Antimicrobial Activity - Science topic

Hell Dear, just read it

Increasing the bacterial concentration in a formulation to compensate for inhibition by a Lactobacillus strain is a complex issue that needs careful consideration from both scientific and ethical perspectives. Here are some key points to consider:

The initial step is to centrifuge the bacillus broth and use the supernatant. Then, take a filtered culture of Bacillus and add the filtered broth to the cultured S. aureus well. (Remove the bacillus)

It is preferable to use the nanoparticles as dispersed in the dispersion media of the synthesis, together with the stabilizers used. Every change in dispersion media likely will lead to destabilization because the stabilizer, solvation layer around the particles is less compatible with the dispersion media.

Ultrasonication may disperse agglomerated particles, but may induce also agglomeration. Using solvents deactivating or killing microbes you do not need nanoparticles with antimicrobial activity, which work usually in aqueous media, often through ions released.

it's important to consider specific proteins that play crucial roles in the survival and reproduction of microorganisms. Enzymes involved in cell wall synthesis: Proteins like penicillin-binding proteins (PBPs) are crucial for bacterial cell wall formation.

DNA gyrase and topoisomerases: Involved in DNA replication and repair, these are essential targets for antimicrobial compounds.

Ribosomal proteins: Targeting bacterial ribosomes can disrupt protein synthesis. Utilize databases like the Protein Data Bank (PDB) to find crystal structures of your selected proteins. Molecular docking predictions should be validated through in vitro and in vivo experiments.for in vitro evaluation you can use microorganisms directly.

Abrar Hussain

"... it will give you better result then other solvent" Note solvent.

Bacterial metabolism is abroad term,fist of all put your isolate on group of classification according to Bergy .manual for clssifactin of bacteria

As long as the DMSO is diluted to a level that does not significantly affect the bacterial growth, then it is OK to make a concentrated stock solution in DMSO. The final DMSO concentration to which the bacteria are exposed should be no more than a few percent. You could do a test to see how sensitive your bacteria are to DMSO itself in order to set the upper limit on DMSO concentration.

I don't think it matters whether you do your assays in 96 well plates, 24 well plates or tubes. But if you can not measure optical density or turbidity, then the easiest thing may be to measure surviving CFUs. You can plate out a sample of the culture after treating, but you might need to also do a dilution series. You can just do a series of 10 fold dilutions and spot 5 ul of each onto your plates (you can usually get a number of spots per plate). Otherwise plating directly after 24 hours is likely to have too high a cell density to count directly, unless the compound is highly inhibitory.

So the MBC is defined as the lowest concentration of the antimicrobial which reduces the initial inoculum of bacteria by at least 3 logs (>=99.9%).

Normally, in the tests performed for determination of MBC you would have an initial concentration of bacteria ca. 10⁵ cfu/ml that would be reduced to below 10 cfu/ml, thus you would have a reduction of 4 logs (99.99%).

Therefore, the MBC would be calculated from the lowest concentration of the substance at which no colonies would be formed on the surface of a general purpose agar (i.e., TSA). For this to happen, you should remove a volume of 0.1 ml from the lowest concentration of antimicrobial inhibiting visible bacterial growth (MIC) and then spreading onto the surface of the agar. Following incubation for a defined optimum time-temperature combination depending on the medium used (e.g., 37oC x 48 h for TSA), MBC is calculated from the plates with no visible colonies, as previously mentioned.

Hope this helps a bit Mona Mahmoud!

There are several factors that can greatly influence the action of antimicrobial agents. Aside from the popularly known factors (population size, concentration of the agent, time of exposure, temperature and microbial composition/ susceptibility), I wish to talk about the following in addition to the suggestions of other researchers:

the content of added test assay may adsorb at the CNT. It seems possible that first dispersing with added polyphosphate may avoid this.

I grow multi-drug resistant MRSA and multi-drug resistant P. aeruginosa on rich media (BHI or TSA) in the presence of at least one of the antibiotics that the strain is resistant to. For MIC testing, I grow it on CAMH plates.

Jessie Gu your strategy is fine, although I suggest doing this slightly differently.

If you want to do an inoculum directly from broth to Sensititre assay, I would avoid TSB, since the presence of cations in the media is important for accurate readings of some antibiotics.

Sensititre has their commercial CAMHB buffered with TES and you can use this same broth for the overnight culture of S. aureus. From that, you can use your broth to make an inoculum in DI water (supplied with the system) until densitometer shows 0.5 MFa and dillute as recommended by the Sensititre in their media.

It is important to use densitometer or at least OD600 nm. to reach the CLSI defined inoculum. Too many or too little bacteria will show different MIC values.

Please eliminate uncontrolled variables. Note Kirby Bauer uses nonselective medium.

You do not know what methicillin carry over, alone or in combination with unknown materials, may be, and there is no reason for MSA in the 1st place. On what basis do you assume an isolate through one culture sequence will lose resistance? As far as I know, the record does not defend that phenomenon

Even if it were a concern, are you sure methicillin in MSA is working? I'm not familiar with its validation. Why not use ORSAB - the medium typically used for isolation of MRSA.

If application - you'll prob run more testing in context but be aware, composition of plant extracts, EO's etc. vary greatly from batch to batch. Ask the supplier both for data showing what constituents are "active" and to establish standard concentrations and establish relevant CoA on a batch basis.

Without knowing your setup, i figure in the 384 well you will only see growth or no growth, so you only have very limited information and might have false negative events. You could in fact use it as a pre-screen, but you might falsly exclude candidates. Kirby Bauer ist very robust and straightforward.

Sure you can do it in 384, but I'd prefer to see the zone of inhibition.

Sonication or evaporation with T control

Alessandra Serain

MIC is more common and more suitable for antimicrobial testing. IC50 is only available when you use microbroth dilution method. For your extract and if you are using mircobroth dilution method, it is better to go further in testing to get MIC and MBC. then you test these concentrations on eukaryotic cells to confirm their selectivity toward microbs.

you can read this article:

Dear Noor Ul Ain, yes and in addition to other parameters, quaternization enhanced antibacterial activity. Please have a look at the following documents. My Regards

10.1039/9781788012638-00001

That could be a different mechanism of the tested compound. The antimicrobial agent could work by inhibiting cell wall synthesis, depolarizing the cell membrane, inhibiting protein and nucleic acid synthesis, and inhibiting or blocking metabolic pathways in bacteria. Otherwise, the mechanism of antioxidant activity using the DPPH assay is by scavenging the DPPH radical. Your tested compound may have a high capacity to scavenge the DPPH radical, but it has not any activities to kill microbes.

Actually, I am still confused about your question because the results of the DPPH assay can be presented by both % inhibition and IC50.

Dear Noor Ul Ain, please have a look at the following recent review document and the references therein. My Regards

Mice are generally not the best model foremost of the E. coli-related intestinal diseases. In fact, it is often quite challenging to induce the clinical symptoms in mice, despite the culture-proven colonization.

Rabbits on the other hand serve as more robust E. coli diarrhea model

in this case, u assume that molecules bind to DNA and cause DNA damage and lead to Bacterial cell death.

u can study various ways like comet assay commonly used for DNA Damage mechanism.

Hi Sofia,

Although OD595-600 and McFarland standards are somewhat standardised methods for estimating the cfu of bacterial cultures, different species can result in different final numbers. OD600 is a turbidity measurement, therefore differences in cell size (even slightly) can have different turbidity measurements. The best approach is to optimize the growth of your cultures under your laboratory conditions. This can be achieved by drawing up a curve comparing OD600 (y =-axis) with counted CFU/mL (x-axis) for each bacterial strain. You will only have to do this once per strain. This will allow you to prepare standardised bacterial inoculums and have approximately the same number of cells in your assay for each bacterial species. It is also worth mentioning that slight variations in cfu/mL should not have a large effect on the MIC values, and the value will stay the same regardless of the bacterial concentration. However, MIC shifts can be observed at bacterial inoculums one to three orders of magnitude larger.

All the best.

I think all the medical plants have this activity.

check this out

For example, copper oxide nanoparticles, Mg2+, silver as well as cationic starch have antimicrobial properties that can help in eliminating fungal and bactrial growth. In some microorganisms that use respiratory enzymes, AgNPs interfere with the respiratory mechanism, leading to the ultimate death of the organism, by destroying enzymes. Silver ion blocks the cytochrome oxidase in the bacterial respiratory chain. Silver nanoparticles have different antimicrobial properties, For example, they have potentials for disruption of cellular adhesion, lipopolysaccharide decomposition (LPS), intracellular penetration and DNA degradation via molecular electron binding and ion release.

It's a good idea. I'm interested in working with you

Please attach the plate image. Usually, a partial clear zone can not consider as an inhibition zone. But if the result is coming the same for the replicates or the result is reproducible we have to look detail into it. Since it is a natural product we can't expect a very good inhibition zone like other antibiotics. And proper swabbing and preparation of disc with the compound is a little tricky. You can repeat the experiment either by well diffusion or try a higher concentration of the extracted compound. I have done a similar experiment with natural products with well diffusion assay and got a good and reproducible result.

Hi Furkan,

did you try to increase the amount of glycerol? instead of 40%,@ you can use 50%.

and why you use pH 12 and heating processes together?

Yes, All Paan varieties have one scientific name "Piper betle L. "

Please go through the following PDF attachments.

It is advisable to use one interpretive criteria. No two standards are exactly the same. Therefore using more than one criteria may complicate interpretation of results by generating contentions.

In case you have been using the standard set by Federal Drug Authority (FDA) there is an exception where by FDA recognizes the standard published in "Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 29th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2019". In this case you may use more an additional interpretive criteria. Note that modifications to the list of recognized consensus standards: Publications in the Federal Register to the list of recognized consensus standards can be accessed at http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/Standards/ucm123792.htm. Thank you.

If you autoclave tween it will degrade.

I would filter. If you filter tweens, it can adsorb to filters. And of course, filtration will be difficult due to viscosity.

Analysis of plant extracts showing efficacy often finds the usual suspects. For example, journals such as Int. Biodegradation Biodeterioration require analysis and novelty of composition for publication of reports of efficacy of extracts from novel plants.

But to Nelson's point - media-based testing has limitations but has some relevance to application. Academically, we may wonder at an assay that evaluated efficacy in low water context but methodology here would prob be even less relevant to application.

Yes

Dear F. Romain Fouelefack you can try link below:

Regards

Thank you

Yes !

Vaiya,

u can see also for antibacterial activity evalution: GB/T 20944.3-2008 (eq. ISO 20743-2007).

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?

The phyto -constituents of any plant part may vary both in quantity as well as quality depending on the soil, stage of maturity of plant, season, breed of plant etc. factors.

The researchers want to work on phyto-constituents of one or more plant should be aware of the factors before planning for the research.

Regards,

This is a qualitative method but the results should be reproducible. You need to make sure that the sample is in contact with the inoculated agar surface. If the treated fabric is hydrophobic, they may not be in contact with the agar surface. You can soak the fabric in a solution containing few drops of wetting agent before placing it on the inoculated agar surface. You will need to make sure that the wetting agent does not affect the growth of bacteria. You also need to make sure that the agar surafce does not become dry. For quantitative results, you should use AATCC Test Method 100 if you antibacterial agent is leaching type.

MIC (minimal inhibitory concentration) does not mean it should be calculated somehow specifically. It just means the minimal concentration of compound at which you can not see the bacterial growth. However, it is always more accurate if you may determine MIC by using a quantitative method. Scientists consider different inhibition % as MICs, usually 90% or 95%, sometimes 99%, but it should be totally fine to use any of those values (let's say 95%) if you provide your explanation of MIC calculation in the description of methods.

Hey, for a real time update on classification of beta-lactamases in general, you got to always check this online database

From the website you have all the known abd emerging beta-lactamases list and classifications

Cheers

Alternatively organic precipitation is suggested to be used. For instance, 2-propanol of acetone followed by solid-phase extraction on hydrophobic of ion-exchange immobile phase.

If some degree is your target, then research of repetitive type is O.K. But so far I understand, such copy paste research or research following the same pattern of another 50 - 100 published paper of no use.

The developed countries always want to continue their business by selling newer technologies. So, why they should accept research papers on the work done on so called old technology?

I am from India, a developing country. I feel that we should not follow the researches of the first world with our inferior laboratory, as it is not possible to do something better than the workers of first world. Imported instruments, imported chemicals and imported ideas can give us actually nothing in basic research.

We should concentrate on the researches we need in our own country. It should be planned as per infrastructure and resources available in our country at maximum level.

This is the only way to be followed, as per my idea.

Thanks.

Hi Mohamed,

I agree with checking the CLSI protocols of microdilution methods. Also, you can slightly modify the protocol by using resazurin, a blue dye which turns bright pink and then transparent when reduced by bacterial activity. I recommend you to use the standard microdilution method from CLSI adding, after incubation and turbidity measurment, 10 uL of resazurin solution at 0.01% (filter sterilized water solution of resazurin). Then, re-incubate the plate for one hour and then read the MIC. In this way MIC is defined as the minimal concentration of your extract in which there was not color change from blue to pink or transparent.

Also, the pink dye resulting from resazurin reduction is fluorescent, so you can use it for quantification.

Best regards!

The reason is that when you streak the sensitive strain across the inhibitor producing strain, then you are carrying inhibitor cells along. So the sensitive strain will mostly die the entire length of the streak after it crosses the inhibitor strain. 

One important thing is whether you are trying to be quantitative or just screening lots of samples. If you are screening for possible inhibitor strains against a single sensitive strain, then you want to use some method that makes it easy, such as spots on a sensitive lawn. If you are screening multiple sensitive strains against a single inhibitor strain, then cross streaking works well. But if you want to be more quantitative then some type of disk assay where you can measure the zone of inhibition might be best. 

Dear Sohel,

Antimicrobial (Antibacterial and Antifungal) activities of Lawsonia inermis are widely studied and reported. Pl refer following threads:-

Dear

Please check interestings methods in these links:

Disc or well diffusion tests are both qualitative assays. Hence, they wont give an accurate estimation of the effect of the antimicrobial activity of plants extracts or any other antimicrobial agent. Minimal Inhibitory Concentration (MIC) and IC50 values must be investigated for quantitative estimation. It all depends on what you are testing e.g. crude extract, protein extract, pure compounds or mixture of phenol-like compounds...doing well diffusion or disc diffusion test of crude or non-purified compound is meaningless... any plant extract would have some inhibitory activity due to the phenolic substances...  

To evaluate the antibiofilm efficacy of different matherial such as titanium surfaces, the whole biomass present on each disc was detected. Briefly, biofilms grown on titanium

discs were air-dried and stained by disc immersion in a 5% crystal violet solution for 15 minute and, after several washing , air dried again. The estimation of biofilm biomass was performed by elution of the biofilm bound crystal violet with ethanol (96%)

followed by the determination of the absorbance of 100 ll of eluted dye solution at 595 nm using a microplate photometer (Multiskan FC, Thermo Scientific; Milan, Italy). Measurements for each discs, were carried out in triplicate. For each bacteria and

surface, mean values and standard deviation of absorbance value were computed. Un-paired Student’s T test was used to compare data between experimental surfaces.

I hope this protocol help you

Rongfeng li see the link!

Antimicrobial susceptibility testing always should be performed strictly according to the approved standards and so only with the standard medium.

Dear Pitchumani

According to me  antimicrobial activities are commonly be refitting to the specifity of biactives molécules. Study of different interactions  requires strong knowledge and large time of investigation. Quantum chemical methods will recommen hard regard.

Best regards

Marius

https://www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)

You could run a trial to test this by comparing previously frozen leaves with fresh leaves. A simple t-test or ln-odds ratio test will be sufficient, with maybe 30 samples of each. I know it's extra work, but it will make your research more robust. I think slow freezing causes less damage than rapid freezing :-)

Micromyx in Kalamazoo, MI does excellent work. 

Thank you so much Dr.Mahajan

I think that this question should be a subject of research. However, many phenolic agents exhibit both antioxidant and antimicrobial properties. In theoretical science,antioxidant activity is all about the reduction or removal  of free radicals and nascent oxygen from an environment. On the other other hand, many bacteria could also depend on these nascent oxygen for survival in any given environment. Thus by implication, reduction or removal of free radicals or oxidants could relate to antimicrobial activity. This could explain in parts  why antioxidant properties could correlate positively with antimicrobial activity.  

The Epsilometer test......I had to look it it since I have always known it as the "Etest" and forgotten what it stood for. 

Cheers,

Tasha

Fermentation process definitely add functional value to herbal juice or decoction.  Specific bioactives come into play and initiate various changes in the formulation. also refer to the articles attached below.

Dear Ramani,

Please, find in attach an article on: Antibiotic development challenges :the various mechanisms of action of antimicrobial peptides and of bacterial resistance

Fernanda Guilhelmelli 1†, NatháliaVilela 1†, PatríciaAlbuquerque1, LorenadaS.Derengowski1, IldineteSilva-Pereira1 and CynthiaM.Kyaw2*

Hope to be useful

Good luck

Dear DAAS Mohamed Seghir,

I intend to help you. Please send me your contact address.

regards,

Ahmed

Candida and other pathogenic yeast strains would be good for human therapeutics, but if you target then plant pathogens affecting crops would be good too starting from Phytophthora, Fusarium, mildews, smuts, blights, rusts, mildews, Verticillium would be good targets too. Depends if you wanna direct them against human or plant pathogens!

Thanks,

Biswa

Dear you read my publications. Good Luck!

I feel that Abhishek's answer is very appropriate

Use freshly prepared nanoparticles along with plant extract

Dear Edison,

I think that chlorhexidine is used as a positive control for antimicrobrial testing since it has a broad spectrum of activity;  it is effective in vitro against both Gram-positive and Gram-negative bacteria including aerobes and anaerobes and yeasts and fungi.

For more on this antimicrobial agent, please use the following link:

 http://www.nature.com/bdj/journal/v186/n6/full/4800090a.html

Hoping this will be helpful,

Rafik

It will be better if you use freshly prepared silver nanoparticles for antimicrobial activity along with their positive and negative control.

Overall pure ethanol extraction is fine, but you do need to make sure all ethanol is evaporated before reconstituting the extract. Also, your extract might diffuse slower than pure ethanol, which could have an effect. You should do a few more repetitions. The disk method is notoriously inexact, and many journals do not accept papers based on this method only.

Hi Abdul, Yes the salts became cloride salts, but the important part is the cation. I use 0.2M HCl for  Fe and Mo cations to add 10e-4 in the culture of Azotobacter strains.  Good day

Most of the time when culture medium is highly nutritional the metabolite activity of bacteria/fungi is not optimal for this reason depending to the concentration of nutrients in this medium 2,3,4...times dilution can be made at the preparation step before used as growth media for metabolite activity.

Thus, to best of my knowlegde ther is no specific protocol to remove nutrients to a formulated medium instead of this you can dilute before use.

The diliued medium is stressful for the microorganism and this condition is good for expression of metabolic activity.

Good luck

I agree with Dr.Ali

with  the require answer

Thanks all for your answers. I decided to go with the FC test. I now just need to understand how to use it with my ethanol extracts. Also this is is not a thesis neither I will publish this, but I appreciate your kind advice :)

Thanks 

It clearly has to do with log reduction, as you correctly pointed out. In order for a chemical disinfectant or antiseptic product to be characterized as such and prove its antimicrobial activity, an at least 5 decimal log reduction shall be demonstrated for this product. This is why you see "it kills 99.9999% of bacteria" written on the label of relevant products...

So, in terms of performance, the two antimicrobials tested with 99.99% and 99.9999% respective reduction values should have approximately a 2 log difference in antimicrobial activity.

I'm assuming it is a local designation for a particular strain. It is not the abbreviation of a known and registered culture collection (that list is on http://www.wfcc.info/index.php/collections/display/).

If you are seeing activity from an agar overlay assay it is most likely an extracellular compound, which are most common as several of the other answers have said.  But also be careful in your terminology, if the compound is excreted it is extracellular, if is 'intra'cellular it could not affect other organisms unless they phagocytize the cells, but it could be some compound that is put into the membrane that could affect other bacteria when in contact.  Also, if you simply sonicate and lyse a bacterial culture you then have both intra- and extra- cellular chemicals in the assay and cannot tell which is the cause (separate the bacteria first, then sonicate/lyse and test that material).

A number of studies have confirmed the antimicrobial efficacy of Oxalis corniculata. You may refer attached papers.