Antimicrobial Susceptibility Testing - Science topic

The first new gonorrhoea antibiotic in decades could address a worrying rise of a drug-resistant form of the bacterium. The disease is often symptomless, and can cause infertility if left untreated. In a trial with 930 participants, the new drug, called zoliflodacin, was as effective and safe as two older drugs in curing infections. Researchers warn that zoliflodacin will have to be used wisely to avoid the bacterium developing resistance to it, too...

Best practices for antimicrobial susceptibility testing using disc diffusion involve standardization, quality control, inoculum consistency, appropriate media, quality antibiotic disks, controlled incubation, and accurate interpretation. Challenges include operator-dependent variability, potential false results, lack of automation, issues with fastidious organisms, and a limited range of antibiotics.

The agar-well diffusion method enhances accuracy by utilizing wells for antibiotic/sample placement. The microtitre plate method, based on resazurin, allows high-throughput testing and minimum inhibitory concentration (MIC) determination. However, both methods demand precision and standardization. Overall, while the disc diffusion method remains valuable, alternative methods offer advantages but come with specific technical requirements and challenges. For further assistance, you can follow the following research articles of mine: https://www.researchgate.net/publication/374223210_Bioefficacy_of_Sida_cordifolia_L_phytoextract_against_foodborne_bacteria_optimization_and_bioactive_compound_analysis

An important question, I am waiting for the answer, too...

The common symptoms of Neisseria gonorrhoeae in males are; greater frequency or urgency of urination, a pus-like discharge or drip from your penis that could be yellow, white, beige, or greenish, discoloration and swelling at the penis opening, testicular swelling or pain, itching and soreness in your anus, rectal bleeding or discharge, and pain when having bowel movements. While for females the common symptoms are; watery, creamy, or greenish vaginal discharge, pain or burning while urinating, an urge to urinate more frequently, heavier periods or spotting between periods, pain during penetrative vaginal sex, sharp pain in your lower abdomen, itching and soreness in your anus, rectal bleeding or discharge, and painful bowel movements (Murphy & Raypole, 2023).

The treatment for males and females is somewhat similar because taking a 500 mg intramuscular dose of ceftriaxone is recommended for the treatment of gonorrhea. Though this medication only stops the infection it does not restore the permanent damage done by the disease (CDC, n.d.). The treatment differs for both sexes due to having different genitals. Females may experience cervicitis which if undiscovered and untreated, arising gonococcal infection can result in upper reproductive tract involvement, such as salpingitis and pelvic inflammatory disease. Pelvic inflammatory disease can be proven with pelvic pain, infertility, and increased risk of ectopic pregnancy. Males, on the other hand, may experience urogenital gonococcal which has complications that include orchitis, epididymitis, penile lymphangitis, penile edema, and post-infectious urethral strictures (Volk, 2023).

References

CDC. (n.d.). Detailed STD Facts - Gonorrhea. CDC. Retrieved September 8, 2023, from https://www.cdc.gov/std/gonorrhea/stdfact-gonorrhea-detailed.htm

Murphy, M., & Raypole, C. (2023, June 20). Gonorrhea: Symptoms, Treatment, Causes, and More. Healthline. Retrieved September 8, 2023, from https://www.healthline.com/health/gonorrhea#symptoms

Volk, J. (2023, April 17). Gonorrhea - StatPearls. NCBI. Retrieved September 8, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK558903/

The EQA program contributes to the long-term monitoring of antimicrobial resistance trends in N. gonorrhoeae by promoting data accuracy, benchmarking laboratory performance, detecting emerging resistance, identifying geographic variations, assessing trends over time, informing public health policies, supporting research, and fostering international collaboration. This comprehensive approach helps address the ongoing challenge of antimicrobial resistance in gonorrhea. Based on consolidated guidance by Pan American Health Organization 2020, Antibiotic resistance in N. gonorrhoeae — given that there are therapeutic alternatives for fever, gonorrhoeae is quickly becoming a frequent antibiotic, raising concerns on a global scale. When local AMR surveillance is not available to support national treatment guidelines, the WHO has revised its advice for empiric treatment to dual antibiotic therapy (ceftriaxone + azithromycin). In addition to the lack of comprehensive gonorrhea surveillance to provide a national-level burden of illness, therapy for gonococcal infections in Latin America and the Caribbean primarily relied on clinical diagnosis. PAHO/WHO to support nations in creating or enhancing AM monitoring systems for N. gonorrhoeae. For the control and eradication of N. gonorrhoeae, regular preventative measures, early diagnosis, and efficient treatment considering local levels of AMR are crucial. By 2030, gonorrhea will be a serious public health issue. PAHO/WHO reiterates their advice to Latin American and Caribbean nations to give priority to the establishment of AMR monitoring in N. gonorrhoeae as a crucial part of their STI surveillance system.

Reference:

Neisseria gonorrhoeae Antimicrobial Resistance Surveillance: Consolidated Guidance. Pan American Health Organization. (2020). https://iris.paho.org/bitstream/handle/10665.2/52307/9789275122372_eng.pdf?sequence=1

A comprehensive approach is vital to validate the detection of Neisseria gonorrhoeae. This includes culture confirmation and nucleic acid amplification tests (NAATs) for diagnosis, with NAATs validated using control samples. Immunochromatographic tests must compare results with culture or NAATs and employ control samples. MALDI-TOF mass spectrometry identifies the bacteria, validated by comparing results with established methods. For antimicrobial susceptibility testing (AST), validation involves using reference strains and adhering to guidelines like CLSI or EUCAST. Regular quality control strains ensure testing accuracy. Participating in external quality assessment (EQA) programs and adhering to recognized standards are crucial for accurate diagnosis and antibiotic resistance surveillance.

Reference:

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., Siatravani, E., … Unemo, M. (2019). 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), 281. https://doi.org/10.1186/s12879-019-3900-z

Work from stock lyophil with limited transfer - ala USP 51.

According to the journal mentioned appropriate prevention, dignostics, and surveillance is needed to be managed, especially when there is an emergence and spread of antimicrobial resistance of Neisseria gonorrhoeae. Participating in an External Quality Assessment (EQA) program for Neisseria gonorrhoeae antimicrobial testing is significant because failing to do so can produce several consequences for both laboratory and public health.

The consequence of not participating the EQA program are; lack of information of the Euro-GASP regarding the resistance of N. Gonorrhoeae, possibly not able to provide the right treatment due to not being aware of the possible resistance of the said causative agent, and losing the opportunity for the laboratory to be evaluated if it has the capacity to detect occuring new, rare, and increasing antimicrobial resistance phenotypes.

Reference:

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

Molecular tests are the gold standard for diagnosing N. gonorrhoeae which can be performed in the lab or at the point of care. The agar dilution method is said to be the gold standard method for antimicrobial susceptibility testing in Ng. according to WHO.

Meyer T, Buder S. The Laboratory Diagnosis of Neisseria gonorrhoeae: Current Testing and Future Demands. Pathogens. 2020 Jan 31;9(2):91. doi: 10.3390/pathogens9020091. PMID: 32024032; PMCID: PMC7169389.

According to the European Committee on Antimicrobial Susceptibility Testing, inoculation agar plates, should be at room temperature before inoculation. The first 15 minutes is about the usage of adjusted inoculum suspension within 15 minutes of preparation. The second 15 minutes state that the disks should be applied within 15 minutes of inoculation because if it is left at room temperature for a long period before disks are applied, the organism might begin to grow which will result in an erroneous reduction in sizes of inhibition zone diameters. With that being stated, according to Anna King and Derek F.J. Brown in their journal entitled Quality Assurance of Antimicrobial Susceptibility Testing by Disc Diffusion, if the problem includes too large or too small zone diameters with all antimicrobials, the following should be observed:

1. Depth of agar should be checked, if it is incorrect, the amount of agar should be adjusted.

2. Inoculum size should be checked.

3. Ensure that the discs are applied to the inoculated plates within 15 minutes and that the plates are incubated within 15 minutes of the application of the discs.

The inclusion of the 15-15-15 rule in the troubleshooting controls proves that it is done to reduce errors in susceptibility testing in disc diffusion.

There are plenty of relevant articles on Google Scholar - e.g. https://academicjournals.org/journal/AJMR/article-full-text-pdf/15879FE11467.pdf

Yes it is ESBL pos.

I think that this study by Yudiati, E. et al. may somewhat help you.

Silver nitrate has a substantial impact on metabolism in E. coli so it is likely that even at sub-lethal doses its metabolism is simply less efficient and a lower OD is attained.

@Rohan krishnan yes we generally prefer the above mentioned species because that generally found infections are because of them. Aim of antimicrobial coating /surface or drug is to protect against the micro-organisms it is formulated for. General testings are based on generally found bacteria so that antimicrobial drug, coating/surface should atleast work against them.

Yes it is possible that microbes will develop antimicrobial resistance (AMR) because of long exposure & therefore need to develop new antimicrobial agents.

Sarita Otta Thank you for your reply

Hello,

I send you CLSI 2020 30th edition.

Best regards,

Of course you are right , PK/PD parameters are very important and I agree that the best option is nitrofurantoin, because it concentrates in urine, and of course because we have to make a prudent use of antimicrobials in our fight against antimicrobial resistance

The MIC’s of the tested essential oils were determined by the Clinical Laboratory

Standards Institute’s method of broth microdilution.

"Clinical and Laboratory Standards Institute. (2006) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Seventh Edition. Clinical and Laboratory Standards Institute document M7-A7 [ISBN 1-56238-

587- 9]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA."

Try to perform the bacterial peak in mueller hinton agar or some other transparent agar, wait over nyght and dilute 5 colonies in 1.5 ml of sterile saline, homogenize and observe the turbidity and perform the antibiogram.

Free online HTML access is available at the CLSI site: https://clsi.org/standards/products/free-resources/access-our-free-resources/

However, to get the PDF seems to be expensive, so if you want this, check with your institution to see if they already have it. (Also, the link to get the M100 PDF doesn't seem to work now, even though the adjacent link for the M60 does.)

Hello Emmanuel,

Based on the experience of working with various bacteria isolates and testing the activity of various antimicrobial agents against bacteria isolates, I recommend the use of MHB (Mueller Hinton Broth-Oxoid) in your micro-dilutions. This media is known to support the growth of bacteria as well as its constituents do not react with antimicrobial drugs.

You can serial dilute out your antimicrobial drugs (to be tested) using the broth and inoculate standardized bacterial cell suspension. After incubation at 37 degrees Celsius for 18-24 hours, you can subculture by pour plating all wells showing no turbidity (growth) using Trytone Soy' Agar and incubate to determine MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration). All the best.

Hanna is correct, there is a big difference between the MIC and the practical useful concentration for molecular biology applications like plasmid transformation. I would also add that ampicillin in solution does not have a long shelf life, so be sure to use fresh stock solutions if you need to measure MIC's and such with accuracy.

According to CLSI guidelines, MIC, broth dilution, agar dilution is more reliable than DDT. for example, Susceptibility to Vancomycin is recommended only when tested by determination of MIC but not by DDT, since the latter is not reliable.

Regards

For determination of AST, researchers should follow the CLSI guidelines strictly.

The method you described above resemble "Agar dilution method" which recommended by CLSI (document CLSI- M02-A12).

In this method, serial dilutions of the drug are prepared in agar and poured into plates. The Advantage of this method over The traditional disk diffusion method that many strains can be inoculated on each plate containing an antibiotic dilution.

Regards

Hello Nirvana Jaganath

Just like what others have said, you have to load antibiotic solution onto the disc and let it dry first before you place it on agar. As for the volume to be added to the disc, it depends on the disc concentration you desire. For example, you have antibiotic stock solution of concentration 10 ug/ul. If you want to prepare prepare disc concentration of for example (30 ug/disc) then you need to add 3 ul of the stock solution per disc.

10 ug/ul x 3 ul/disc = 30 ug/disc

The determination of antibiotic discs concentration and types of antibiotics used for susceptibility testing could vary depending on your organism. You can always refer to guidelines by Clinical and Laboratory Standard Institute (CLSI). Always refer to the latest version

Attached herewith is the CLSI guidelines for 2016.

Hope it helps

we use the agar dilution methods according to the CLSI.

Chocolate Mueller Hinton Agar has been developed for fastidious organism susceptibility testing , originally for Haemophilus species and A. pleuropneumoniae and H. somni (both veterinary pathogens).

Currently the CLSI suggests the use of Haemophilus test medium for H. influenza and H. parainfluenzae species.

If you are testing streptococci I would suggest that it is not appropriate as it has X and V factors which were added to this media specifically to allow the organisms above to grow.

CLSI quality control ranges and interpretive criteria for Streptococci, Listeria and N. meningitidis were developed using 5% sheep blood Mueller Hinton agar . So I would not recommend the use of chocolate Mueller Hinton for these species.

My Dear, please read in down pdf

You will find what you want in the updated version of CLSI 2016 document (attached file).

Regards

 

Hi:

You can use the following practical reference (attached file). It is very helpful: SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA

Regards

Hi:

If you have a pure substance, CLSI guidelines say you should apply two-fold agar dilution method for determination of MICs (ranging from 0.008 mgl/l to 128 mg/L).

Regards

Microdilution  is the best. It is recommended by  CLSI and EUCAST guidelines. It is   advantageous by  its easy to handle, cost effective, and most standard method.

Thanks for your suggestion, Dr. Hölzel. You are right, I only need the MICs..I´ve made an error in reasoning for I used color change as indicator. Here I also have semi-positives and I usually marked positives, semi-positives and negatives manually on a default sheet with the panel design, same as you do with the MICs. Then I transferred the results to the office computer later. I will modify my documentation according to your suggestion, thanks for pointing that out.

Apart the good properties of DMSO, as early reminded by Dr Gil and Dr Elgailani, it has also a very bad smell. You may check it directly by sniffing from the bottle. This bad odour might have a bad impact on the sensorial impressions of your panel test. I would suggest to use a different solvent, more volatile than DMSO so its own odour will be readily eliminated from the treated materials.

I'd suggest to use different concentrations. If you still want the roots to grow considerably, go for a range of 15nM-100nM. In our hands, everything higher than 100 nM NAA strongly inhibits primary root growth. 

Hi Suzan M. Ragheb:

EUCAST differentiates between clinical resistance and the associated clinical breakpoints, and microbiological resistance and epidemiological cut-off values. 

Clinical resistance and clinical breakpoints terms are common to EUCAST and CLSI.

Both EUCAST and CLSI provide for three categories of identification: susceptible, intermediate and resistant.

EUCAST introduced the term, ‘microbiological resistance’, and it also presents epidemiological cut-off values (ECVs, also referred to as ‘ECOFF’) for antimicrobials against a wide range of bacteria. These ECVs are determined on the basis of the distribution of MICs for an antimicrobial and a bacterial species.

The differences between ECVs and clinical breakpoints, the principles of which apply to all antimicrobial compounds, have been adequately addressed by several authors (1, 2, 3).

1. Bywater R., Silley P. & Simjee S. (2006). Antimicrobial breakpoints – definitions and conflicting requirements. Vet. Microbiol., 118 (1–2), 158–159. E-pub.: 17 October 2006.

2. Silley P., de Jong A., Simjee S. & Thomas V. (2011). Harmonisation of resistance monitoring programmes in veterinary medicine: an urgent need in the EU? Int. J. antimicrob. Agents, 37 (6), 504–512. E-pub.: 3 February 2011.

3. Simjee S., Silley P., Werling H.O. & Bywater R. (2008). Potential confusion regarding the term ‘resistance’ in epidemiological surveys. J. antimicrob. Chemother., 61 (1), 228–229. E-pub.: 20 November 2007.

Yes, you can read results of microdilution sensitivity test  without using microtiter plate reader, if you don't have the latter.

I think it depends on the value of "I". If it was closer to the breakpoint of "Sensitive" , it should considered Sensitive, otherwise If it was closer to the breakpoint of "resistant" , it should considered Resistance. However, you have first to re-test your organism three times by DDt before you determine it was "I" or not.

Please find attached ClSI M07-A9 2012. In fact the CLSI 2015 version has no greater differences from version CLSI 2012

Follow the guidelines of CLSI M-02 document (attached)

Hi, for order, selection, and application of antibiotic disks, you should follow the guidelines of CLSI document:

Hi, I think that the attached article would be helpful:

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

I agree with you that an MIC makes more sense in this context.

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 Fahad. You can try Clinical and Laboratory Standards Institute (CLSI) latest guideline in 2016 here: http://em100.edaptivedocs.net/dashboard.aspx

Please let me clarify: Although the links and documents provided above are generally very useful, you need to know two basic facts:

i) you should follow the instructions of the source of your breakpoints or vice versa - breakpoints are only valid under specific conditions and MICs could be altered by methodological parameters. Thus, if you decide to follow CLSI instructions, then do not refer to the EUCAST breakpoints.

(Personally, I would suggest to choose the European standard for testing of European isolates, bit CLSI is internationally valid, of course. It could be worth checking the actual differences of the methods for your genus / species of interest; sometimes there is an "overlap" in the allowed range of parameters (inoculum, time, temperature), so you can manage to follow both standards at once by carefully adjusting your parameters to this "overlap".)

ii) EUCAST follows the ISO standard now (ISO 20776-1 (2006)), I doubt that the 2003 document is still up to date, but I did not check.

iii) Unfortunately, this is not a public document, so I doubt that it will be uploaded here. This is the link to the distributor: http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=41630

0.5 MacFarland standard is recommended for standardizing the orga

We use DMSO and dissolve compounds at 25mM all the time. We will not choose any compounds that have activity at higher than 100uM final concentration, so we don't use much of the compounds/experiment with this stock concentration. 

In my experience, IC50 is not used to measure inhibition of bacterial cell growth. It may be used for other microorganisms. The reason is that the antibacterial compound is normally tested as a series of 2-fold dilutions, and the growth inhibitory effect of the compound is usually observed over a very narrow concentration range, one or two dilutions. A plot of bacterial growth versus concentration of compound often is too steep to calculate IC50 meaningfully. That may be why the MIC is used instead with bacteria. For bacteria, the MIC is more of an observation than a calculation. In a broth microdilution assay, one simply observes the lowest compound concentration in the set of 2-fold serial dilutions at which the bacteria do not grow to stationary phase in one day from a dilute starter culture. MICs are normally reported in µg/ml.

By the way, MBC, minimal bactericidal concentration, is the lowest concentration of the compound that kills a culture of bacteria. This is distinct from MIC, which is the minimal concentration that inhibits growth of a dilute culture of bacteria.

Philippines experience may be helpful

Dear Dhanshree

To interpret the MIC you have to see the absorption of blank/media control in comparison to growth control. And you have to make sure that when you added the inoculum there was zero or absorption must be equivalent to blank media. Now see the absorption of compound MIC line which must have difference of 90% (MIC90) and 99% (MIC 99).

Say if growth control is giving an absorption of 1.0 O.D and MIC well is giving 0.1 then there is 90% difference (MIC90) and if there is 0.01O.D  then this is MIC99.

I hope this would help.

Hi,

For microbroth dilution of antimicrobial susceptibility test you can perform the broth dilution test as recommended by  "Clinical Laboratory Standard Institute  Guidelines" (CLSI). 

or you can see this ariticle :

"Determinination of MICs by Jennifer M. Andews" Journal of Antimicrobial Chemotherapy 2001, 48, suppl, S1, 5-16.

The article is attached herewith. Please find the attachment.

The peptide is pretty large, more a protein than a peptide, so my first thought was that it has a tertiary structure that was denatured by the reverse-phase chromatography conditions. However, since you already did organic extraction and acetone precipitation with retention of activity, this seems unlikely. My second thought is that the peptide simply stuck to the reverse phase column and never eluted. It might require methanol to get it off. Maybe you should switch to a C3 or C4 column.

Another possibiity is that the activity of the peptide was destroyed due to exposure to the ion-pairing reagent, particularly if it was a strong acid like TFA. This might happen if the peptide has an unusual amino acid or acid-labile post-translational modification.

For this small protein, you might try using aqueous gel filtration chromatography and hydrophobic interaction chromatography for purification at neutral pH, rather than organic solvent-based methods like organic extractions and reverse-phase chromatography.

You could consider a luminogenic high throughput system, that utilizes ATP of living organisms in order to produce oxyluciferin from Luciferin. The amount of light produced would then correlate with your living microbes. There are tests out there that use this system to analyze the microbial contamination of food.

So why not put this into a 96 well format?

Have a look at this:

and this:

If your compounds are insoluble in water the best way to perform is to use DMSO ( see Vijay answer's) but you have to be very careful because DMSO is toxic. So you have to test the maximum DMSO concentration you used as control condition to evaluate the impact on bacterial growth. In my lab we only used broth method, because with agar plates you sometimes have troubles of diffusion especially with insoluble compounds

Hi Simone,

What about sinking of cells on the bottom of well? This is a problem with bigger cells (e.g. yeast) - is it posible that some of your cells sink to the bottom? Because this would then have a great influence, since 10ul of cells on the bottom of well would result in different cell concetration (relatively for the first milimeter above the bottom) when compared to 5 ul of cells (I assume MIC is higher in bigger volume?). However, if you shake, than I would count on oxygen transfer (surface/volume ratio) as others have suggested.

Oh, another thought - evaporation reduce smaller voume relatively more than bigger volume if both have same surface.

Regards, Jure

Read the article "The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic Susceptibilities of Bacterial Biofilms".

It is a9 6 well plate with 96 pegs attached to the lid of the plate where you can grow biofilm. The plates are available from innovotech and the assay is called MBEC i.e. Minimum Biofilm Eradication assay.

You can follow gudelines of CLSI, or there are documents of EUCAST and BSAC available online at:

You can find there also acceptable MIC limits for antibiotics against recommended quality control strains

Very good the answer of Sebastian. You should know what is the best solvent for your antimicrobial compounds. Do you try to use a Soxhlet?

Best regards

Thank you Mr. Ali 

Il existe un document émanant de la Société Française de Microbiologie concernant la réalisation et l'interprétation des antibiogrammes dans le domaine vétérinaire. Vous pouvez le trouver sur le site de la CASFM.

maybe you could try with cylinder plate by difusion method, if you have trouble with the disk.

There are serious limitations to the use of disk diffusion method.  Results may be unexpected or borderline.  In such cases another method of testing may be required or the test may need to be repeated for confirmation.

Although widely used for antimicrobial susceptibility testing, the disk diffusion method is not applicable to some organisms.  The following are examples of organisms to which disk diffusion method cannot be applied

1. Microorganisms that are fastidious, slow growing or have special growth requirements cannot be tested by disk diffusion method

2. Mycobacterial and fungal susceptibility testing requires specialised technique that are usually available only in reference laboratories

3.  Special techniques may be required to detect penicillin resistant Streptococcus pneumoniae, methicillin resistant Staphylococcus aureus, and aminoglycoside resistant Enterococcus faecalis,

In addition, disc diffusion methods may indicate in vitro susceptibility of certain agents for some organisms, despite lack of therapeutic efficacy in actual practice, e.g. Salmonella typhi susceptibility to aminoglycosides and enterococcus susceptibility to cephalosporins.

So the answer to your question is yes, except that certain antibiotics can be problematic to test with the disk diffusion method because of the specific physiochemical properties of the molecules. Vancomycin, colistin, and macrolides such as clarithromycin have higher molecular weights and therefore diffuse very slowly in agar.  The limited diffusion and poorly resolved concentration gradient around these disks result in only a few millimetres of difference in zone sizes between susceptible and resistant strains, which could result in a potentially ambiguous reading.  Results can also be influenced by the positioning of the light source on the plate. Plates are often read with use of reflected light, with the exception of linezolid, oxacillin, and vancomycin for both S. aureus and Enterococcus species. In these cases, the zones of inhibition should be measured by using transmitted light in order to ensure an accurate measurement of zone diameter. If results are unexpected or borderline, another method of testing may be required or the test repeated for confirmation.

Professor Sayed S Bukhari MD FRCPath

I agree very well with Thavasi, but I work on protozoan parasites and drug resistance/drug development rather than bacterial, but the issues are surprisingly similar. In short, you can get any variation: some dugs are highly specific for a subspecies. For instance pentamidine is a drug with EC50 value of 2-3 nanomolar against Trypanosoma brucei brucei but is 100-fold less active against Trypanosoma congolense. The reason is unique transport proteins expressed in brucei but not congolense species, or the also closely related Leishmania species. On the other hand some driugs have good activity against a large number of species, for instance nito-heterocycles such as metronidazole. In that case the activity is often related to a more general cellular toxicity, however, less specific.

Finally, I agree with the earlier posters that resistance to many drugs can be induced very quickly. But yet again there is no universality here. For instance, we were unable to induce any level of resistance to curcumin in trypanosomes, even after a year of culturing with exposure, using protocols that worked rapidly with several other drugs. 

The current breakpoint criteria remain acceptable in minimizing intermethod discords, the alternative susceptible breakpoint criteria proposed by combining pharmacokinetic/pharmacodynamic (PK/PD), microbiology MIC population analyses, and clinical success parameters possess improved intermethod agreement for the ESBL screening drugs.

Hi!

The concept of MIC 90 and MIC 50 are terms widely used in standards such as CLSI. In the case of fungi, is considered to antifungal treatment success not only depends on the agent itself, but also the host immune system, eg. MIC50 is determined assuming that the other 50% of the elimination of the fungus will make the body itself.

Another important aspect to the use of MIC 50 and MIC 90 depends on the nature the agent: For fungicides (eg antotericina B) is used MIC90 and fungistatic (eg fluconazole) is used MIC50

I know of a FACS based method as well as a method that works on a plate reader after some validation, but neither of these is automated.

found this link to second edition http://isoforlab.com/phocadownload/csli/M31-A2.pdf

Hi Dima,

Why don't you test the growth of each of your strain (ie do it three times for each strain). For example, at each OD measurement check the CFU by plating. Also record the time each measurement is taken. Plot on graph to determine the the growth characteristics of your bacteria, ie lag, log, stationary phase. You would want to use each of your strain at the same phase. If you have good methodology (good dilution and plating techniques, keeping conditions similar for all strains [ie. not leaving some cultures sitting around while testing others])and always use culture at the same determined OD, then you should get similar numbers of bacteria in your standard inoculum (though always check inoculum bacterial numbers by plating). Many strains can be in the declining phase by 24 hours (depending on many factors) and hence will give variable results. What phase you want to use depends on your aim but often mid-log phase growth is used. So, if you know your growth characteristics, you can adjust the bacterial numbers to 108CFU/ml safely by diluting. Once diluted you can check whether the density is similar to your McFarland but don't forget, visual checking is subjective especially as the colour of the culture broth can influence your perception. It takes a bit of work initially but then later you save time and you can be sure that you have a solid system.

I performed the Pearson coeficiient between MAR value of each strain and the MIC value of each oil sample.

For environmental friendliness then supercritical CO2 and H2O are handy and trendy as you rightly mention. I personally have been working on extraction of chemicals from biomass using SC-CO2 too. It is top on our considerations in view of the friendliness of the technique to the environment. Thanks.