Chemical Composition and Quorum Sensing Inhibitory Effect of Nepeta curviflora Methanolic Extract against ESBL Pseudomonas aeruginosa

Haitham Qaralleh KOREAN PHARMACOPUNCTURE INSTITUTE 2023 Journal of pharmacopuncture Vol.26 No.4

Objectives: Bacterial biofilm is regarded as a significant threat to the production of safe food and the arise of antibiotic-resistant bacteria. The objective of this investigation is to evaluate the quorum sensing inhibitory effect of Nepeta curviflora methanolic extract. Methods: The effectiveness of the leaves at sub-inhibitory concentrations of 2.5, 1.25, and 0.6 mg/mL on the virulence factors and biofilm formation of P. aeruginosa was evaluated. The effect of N. curviflora methanolic extract on the virulence factors of P. aeruginosa, including pyocyanin, rhamnolipid, protease, and chitinase, was evaluated. Other tests including the crystal violet assay, scanning electron microscopy (SEM), swarming motility, aggregation ability, hydrophobicity and exopolysaccharide production were conducted to assess the effect of the extract on the formation of biofilm. Insight into the mode of antiquorum sensing action was evaluated by examining the effect of the extract on the activity of N-Acyl homoserine lactone (AHL) and the expression of pslA and pelA genes. Results: The results showed a significant attenuation in the production of pyocyanin and rhamnolipid and in the activities of protease and chitinase enzymes at 2.5 and 1.25 mg/mL. In addition, N. curviflora methanolic extract significantly inhibited the formation of P. aeruginosa biofilm by decreasing aggregation, hydrophobicity, and swarming motility as well as the production of exopolysaccharide (EPS). A significant reduction in AHL secretion and pslA gene expression was observed, indicating that the extract inhibited quorum sensing by disrupting the quorum-sensing systems. The quorum-sensing inhibitory effect of N. curviflora extract appears to be attributed to the presence of kaempferol, quercetin, salicylic acid, rutin, and rosmarinic acid, as indicated by LCMS analysis. Conclusion: The results of the present study provide insight into the potential of developing anti-quorum sensing agents using the extract and the identified compounds to treat infections resulting from quorum sensing-mediated bacterial pathogenesis.

Effect of salinity and incubation time of planktonic cells on biofilm formation, motility, exoprotease production, and quorum sensing of <i>Aeromonas hydrophila</i>

Jahid, Iqbal Kabir,Mizan, Md. Furkanur Rahaman,Ha, Angela J.,Ha, Sang-Do Elsevier 2015 FOOD MICROBIOLOGY Vol.49 No.-

<P><B>Abstract</B></P> <P>The aim of this study was to determine the effect of salinity and age of cultures on quorum sensing, exoprotease production, and biofilm formation by <I>Aeromonas hydrophila</I> on stainless steel (SS) and crab shell as substrates. Biofilm formation was assessed at various salinities, from fresh (0%) to saline water (3.0%). For young and old cultures, planktonic cells were grown at 30?°C for 24?h and 96?h, respectively. Biofilm formation was assessed on SS, glass, and crab shell; viable counts were determined in R2A agar for SS and glass, but <I>Aeromonas-</I>selective media was used for crab shell samples to eliminate bacterial contamination. Exoprotease activity was assessed using a Fluoro™ protease assay kit. Quantification of acyl-homoserine lactone (AHL) was performed using the bioreporter strain <I>Chromobacterium violaceum</I> CV026 and the concentration was confirmed using high-performance liquid chromatography (HPLC). The concentration of autoinducer-2 (AI-2) was determined with <I>Vibrio harveyi</I> BB170. The biofilm structure at various salinities (0–3 %) was assessed using field emission electron microscopy (FESEM). Young cultures of <I>A.?hydrophila</I> grown at 0–0.25% salinity showed gradual increasing of biofilm formation on SS, glass and crab shell; swarming and swimming motility; exoproteases production, AHL and AI-2 quorum sensing; while all these phenotypic characters reduced from 0.5 to 3.0% salinity. The FESEM images also showed that from 0 to 0.25% salinity stimulated formation of three-dimensional biofilm structures that also broke through the surface by utilizing the chitin surfaces of crab, while 3% salinity stimulated attachment only for young cultures. However, in marked contrast, salinity (0.1–3%) had no effect on the stimulation of biofilm formation or on phenotypic characters for old cultures. However, all concentrations reduced biofilm formation, motility, protease production and quorum sensing for old culture. Overall, 0–0.25% salinity enhanced biofilm formation and expression of quorum sensing regulatory genes in young cultures, whereas these responses were reduced when salinity was >0.25%. In old cultures, salinity at any concentrations (0.1–3%) induced stress in <I>A.?hydrophila</I>. The present study provides insight into the ecology of <I>A.?hydrophila</I> growing on fish and crustaceans such as shrimp and crabs in estuarine and seawater.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Salinity controls biofilms formation by <I>Aeromonas hydrophila</I>. </LI> <LI> Planktonic age is important for controlling biofilms and quorum sensing. </LI> <LI> Salinity modulates quorum sensing, motility and exoprotease activity. </LI> <LI> Water salinity contributes to biofilms formation on crab shell by <I>A.?hydrophila</I>. </LI> </UL> </P>

In Silico Development of Quorum-Sensing Inhibitors

변재영,심준,류은주,심재현,이환,조광휘,최봉규,이주련 대한화학회 2017 Bulletin of the Korean Chemical Society Vol.38 No.7

Quorum sensing (QS) is a chemical communication between bacteria, with which bacteria sense the population of their own species. Autoinducer-2 (AI-2) is a class of universal quorum-sensing molecules, which is used by both Gram-negative and Gram-positive bacteria. The inhibition of AI-2-mediated QS has various practical applications, including the prevention of the formation of biofilm in dental gums. In this work, we develop a computational protocol for developing AI-2 inhibitors. A challenging aspect of such an endeavor is that the receptor undergoes a large conformational change upon ligand binding. We combine several methods such as molecular docking with multiple conformations, molecular dynamics simulations, and molecular mechanics Poisson–Boltzmann computation, in order to estimate binding affinity of candidate molecules to a quorum-sensing receptor. We apply our method to rank the substances in a chemical library. We indeed find a molecule that has a higher affinity than previously known ligands, thus showing the feasibility of the protocol for the development of quorum-sensing inhibitors.

생물방제균 Pseudomonas fluorescens 2112의 고추 근권정착능과 Quorum-sensing 기능

정병권 ( Byung Kwon Jung ),김요환 ( Yo Hwan Kim ),김상달 ( Sang Dal Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2013 한국미생물·생명공학회지 Vol.41 No.1

다기능 식물생장촉진근권세균(PGPR)인 P. fluorescens 2112 균주가 고추의 생물방제와 성장촉진에 긍정적인 영향을 주기 위해서는 생물막을 형성하여 근권에 정착하는 colonization이 필수조건이다. 따라서 근권정착능에 주요한 생물막 형성에 필요한 quorum sensing의 신호분자인 AHLs의 생산 유무를 조사한 결과, petri dish bioassay에서 AHLs를 생산하여 푸른색환을 형성하는 것을 확인할 수 있었으며 아울러 P. fluorescens 2112 균주의 생장곡선에서 대수증식기 중반에서 정체기 초반에 가장 많은 AHLs를 생산함을 확인하였다. 또한 탄소길이가 6개인 AHLs를 생산한다는 사실을 TLC bioassay를 통해 확인하였다. 그리고 고추의 뿌리 및 근권토양에서의 정착밀도를 Double layer filter paper와 Ahmad와 Baker 법으로 분석하여 확인하였다. 그 결과, 뿌리 상단과 말단에서 각각 3 × 105 CFU/g root와 8 × 103 CFU/g root로 확인되었으며, 근권토양에서 균주는 표면으로부터 가까운 1 cm 깊이에서는 3.5 × 106 CFU/g soil의 높은 밀도로 존재 하였으나, 먼 5 cm 깊이의 근권토양에서는 1.1 × 10 CFU/g soil의 낮은 밀도로 존재하였다. 그리고 주사전자현미경을 통해 고추 뿌리의 표피 및 말단에서 처리한 균주가 생물막 형태의 군집을 형성하는 것을 확인하였다. 결과적으로 P. fluorescens 2112 균주가 AHLs를 생산하여 quorum sensing이 이루어졌으며, 이로 인해 고추의 뿌리에 생물막과 유사한 군집을 형성하여 고밀도로 colonization이 일어났을 것으로 생각된다. 따라서 P. fluorescens 2112 균주의 특징인 다양한 항진균 물질과 auxin을 생산함과 동시에 colonization 을 통해 고추의 생육촉진이나 생물방제에 긍정적인 효과를 줄 수 있을 것이다. Biofilm formation of multifunctional plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens 2112 is necessary for P. fluorescens 2112 to have a positive impact on the rhizosphere of red-pepper. This study investigated whether signal molecules of the quorum sensing AHLs are produced in order to confirm biofilm formative ability. Through the use of Petri dish bioassays a blue circle formed evidence of AHLs. It was confirmed that P. fluorescens 2112 produced six-carbon-chai -long AHLs by TLC bioassay. The bacterial density of P. fluorescens 2112 on the top and bottom of pepper plant roots was estimated as 3 × 105 and 8 × 103 CFU/g root, respectively. P. fluorescens 2112 exist more with high-density of 3.5 × 106 CFU/g soil at a depth of 1 cm but at a low-density of 1.1 × 10 CFU/g soil at a depth of 5 cm, from the surface of rhizosphere soil. In addition, biofilm formation of P. fluorescens 2112 on the epidermises and the tips of the red-pepper roots were confirmed visually by SEM. Thus, the production of AHLs by P. fluorescens 2112 brings about quorum sensing signaling and the formation of biofilm on the roots which has a positive effect on economically important crops such as red-pepper by additionally producing a variety of antifungal substances and auxin.

Inhibition of Quorum Sensing Regulated Virulence Factors and Biofilm Formation by Eucalyptus globulus against Multidrug-Resistant Pseudomonas aeruginosa

Pankaj Kumar Sagar,Poonam Sharma,Rambir Singh 대한약침학회 2022 Journal of pharmacopuncture Vol.25 No.1

Objectives: The quorum-sensing–inhibitory and anti-biofilm activities of the methanol extract of E. globulus leaves were determined against clinically isolated multidrug-resistant Pseudomonas aeruginosa. Methods: The preliminary anti-quorum–sensing (AQS) activity of eucalyptus was investigated against a biosensor strain Chromobacterium violaceum ATCC 12472 (CV12472) by using the agar well diffusion method. The effect of sub-minimum inhibitory concentrations (sub-MICs) of the methanol extract of eucalyptus on different quorum-sensing–regulated virulence factors, such as swarming motility, pyocyanin pigment, exopolysaccharide (EPS), and biofilm formation, against clinical isolates (CIs 2, 3, and 4) and reference PA01 of Pseudomonas aeruginosa were determined using the swarm diameter (mm)-measurement method, chloroform extraction method, phenol (5%)-sulphuric acid (concentrated) method, and the microtiter plate assay respectively, and the inhibition (%) in formation were calculated. Results: The preliminary AQS activity (violacein pigment inhibition) of eucalyptus was confirmed against Chromobacterium violaceum ATCC 12472 (CV12472). The eucalyptus extract also showed concentration-dependent inhibition (%) of swarming motility, pyocyanin pigment, EPS, and biofilm formation in different CIs and PA01 of P. aeruginosa. Conclusion: Our results revealed the effectiveness of the E. globulus extract for the regulation of quorum-sensing–dependent virulence factors and biofilm formation at a reduced dose (sub-MICs) and suggest that E. globulus may be a therapeutic agent for curing and controlling bacterial infection and thereby reducing the possibility of resistance development in pathogenic strains.

Vibrio parahaemolyticus cqsA controls production of quorum sensing signal molecule 3-hydroxyundecan-4-one and regulates colony morphology

Kui Wu,Yangyun Zheng,Qingping Wu,Haiying Chen,Songzhe Fu,Biao Kan,Yongyan Long,Xiansheng Ni,Junling Tu 한국미생물학회 2019 The journal of microbiology Vol.57 No.12

In order to adapt to different environments, Vibrio parahaemolyticus employed a complicated quorum sensing system to orchestrate gene expression and diverse colony morphology patterns. In this study, the function of the putative quorum sensing signal synthase gene cqsA (VPA0711 in V. parahaemolyticus strain RIMD2210633 genome) was investigated. The cloning and expression of V. parahaemolyticus cqsA in Escherichia coli system induced the production of a new quorum sensing signal that was found in its culture supernatant. The signal was purified by high performance liquid chromatography methods and determined to be 3-hydroxyundecan- 4-one by indirect and direct mass spectra assays. The deletion of cqsA in RIMD2210633 changed V. parahaemolyticus colony morphology from the classical ‘fried-egg’ shape (thick and opaque in the center, while thin and translucent in the edge) of the wild-type colony to a ‘pancake’ shape (no significant difference between the centre and the edge) of the cqsAdeleted colony. This morphological change could be restored by complementary experiment with cqsA gene or the signal extract. In addition, the expression of opaR, a well-known quorum sensing regulatory gene, could be up-regulated by cqsA deletion. Our results suggested that V. parahaemolyticus used cqsA to produce 3-hydroxyundecan-4-one signal and thereby regulated colony morphology and other quorum sensing-associated behaviors.

Acyl-Homoserine lactone Quorum Sensing in Bactreria

Greenberg, E.Peter The Microbiological Society of Korea 2000 The journal of microbiology Vol.38 No.3

Recent advances in studies of bacterial gene expression and light microscopy show that cell-to cell communication and communication and community behavior are the rule rather than the exception. One type of cell-cell communication, quorum sensing in Gram-negative bacteria involves acyl-homoserine lactone signals. This type of quorum sension represents a dedicated communication system that enables a given species to sense when it has reached a critical population density. and to respond by activating expression of specific genes. The LuxR and LuxI proteins of Vibrio fisheri are the founding members of the acyl-homoserine lactone quorum sensing signal receptor and signal generator families of proteins. Acyl-homeserine lactone signaling in Pseudomonas aeruginosa is one model for the relationship between quorum sensing community behavior, and virulence. In the P. aeruginosa model. quorum sensing is required for normal biofilm maturation and virulence. There are multiple quorum-sensing circuits that control the expression of dozens of specific genes in P. aeruginosa.

A common evolutionary pathway for maintaining quorum sensing in Pseudomonas aeruginosa

Bai-min Lai,Hui-cong Yan,Mei-zhen Wang,Na Li,Dongsheng Shen 한국미생물학회 2018 The journal of microbiology Vol.56 No.2

In the bacterium Pseudomonas aeruginosa, the synthesis and secretion of extracellular protease is a typical cooperative behavior regulated by quorum sensing. However, this type of cooperative behavior is easily exploited by other individuals who do not synthesize public goods, which is known as the “tragedy of the commons”. Here P. aeruginosa was inoculated into casein media with different nitrogen salts added. In casein broth, protease (a type of public good) is necessary for bacterial growth. After 30 days of sequential transfer, some groups propagated stably and avoided “tragedy of the commons”. The evolved cooperators who continued to synthesize protease were isolated from these stable groups. By comparing the characteristics of quorum sensing in these cooperators, an identical evolutionary pattern was found. A variety of cooperative behaviors regulated by quorum sensing, such as the synthesis and secretion of protease and signals, were significantly reduced during the process of evolution. Such reductions improved the efficiency of cooperation, helping to prevent cheating. In addition, the production of pyocyanin, which is regulated by the RhlIR system, increased during the process of evolution, possibly due to its role in stabilizing the cooperation. This study contributes towards our understanding of the evolution of quorum sensing of P. aeruginosa.

The Effects of Betonicine, Floridoside, and Isethionic Acid from the Red Alga Ahnfeltiopsis flabelliformis on Quorum-Sensing Activity

Hai Bo Liu,고경표,김정선,서영완,박성훈 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.4

A mixture of three compounds (betonicine, floridoside, and isethionic acid) purified from the red alga Ahnfeltiopsis flabelliformis, was reported to have an inhibition activity on bacterial quorum sensing mechanism mediated by N-octanoyl-DL-homoserine lactone (OHL) and the TraR transcriptional activator protein. We subsequently conducted a more detailed investigation of the three compounds using chemically synthesized (betonicine and floridoside) or commercially available (isethionic acid) compounds, individually or in various combinations. When tested alone, none of the three compounds inhibited OHL, but a mixture of floridoside and isethionic acid exhibited a dose-dependent inhibitory effect on the function of OHL. In contrast, betonicine or its isomer exhibited a dose-dependent stimulatory effect, similar to OHL, at concentrations between 10-³and 10-6M. These three compounds will be useful for elucidating the interaction between OHL and TraR, and for developing novel bacterial quorum-sensing inhibitors.

Quorum sensing 결핍 세균에서 생물막 형성의 시간적 추이 분석

김수경,이미난,이준희,Kim, Soo-Kyoung,Lee, Mi-Nan,Lee, Joon-Hee 한국미생물학회 2014 미생물학회지 Vol.50 No.2

녹농균(Pseudomonas aeruginosa)과 비브리오 불니피쿠스균(Vibrio vulnificus)은 그람 음성의 병원균들로써, quorum sensing(QS) 기전을 통해 병원성을 발현하는 세균들이다. 이들 병원균의 감염은 많은 경우 생물막 형성에 의해 매개된다고 알려져 있는데, 이에 본 연구에서는 P. aeruginosa와 V. vulnificus를 대상으로 QS 기전의 유무에 따른 생물막 형성의 시간적 추이를 분석해 보았다. 그 결과 P. aeruginosa의 경우 QS 기전이 결핍된 균주가 야생형에 비해 초기 부착은 더 잘 하였으나, 이후 생물막 구조의 성숙 능력은 야생형에 비해 현저히 떨어짐을 알 수 있었다. 이러한 특성 때문에 야생형과 QS 결핍 균주의 생물막 형성을 시간의 추이에 따라 정량적으로 비교해 보면 초기 10시간 정도 까지는 QS 결핍 균주가 더 많은 생물막을 형성하다가, 이후 야생형이 더 많이 생물막을 형성하는 역전 현상이 관찰되었다. V. vulnificus는 P. aeruginosa와는 달리 QS 결핍 균주가 야생형보다 더 많은 생물막을 형성한다고 보고된 균주이다. 이 균주에서 같은 방식으로 생물막 형성을 조사해 본 결과, 108시간의 장시간 동안에도 항상 QS 결핍 균주가 야생형 보다 더 많은 생물막을 형성하여, 역전 현상은 관찰되지 않았다. 이 결과는 P. aeruginosa의 경우에는 QS 기전이 초기 부착은 저해하는 방향으로, 성숙과정은 촉진시키는 방향으로 작용하며, V. vulnificus에서는 일관되게 생물막 형성을 저해하는 방향으로 작용함을 보여주는 것이다. 따라서 생물막 제어를 위한 타겟으로 QS기전을 이용할 때에는 제어하고자 하는 생물막 형성 단계와 세균 종을 함께 고려하여야 한다고 제안한다. Pseudomonas aeruginosa and Vibrio vulnificus are Gram-negative human pathogens, which exert their virulence through quorum sensing (QS) regulation. The infection of these pathogens have been known to be mediated by biofilm formation in many cases and this study carried out the time-course analysis of biofilm formation depending on the QS regulation in P. aeruginosa and V. vulnificus. In P. aeruginosa, our results demonstrated that QS-deficient mutant better attached to surface at initial stage of biofilm formation, but poorly proceeded to the maturation of the biofilm structure, while wild type less attached at initial stage but developed highly structured biofilm at late stage. Because of this, the quantitative comparison of biofilm formation between wild type and the QS mutant showed the reversion; the QS mutant formed more biofilm until 10 h after inoculation than wild type, but wild type formed much more biofilm after 10 h than QS mutant. V. vulnificus has been reported to form more biofilm with the mutation on QS system. When we performed the same time-course analysis of the V. vulnificus biofilm formation, the reversion was not detected even with prolonged culture for 108 h and the QS mutant always forms more biofilm than wild type. These results indicate that the QS regulation negatively affects the attachment at early stage but positively facilitates the biofilm maturation at late stage in P. aeruginosa, while the QS regulation has a negative effect on the biofilm formation throughout the biofilm development in V. vulnificus. Based on our results, we suggest that the developmental stage of biofilm and bacterial species should be considered when the QS system is targeted for biofilm control.