Quorum Sensing - Science topic

Hello Lim Wei Gene,

There are a few other ways to show the anti-QS effect of a molecule. The CV strain makes use of short-chain AHLs (C4- and C6 AHLs) for QS. You can also try using the Agrobacterium tumefaciens NTL4 or NT1 reporter strain which detects the presence of long-chain AHLs (C8- and C-12 AHLs). The latter contains a plasmid that induces the expression of the B-Gal gene (due to the presence of AHL molecules) resulting in the hydrolysis of X-Gal on a solid medium, producing light-blue colored colonies or bacterial growth. While in the absence of this enzyme (no QS as a result of no or low concentration of AHL molecules), the colonies appear white in color. You can also quantify this activity by extracting AHLs from your culture, then inducing NTL4 cells with the isolated AHLs, and performing a B-galactosidase assay to determine the activity of B-gal produced as a result of induction by the AHL molecules. Thus, you can detect QS by the virtue of AHL production through the B-gal activity.

For more reference, you can read these 2 articles published by my colleagues Dr. Sunil Kumar Bose and Dr. Karuna Sharma.

Terpinen-4-ol attenuates quorum sensing regulated virulence factors and biofilm formation in Pseudomonas aeruginosa.

Sustained release of Zingerone from polymeric nanoparticles: An anti-virulence strategy against Pseudomonas aeruginosa.

Good luck!

Detection of Quorum-Sensing Molecules for Pathogenic Molecul...

If you are going to screen the plant bioactive for quorum sensing activities means, go with Gram-negative bacterial pathogens.

The Gram-negative bacterial pathogens are ideal for quorum sensing inhibition studies. Because the Gram-negative bacterial pathogens regulates their virulence factors production through signal molecules called acyl homoserine lactone (AHL). Generally, these AHL molecules go and bind with the receptor protein and these complex triggers virulence gene expression. The chemical compounds (including plant bioactive) may act as the analogous for the receptor protein. Therefore, it competitively binds with the AHL and prevents the formation of AHL-receptor protein complex. This leads to prevent the virulence gene expression. So, the possibility is many of the plant or natural compounds can act as the analogous for the receptor protein of Gram-negative bacteria.

Further, the QS system in Gram-negative bacterial pathogens such as Pseudomonas aeruginosa, Serratia marcescens, Acinetobacter baumannii, Vibrio harveyi, Aeromonas hydrophila etc were studied well.

But, the Gram-positive bacterial pathogens were mainly reported for their biofilm formation. More, only QS contributed meagre in virulence factors production in Gram-positive bacterial pathogens. The Gram-positive bacterial pathogens are most apt for biofilm inhibition studied and not for QS inhibition. Furthermore, the QS system in Gram-positive bacterial pathogens is totally different from Gram-negative bacterial pathogens.

More, the biofilm is the important virulence factors for bacterial pathogens. The biofilm formation in many Gram-negative bacterial pathogens are also comes under QS system. In contrast, the biofilm formations in Gram-positive bacterial pathogens are mostly not related to QS system. Only some Gram-positive bacterial pathogens are controlling their biofilm under QS.

So, my opinion is go with Gram-negative bacterial pathogens especially Acinetobacter baumannii. Because, now a days these pathogens are emerge as the global threat. The WHO categorized these bacterial pathogens as the critical pathogen, means need to find effective drug to prevent their infections. If you like to screen the plant bioactive for mainly biofilm inhibition means go with Gram-positive bacterial pathogens.

Also, my another opinion is mostly don’t go with the Chromobacterium violaceum for screening the plant bioactives. Because, after scrutinize the compounds, you need to go with the other virulent Gram-negative bacterial pathogens for assessing the activity (Note that the C. violaceum is not pathogen, it is only a marker strain and soil bacterium). The scrutinized compounds mostly will not work against another virulent Gram-negative bacterial pathogens. Because, the AHL signal molecules are vary in every Gram-negative bacterial pathogens. So, the scrutinized compounds by the C. violaceum may not work with particular Gram-negative bacterial pathogens. Therefore, my suggestion is if you have limited plant extracts or bioactive compounds means go directly with the screening of the compounds or extracts for their biofilm inhibition against your selected Gram-negative bacterial pathogens. It will be the effective way and give the ideal QS inhibitors against particular Gram-negative bacterial pathogens.

Thank You.

Interesting question ! But I don't think they quorum sense because there is no auto inducer like molecule or peptide found till date in their genome.

But Silpe, J. E. & Bassler, B. L. 2019 and Xiaolong et al., 2020 found that there is link between quorum sensing signals and viral growth which means secondary infection in viral infections would lethal especially in case of COVID.

DOI:https://doi.org/10.1016/j.cell.2018.10.059

best wishes

VK

Bacteria communicate with one another using chemical signal molecules. ... This process, termed quorum sensing, allows bacteria to monitor the environment for other bacteria and to alter behavior on a population-wide scale in response to changes in the number and/or species present in a community.

That is because quorum-sensing is threshold-dependant: every cell produce an amount of molecules that cannot trigger it inside the same aforementioned cell. So, basically, if a lot of bacteria produce those molecules that means also that their intake will exceed the threshold value and cause the phenomenon to be activated. It is a fine and logic way for bacteria to communicate the size of the microbial population. Hope that helped

Quorum sensing is mediated by QS-autoinducers (AI). Most of them are known. Just take AI of various types (I-IV) and work with them, trying to induce resistance. However, it doesn't work that way, I think. Often, direct damaging contact of the antibiotic`s molecule with the molecular target is necessary for resistance development. QS is likely to be involved indirectly. For example, "some hydrophobic QS-autoinducers such as PQS are trafficked between cells via membrane vesicles. In this case, the peptide’s (antimicrobial peptides) membrane-permeabilizing action releases accumulated PQS molecules, which can trigger the expression of the virulence genes associated with quorum sensing" (doi:10.3389/fmicb.2019.01160)

Nevertheless. you can study one of the existing editions of QS: Methods and Protocols, available at https://link.springer.com/book/10.1007%2F978-1-4939-7309-5

QS systems are strain dependent. Sometimes, but not always, this fact is related to the acquisition of the QS genes through horizontal transfer.

It has to be a screen that does not take energy from the particles being diffracted. If it does take any energy then the pattern is lost. In that case it is also possible to tell which slit a particle went through (because you can in principle detect where the energy appears). When the screen is massive enough then no energy exchange occurs - like an elastic ball bouncing from the earth.

Hello Ashish,

We are using pure grade methanol for storing wide range of AHLs in our LAB, at -20 °C.

Best,

S.S.A. SHAH

Bioassay using Vibrio harveyi BB strain may be less reproducible depending on the sample and microbial state. Therefore, it is recommended to proceed simultaneously with bioassay and other analytic methods (e.g., LC-FID or LC-UV) to detect AI-2 or DPD.

QS is cell-to-cell communication protocol by which bacteria respond to its local environment, i.e., pathogenic activation, resistance to antibiotics etc. QS carried out when signal molecule like acyl homoserine lactone (AHL) binds to LasR protein of the QS system. Enzyme mediated disruption of this process is called QQ where enzyme inhibits signal molecules through variety of process. For example, lactonase inactivate the process by lysing lactone ring of AHL molecule while acylase cut off its acylase chain.

(1) Nobody has solved the structure yet or (2) somebody has solved the structure but has not made it available to the public.

Enzymes like lactonases, acylases which target the AHLs, various oxidoreductases, use of antibiotics, synthetic molecules that sequester the signal can be used. (Interference With Quorum-Sensing Signal Biosynthesis as a Promising Therapeutic Strategy Against Multidrug-Resistant Pathogens by Martinez et al., 2019)

Also some methanol extracts of roots of Albiza schimperiana (ASRM), Solanum torvum and petroleum ether extract of seed of Justica schimperiana (JSSP) and many more have been used and good results have been seen.

Dear Miguel,

Thank you very much for you kind, valuable suggestion. However, to the best of my knowledge we can use your method of evaluation for Gram-negative bacteria not for Gram-positives' particularly S. aureus. Thanks

We have Chromobacterium violaceum isolated from Tripura soil.

Recently, violacein biosynthesis from C. violaceum in the presence of antibiotics was studied ( Liu et al., 2013 ). Violacein production was enhanced by 25%

using sub-inhibitory concentrations of amikacin (1/4 MIC), erythromy-

cin (1/8 MIC), gentamicin (1/2 MIC), kanamycin (1/6 MIC) and tetracy-

cline (1/16 MIC) without bacterial growth inhibition.

.

PubChem

URL: https://pubchem.ncbi.nlm.nih.gov

Data deposited in or computed by PubChem

Added file.

I suggest you to read the following

Autoinducer Bioassay Details of the method is described in this paper

I think the best method is HPLC-MS-MS. I tried to quantify C4 and C12-HSL of Pseudomonas aeruginosa and it worked w

Recent and extraordinary advances in our understanding of the genetics, genomics, biochemistry, and signal diversity of QS is helping researchers to understand the connections between QS and bacterial sociality; a foundation that places us at the beginning of a new era in which researchers will be able to work towards new medicines to treat devastating infectious diseases, and use bacteria to understand the biology of sociality.

Bassler and colleagues have found that bacteria lacking RhlI, which are therefore unable to synthesize the C4-HSL autoinducer, still secrete a molecule capable of activating RhlR. The researchers do not yet know what this molecule is, but it seems to be quite different from C4-HSL. “We are currently working to purify and identify this molecule". https://blogs.princeton.edu/research/2017/08/21/researchers-find-an-alternative-mode-of-bacterial-quorum-sensing/. Most importantly see these articles. They are phenomenal.

This paper discuss the problem in designing the universal primer of LuxI.

Hello, Quorum sensing of bacteria is the regulation of gene expression in response to fluctuations in cell-population density. Quorum sensing bacteria produce and release chemical signal molecules called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. But each of cell is individual organism.

Eukaryotic gene expression is more complex than prokaryotic gene expression because the processes of transcription and translation are physically separated. Unlike prokaryotic cells, eukaryotic cells can regulate gene expression at many different levels. Epigenetic changes are inheritable changes in gene expression that do not result from changes in the DNA sequence. Eukaryotic gene expression begins with control of access to the DNA.

Dear Hanna Simpson,

Yes, LC-MS is the most effective method as chemical analysis in your possibly study.

I am in consensus with Anthony Ayodeji Adegoke. However, in my experience studies with CV is not enough to conform quorum sensing inhibitory property. In our lab we have identified compounds with significant anti-QS property who exhibited no reduction in violacein production and vice versa.

Dear Elisa,

It is always difficult to go from growth experiments to the identification of an active compound, especially if your model organism is hard and slow to grow, you lack a fast and simple assay for the activity, and if you are not based in a biochemical lab. However, there are some simple experiments that you can do to narrow down the nature of your active compound.

I would suggest that you try some simple growth addition experiments to demonstrate that the effect is real, and that cells are responding to this compound in a dosage-dependent manner. The easiest experiment is to add some filter-sterilised (FS) culture supernatant from cultures in which you see cell death or growth-inhibition to a fresh culture - you would expect to be able to inhibit the growth of this or see cell death occurring earlier than if you did not add FS culture supernatant.

If this works, then there are some simple tests you can do : test FS culture supernatants from cultures grown in different media, grown for different lengths of time, and grown to different cell densities. Try fractionating your FS culture supernatant using dialysis membrane or filters (with different pore sizes) to see if you can put a size limit on the active compound; try heat inactivating the compound, and precipitating or extracting it with alcohol, phenol, ammonium sulphate, etc.

The aim of all of this is to produce a semi-purified concentrated extract that you could then consider fractionating by HPLC and then identifying the compound by mass spectroscopy (if you had access tho this type of analytical equipment). Being able to demonstrate that you have a semi-purified extract plus a simple phenotype you can assay activity with would be a good end-point for a project where you could not do any further biochemical analyses.

Regards, Andrew.

In addition to AHL, AI-2, and DSF , the following database lists DKPs (Diketopiperazines) and HAQs (4-hydroxy-2-alkylquinolines)(, and "others") as categories of signaling molecules. You can search the database by organism, too.

I also find layer of bubbles as stated by Uma. Can anyone please tell if it is fine to spool out that layer before subjecting the entire solvent extract for drying in a rotary evaporator?

Dear Zaduryan

Please, find in the attached file a review article titled "Messing with Bacterial Quorum Sensing".

I think and hope it's benefit for you. Regard

Even, ıt is not an answer. please look my reference

Dear Artur Zaduryan,

Kindly purify the A. tumefaciens again by simple streak plate method, pickup single colony, grow overnight in LB with antibiotics and try the biosensor assay once again.

I have sent the TLC procedure in detail by personal email.

Hope this will help.

Best wishes,

HARSHAD

It's better if you use the conc. within 0.5 to 1 μg/ml.

Thank you dear professor for your interesting response. If you have articles concerning the subject, please propose me.

Hi Rok,

The answer is 'yes' and 'no': take a look at the review of Pereira et al. (2013) https://academic.oup.com/femsre/article/37/2/156/623107/AI-2-mediated-signalling-in-bacteria.

Authors claim that "although the signal synthase, corresponding biosynthetic pathway and chemical products are the same in every AI-2-producing bacterium tested thus far, these studies demonstrate that the molecule ultimately detected by these bacteria can differ". There are a few intermediates and their derivatives which apparently also bind to receptors, thus opening the possiblity that other (closely related) molecules could serve as signals. There might be some update on this in the more recent literature, but this is not really my narrow field, so I hope you will get more hints on this.

Best regards,

Marko

The enzyme database BRENDA (http://www.brenda-enzymes.info) would have that type of info if it is available at all.

PubMed: "efflux"[title/abstract] and "quorum sensing"[title/abstract] 

Since your question is still open, you may want to add some details on the specific AHLs (HSLs) you are interested. The HSL classes involve quite a number these days and only the P. aeruginosa system is well characterized. I would not rule out your request for “primers” and I am again guessing you meant routine PCR, as valid. Indeed PCR may be used for HSL determination but one would usually employ qPCR be more definitive. For example in the P. aeruginosa if you run a qPCR for LasI and LasR (using a baseline gene off course), you will derive a highly sensitive information of the quantitative expression of the HSL, N-3-oxo dodecanoyl-L-homoserinelactone. The primers and PCR conditions are available online, but we will be happy to help.

Therefore, PCR may very well be used for studying QS expression but restricted to specific related genes. However if one would need a truly unbiased, global approach for HSL detection (please note nearly all HSL reporter strains and sensitive to specific classes and not ALL HSLs), one would choose LCMS (Cataldi et al. 2008) or LDI-MS (Ghosh et al. 2012) . If you have either of these technologies available, you can detect nano-molar levels of a-n-y HSL of choice, irrespective of their classes or bacteria . Hope this helps!

Suggested reference:

1. Cataldi, T. R., et al. (2008). "Profiling of N‐acyl‐homoserine lactones by liquid chromatography coupled with electrospray ionization and a hybrid quadrupole linear ion‐trap and Fourier‐transform ion‐cyclotron‐resonance mass spectrometry (LC‐ESI‐LTQ‐FTICR‐MS)." Journal of mass spectrometry 43(1): 82-96.

2. Ghosh, D., et al. (2012). Selective Detection and Analysis of Small Molecules, Google Patents. EP2676287B1

Thank you Dr. Aamal.

I discuss about other things. EDF is a quorum sensing signals that starting programmed cell death in bacteria and it isn't extra cell toxin. EDF in some way caused unstable toxin-antitoxin complex in bacterial cell.   

Dear Shamas,

I do not know the reason you need so much concentration but I agree with Uelinton about the range of nM to microM is enought to activate the phenotypes depending on QS molecules. 

As an example you can see the natural range of AHL production by the pathogen bacteria Edwarsiella tarda.

You can use HPLC methodology to detect and quantify AHLs quorum sensing molecules in your samples.

Unfortunately, (Nonomura, H. (1974), is not available on the net. However, for identification of Streptomyces spp. , Shirling and D. Gottlieb, (attached below), would be so helpful, and for identification of other actinomycetes, the following attached article may be helpful: 

Hi Ewelina,

Firstly you have to identify the homologs of known receptor present in all the susceptible strains. You can address this by identify the known receptor in all strains using protein-BLAST using non-reduntant database.

If these protein sequences are present in other strains having simiarity higher than 40%. Then there is strong possibility that the bacteriocin will act against other strains as well.

To further confirm the possibility of interaction of bacteriocin with other strain proteins, you have to perform Multiple sequence alignment (MSA) of known receptor in all strains. If the binding site residues of bacteriocin which may possible interact with your known receptor (through X-RAY, NMR or docking studies) you can look for similar residues in other homologs. If they are conserved (indicated by Astrix or dot sign in MSA), then most possibly the inhibition will occur in other strains.

@Sergey Dobretsov, Sir I am using Marin Agar on which both A136 and my test strains grows well but not responding to X-Gal even to positive control. sir my protocols are as follow

1. Culturing of Reporter strains using Marine Agar.

2. Culturing of Positive and test strains > incubating them for 24/48 hours > culturing them with repoter strain A136 in T manner> incubatin them for 24/48 Hours at 28 °C > adding X-Gal > Cheking for QS activity after 12 hours then after 24 hours then after 36 hours then after 48 hours.

Sir is there any thing wrong in this protocol? if yes kindly guied me.

Respectful Regards,

Thank you very much Pendru Raghunath and Gamal Enan for the kind information.

Is this associated with the phenomena of  SCV? 

My opinion is that it is part of Population diversity/heterogeneity you should look for Phenotypic Switching and Small Colony Variants in those showing different exponential phases.  Generation of microbial heterogeneity in response to stress. Also did you consider the age  of the parent inocula? 

Synergy is possible when the desired biological activity or pharmacological activity has several sites of action or targets.If the used compounds act on different targets , similar activity can b observed

Have you checked the KEGG Compound database? 

There you can search for a compound and then select it to see their associated reactions and pathways. 

I hope that helps.

The vast majority of gram negative Quorum sensing systems that have been studied thus far utilize N- AHLs as signaling molecules. When in high enough concentrations, these molecules can bind to and activate a transcriptional activator, or R protein, which in turn induces expression of target genes. The use of biosensors to screen spent culture supernatants has led to the discovery that AHLs are produced by a plethora of unrelated bacteria. Biosensors typically consist of a QSC promoter fused to a reporter such as lac Z or the lux operon

In gram positives, Quorum sensing systems typically make use of small post-translationally processed peptide signal molecules. These peptide signals interact with the sensor element of a histidine kinase two-component signal transduction system

Methods have been developed for determination of the AHLs using TLC  Separations were usually performed on fused silica capillary columns 20 m long with hexane as mobile phase and with temperature gradients from 100 to 300 °C. Sometimes, however, sensitivity was lower than expected. There could be several reasons for this, for example masking effects by matrix constituents, e.g. diketopiperazines, the polar nature of the AHLs.

@Adam B Shapiro: Dear the Compound is Beta-amyrin and is produced in some plants. But their is no known Transporter responsible to efflux it from the Cell. 

Some other Suggestion please because we already check the related Transporter but no reported todate.

Regards

I agree with all answers above ...good luck.

 Thank you so much.

 Dear Alice,

You have made your microbe very very specific with the strain number 'S178S (JF513150)'. I googled the name of the microbe Pseudomonas mendocina and found some literature (old as well as current) on it. Since the species (mendocina) of the genus (Pseudomonas) is studied and reported in those literature you can have some clear idea on growth conditions if you compare the conditions used in different literature. In my opinion you can easily check it is working with your specific strain or not. For habitat and pathogenesis, exceptions and variations will always be there though. The competitive behaviour can only be assessed if it is grown with another microbe with similar nutrient requirement. I am listing the links of the literature I found for you, but think you will find more of them if you search further (in Sciencedirect, Google Scholar etc.).

1. https://microbewiki.kenyon.edu/index.php/Pseudomonas_mendocina

2. http://jcm.asm.org/content/30/6/1583.full.pdf

3. http://onlinelibrary.wiley.com/doi/10.1046/j.1198-743x.2001.00331.x/full

4. J Clin Case Rep 5:598. doi:10.4172/2165-7920.1000598

5. doi: 10.1097/IPC.0000000000000394

6. jmedicalcasereports.biomedcentral.com/articles/10.1186/s13256-016-1057-6

Hope these will be helpful to you.

Regards,

Shiladitya

Hello Vivek.

My partner worked with the same AHL, and instead of glacial acetic acid, we used 0,05% formic acid with ethyl acetate to prepare our stocks of all AHLs that we use. We had the same situation (white precipitation). Once we prepare the stocks, we put at -20°C, then before of dissolving with the broth, we just resuspend  in a vortex. We didn't notice a precipitation in our test, probably because the volume we used was minimal. Still it would be great to know if there is a properly way to dissolve this particular AHL since it is hydrophobic.

I found this, hope it helps...

We find that AHLs are best maintained as stock solutions in

ethyl acetate acidified with glacial acetic acid (1 ml of acetic

acid per 1 L of ethyl acetate). We store them at −20°C in glass

vials. If borosilicate vials are used for this purpose, prior to

adding AHL solutions, glass vials are first treated with 0.2 N

HCl, and then rinsed extensively with deionized water.

Teflon-lined caps should be used for capping vials containing

organic solutions of AHLs. As needed for experiments, serial

dilution of 3-oxo-C12-HSL can be prepared in other organic

solvents (including ethanol, methanol and acetonitrile), however

stability of the compound in these solutions is not known.

3-oxo-C12-HSL is not stable in aqueous solutions where it

converts into tetramic acid, an antibiotic (22). At neutral or

basic pH, AHLs undergo spontaneous lactonolysis (23).

From Chapter 9 "Luminescent Reporters and Their Applications for the Characterization of Signals and Signal-Mimics that Alter LasR-Mediated Quorum Sensing" - Quorum Sensing: Methods and Protocols

Best regards.

In my opinion that much high amount of compound to determine MIC is not worth for drug discovery related process. Because there are limitation to achieve these concentrations inside the invivo systems. Nowadays people are looking for nanomolar activities with biologicaly safe molecules, So I suggest you to look for active molecule having fractional MIC in micrograms. 

Already established that pseudomonas nearly always mutates into a mucoid strain. The alginate-containing matrix of the mucoid strain is thought to allow the formation of protected microcolonies and provide increased resistance to opsonization, phagocytosis, and destruction by antibiotics. Its mutation which increase the adaptation capability of strain. It can be virulent pulmonary pathogen causing cystic fibrosis, be careful !

 Bacteria are social organisms capable of interacting with

each other and their surroundings. Particularly well

described is the ability to coordinate gene expression in

accordance with population density, a process termed

quorum sensing (QS) (Fuqua et al., 1994)

Read 1. Quorum sensing inhibitors: a bargain of effects article review,

2. Advances in Applied Microbiology, Volume 33

Hi Dear

pls. see attached manuscript that may help you 

I just found your posting by chance today. Although your question is quite long ago, I would like to answer it as no one did this before. You might simply prepare stocks with much higher AHL concentrations. What is your currently used concentration? In my paper I wrote: "Stock solutions of C6-HSL and C8-HSL (400 mM) were prepared in anhydrous dimethyl sulfoxide (DMSO) containing 0.2% glacial acetic acid and stored in aliquots at -20°C." Even when I used 2 mM AHL for biocatalysis, the DMSO concentration was negligible. For long-chain AHLs you might have to use lower stock concentrations. The activity assay I published might be an alternative to the OPA method if it is not working properly. Good luck for your research!

Extract the carotenoids, analyse by TLC; or HPLC using RP C18 column. Best...

Dear Ahmed

I have CV026 and NT1 biosensors, but according the regulation of post in Iran it is impossible to post the bacteria. do you have any suggestion for it?!

I want to make joined work. I can make some of the work in my lab. and you can complete the experiments I can not make due to lack of equipments for example or some standard strains and we can publish together. 

Hi there,

Gelatinase which participates degradation of collagen is involved in the ECM degradation process in order to generate peptides to be uptaken by the bacteria for  supplying substrates for cell metabolism. It is one of the essential activities for bacterial survival during infection.

Dear Cat,

With regard to biofilm infection I have a paper which might be of interest for you. See attachment.

Kind regards

Stig Jeansson

Attached: Bacteria, biofilm and honey_a study of the effects of honey on 'planktonic' and biofilm-embedded chronic wound bacteria 

I

For $185, you can purchase from ATCC (kind of a steep price, if you wanted my opinion on the matter): 

An alternative suggestion is to find a source of C. violaceum that is not an ATCC strain. Academic groups often will distribute these freely.

Pyocyanin, one of the many virulent factors produced by Pseudomonas aeruginosa, is a zwitter ion whose activity in in vitro studies suggest it interferes with multiple cellular functions. But few cellular pathways affected by pyocyanin activity are known. However, high concentrations of pyocyanin have been deemed crucial for most related infections. So it's possible that the crude plant extract with increasing concentration interfered with QS interactions, as a result lowering the virulent factor (pyocyanin).   

The ideal situation would be to run on same plate at same time.  It is possible for different lots and plates in same lot to have slight differences that may affect Rf values.  If you have confidence in standard and the match is valid, then it should be okay .  I am a forensic scientist so I have a bit more sensitivity to slight variations than a research chemist, but you should still show that the match is valid each time you open a new box of plates.  An easy way to do this is once you have a pure substance as discussed above such as shown by GC, LC, MS, etc, save a portion of that material as secondary standard to use with TLC.  It will also show if degradations  occur under your conditions.

The term antibacterial can apply to both bacteriocidal or bacteriostatic agents. So the reports that say the QS agents are antibacterial may have just not defined whether they are bacteriostatic or bacteriocidal.

Some QS inhibitors are bacteriocidal at higher concentrations, while quorum sensing inhibitory at lower concentrations. Given the nature of quorum sensing, I would also have thought that QS inhibitors (when acting as inhibitors, not bacteriocides) would be bacteriostatic. However, if the process that is regulated by QS is essential to the survival of the bacteria, then inhibition of this process would lead to mortality. However, this would mean that the bacteria would not be able to survive in sparse populations, which seems like something that would be selected out pretty quickly. Unless of course this was an inter species interaction that enhances competitiveness of one species (the QS inhibitor producer) over another (the species that needs the QS process for survival).

Hi

Most of essential oils have photodynamic activity, with a simply assay diffusion disc, exposing the plate to different lights you'll find interesting differences.

best regards

Dear @Sidartha, I find this article is very relevant for your issue.

"Dr. Louis Rosenberg, CEO of Unanimous AI, is building a software platform, UNU, that assembles groups to make collective decisions. "What's different about this is that it fundamentally keeps people in there," he said. "We're focused on using software to amplify human intelligence."...

UNU is an online platform where anyone can log in and answer questions as a swarm. "People benefit when they form a real-time system the way bees, fish, and birds do, as opposed to people just casting a single vote the way people do," said Rosenberg..."

The good thing about ethyl acetate is that you can add it to your test tube and wait for it to evaporate, leaving only the AHL behind. However, the same principle applies when storing your working solution. Ethyl acetate will evaporate even in freezer at -18C. So, after a while, the concentration of AHL will change.

That does not happen when using acetonitrile. So, say you stock it in acetonitrile, at high concentration, you can then dissolve it in sterile distilled water (or your growth medium) at a more suitable concentration before using it in your media. 

However care must be taken in order to avoid growth inhibition due to acetonitrile. So, when adding it to your media, make sure it is only a tiny fraction (10 to 100 microL of AI in acetonitrile to 100 mL of media).

You may consider using a zymography analysis with the introduction of specific substrates (gelatin, for example) into gel. Use conditioned media that contains secreted products for the analysis. This will allow you to identify conditions (quorum sensing, inducing factors) that promote the expression of proteolytic phenotype in P. aeruginosa. The sequence analysis of the eluted protein will help you to identify the protease that is inhibited by the plant extract. Specifically to elastase, you can use its capacity to degrade collagen. Using commercially available fluorescein-labeled collagens, you can perform high throughput screening analysis in 96 well plates (see Shogan et al., STM 2015, Collagen degradation and MMP9 activation by Enterococcus faecalis contribute to intestinal anastomotic leak) for the analysis of your plant extracts