要的是,固体 平板实验证明该发酵滤液对P.aeruginosa 的生长没有影响。结论： 在不影响病原菌生长的前提下,对病原菌的多细胞 行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药 性方面有潜在应用价值。     

The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional

The role of quorum sensing in Pseudomonas aeruginosa biofilm formation is unclear. Some researchers have shown that quorum sensing is important for biofilm development, while others have indicated it has little or no role. In this study, the contribution of quorum sensing to biofilm development was found to depend upon the nutritional environment. Depending upon the carbon source, quorum-sensing mutant strains (lasIrhlI and lasRrhlR) either exhibited a pronounced defect early in biofilm formation or formed biofilms identical to the wild-type strain. Quorum sensing was then shown to exert its nutritionally conditional control of biofilm development through regulation of swarming motility. Examination of pilA and fliM mutant strains further supported the role of swarming motility in biofilm formation. These data led to a model proposing that the prevailing nutritional conditions dictate the contributions of quorum sensing and swarming motility at a key juncture early in biofilm development.     

The carboxy terminal domain of Epr, a minor extracellular serine protease, is essential for the swarming motility of Bacillus subtilis 168

In this study we have investigated the role of Epr, a minor extracellular serine protease, in the swarming motility of Bacillus subtilis 168. We identified that the protease activity of Epr was dispensable for swarming. Since the protease activity of Epr was confined to its N-terminal domain, we hypothesized instead that its C-terminal domain (CTD) could be critical for swarming. Our study showed that not only the expression of Epr-CTD was necessary, but also its secretion was crucial for the swarming motility of B. subtilis 168.     

Tannin derived materials can block swarming motility and enhance biofilm formation in Pseudomonas aeruginosa

Surface-associated swarming motility is implicated in enhanced bacterial spreading and virulence, hence it follows that anti-swarming effectors could have clinical benefits. When investigating potential applications of anti-swarming materials it is important to consider whether the lack of swarming corresponds with an enhanced sessile biofilm lifestyle and resistance to antibiotics. In this study, well-defined tannins present in multiple plant materials (tannic acid (TA) and epigallocathecin gallate (EGCG)) and undefined cranberry powder (CP) were found to block swarming motility and enhance biofilm formation and resistance to tobramycin in Pseudomonas aeruginosa. In contrast, gallic acid (GA) did not completely block swarming motility and did not affect biofilm formation or tobramycin resistance. These data support the theory that nutritional conditions can elicit an inverse relationship between swarming motility and biofilm formation capacities. Although anti-swarmers exhibit the potential to yield clinical benefits, it is important to be aware of possible implications regarding biofilm formation and antibiotic resistance.     

Inhibition of swarming motility of Pseudomonas aeruginosa by branched-chain fatty acids.

Pseudomonas aeruginosa is capable of moving by swimming, swarming, and twitching motilities. In this study, we investigated the effects of fatty acids on Pseudomonas aeruginosa PAO1 motilities. A branched-chain fatty acid (BCFA)--12-methyltetradecanoic acid (anteiso-C15:0)--has slightly repressed flagella-driven swimming motility and completely inhibited a more complex type of surface motility, i.e. swarming, at a concentration of 10 microg mL(-1). In contrast, anteiso-C15:0 exhibited no effect on pili-mediated twitching motility. Other BCFAs and unsaturated fatty acids tested in this study showed similar inhibitory effects on swarming motility, although the level of inhibition differed between these fatty acids. These fatty acids caused no significant growth inhibition in liquid cultures. Straight-chain saturated fatty acids such as palmitic acid were less effective in swarming inhibition. The wetness of the PAO1 colony was significantly reduced by the addition of anteiso-C15:0; however, the production of rhamnolipids as a surface-active agent was not affected by the fatty acid. In addition to motility repression, anteiso-C15:0 caused 31% repression of biofilm formation by PAO1, suggesting that BCFA could affect the multiple cellular activities of Pseudomonas aeruginosa.     

Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.     

Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa

Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 g/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20–40 g/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20–100 g/ml. Similarly, 100 g/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).     

粘质沙雷氏菌AS-1中SpnR功能及卤化呋喃对其群体感应的抑制

通过分泌和感知一系列信号分子,细菌能够根据自身菌体密度的变化调控基因的表达,从而控制一系列重要的表现型,包括毒力因子的产生,生物膜的形成以及菌体发光等.这种广泛存在的信号机制被称为群体感应.在沙雷氏菌种中已经发现了多套群体感应机制.粘质沙雷氏菌AS-1从土壤中分离,其中含有LuxI/LuxR的同类蛋白,被称为SpnI/SpnR.粘质沙雷氏菌AS-1合成AHLs分子N-hexanoy1-L-homoserinelactone(C6-HSL)和N-(3.oxohexanoyl)-L-homoserine lactone(3-oxo-C6-HSL)作为其信号分子,通过群体感应感知菌体密度来控制基因的表达.通过基因替代的方法制得了spnR基因破坏的变异株,命名为粘质沙雷氏菌AS-1R.对粘质沙雷氏菌AS-1R的研究表明SpnR蛋白消极的调控沙雷氏菌红色色素的产生,运动性以及生物膜的形成等一系列由群体感应控制的性状:另一方面,作为一种天然的群体感应抑制剂,卤化呋喃能够有效的抑制粘质沙雷氏菌AS-1的群体感应,但并不干扰AHL-SpnR的相互作用.为运用粘质沙雷氏菌群体感应调节抑制其致病性提供了方法和依据,同时也为卤化呋喃对群体感应抑制机理的研究提供了新的思路.