Antimicrobial Susceptibility Testing - Science topic
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Questions related to Antimicrobial Susceptibility Testing
European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP) has been monitoring the antimicrobial susceptibility in Neisseria gonorrhoeae since 2004. Its EQA scheme is necessary for the quality assurance that the laboratory provides in the N. gonorrhoeae antimicrobial susceptibility testing. Participants in this program undergo a 10-year EQA to evaluate N. gonorrhoeae's antimicrobial susceptibility.
To know more about the article entitled "Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data" click on the link below
Are there any difficulties in using disc diffusion method as a test for antimicrobial susceptibility?
if i dissolve the test material in ethanol 70%, and use the ethanol as negative control ? What do you think about this study design?
According to the journal cited below, methodology should be investigated whenever zone diameters are consistently not within the mean range because there might be an error. Control charts such as Shewhart Diagram, and Westgard rules were mentioned to be helpful in interpreting changes and monitoring the performance, aside from those two, what are the other useful methods that we can utilize to assess the zone diameter issues?
King, A., & Brown, D. F. J. (2001). Quality assurance of antimicrobial susceptibility testing by disc diffusion. Journal of Antimicrobial Chemotherapy, 48(suppl_1), 71–76. https://doi.org/10.1093/jac/48.suppl_1.71
Based on the research article, “Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data” by Cole et al. (2019) ensures the quality of data used for patient care and public health efforts. It supports accurate diagnosis, treatment optimization, surveillance, research, and international collaboration in the fight against gonorrhea. With that, answering this question would help to further know how to give the patients appropriate antibiotics, improving their chances of successful treatment and reducing the risk of antibiotic resistance. Here’s the link to the research article:
This relates to the research article titled 'Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidates the high reliability of data.' You can access the research journal at this link: https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-019-3900-z.
The validation of diagnostic and antimicrobial susceptibility tests for detecting Neisseria gonorrhoeae, a sexually transmitted bacterium, is of paramount importance in the field of healthcare. Ensuring the accuracy and reliability of these tests is crucial for accurate diagnosis and appropriate treatment, given the growing concerns about antibiotic resistance. This discussion aims to explore the various methods and strategies that can be employed to effectively validate the detection of Neisseria gonorrhoeae through diagnostic and antimicrobial susceptibility tests.
Source: Cole, M.J., Quaye, N., Jacobsson, S. et al. Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data. BMC Infect Dis 19, 281 (2019). https://doi.org/10.1186/s12879-019-3900-z
The journal entitled Quality assurance of Antimicrobial Susceptibility Testing by Disc Diffusion by King, A., & Brown, D. F. (2001) stated that resistant organisms are necessary when testing particular resistance mechanisms. These mechanisms are said to be unstable and should be monitored carefully. How can we observe these organisms' resistance loss, which is essential for quality control procedures?
Reference: King, A., & Brown, D. F. (2001). Quality assurance of antimicrobial susceptibility testing by disc diffusion. The Journal of antimicrobial chemotherapy, 48 Suppl 1, 71–76. https://doi.org/10.1093/jac/48.suppl_1.71
How might the decision of laboratories or healthcare facilities to skip participation in an External Quality Assessment (EQA) program impact the accuracy and reliability of Neisseria gonorrhoeae antimicrobial susceptibility testing results? This question came to mind while reading this research article entitled "Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data".
In 2012, there were 78 million new instances of gonorrhea among adults, making it the second most prevalent bacterial STI in the world. Infertility, pelvic inflammatory disease, ectopic pregnancy, and other consequences and sequelae of gonorrhoeic can occur if it is left untreated. Due to this, it's critical to understand what the most accurate method is for identifying Neisseria gonorrhoeae infection in a certain person. In this manner, an accurate diagnostic and quality control for the test in question can be accomplished.
Cole, M. J., Quaye, N., Jacobsson, S., Day, M., Fagan, E., Ison, C., Pitt, R., Seaton, S., Woodford, N., Stary, A., Pleininger, S., Crucitti, T., Hunjak, B., Maikanti, P., Hoffmann, S., Viktorova, J., Buder, S., Kohl, P., Tzelepi, E., . . . Unemo, M. (2019, March 25). Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data. BMC Infectious Diseases, 19(1). https://doi.org/10.1186/s12879-019-3900-z
I'm currently working in a microbiology lab at a hospital, and there's a lot of antimicrobial susceptibility testing (disk diffusion). We're following Ecucast's recommendations, included this 15-15-15 rule. I can understand why it's important to respect the first and the last "15", but I'm having a hard time seeing the meaning of the second "15"... Anyone who can help?
I want to screen for antimicrobial activities of spices extract against fungal isolates from food. what method /methods is best for it?
The 0 mm inhibition zone was an E. coli isolate from an influent of Domestical WWTP (SBA)
the other one (SCA-2) that showed synergy between CTX - AMC and CAZ - AMC was an isolate from the effluent
how do i interpret the result of the influent E. coli? is it ESBL positive?
I want to perform antimicrobial susceptibility testing of Vibrio spp. I want to use CLSI recommended antimicrobials for AST. That's why I badly need the list of antimicrobials along with their interpretive value.
I am treating E. coli at different AgNO3 concentrations in order to follow bacteria kinetic growth. The slope of the bacterial growth curve continuously decreased with increasing AgNO3 concentration. At low concentrations, the growth of bacteria was delayed and at higher concentrations, growth was completely inhibited.
My question is related to why bacteria are not reaching the same Max. OD600 after long-term silver exposure for concentration below MIC. Assuming that the stationary phase is often due to a growth-limiting factor such as the depletion of an essential nutrient, I would expect that all the curves reached the same OD600 at a certain point after bacteria re-growing. I was thinking one possible explanation could be that AgNO3 is inducing the expression of new genes required for survival under stress (e.g, high AgNO3 concentration, lack of nutrients or toxic byproducts).
Thank you in advance,
A colleague recently asked me this trivial question of why we only prefer E. coli, P. aeruginosa, A. niger, F. oxysporum for antimicrobial susceptibility testing. My answer was that they're the generalist representative species we know much about. Still, it made me wonder if years of antimicrobial testing would've made these strains less susceptible to various antimicrobial compounds, synthetic and natural. Please do enlighten.
I tested a number of isolates for antimicrobial susceptibility testing (agar dilution method) manually and same isolates in VITEK 2 Compact. Some results didn't match between manual agar dilution and in VITEK 2. Is VITEK 2 Compact 100% reliable to MIC?
I would like to have access to the pdf "Clinical and Laboratory Standards Institute. 2018. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 11th Edition, CLSI guideline M07", since I couldn't get it on Sci Hub.
I just did an antimicrobial susceptibility testing for E. coli isolated from the Urinary tract. Nitrofuranthoin recorded 22mm zone diameter of inhibition (sensitive if >17mm). Amikacin had 18mm zone diameter of inhibition (sensitive if >17mm). Now does this mean that Nitrofuranthoin is a better choice of antibiotic for the elimination of this pathogen in the patient?
I am trying to grow S.aureus NCTC 6571 in Cation-adjusted Mueller Hinton Broth. I need this to be at a starting inoculum of 10^5CFU/ml for antimicrobial susceptibility testing. I am diluting this from an overnight culture to 10^8CFU/ml by using a 0.5 McFarland Standard, then to 10^5CFU/ml in 3 1 in 10 serial dilutions. However, I am not getting any growth what-so-ever. Has anyone had a smiliar issue or is there anything you can suggest?
Hello everyone. I want to improve my knowledge on Antimicrobial Susceptibility Testing. Please help me. I was recommended to study CLSI M100 guideline.
I would appreciate if researchers could share the CLSI guideline to me. Your help is highly appreciated.
Hello everyone, I want to carry out an antimicrobial Susceptibility Testing using broth microdilution method with 96-well microtiter plate to test the antimicrobial activity of standard anti-inflammatory drugs on Streptococcus pyogenes , However, I want to know the best media for the growth of Streptococcus pyogenes and how to prepare the media.
I am looking foreword to reading your suggestions.
Thanks and Kind regards
I was looking for data on the MIC of ampicillin for the DH10B and DH5a strains of E. coli however could not find any data relating to these two strains.
A clear disk inhibition diameter can be observed but for the same concentration of the antibiotic, the bacteria is able to grow in the broth microdilution.
I have thought of a couple of possible reasons:
1. Mishandling during the experiment
3. Insufficient antibiotic conc. in the broth microdilution due to errors while handling
What are the other possible reasons?
When I read some article about antimicrobial susceptibility test practice, some researcher perform the test too different. For example, a researcher prepare agar medium (Mueller-Hinton agar) with adding bacteria culture suspension (0.5 McFarland density) into the agar medium before get cold and pour in agar plate. The researchers mix bacteria suspension with agar medium as 100ul/100ml (bacteria suspension/agar). Especially researchers do that when agar medium is pouring temperature (this is about 45-50 ºC). After agar plates get cold, place antimicrobial disc on the plates.
In this respect, I wonder that is there any reference to this method? Does bacteria die when the suspension mixed with agar medium that is pouring temperature? And finally could we determine antimicrobial zone diameter without spread antimicrobial suspension on agar plate by swab?
I would like to test certain bacteria, yeast and mould against various liquid anti-microbials using the disk diffusion test. The anti-microbials are made in-house and are thus not available in commercial impregnated disks. The current way of testing is just to place the blank sterile disk in the middle of an inoculated agar plate and then apply the anti-microbial liquid to the disk using a pipette. The problem with this method is that only 10 ul of solution can be added to the disk before it floods (I think this is because the disk starts to absorb moisture from the agar as soon as it makes contact thus reducing how much liquid it can hold). In some cases using only 10 ul results in very small zones of inhibition. Can I maybe place the disks in a empty sterile petri dish or such and then apply the antimicrobial solution to the disk and allow it to absorb before placing on the plate? Also if I do it that way should I allow the disk to dry first or apply it to the plate while still moist?
Hi, in my project I am doing antimicrobial susceptibility testing. I have done both disk diffusion and broth microdilution for amikacin susceptibility testing for Acinetobacter baumannii and my results does not agree. My disk diffusion states all three my isolates are resistance and my broth microdilution states that the 1st isolate is susceptible, the 2nd is susceptible/intermediate and the 3rd is resistant. I have performed the disk diffusion twice and BMD 4 times. The 1st and 2nd isolates results vary in their MIC in the 4 BMD, they are not consistent. I did this BMD the same way as the CSLI recommend and for gentamicin and tobramycin and they worked. Any idea why my amikacin BMD will not work?
Fisher is out of Mueller Hinton with 5% Sheep blood, will using Chocolate Mueller Hinton be acceptable?
I take maybe two weeks try to solve this problem but it's should be better to ask in this community. My work is to do the agar dilution for my organism Neisseria gonorrhoeae using GC agar base + 1% isovitalex according to CLSI guideline. I bought GC agar base from Oxoid company and isovitalex from BD.
I prepare my GC agar base as suggested in the label of GC agar bottle by adding 18 g of GC agar base to 235 ml of distilled water (my water is RO) and boiling with stirring to dissolve the agar. After that I sterilize it by autoclaving and put it out to 50C water bath for cooling and adding isovitalex to be a 1% final concentration. This is how I make this medium.
But what I encounter for now is that my ATCC49226 that use for quality control in antimicrobial susceptibility testing cannot grow well after incubation 24 hrs in 5% CO2 at 37C and also my clinical isolates too. I ask this with the routine staff doing her job for preparing this media and she also told me that her media sometimes cannot grow too.
So I wonder what I do a mistake or it is just typically happened in this media. does anyone has some suggestion? I try to do every step elaborately as suggested in CLSI guideline and manufacturer' instruction but still not good.
I've prepare 100 ml of GC agar base and isovitalex 1 ml to make a 1% final concentration. BD will give you like 2 bottle, one for diluent and another one for powder. You have to reconstitute using 10 ml of diluent to dissolve the powder.
From my experience, ATCC 49226 and clinical isolates of Neisseria gonorrhoeae will grow well after 48 hrs incubation not 24 hrs. When incubation with 24 hrs, I can see colonies only first lane of streaking and sometimes I cannot see any grown colonies. What I should do because CLSI recommended 24 hrs of incubation.
Thanks for every suggestions.
Hello everyone. I'm getting confused about my work. please help me. I put a few Antimicrobial disks including Imipenem, Amoxicillin/tazobactam,
piperacillin/ tazobactam and cefuroxime for measuring the Susceptibility (S,R,I) for Staphylococcus aureus. The problem is here, because the CLSI 2013 and older version haven't any guidelines. I don't know what I am suppose to do? Thanks.
To test the antibiotic effect on anaerobic oral bacteria in biofilm setting, which methods are most acceptable and recommended?
I am working with some fern antimicrobial properties and I would like to know the range of the best concentrations that I can prepare my plant extracts before I can be sure they have performed best and can compete with conventional drugs for such microbial conditions.
According to CLSI standards we can use both for finding the MIC of an antibiotic so, which is more reproducible? Although macrodilution method is laborious it seems to work for me.
I am performing 96-well microtiter plate antimicrobial susceptibility testings. Right now, I already have a huge load of positive and negative results. I´ve been documenting these results through tables in Microsoft word files.
However, I´d like to save my results more efficient with less time required . Therefore, I am looking for an excel file - if possible with a "check function" for positive hits at the determined position/concentration of the 96-well design (like a classical checklist) - that i can modify according to my needs.
In the past, I have designed excel files with automatic check-lists, so I know how much work it is and I´d like to skip this step. There are so many people working on susceptibility tests with 96-well plates, so I am hoping that there is someone who can help me out with a crude template.
Preferably with A-H on top and 1-12 on the side.
I am working on the food applications of some essential oils. as part of the project and antimicrobial susceptibility test was conducted, where EO's were dissolved in DMSO before their use [in vitro]. Now I want to conduct a consumer sensory evaluation of the essential oil in some selected foods. Must I still dissolve the essential oils in the DMSO before I apply them to the food?
I'd like to ask about the differences between two terms breakpoint and cut-off values. Are the two terms interchangeable? Thank you.
Is it acceptable to read results of microdilution sensitivity test (antimicrobial sensitivity test using microdilution method) without using microtiter plate reader even though coumpound under investigation are water insoluble and may cause some turbidity with the broth?
As you know, antibiogram resulted as susceptible (S), intermediate (I) or resistant(R). i also want to detect gene expression between resistant and susceptibel strains. Can i think intermediates as a resistant?
Is there a reference that describes the disc diffusion method of antimicrobial susceptibility test in details (the steps , problem troubleshooting, the discs choice and interpretation of the results) Thanks.
Are there recommendations for the order of antibiotic discs applied on Miller Hinton agar to perform disc diffusion antimicrobial sussceptibility test? Thanks.
Could any one say the simple procedure for Anti-Fungal Susceptibility (AFS) testing, like MIC. If you know, please say the material and methods used for AFS testing
I would like to go for mode of action studies for bio active compounds from plant source, for that it will be helpful if i get some studies involving methods other than, identification of membrane disruption against bacterial test pathogens.
I heard about the use of IC50 to test the susceptibility of a bacterial strain to a given drug. I didn' t understand very well why people use it to test the acceptor strain before performing transformation experiments,.Would it not better to perform a minimal inhibitory concentration (MIC) instead? Why do you need to know the half minimal inhibitory concentration of your acceptor strain? Maybe it makes more sense to know the concentration of the drug that completely inhibits the drug doing a MIC...Sorry, It is completely new for me, so maybe I can't to understand this fully. Do you have an idea?
Thank you very much
I try to make antimicrobial test for plant extract with disk diffusion method. I macerated grinded plant in ethanol 96%. This ethanol extract is tested for antimicrobial activity against E.coli. Surprisingly, I got zone of inhibition of pure ethanol as control 23 mm and ethanol plant extract was less than 23 mm (faint haze zone) where clear zone i got is around 13 mm. Could anyone tell my why?
I have 10 bacterial isolates, and I examined them against 12 antibiotics according to British Society Antimicrobial Chemotherapy (BSAC), after I got the zone diameter breakpoints I need to compare them with BSAC to determine if the bacterial isolates are resistant or sensitive to those antibiotics but the breakpoints in the guideline are applicable for some species and some antibiotics so how can I get the breakpoints for other bacteria and other antibiotics?
Providencia rettgrei, E.coli, Pantoea, Citrobacter, Acinetobacter, Alcaligenes, Aeromonas, Klebsiella, Shigella.
Penicillin, Ampicillin G,Imipenem,Streptomycin, Chhloramphenicol, Erythromycin, Meropenem,Tetracycline,Rifampicin,Gentamicin, Nalidixic acid, Amikacin.
I am going to perform antimicrobial susceptibility testing on a pseudomonas spp and need the guidelines. Thank you.
In occasions, we carry out the antimicrobial susceptibility test, what is the proper intensity for a bacterial (for example Escherichia coli )?
we are trying to evaluate antimicrobial activity of some synthetic compounds using well diffusion method, they are water insoluble, and they were dissolved in pure DMF, but after application of compounds solutions with few seconds the liquid appears to get out from the well to the surface of the agar (as if the volume increased,although when applied they didn’t reach edges of the well), and after 24 hour there is still some liquid that didn’t diffuse remaining in the well, and there is something like precipitate around the well from inside and outside. And when inhibition zone occurred it is the same or smaller than the zone of the solvent, I tried to filter them with 0.22 µl filter, compounds gave inhibition zone similar to that of DMF, and there was less precipitate in the well. (Standard antibiotics gave good reproducible results without problems)
Should I solve the precipitation problem first to take results or is it normal for synthetic compounds and I should take it as negative result.
And I’m afraid to do broth dilution method after filtration sterilization so compound activities decrease.
Can anyone help me in finding the approved source of antibiotic powder for disk preparation to be used for antimicrobial susceptibility test.
The chemical suppliers charge heavy price, and could not be affordable for disk preparation.
Further, I also request you to tell me the quality control measures should I follow (apart from testing with ATCC QC strains) ?
Hello, I have done 96 well plate assay for detection of MIC of antibiotic for particular organism. I took the O.D of the plate and I understood the concentration of antibiotic which inhibits the growth. But i'm not getting how to interpret the results. Is anyone can help me to sought out this problem.
Kindly provide me an information regarding performance of microbroth dilution of antimicrobial susceptibility test.....
Mohan Kumar S
I am trying to purify a antimicrobial peptide having mass greater than 5KDa. After removing biomass from fermentation broth, first I did organic extraction using diaion HP resin and elution with methanol. then I went for acetone precipitation and then cation exchange chromatography. Activity is there till this step. As I load (after dialysis) it onto semiprep C18 or C8, no fraction shows activity. I use 5 -95% gradient of Acetonitrile-water. Two regions shows peaks at 230 nm (Less or negligible absorbance at 254 nm or above) but none of the fractions shows activity. Even the area which has no peak in chromatogram does not show any activity. Can anyone tell me what is actually happening and how to troubleshoot this kind of problem ?
Most studies utilise the checkerboard technique or time kill assays to investigate synergistic relationships but neither of these are particularly high throughput. Can anyone suggest a method for testing hundreds of different combinations that is both quick and reliable. My initial idea is to you a modified disk diffusion assay (one antimicrobial in the culture media, the other on a disc) and then following up the best hits with a checkerboard / time kill assay , however my feeling is as these techniques are so different the results may not correlate well. Are there any other techniques that people use?
I need to find out MIC50 and MIC90 for my clinical isolates using E-test, but by reading reference papers I couldn't make out the step by step procedure. Does anyone know how to calculate both MICs or please suggest some examples and references? Thank you.
We usually estimate the MIC in 96-well microplate with the final volume of 100 uL (95 uL of appropriate diluted antibacterial compound and 5 uL of 0.5 McFarland cultured cells). We also tried with 190 uL of diluted antibacterial compound and 10 uL of 0.5 McFarland cultured cells.
Our results were slightly different.
Can someone explain me why it happened?
I intend to evaluate the quality of few commercial antibiotic preparations in lab against known bacteria. But to keep myself on right track, I need some workflow document to follow the required steps. We can use the disc diffusion or/and agar dilution method. The aim is to confirm that antibiotic preparation contains sufficient conc. of active antibiotic or not?
I'll be extracting compounds from Asparagus racemosus powder to determine synergism with antibiotics on a MIC plate using acetone, chloroform, methanol, and DMSO.
I'm planning on putting 100 g of root in an Ehrlenmeyer flask closed to air in 0.5 L of solvent for each of the solvents, 4 flasks in total. I'll keep them at room temperature for seven days, periodically moving the material around. After, I'll strain the undissolved material, dry it, determine weight and thus concentration of the extract.
Do I have my facts straight? If anyone had any suggestions, I'd greatly appreciate it!
It is the method used to give rapid presumptive information about antimicrobial susceptibility without identification of bacteria. Diarectly from organs.
I want to test bacterial resistance of hand sanitizer by using disk diffusion method. Can I just soak sterile paper disks with hand sanitizer and place them on a culture plate?
I would like to read opinions about this. Because most of the studies use other techniques, using MIC, to assess the antimicrobial susceptibility? Is there a restriction from CLSI to use disk diffusion on this?
There are some reports on specific antimicrobials for particular strains. On the other hand they are not tested to other species of the same genus. So, is it possible to use the same antimicrobial for other species of same genus.
I would like to know why isn't the MIC-Minimum Inhibitory Concentration measured as MIC 50 or MIC 90, instead of just 99.9% MIC or complete inhibition? Is MIC 50 acceptable for new antimicrobial? Do companies buy MIC 50 new antimicrobial to use to develop their products?
If it kills only 50%, what about the other 50%?
I would appreciate getting information on automated bacterial cell viability counters that are available in the market or that you use in your research and recommend for reliable and reproducible bacterial cell viability counts for antimicrobial susceptibility testing.
I am not able to get the interpretation criteria for disc diffusion method as suggested by Clinical and Laboratory Standards Institute (CLSI) for animal pathogens. Can anyone provide me the chart?
Performance standards for antimicrobial disk and dilution
susceptibility tests for bacteria isolated from animals; M31-A3
approved standard, CLSI,
2008 version or its advance version published in year 2012
It would be great to have your opinion in that: what is the valid approach to prepare a standard the inoculum with known cfu/.ml? I prepared an overnight broth culture (18hrs) at 37 C, afterwards the turbidity of culture is adjusted to match that of a 0.5 McFarland standard (108 CFU/ml). How accurate is this method when we are using different strains for same antibacterial testing? Standard methods indicate the use of growth method (2-6 hrs broth culture until turbid) or direct suspension method (from an overnight colonies on agar plate) when preparing the inoculum, but in some publications, a 24 hrs overnight culture is being used then later its turbidity is adjusted to Mcfarland standard. So is the later method valid? I also tried to plot the serially diluted cfu/ml with the OD readings after 24 hours incubation of an overnight culture at 37C, but it didn't give convincing results, there were variations.
I want to associate the results of different antibiotics against S. aureus strains (both MRSA and MSSA). What is the best correlation test? I also want to compare these results with the results of two plant oils.