群体感应抑制剂对海洋生态功能菌生物膜形成的影响

[目的]研究天然群体感应抑制剂(Quorum sensing inhibitors,QSI)分子对海洋生态功能菌生物膜形成的影响.[方法]以对污损生物幼虫附着具有诱导作用的海洋细菌为目标菌,通过在其生物膜的形成过程中添加天然群体感应抑制剂,研究其对目标菌成膜细菌数和浮游细菌数、生物膜形态以及生物膜表面胞外多糖含量的影响.[结果]呋喃酮和吡啶在50 mg/L时,对8株目标菌的成膜有显著的抑制作用,抑制率在80％左右,吲哚、青霉烷酸和香豆素在较高浓度800 mg/L才有比较好的抑制活性.生长抑制实验结果显示,同等浓度下,QSI分子对目标菌成膜的抑制活性明显高于其对浮游细菌生长的抑制活性.结果表明,QSI分子主要通过干扰目标菌群体感应系统以抑制生物膜的形成.[结论]研究证实QSI分子在海洋菌生物膜形成过程中具有一定的调控作用.通过添加QSI可能能够间接抑制由生物膜诱导的污损生物附着,从而以新的角度研制新型抗污损物质.     

A mathematical model of quorum sensing in a growing bacterial biofilm

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. This paper describes a one-dimensional model of a growing Pseudomonas aeruginosa biofilm. Quorum sensing has been included in the model by the addition of equations describing the production, degradation, and diffusion of acyl-homoserine lactones in the biofilm. In order for quorum sensing to initiate near the substratum, in accordance with experimental observations, model results suggest that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. This result highlights the importance of careful study of the relationship between metabolic activity of the bacterium and signal synthesis. Received 11 March 2002/ Accepted in revised form 01 August 2002     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.     

Inhibition of biofilm formation,quorum sensing activity and molecular docking study of isolated 3, 5, 7-Trihydroxyflavone from Alstonia scholaris leaf against P.aeruginosa

The current study is to evaluate the inhibition of biofilm formation and quorum sensing activity of isolated 3, 5, 7-Trihydroxyflavone (TF) from A.scholaris leaf extract against Pseudomonas aeruginosa. The effects of isolated TF on quorum sensing-regulated virulence factors production such as swimming motility, pyocyanin production, proteolytic, EPS, metabolic assay and inhibition of biofilm formation against P.aeruginosa was evaluated by standard protocols. In addition, the interaction between the isolated TF and active sites of QS- gene (LasI/rhlI, LasR/rhlR, and AHLase) in P.aeruginosa was evaluated by molecular docking studies using AutoDock Tools version 1.5.6. Based on the structural elucidation of the isolated compound was identified as 3, 5, 7-Trihydroxyflavone. Consequently, the isolated TF shows a significant reduction of biofilm formation through the inhibition of QS-dependent phenotypes such as pyocyanin production, proteolytic, swimming motility, EPS activities against P.aeruginosa in a dose-dependent manner. Molecular docking analysis of isolated TF can interfere the signaling [N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL)] molecules in P.aeruginosa by QS genes (LasI, LasR, rhlI, and AHLase) regulation. The isolated TF compound from A.scholaris reveals a greater potential to inhibit biofilm and QS dependent virulence factor production in P.aeruginosa. Docking interaction studies of TF-LasR complex express higher binding affinity than the other QS gene in P.aeruginosa.     

The role of quorum sensing and the effect of environmental conditions on biofilm formation by strains of Vibrio vulnificus

Abstract

It has been suggested that Vibrio vulnificus attaches to plankton and algae and is found in large numbers in the environment. Factors affecting attachment, biofilm formation and morphology of V. vulnificus have not been thoroughly investigated. This study evaluated the role of quorum sensing (QS) and environmental conditions on biofilm development of V. vulnificus. It was found that biofilm development by V. vulnificus was affected by nutrient and glucose concentration, but not by NaCl concentration or temperature under the conditions used here. Moreover, biofilm development of a QS mutant strain proceeded rapidly and sloughing occurred earlier than for the isogenic parent strain. There was a significant loss of viability for the QS mutant biofilm early in development. Hence, it is hypothesised that factors regulated by the QS system play a role in proper biofilm development and maintenance of V. vulnificus. Furthermore, it is shown that biofilm development varied among isolates.     

Phenol, 2,4-bis(1,1-dimethylethyl) of marine bacterial origin inhibits quorum sensing mediated biofilm formation in the uropathogen Serratia marcescens

Intercellular communication in bacteria (quorum sensing, QS) is an important phenomenon in disease dissemination and pathogenesis, which controls biofilm formation also. This study reports the anti-QS and anti-biofilm efficacy of seaweed Gracilaria gracilis associated Vibrio alginolyticus G16 against Serratia marcescens. Purification and mass spectrometric analysis revealed the active principle as phenol, 2,4-bis(1,1-dimethylethyl) [PD]. PD affected the QS regulated virulence factor production in S. marcescens and resulted in a significant (p < 0.05) reduction in biofilm (85%), protease (41.9%), haemolysin (69.9%), lipase (84.3%), prodigiosin (84.5%) and extracellular polysaccharide (84.62%) secretion without hampering growth, as evidenced by XTT [2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. qPCR analysis confirmed the down-regulation of the fimA, fimC, flhD and bsmA genes involved in biofilm formation. Apart from biofilm inhibition and disruption, PD increased the susceptibility of S. marcescens to gentamicin when administered synergistically, which opens another avenue for combinatorial therapy where PD can be used to enhance the efficacy of conventional antibiotics.     

New tryptophanase inhibitors: Towards prevention of bacterial biofilm formation

Tryptophanase (tryptophan indole-lyase, Tnase, EC 4.1.99.1), a bacterial enzyme with no counterpart in eukaryotic cells, produces from L-tryptophan pyruvate, ammonia and indole. It was recently suggested that indole signaling plays an important role in the stable maintenance of multicopy plasmids. In addition, Tnase was shown to be capable of binding Rcd, a short RNA molecule involved in resolution of plasmid multimers. Binding of Rcd increases the affinity of Tnase for tryptophan, and it was proposed that indole is involved in bacteria multiplication and biofilm formation. Biofilm-associated bacteria may cause serious infections, and biofilm contamination of equipment and food, may result in expensive consequences. Thus, optimal and specific factors that interact with Tnase can be used as a tool to study the role of this multifunctional enzyme as well as antibacterial agents that may affect biofilm formation. Most known quasi-substrates inhibit Tnase at the mM range. In the present work, the mode of Tnase inhibition by the following compounds and the corresponding Ki values were: S-phenylbenzoquinone-L-tryptophan, uncompetitively, 101 μM; α-amino-2-(9,10-anthraquinone)-propanoic acid, noncompetitively, 174 μM; L-tryptophane-ethylester, competitively, 52 μM; N-acetyl-L-tryptophan, noncompetitively, 48 μM. S-phenylbenzoquinone-L-tryptophan and α-amino-2-(9,10-anthraquinone)-propanoic acid were newly synthesized.     

The Freter model: A simple model of biofilm formation

A simple, conceptual model of biofilm formation, due to R. Freter et al. (1983), is studied analytically and numerically in both CSTR and PFR. Two steady state regimes are identified, namely, the complete washout of the microbes from the reactor and the successful colonization of both the wall and bulk fluid. One of these is stable for any particular set of parameter values and sharp and explicit conditions are given for the stability of each. The effects of adding an anti-microbial agent to the CSTR are examined.Supported by NSF Grant DMS 0107439 and UTA Grant REP 14748717Supported by NSF Grant DMS 0107160     

Antigen forks: bispecific reagents that inhibit cell growth by binding selected pairs of tumor antigens

Bispecific antibodies of a new category, termed antigen forks, were constructed by crosslinking antibodies that recognized pairs of distinct tumor cell surface antigens. At concentrations of 1–100 nM, several such forks inhibited the growth of human tumor cell lines bearing both relevant antigens. The same cells were not inhibited by unconjugated component antibodies, and the active conjugates did not inhibit the growth of human cell lines that expressed lower levels of relevant antigens. The three most active antigen forks all contained monoclonal antibody 454A12, which recognizes human transferrin receptor. This antibody was conjugated respectively to antibodies 113F1 (against a tumor-associated glycoprotein complex), 317G5 (against a 42-kDa tumor-associated glycoprotein), or 520C9 (against the c-erbB-2 protooncogene product). The 317G5-454A12 fork strongly inhibited the HT-29 and SW948 human colorectal cancer cell lines, while the 113F1-454A12 fork was also effective against SW948. By designing forks against antigens of incompatible function that are co-expressed at high levels on tumor cells but not on normal tissues, it may be possible to generate reagents that inhibit tumor growth with enhanced selectivity.     

A new phenothiazine structural scaffold as inhibitors of bacterial quorum sensing in Vibrio harveyi

Quorum sensing has attracted much attention due to its involvement in pathologically relevant events such as biofilm formation, virulence factor production, and sporulation. Inhibitors of quorum sensing are important research tools and potential therapeutic agents. In this paper, we describe a phenothiazine structural scaffold as a new type of quorum sensing inhibitors with IC₅₀ values in the single digit micro molar range in Vibrio harveyi.     

A different path: Revealing the function of staphylococcal proteins in biofilm formation

Staphylococcus aureus and Staphylococcus epidermidis cause dangerous and difficult to treat medical device-related infections through their ability to form biofilms. Extracellular poly-N-acetylglucosamine (PNAG) facilitates biofilm formation and is a vaccination target, yet details of its biosynthesis by the icaADBC gene products is limited. IcaC is the proposed transporter for PNAG export, however a comparison of the Ica proteins to homologous exo-polysaccharide synthases suggests that the common IcaAD protein components both synthesise and transport the PNAG. The limited distribution of icaC to the Staphylococcaceae and its membership of a family of membrane-bound acyltransferases, leads us to suggest that IcaC is responsible for the known O-succinylation of PNAG that occurs in staphylococci, identifying a potentially new therapeutic target specific for these bacteria.     

Phages of Pseudomonas aeruginosa: response to environmental factors and in vitro ability to inhibit bacterial growth and biofilm formation

Aims: To examine effects of various environmental factors on adsorption and inactivation of Pseudomonas aeruginosa‐specific phages: δ (family Podoviridae), J‐1, σ‐1 and 001A (family Siphoviridae) and their ability to inhibit bacterial growth and biofilm formation. Methods and Results: The phages examined in the study were clonally different, as revealed by RFLP. The temperature in the range 7–44°C had no influence on the adsorption of Podoviridae, but did affect Siphoviridae adsorption, particularly 001A. All phages were significantly stable at pH 5–9, and phages δ and 001A even at pH 3. Most of the examined carbohydrates and exopolysaccharides of the original host efficiently inactivated phage δ, while phages σ‐1 and J‐1 were inactivated considerably only by the amino acid alanine. Silver nitrate efficiently inactivated all the phages, while Siphoviridae were more resistant to povidone‐iodine. Serum of nonimmunized rats had no influence on phage inactivation and adsorption. Only phage δ showed ability to effectively inhibit in vitro bacterial growth and biofilm formation. Conclusions: The examined environmental parameters can significantly influence the adsorption and viability of Ps. aeruginosa‐specific phages. The phage δ is a good candidate for biocontrol of Ps. aeruginosa. Significance and Impact of the Study: The study provides important data on Ps. aeruginosa‐specific phage adsorption, inactivation and in vitro lytic efficacy.     

Antibiotic susceptibility pattern of fish pathogens: A new approach of emerging the bacterial resistance through biofilm formation in in-vitro condition

BackgroundThe ability of many bacteria to adhere on the host surfaces and forming biofilms has major implications in a wide variety of industries including the food industry, where biofilms may create a persistent source of contamination. In the same environmental condition, the multiple bacterial species can closely interact with each other and may easily enhance their drug resistance capability, which finally increases the multi-drug resistant (MDR) attribute of the species.ObjectiveThe present study examined whether the mixed-species biofilm possesses any impact on the enhancement of the antibiotic resistance of the planktonic or single-cell bacterial isolates present in the fish samples.MethodsIn this regard, Cyprinus rubrofuscus (Koi), Heteropneustes fossilis (Shing) and Mystus vittatus (Tengra) fishes were collected and subjected to form an in vitro biofilm by shaking condition into the wise bath. The drug-resistant pattern was determined by the Kirby Bauer technique.ResultsAll the samples exhibited a huge array (up to 10⁷ cfu/ml or g) of bacteria such as E. coli, Klebsiella spp., Vibrio spp., Salmonella spp., Proteus spp. and Staphylococcus spp. The isolates from both the bulk samples and their corresponding biofilms were subjected to antibiogram assay using antibiotics such as Ampicillin (10 µg), Erythromycin (15 μg), Streptomycin (STP 10 μg), Oxacillin (10 µg), Nalidixic acid (30 µg). Before biofilm formation, few of the isolates were found to be sensitive and few were resistant against the antibiotics. But when the species were isolated from the biofilm the sensitive one acquired drug resistance and resistant strain unveiled more resistance towards the same antibiotics. The present study revealed extensive bacterial contamination in fish samples among those some were resistant against the supplied drugs.ConclusionAfter the formation of multi-species biofilm, the isolates became more resistant against the same drugs that is alarming for consumers and major obstacles to maintain sustainable health.     

Acylureas: A new class of barbiturate-like bacterial cytochrome P-450 inducers

The soluble, cytochrome P-450-dependent fatty acid monooxygenase of Bacillus megaterium ATCC 14581 is induced by phenobarbital and at least twelve other barbiturates (Kim, B.-H. and Fulco, A.J. 91983) Biochem. Biophys. Res. Commun. 116, 843–850). We have since found that the inducer potency of phenobarbital and of six other of these barbiturates was enhanced by adding them to growth medium prior to sterilization by autoclaving. A similar ‘activation’ was effected simply by autoclaving these barbiturates in distilled water at pH 8.0. When the hydrolytic products resulting from such treatment of phenobarbital were identified and screened for inducer activity, the major product, 2-phenulbutyrylurea, was found to be 3–5-times more potent than phenobarbital itself. The racemic mixture, (±-)-2-phenylbutyryluera was somewhat more active as an inducer than was either of the enantiomers (±) or (?) tested singly. Of the other hydrolytic products of phenobarbital, only 2-phenylbutyramide had significant inducer activity (about the same as phenobarbital). Among other ureides tested, tow monosubstituted acetylureas (phenylacetylurea and dodecanoylurea) were inactive as inducers, but six of seven disubstituted acetylureas were better inducers than 2-phenylbutyrylurea.     

A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.