Synthesis,in Silico Study and Biological Evaluation of N-(Benzothiazol/Thiazol-2-yl)benzamide Derivatives as Quorum Sensing Inhibitors against Pseudomonas aeruginosa

The development of bacterial resistance to chemical therapy poses a severe danger to efficacy of treating bacterial infections. One of the key factors for resistance to antimicrobial medications is growth of bacteria in biofilm. Quorum sensing (QS) inhibition was created as an alternative treatment by developing novel anti-biofilm medicines. Cell-cell communication is impeded by QS inhibition, which targets QS signaling pathway. The goal of this work is to develop newer drugs that are effective against Pseudomonas aeruginosa by decreasing QS and acting as anti-biofilm agents. In this investigation, N-(benzo[d]thiazol-2-yl)benzamide/N-(thiazol-2-yl)benzamide derivatives 3a-h were designed and synthesized in good yields. Further, molecular docking analyses revealed that binding affinity values were founded −11.2 to −7.6 kcal/mol that were moderate to good. The physicochemical properties of these prepared compounds were investigated through in-silico method. Molecular dynamic simulation was also used to know better understanding of stability of the protein and ligand complex. Comparing N-(benzo[d]thiazol-2-yl)benzamide 3a to salicylic acid (4.40±0.10) that was utilised as standard for quorum sensing inhibitor, the anti-QS action was found greater for N-(benzo[d]thiazol-2-yl)benzamide 3a (4.67±0.45) than salicylic acid (4.40±0.10). Overall, research results suggested that N-(benzo[d]thiazol-2-yl)benzamide/N-(thiazol-2-yl)benzamide derivatives 3a-h may hold to develop new quorum sensing inhibitors.     

Manipulation of pyrite colonization and leaching by iron-oxidizing <Emphasis Type="Italic">Acidithiobacillus</Emphasis> species

In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4–5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H₂O₂, indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans ^T. A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans ^T. In addition, sodium glucuronate addition enhanced pyrite dissolution by 25 %.     

青岛近海沉积物生物被膜中密度感应及密度感应淬灭细菌多样性

【背景】细菌密度感应(Quorum sensing,QS)是指细菌利用分泌的信号分子进行相互交流的现象,而密度感应淬灭(Quorumquenching,QQ)是指通过干扰信号分子的产生、释放、积累或应答从而阻抑密度感应通路。【目的】探究青岛近海沉积物生物被膜中密度感应和密度感应淬灭细菌的多样性。【方法】采用海水培养基2216E从青岛近海沉积物生物被膜中分离获取可培养细菌,采用平板交互划线和高通量筛选的方法分别筛选具有密度感应和密度感应淬灭的菌株。【结果】共分离获得83株共54种具有密度感应和密度感应淬灭的细菌,分属于四大细菌门类:变形菌门、拟杆菌门、厚壁菌门和放线菌门。其中,38株(45.8%)可以产生酰基高丝氨酸内酯(Acyl-homoserine lactone,AHL)类信号分子,它们分属于变形菌门(37株,15种)和拟杆菌门(1株,1种),优势属为弧菌属和鲁杰氏菌属;能够降解AHL类信号分子的有57株(68.7%),在变形菌门(41株,23种)、拟杆菌门(14株,10种)、厚壁菌门(5株,5种)以及放线菌门(1株,1种)中均有分布。【结论】在青岛近海沉积物生物被膜可培养细菌中,具有密度感应和密度感应淬灭现象的菌株具有很高的丰度和多样性,为后续生态学意义的研究与海洋微生物的开发提供了参考。     

黄河鲤水产细菌的分离及其毒力因子和群体感应信号分子的检测

【背景】水产细菌病害制约水产养殖业健康发展,群体感应与细菌毒力因子的产生密切相关,群体感应调控细菌的毒力因子特性值得进一步研究。【目的】探究群体感应与黄河鲤细菌病害的关系,明确群体感应对细菌毒力因子特性的影响。【方法】通过16S rRNA基因测序并构建系统进化树确定筛选菌株的进化地位,通过脱脂牛奶平板法和偶氮酪蛋白法检测菌株胞外蛋白酶活力,采用结晶紫染色法对菌株的生物膜形成能力进行测定,通过报告菌株BB170和CV026分别测定菌株产信号分子AI-2和高丝氨酸内酯的能力,外源添加高丝氨酸内酯检测信号分子对菌株胞外蛋白酶活力和生物膜形成能力的影响。【结果】哈夫尼亚菌(Hafnia sp.) Z11和气单胞菌(Aeromonas sp.) Z12具有高水平的胞外蛋白酶活力和生物膜形成能力,能够分泌AHLs信号分子且具有菌体密度依赖性。外源添加HSL对菌株毒力因子特性有不同程度的影响,外源添加高浓度的N-丁酰基高丝氨酸内酯(C4-HSL)和N-己酰基高丝氨酸内酯(C6-HSL)能够分别提高菌株Z11和Z12的胞外蛋白酶活力和生物膜形成能力。【结论】高浓度群体感应信号分子AHLs对哈夫尼亚菌和气单胞菌胞外蛋白酶活性有促进作用,说明该2种菌的群体感应现象可能会影响其毒力。     

Microbial population dynamics and proteomics in membrane bioreactors with enzymatic quorum quenching

Quorum sensing gives rise to biofilm formation on the membrane surface, which in turn causes a loss of water permeability in membrane bioreactors (MBRs) for wastewater treatment. Enzymatic quorum quenching was reported to successfully inhibit the formation of biofilm in MBRs through the decomposition of signal molecules, N-acyl homoserine lactones (AHLs). The aim of this study was to elucidate the mechanisms of quorum quenching in more detail in terms of microbial population dynamics and proteomics. Microbial communities in MBRs with and without a quorum quenching enzyme (acylase) were analyzed using pyrosequencing and compared with each other. In the quorum quenching MBR, the rate of transmembrane pressure (TMP) rise-up was delayed substantially, and the proportion of quorum sensing bacteria with AHL-like autoinducers (such as Enterobacter, Pseudomonas, and Acinetobacter) also decreased in the entire microbial community of mature biofilm in comparison to that in the control MBR. These factors were attributed to the lower production of extracellular polymeric substances (EPS), which are known to play a key role in the formation of biofilm. Proteomic analysis using the Enterobacter cancerogenus strain ATCC 35316 demonstrates the possible depression of protein expression related to microbial attachments to solid surfaces (outer membrane protein, flagellin) and the agglomeration of microorganisms (ATP synthase beta subunit) with the enzymatic quorum quenching. It has been argued that changes in the microbial population, EPS and proteins via enzymatic quorum quenching could inhibit the formation of biofilm, resulting in less biofouling in the quorum quenching MBR.     

Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Azithromycin Reduces the Production of α-hemolysin and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases which are controlled by quorum sensing. In this study, we show that azithromycin was active against methicillin-resistant Staphylococcus aureus (MRSA) strains with MICs ranged from 32 to 64 μg/mL. Azithromycin at subinhibitory concentration, markedly reduced the production of α-hemolysin at (1/16MIC, 1/8MIC) and biofilm formation at (1/16MIC, 1/8MIC), respectively. The results indicated that sub-inhibitory concentrations of azithromycin decreased the production of α-hemolysin and biofilm formation in MRSA in a dose-dependent manner. Therefore, azithromycin may be useful in the treatment of α-hemolysin producing and biofilm formation MRSA infections.     

The dependence of quorum sensing on the depth of a growing biofilm

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. In this paper, a one-dimensional model of a growing Pseudomonas aeruginosa biofilm is examined. Quorum sensing has been included in the model through equations describing the production, degradation, and diffusion of the signaling molecules, acyl-homoserine lactones, in the biofilm. From this model, we are able to make some important observations about quorum sensing. First, in order for quorum sensing to initiate near the substratum, in accordance with experimental observations, the model suggests that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. Second, the induction of quorum sensing is related to a critical biofilm depth; once the biofilm grows to the critical depth, quorum sensing is induced. Third, the critical biofilm depth varies with the pH of the surrounding fluid. Of particular interest is the prediction of a critical pH threshold, above which quorum sensing is not possible at any depth. These results highlight the importance of careful study of the relationship among metabolic activity of the bacterium, signal synthesis, and the chemistry of the surrounding environment.     

基于Lux型群体感应系统干预的生物被膜调控在污水处理中的研究进展与前景

基于Lux型群体感应系统的生物被膜调控在污水处理中的研究备受关注,群体感应系统的干预包括正向强化和负向削弱两类。群体感应系统的正向强化作用可提高生物膜法污水处理中的挂膜速度,提高污水处理效率,促进活性污泥中胞外聚合物(Extracellular polymeric substance,EPS)和可溶性微生物产物(Soluble microbial products,SMP)的生成,提高生物被膜的产量;群体感应的负向削弱作用可以降解生物被膜形成过程中所需要的信号分子,切断生物被膜形成的基因表达过程,有效抑制MBR膜表面生物被膜的形成,防止膜污染。对信号分子酰基高丝氨酸内酯(N-acyl homoserine lactone,AHLs)的结构和作用机理的进一步研究、群体感应淬灭菌的固定化技术与应用、多种防治膜污染方法的协同效果验证及群体感应干预在更多污水处理领域的应用可行性是该领域要研究的几个重要方向。     

Fungal elicitor protein PebC1 from Botrytis cinerea improves disease resistance in Arabidopsis thaliana

We previously identified a novel protein elicitor, PebC1, from Botrytis cinerea and described its enhancement of plant growth, drought tolerance and disease resistance in tomato. Here, we have investigated the defense-associated molecular responses in Arabidopsis thaliana after treatment with recombinant PebC1. PebC1 was expressed in Escherichia coli. Recombinant protein treatments improved plant resistance to Botrytis infection and maintained plant defenses for more than 21 days. The purified protein at 10 μg ml^?1 activated extracellular medium alkalization (pH) and reactive oxygen species and nitric oxide generation and also induced defense gene expression. Arabidopsis mutants that are insensitive to salicylic acid had increased resistance to Botrytis infection after PebC1 treatment but PebC1 did not affect the resistance of mutants with jasmonic acid and ethylene transduction pathways. The results suggest that PebC1 can function as an activator of plant disease resistance and can promote disease resistance to Botrytis in A. thaliana through the ethylene signal transduction pathway.     

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

Ionic homeostasis disturbance is involved in tomato cell death induced by NaCl and salicylic acid

The ability of salicylic acid and NaCl to induce programmed cell death by disturbing ionic homeostasis was investigated using tomato suspension culture cells. NaCl (300?mM) and salicylic acid (1?mM) inhibited cell growth and caused cell death within 1?wk of exposure. Treatment with NaCl increased the production of reactive oxygen species and the permeability of plasma membrane, but it also led to a reduction in the pH of the culture medium and resulted in a disturbance in ionic homeostasis of the cells. Salicylic acid-induced cell death in tomato suspension culture was also accompanied by production of reactive oxygen species and increases in both electrolyte leakage and pH of the culture media. However, reactive oxygen species production was not significantly different in cultures treated with a lethal salicylic acid concentration and 100?mM NaCl, in which most of the cells survived. A decrease in the K⁺/Na⁺ ratio was observed only in those cell cultures in which the salicylic acid treatment induced the death of cells. These results suggest that the decrease of the intracellular K⁺ concentration and K⁺/Na⁺ ratio is a common phenomenon in triggering programmed cell death by lethal concentrations of salicylic acid and NaCl.     

Subtilosin Prevents Biofilm Formation by Inhibiting Bacterial Quorum Sensing

Subtilosin, the cyclic lantibiotic protein produced by Bacillus subtilis KATMIRA1933, targets the surface receptor and electrostatically binds to the bacterial cell membrane. In this study, subtilosin was purified using ammonium sulfate ((NH₄)₂SO₄) precipitation and purified via column chromatography. Subtilosin’s antibacterial minimum and sub-minimum inhibitory concentrations (MIC and sub-MIC) and anti-biofilm activity (biofilm prevention) were established. Subtilosin was evaluated as a quorum sensing (QS) inhibitor in Gram-positive bacteria using Fe(III) reduction assay. In Gram-negative bacteria, subtilosin was evaluated as a QS inhibitor utilizing Chromobacterium voilaceum as a microbial reporter. The results showed that Gardnerella vaginalis was more sensitive to subtilosin with MIC of 6.25 μg/mL when compared to Listeria monocytogenes (125 μg/mL). The lowest concentration of subtilosin, at which more than 90% of G. vaginalis biofilm was inhibited without effecting the growth of planktonic cells, was 0.78 μg/mL. About 80% of L. monocytogenes and more than 60% of Escherichia coli biofilm was inhibited when 15.1 μg/mL of subtilosin was applied. Subtilosin with 7.8–125 μg/mL showed a significant reduction in violacein production without any inhibitory effect on the growth of C. violaceum. Subtilosin at 3 and 4 μg/mL reduced the level of Autoinducer-2 (AI-2) production in G. vaginalis. However, subtilosin did not influence AI-2 production by L. monocytogenes at sub-MICs of 0.95–15.1 μg/mL. To our knowledge, this is the first report exploring the relationship between biofilm prevention and quorum sensing inhibition in G. vaginalis using subtilosin as a quorum sensing inhibitor.     

Differential effects of elicitors on the viability of rice suspension cells

We have compared the effects of two elicitors of defense-related processes on rice (Oryza sativa L.) suspension cells. Both chitosan and salicylic acid induced the accumulation of extracellular chitinase, thickening of the cell wall, and a variety of cytological changes in treated cells. Chitosan also induced the production of a brown pigment and cell death. Both of these effects depended on the availability of reactive oxygen species, because the damage was greatly reduced by either catalase or free-radical scavengers. Pretreating cells with salicylic acid also protected them from the cytotoxic effects of chitosan. This type of induced tolerance persisted when salicylic acid was removed and was not simply due to the release of extracellular substances, because salicylic acid-treated cells did not protect untreated cells from chitosan-induced death. Salicylic acid also stimulated the production of a 10-kilodalton subtilisin inhibitor that was not produced by chitosan-treated cells. Most of these changes are associated with the hypersensitive response of many plant species, including monocotyledons, and may serve as an in vitro model for investigating the biochemistry of some diseases.     

A multi-phase mathematical model of quorum sensing in a maturing Pseudomonas aeruginosa biofilm

It is well known that sessile bacteria have a strong tendency to exist in a biofilm phenotype, whereby bacterial cells aggregate and produce a gel-like extracellular matrix, which, in an infection scenario, offers a significant barrier to attack by conventional antibiotics and the immune system. In this paper we develop a multi-phase model of a maturing Pseudomonas aeruginosa biofilm, allowing for the production and secretion of exopolysaccharide (EPS). The primary quorum-sensing system of P. aeruginosa (namely the lasR system) is believed to be required for full biofilm development, and we thus take the synthesis of EPS to be regulated by the cognate signal molecule, 3-oxo-C12-HSL. We also take EPS and signal production, along with bacterial growth, to be limited by oxygen availability, thus factoring in the nutrient poor conditions deep inside the biofilm. We use simulations to examine the role played by quorum sensing in the biofilm maturation process, and to investigate the effect of anti-quorum sensing and antibiotic treatments on EPS concentration, signal level, bacterial numbers and biofilm growth rate. In addition, we undertake analysis of the associated travelling-wave behaviour.     

In vitro assessment of the acaricidal activity of computer-selected analogues of carvacrol and salicylic acid on Rhipicephalus (Boophilus) microplus

Rhipicephalus (Boophilus) microplus is a tick that causes huge economic losses in cattle. The indiscriminate use of acaricides has generated resistance to most compounds present on the market. This makes further investigation on other potential acaricides necessary, the in silico assay being an alternative to the design of new compounds. In the present study a biosilico assay was performed using TOMOCOMD-CARDD (TOpological MOlecular COMputer Design-Computer-Aided Rational Drug Design) and WEKA (Waikato Environment for Knowledge Analysis) software. Two carvacrol and four salicylic acid derivatives, synthesized by conventional methods and evaluated with the larval packet test on larvae of R. (B.) microplus were selected. All evaluated compounds presented acaricidal activity; however, ethyl 2-methoxybenzoate (91.8 ± 1.7 % mortality) and ethyl 2,5-dihydroxybenzoate (89.1 ± 1.6 % mortality) showed greater activity than salicylic acid. With regard to the carvacrol analogues, carvacrol acetate (67.8 ± 2.1 % mortality) and carvacrol methyl ether (71.7 ± 1.6 % mortality) also showed greater activity than carvacrol (35.9 ± 3.2 % mortality). TOMOCOMD-CARDD and WEKA software were helpful tools in the search for alternative structures with potential acaricidal activity on R. (B.) microplus.     

Identification of quorum-sensing-regulated genes of Burkholderia cepacia

Quorum sensing is a regulatory mechanism (operating in response to cell density) which in gram-negative bacteria usually involves the production of N-acyl homoserine lactones (HSL). Quorum sensing in Burkholderia cepacia has been associated with the regulation of expression of extracellular proteins and siderophores and also with the regulation of swarming and biofilm formation. In the present study, several quorum-sensing-controlled gene promoters of B. cepacia ATCC 25416 were identified and characterized. A total of 28 putative gene promoters show CepR-C(8)-HSL-dependent expression, suggesting that quorum sensing in B. cepacia is a global regulatory system.