- Pushpa Philem asked a question:OpenIs there any way I can improve AHL stock preparation for quorum quenching OPA based enzyme assay?
I am working on quorum quenching enzyme assay. My AHL stock is prepared in DMSO. In CV026 bioassay, my enzyme shows quorum quenching activity. For kinetic study, we are performing OPA based assay for detection of HSL. The read out of the OPA assay is very less. As the substrate concentration increases,the DMSO % goes upto 6-7 % in the final reaction mixture of the enzyme and the AHL (in DMSO). Is there any other way to prepare a stock solution with less DMSO without solubility issue ? Any advice on improving the OPA assay is requested.Following
- Vinothkannan Ravichandran added an answer:21Does anyone have Chromobacterium violaceum CV026 and VIR07?I need these cultures for my research.
If anyone want to get CV026 from India, get it done from NCCS, Pune
Thanks to Prof.Paul Williams for the valuable info.Following
- Uelinton Pinto added an answer:11Problem in dissolving AHL (3oC12HSL)I am trying to make a working concentration of 500 uM 3oC12HSL in LB (or any other aqueous medium for bacterial growth) by diluting my stock solution of 100 mM 3oC12HSL in DMF. Every time I add my stock solution to the medium the AHL starts to precipitate. I understand it is sparingly soluble in water. Is there any way to dissolve AHL into aqueous medium?
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).Following
- Eric Omwenga added an answer:5What are the types of Quorum sensing signaling molecules?
May I know the different types of signaling molecules in bacteria and fungi. Do different bacteria have different signaling molecules. Which are intracellular and intercellular molecules
All quorum-sensing systems utilize small, secreted signaling molecules
known as autoinducers (AIs) that are of three categories: acylated homoserine lactones (AHLs), used by Gram-negative bacteria (also sometimes referred
to as autoinducer-1 [AI-1]); peptide signals, used by Gram-positive
bacteria; and autoinducer-2 (AI-2), used by both Gram-negative
and Gram-positive bacteria. There are also other quorum sensing
signals that go beyond these classes, including
Pseudomonas quinolone signal (PQS), diffusible signal factor
(DSF), and autoinducer-3 (AI-3), and new molecules will undoubtedly
be discovered as the study of quorum sensing expands
to species of bacteria yet to be investigated.Following
- Ravindra Pal Singh added an answer:5Can Quorum sensing and Quorum Quenching genes be harbored in the same bacterial genome?
Do you have examples of bacteria using Quorum sensing and Quorum Quenching system?
Yes, there are many examples as above given and more others.Following
- Harshad S. Lade added an answer:6What are the methods/on-line sensors or biosensors used for non-destructive early detection of membrane biofouling in MBRs treating wastewaters?
Early prediction of biofouling in MBR based on quorum sensing molecules and extracellular polymeric substances.
Thank you very much for the information.Following
- Uma Kranthi added an answer:18Has anyone performed analysis for N-acylhomoserine lactones (AHLs) using TLC and LC-MS?I am working on bacterial quorum sensing and biofilm. I tried TLC for AHLs assay using Agrobacterium bioreporter, but it didn't work, although I got positive results with T-streak method. Does anybody know how to detect AHLs, by TLC and LC-MS?
Hello Pramal Biswa,
LCMS you can use a C18 column for seperation in an Acetonitrile(Acn) water gradient (The column is initially eluted with 10 % acetonitrile (ACN) at 1ml/min for 10 mins followed by a linear gradient to reach 100% ACN in 65 minutes and an additional 15 min at 100% ACN.)
In the generated MS data, check for m/z peak corresponding to 101/102(lactone fragment) or 143/144 (Mclafferty fragment) and the corresponding molecular ion peak.
May I know the reference for this protocolFollowing
- Manmohit Kalia added an answer:1How to quantify Acylhomoserine lactones in biofilm? Any Spectrophotometric method ?Quantification of AHLs and AI-2 in biofilm?
You have to go with the Biosensor strains only then you will be able to quantifyFollowing
- Yannick D N Tremblay added an answer:5Has anyone done biofilm formation experiments for streptococcus canis clinical isolates?
please any one did biofilm formation for streptococcus canis clinical isolates advice me for the best protocol which used in this condition, however I used TSB with glucose and THB broth with glucose i didn't observe biofilm in tissue culture plate.
By chemical defined medium, I mean a medium in which the exact chemical composition is known.
By Minimal media, I mean those that contain the minimum nutrients possible growth.
The medium in the article proposed by Mostafa is a good start. I had success with the medium for Streptococcus mutans biofilm.
We also use something similar for Streptococcuis suis biofilm and the recipe can be found in the following article:
- Eliana Endo added an answer:6How to study quorum sensing inhibitors against S. aureus ?
I would like to test some compounds as quorum sensing inhibitors. Is there any simple method to do this?
- Md. Furkanur Rahaman Mizan added an answer:1Does anyone have access to the Vibrio harveyi Autoinducer 2 biosensor set..BB120, BB152 etc?
My panel from ATCC died due to biofreezer malfunction. I want to test for AI-2 production.
I have BB120Following
- Saheli Ghosh added an answer:3Can anyone help me with for streptococcus canis biofilms?
iI did biofilm for streptococcus canis by using microtiter plate method as following overnight growth of this strains on TSB then diluted the bacterial suspension into 1:100 using fresh TSB+1% Glucose and distributed 100ul in each well then the plate incubated at 37 for 24 hr and 48 hr and didn't observe biofilm, what yours suggestion?
I would suggest you to incubate the culture for more than 48 hrs.Use any positive control i.e any biofilm forming strain alongwith your organism.Then you perform the biofilm assay then check the O.D.Biofilm formation also depends upon media.Also you can experiment by changing the media.Time required for biofilm formation varies in organisms.Following
- Uma Kranthi added an answer:6Does 0.1% acidified ethyl acetate kill all bacteria?
I was doing N-Acyl homoserine lactone (AHL) extraction from bacteria using 0.1% acidfied ethyl acetate. Wondering does certain bacteria tolerance to this so that I might leave bacteria broth and acidfied ethyl acetate mixtures for extended time so that better yield of AHLs?
I agree with Padmavathi. Extracting twice with ethylacetate is enough to get a detectable amount of AHL.
I find a immisicible layer of bubbles in between broth supernatant and ethylacetate when I shake to extract AHL. May I know what that is?
And I use ethylacetate instead of acidified ethylacetate. As there was not much difference using ethylacetate and acidified ethylacetate.Following
- Catriona Anderson added an answer:3Are any quorum sensing inhibitors available for medicinal use in Europe?I am involved in an ongoing study looking at biofilm infection in patients. To my knowledge QSI are not yet used but may be advantageous in the fight against chronic bacterial infection. Any advice in this area gratefully received.
Thank you :-)Following
- Vijay Kothari added an answer:7Are there Quorum sensing inhibition assays?
How to detect quorum sensing inhibitors in Vitro?
Using Chromobacterium violaceum as the model organism is the simplest assay.Following
- Ines Ghali added an answer:5Can Quorum sensing autoinducers such as AHLs and AI-2 destroyed or affected by high temperature?
Quorum sensing autoinducers
Thank you Dear Christian Utpatel : practically saved my life :)Following
- Alireza Japoni-Nejad added an answer:5Are efflux pumps of bacteria the means by which all secretions from the bacterium take place?The first response of bacteria to a noxious agent such as an antibiotic is the induced over expression of efflux pumps. Once the threat of the noxious agent passes, the level of efflux pump expression returns to its normal baseline.
Material secreted by bacteria such as those involved in quorum sensing (QS) and biofilm take place via the efflux pump system of the bacterium (Varga ZG, Armada A, Cerca P, Amaral L, Mior Ahmad Subki MA, Savka MA, Szegedi E, Kawase M, Motohashi N, Molnár J. Inhibition of quorum sensing and efflux pump system by trifluoromethyl ketone proton pump inhibitors. In Vivo. 2012). Because diffusion of other materials does not take place readily across the plasma membrane or outer membrane of bacteria, the means by which these materials (toxins, inducers, inhibitors, etc) are secreted may represent targets that may be exploited for the therapy of infections whose success lies in the materials secreted.
I would like for the members of RG to present their views on this subject for the purpose of expanding the possibility of new targets to be exploited, especially for the therapy of Gram-negative infections.
Dear Dr Leonard Amaral
ATP binding cassette (ABC pumps) transporters is Type one secretion systems (T1SS) . ABC are heterotrimeric complexes consisting of an IM ABC exporter, a membrane fusion protein (MFP) and a pore-forming, outermembrane protein (OMP). T1SS allow the secretion of a wide range of substrates (proteinaceous and nonproteinaceous) from the cytoplasm to the extracellular space in a single step, without a stable periplasmic intermediate.Following
- Fatemah Allam added an answer:4What indicator organisms can I use for simple detection of Quorum Sensing Inhibitors?
I want to screen for QSI from environmental bacterial isolates and plant fragments. I am currently working with Chromobacterium ATCC 12472 by inoculating it on soft agar overlayed on test organisms or plant fragments.
Right now I feel the need to add more indicator organisms to further confirm QSI activity.
Can anyone suggest or recommend other indicator bacterial strains? And can I ask where or how to obtain them?
Dr. Ankita Pagedar,
Could you please add the publications of your answer.
- Trinish Sarkar added an answer:5Which specific nutrient stress induces stationary phase in an E.coli population growing in Luria-Bertani medium?Among several stress conditions, low levels of carbon, nitrogen or phosphorus, as well as amino acid starvation, trigger Rpos cascade,in turn contributing to stationary phase induction.Thank you Surendra for recommending the paper.It was very helpful.Following
- Daniel Deng added an answer:13How do you distinguish/separate dormant bacteria from active bacteria?In bacteria biofilm, a lot of bacteria become metabolically dormant while some are still actively growing. How do you separate them?Dr. Flemming, I would like to have a look at those papers. Please send me PDFs to firstname.lastname@example.org. Thank you very much.Following
- Ganga Viswanath added an answer:1How can we determine AHLs from Biofilm in Microtitre plates?I want to grow pesudomonas aeruginosa PA01 biofilm in microtitre plate, can I measure AHLs or AI-2 in microtitre plate?you cannot measure it directly on microtitre plate. In the plate you can just observe whether AHL is positive or negative. here are some papers which might be helpful for you.
Extraction of violacein from Chromobacterium violaceum provides
a new quantitative bioassay for N-acyl homoserine lactone
Isolation and characterization of acyl homoserine
lactone–producing bacteria during an urban
river biofilm formation
- Mohammad Mahfuz Ali Khan Shawan added an answer:1Biofilms in water systems - reasons and solutions?What are the major reasons for using biofilms in water systems and what are possible solutions?Hi, Najmul,
Micro-organisms thrive in the presence of water and nutrients. The movement of water creates an unstable environment for survival. Consequently, micro-organisms have evolved in a manner that allows them to adhere to a substrate with great tenacity. When they find a suitable surface they attach themselves and then initiate multiplication. Growth can be rapid, with new cells attaching to each other as well as extending laterally across the surface.
There are a variety of nutrient sources within water systems. These include scale, sediment, corrosion products and other trapped organic and inorganic matter. The movement of the water ensures that colonies receive a constant supply of nutrients. When this is combined with specific temperature conditions, which are often found inside heating systems such as calorifiers and heat exchangers, the perfect conditions exist for rapid microbial growth. The population will predominantly consist of aerobic (oxygen seeking) and anaerobic (living without oxygen) bacteria.
Using Chlorine Dioxide as a part of a water hygiene management programme will eliminate biofilm and maintain a safe, efficient and cost effective water system.
Have a nice day.........Following
- Joseph ( Jozsef in Hungarian) -- Molnar added an answer:14If bacteria use 'Quorum Sensing' to engage in intra-and-inter-species communication, how can we leverage on this phenomenon for antibiotics?Different chemicals are produced by bacteria for inter and intra species communication, how do we use these chemicals to our advantage in developing antibiotics?The QS can be a potential target of inhibitor phytochemicals or synthetic compounds. QS inhibitors are able to modify the effect of chemotherapy through blocking acyl-homoserin lactone synthase, other mediators, or second messenger e.g. cyclic-di-GMP resulting in changes in active efflux or diffusion, changes in cell to cell signal transport, secretion of biofilm, swarming, efflux pump systems, PMF, cell adhesion, cohesion etc.Following
- Mohamed ahmed Sebak added an answer:12Do you know any protocols to detect metabolic activity in bacterial biofilm?I grow my bacteria in a define culture medium (C-limited) in 96-well plates. I tried TTC assay, but it doesn't seem to work well. I have to add extra glucose to intensify the reaction and the red precipitate is hard to dissolve for measurement.I think XTT may be useful
- Mesrop Ayrapetyan added an answer:2How do bacteria regulate the production of quorum sensing molecules?Quorum sensing (QS) in bacteria regulates their gene expression in a population-density dependent manner, which controls population behaviors. Once the population reach a high density and so do the QS molecules, the expression of many genes in bacteria are triggered. Do bacteria still keep on producing QS signals once their population is high? How do they regulate QS production?Several types of quorum sensing have been discovered to date. For example: there are species specific autoinducers (QS molecules) known as acyl-homoserine lactones which have been discovered in many bacteria. There is also interspecies quorum sensing through the autoinducer-2 molecule which has been found in several species as well. There are also other quorum sensing molecules that are peptides.
In Vibrios for example, the quorum sensing master regulator (LuxR) which is activated by the presence of a high concentration of AI-2 molecules (QS molecules), regulates many genes (activates or represses). Interestingly, LuxR also increases transcription of the gene that codes for the enzyme that catalyzes the formation of AI-2. This may imply that at high cell density when there are more and more QS molecules around, even more is being produced.
To get a more detailed answer about specific quorum sensing systems I would look at some of the literature. Here's a good paper to give you some idea
I hope this helps.
- Richard H Bentham added an answer:23Is bacterial colony grown on agar plate a biofilm?I know it is more or less a philosophical question, however quite intriquing. Bacterial cells on agar plate adhere to surface and majority of cells is covered by slime layers. Thus it matches the biofilm definition, however I haven't heard anyone who would define "agar" colonies such way.Hi All,
I think a simple answer to this question is that some organisms grown in monoculture on an agar plate are incapable of forming biofilms on other nutrient depleted surfaces. If they can't grow as biofilm on a surface other than nutrient agar then I suppose that they are not truly biofilm forming organisms. Alternatively there are many biofilm organisms that will not grow on agar surfaces. I think we should understand that agar is , for the most part, a very artificial medium.
Organisms like Pseuodmonads will grow happily on agar and are well known biofilm pioneers, others are secondary colonisers (for instance my interest Legionellae) or of course the predators (virus, protozoa and other eukarya). I agree with Andrew Spiers that there are a lot of terms floating around (pun intended!) but I think they tend to refer to physiological adaptation ecological niches. To that extent quorum sensing in response to environment may be a major determinant of biofilm / floc / slime/ granule etc formation within any given ecosystem.
hope this helps,
- Marwan M. A. Mohammed added an answer:10Biofilm Exopolysaccharide determination by fluorescence dyeI am doing biofilm EPS determination using WGA and ConA alexa 488 fluorescence dye but even in positive control, I do not get any fluorescence under spectrofluorometer. I am using the concentration of dye as 10 micolite/mL as recommended by manufacturer of Invitrogen and paper.
Does anybody know why fluorescence is not visible?I stained the proteins in the biofilm matrix using the SYPRO® Ruby Biofilm Matrix Stain, but I didn't try staining of Exopolysaccharide.Following
- Sagar Patil added an answer:2What important things does one has to know before go to bio-sensors fabrication/to do basic science about?
I am synthesizing thin films of some bio-active materials which can sense bio-molecules, I need to know, what can be criteria to choose these mixtures to study in Lab (vitro)? I am Electrochemistry student and know cyclic voltammetry and electrochemical measurements, so in this field how can I do these studies?Thank you sir!Following