Repurposing promethazine hydrochloride to inhibit biofilm formation against Burkholderia thailandensis.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25 µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24 h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis.

An Investigation of Quorum Sensing Inhibitors against Bacillus cereus in The Endophytic Fungus Pithomyces sacchari of the Laurencia sp.

Bacillus cereus, a common food-borne pathogen, forms biofilms and generates virulence factors through a quorum sensing (QS) mechanism. In this study, six compounds (dankasterone A, demethylincisterol A3, zinnimidine, cyclo-(L-Val-L-Pro), cyclo-(L-Ile-L-Pro), and cyclo-(L-Leu-L-Pro)) were isolated from the endophytic fungus Pithomyces sacchari of the Laurencia sp. in the South China Sea. Among them, demethylincisterol A3, a sterol derivative, exhibited strong QS inhibitory activity against B. cereus. The QS inhibitory activity of demethylincisterol A3 was evaluated through experiments. The minimum inhibitory concentration (MIC) of demethylincisterol A3 against B. cereus was 6.25 µg/mL. At sub-MIC concentrations, it significantly decreased biofilm formation, hindered mobility, and diminished the production of protease and hemolysin activity. Moreover, RT-qPCR results demonstrated that demethylincisterol A3 markedly inhibited the expression of QS-related genes (plcR and papR) in B. cereus. The exposure to demethylincisterol A3 resulted in the downregulation of genes (comER, tasA, rpoN, sinR, codY, nheA, hblD, and cytK) associated with biofilm formation, mobility, and virulence factors. Hence, demethylincisterol A3 is a potentially effective compound in the pipeline of innovative antimicrobial therapies.

Diketopiperazine Modulates Arabidopsis thaliana Root System Architecture by Promoting Interactions of Auxin Receptor TIR1 and IAA7/17 Proteins.

Plants can detect the quorum sensing (QS) signaling molecules of microorganisms, such as amino acids, fat derivatives and diketopiperazines (DKPs), thus allowing the exchange information to promote plant growth and development. Here, we evaluated the effects of 12 synthesized DKPs on Arabidopsis thaliana roots and studied their underlying mechanisms of action. Results showed that, as QS signal molecules, the DKPs promoted lateral root development and root hair formation in A.thaliana to differing degrees. The DKPs enhanced the polar transport of the plant hormone auxin from the shoot to root and triggered the auxin-responsive protein IAA7/17 to decrease the auxin response factor, leading to the accumulation of auxin at the root tip and accelerated root growth. In addition, the DKPs induced the development of lateral roots and root hair in the A. thaliana root system architecture via interference with auxin receptor transporter inhibitor response protein 1 (TIR1). A series of TIR1 sites that potentially interact with DKPs were also predicted using molecular docking analysis. Mutations of these sites inhibited the phosphorylation of TIR1 after DKP treatment, thereby inhibiting lateral root formation, especially TIR1-1 site. This study identified several DKP signal molecules in the QS system that can promote the expression of auxin response factors ARF7/19 via interactions of TIR1 and IAA7/17 proteins, thus promoting plant growth and development.

Microwave irradiation followed by zinc oxide based dispersive solid-phase extraction coupled with HPLC for simultaneous extraction and determination of flavonoids in Veronicastrum latifolium (Hemsl.) Yamazaki.

In this study, a sample preparation method involving microwave irradiation followed by dispersive solid-phase extraction was used to extract and separate flavonoids from Veronicastrum latifolium (Hemsl.) Yamazaki. Ethyl lactate was selected as the extraction solvent during microwave irradiation. Zinc oxide and ethanol were used as the adsorbents and the eluent, respectively, in dispersive solid-phase extraction. Several experimental parameters affected the extraction performance, such as the extraction solvent and method, the microwave irradiation time, temperature, and solid-to-liquid ratio, the type of adsorbent, the amount of sample solution, the adsorption time and method, and the type and volume of the eluent, and were investigated by single-factor and Box-Behnken design experiments in the pretreatment process. The recommended method was applied to extract flavonoids in Veronicastrum latifolium (Hemsl.) Yamazaki. The enriched flavonoids were analyzed for the first time by high-performance liquid chromatography, and seven flavonoids-vitexin, luteolin, wogonoside, apigenin, chrysoeriol, acacetin, and pectolinarigenin-were identified. Additionally, the method exhibited good linearity (r2 ≥ 0.9992) for flavonoids under the optimum conditions. Moreover, the relative standard deviation of the intraday and interday precision was less than 3.97%. The mean recoveries for flavonoids were more than 98.10%. This work is the first to report a method to extract, separate, and analyze flavonoids from Veronicastrum latifolium (Hemsl.) Yamazaki. Graphical Abstract Microwave irradiation followed by ZnO-based dispersive solid-phase extraction for the pretreatment of Veronicastrum latifolium (Hemsl.) Yamazaki samples, and high-performance liquid chromatography (HPLC) for analysis of enriched flavonoids.

Dopamine, an exogenous quorum sensing signaling molecule or a modulating factor in Pseudomonas aeruginosa?

Pseudomonas aeruginosa is recognized globally as an opportunistic pathogen of considerable concern due to its high virulence and pathogenicity, especially in immunocompromised individuals. While research has identified several endogenous quorum sensing (QS) signaling molecules that enhance the virulence and pathogenicity of P. aeruginosa, investigations on exogenous QS signaling molecules or modulating factors remain limited. This study found that dopamine serves as an exogenous QS signaling molecule or modulating factor of P. aeruginosa PAO1, enhancing the production of virulence factors and biofilms. Compared to the control group, treatment with 40 µM dopamine resulted in a 33.1 % increase in biofilm formation, 68.1 % increase in swimming mobility, 63.1 % increase in swarming mobility, 147.2 % increase in the signaling molecule 3-oxo-C12-HSL, and 50.5 %, 28.5 %, 27.0 %, and 33.2 % increases in the virulence factors alginate, rhamnolipids, protease, and pyocyanin, respectively. This study further explored the mechanism of dopamine regulating the biofilm formation and virulence of P. aeruginosa PAO1 through transcriptome and metabolome. Transcriptomic analysis showed that dopamine promoted the expression of virulence genes psl, alg, lasA, rhlABC, rml, and phz in P. aeruginosa PAO1. Metabolomic analysis revealed changes in the concentrations of tryptophan, pyruvate, ethanolamine, glycine, 3-hydroxybutyric acid, and alizarin. Furthermore, KEGG enrichment analysis of altered genes and metabolites indicated that dopamine enhanced phenylalanine, tyrosine, and tryptophan in P. aeruginosa PAO1. The results of this study will contribute to the development of novel exogenous QS signaling molecules or modulating factors and advance our understanding of the interactions between P. aeruginosa and the host environment.

Tyramine, one quorum sensing inhibitor, reduces pathogenicity and restores tetracycline susceptibility in Burkholderia cenocepacia.

Burkholderia cenocepacia is an opportunistic respiratory pathogen of particular relevance to patients with cystic fibrosis (CF), primarily regulating its biological functions and virulence factors through two quorum sensing (QS) systems (CepI/R and CciI/R). The highly persistent incidence of multidrug resistant Burkholderia cenocepacia poses a global threat to public health. In this study, we investigated the effects of tyramine, one biogenic amine, on the QS systems of Burkholderia cenocepacia. Genetic and biochemical analyses revealed that tyramine inhibited the production of N-hexanoyl-homoserine (AHL) signaling molecules (C8-HSL and C6-HSL) by blocking the CepI/R and CciI/R systems. As a result, the inhibition of QS systems leads to reduced production of various virulence factors, such as biofilm formation, extracellular polysaccharides, lipase, and swarming motility. Notably, as a potential quorum sensing inhibitor, tyramine exhibits low toxicity in vivo in Galleria mellonella larvae and is well characterized by Lipinski's five rules. It also shows high gastrointestinal absorption and the ability to cross the blood-brain barrier according to SwissADME database and ProTox-II server. Additionally, tyramine was found to enhance the efficacy of tetracycline in reducing the infectivity of Burkholderia cenocepacia in Galleria mellonella larvae infection model. Therefore, tyramine could be a promising candidate for combination therapy with traditional antimicrobials to improve their effectiveness against Burkholderia cenocepacia.

2-Hydroxymethyl-1-methyl-5-nitroimidazole, one siderophore inhibitor, occludes quorum sensing in Pseudomonas aeruginosa.

Siderophore is necessary for the survival of microorganisms and is interregulated with quorum sensing (QS) systems. It is related to growth, proliferation, virulence, and other bacterial social activities as a virulence factor. Thus, we speculated that the QS system could be occluded by inhibiting siderophore production. 2-Hydroxymethyl-1-methyl-5-nitroimidazole (HMMN), one siderophore inhibitor of Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1), was obtained by using the Chromeazurol S (CAS) method. We found that HMMN inhibited siderophore production and influenced the biological effects of QS regulation, including biofilm formation and pyocyanin production. HMMN (150 µg/ml) inhibited the siderophore production of P. aeruginosa PAO1 by 69.37%. In addition, HMMN could inhibit pyocyanin production and biofilm formation and erase the formed biofilm of P. aeruginosa PAO1. HMMN (150 µg/ml) inhibited the biofilm formation of P. aeruginosa PAO1 by 28.24%. The erasure rate of the formed biofilm reached 17.03%. Furthermore, HMMN (150 µg/ml) inhibited P. aeruginosa PAO1 pyocyanin production by 36.06%. Meanwhile, positive-control hordenine (500.0 µg/ml) reduced the biofilm formation and pyocyanin production of P. aeruginosa PAO1 by 14.42% and 34.35%, respectively. The erasure rate of hordenine to the formed biofilm is 11.05% at 500 µg/ml. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that HMMN downregulates not only siderophore-related genes but also QS-related genes, as well as hordenine. These results suggest that a siderophore inhibitor could be used as a QS inhibitor to occlude the QS system and reduce virulence.

3-Phenylpropan-1-Amine Enhanced Susceptibility of Serratia marcescens to Ofloxacin by Occluding Quorum Sensing.

Serratia marcescens (S. marcescens) is an environmental bacterium that causes infections with high morbidity and mortality. Notably, infections caused by multidrug-resistant S. marcescens have become a global public health issue. Therefore, the discovery of promising compounds to reduce the virulence of pathogens and restore antibiotic activity against multidrug-resistant bacteria is critical. Quorum sensing (QS) regulates virulence factors and biofilm formation of microorganisms to increase their pathogenicity and is, therefore, an important factor in the formation of multidrug resistance. In this study, we found that 3-phenylpropan-1-amine (3-PPA) inhibited S. marcescens NJ01 biofilm formation and virulence factors, including prodigiosin, protease, lipase, hemolysin, and swimming. The combination of 3-PPA (50.0 µg/mL) and ofloxacin (0.2 µg/mL) enhanced S. marcescens NJ01 sensitivity to ofloxacin. Based on crystalline violet staining, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM), 3-PPA (50.0 µg/mL) reduced S. marcescens NJ01 biofilm formation by 48%. Quantitative real-time PCR (qRT-PCR) showed that 3-PPA regulated the expression of virulence- and biofilm-related genes fimA, fimC, bsmB, pigP, flhC, flhD, and sodB. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that 3-PPA affected intracellular metabolites of S. marcescens NJ01, leading to reduce metabolic activity. These results suggested that 3-PPA inhibits the pathogenicity of S. marcescens NJ01 by occluding QS. Thus, 3-PPA is feasible as an ofloxacin adjuvant to overcome multidrug-resistant S. marcescens and improve the treatment of intractable infections. IMPORTANCE Multidrug-resistant bacteria have become a major threat to global public health, leading to increased morbidity, mortality, and health care costs. Bacterial virulence factors and biofilms, which are regulated by quorum sensing (QS), are the primary causes of multidrug resistance. In this study, 3-PPA reduced virulence factors and eliminated biofilm formation by inhibiting QS in S. marcescens NJ01 bacteria, without affecting bacterial growth, thus restoring sensitivity to ofloxacin. Thus, the discovery of compounds that can restore antibiotic activity against bacteria is a promising strategy to mitigate multidrug resistance in pathogens.

Efficient extraction of flavonoids from Flos Sophorae Immaturus by tailored and sustainable deep eutectic solvent as green extraction media.

In the face of the many shortcomings of conventional organic solvents in the age of green chemistry, deep eutectic solvents (DESs) appear under the spotlight of natural product extraction because of its outstanding advantages. In this study, the extraction of six compounds from Flos Sophorae Immaturus (FSI) with DES-5 (choline chloride/1,4-butanediol) as its topgallant solvent was determined by screening nine DESs. After single factor test and BBD experiment, the optimum conditions of deep eutectic solvent-based microwave-assisted extraction (DES-MAE) were: choline chloride/1,4-butanediol (molar ratio of 1:2) and water content (25%, v/v), time 20 min, microwave power 600 W, temperature 62 ℃, liquid/solid ratio 26 mL/g. The extraction yields of rutin, nicotiflorin, narcissin, quercetin, kaempferol and isorhamnetin were 116.78, 15.01, 23.85, 27.59, 3.09 and 3.33 mg/g, respectively. The kinetic experiment results showed that DES-MAE has significant advantages in the extraction of six compounds. The experimental results showed that DES-MAE could obtain higher yields of target components in a short time than other methods (DES-HRE, DES-UAE and Ethanol-MAE). In addition, the target components were separated from the DES extraction solution, and the recoveries of the target compounds were in the range of 75.5%-84.1%. Therefore, this paper provides a strategy for extraction and separation, the aim of which is to obtain flavonoids from FSI efficiently. Meanwhile, this study can also be used as an alternative to the traditional methods for obtaining bioactive components from plant sources in biochemistry, food industry and pharmaceutical fields.