Hi,
I am checking if my bacteria are resistant to particular antibiotics. I don't want to do the MIC, I would like to have a rapid result since I am testing several antibiotics. What I have done is to grow bacteria overnight and I streaked 100 ul of this on plates at two concentrations. Actually I did not know what concentrations it is better to test to determine if a bacterium is resistant or not. I have chosen a concentration which is the MIC breakpoint value published by EUCAST to determine if a bacterium is resistant or sensitive. For example, if the breakpoint value was 16 ug/ml for the amikacin I tested my bacteria on plates with 16 and 32 ug/ml...Does this method make sense for you? Now, I don't know how to interpret the data in some cases..For some bacteria it is clear that they are antibiotic resistant because I have a sort of lawn on the plate for both the concentrations.For others, I get separated colonies (see the attachments) and I have no colonies on the plates with higher concentration.. Do I have to consider them resistant for you?
Thanks
I would actually recommend to do some sort of standardized testing. This could be disc diffusion assay (Kirby-Bauer) which allows you to test up to six antibiotics on a standard agar plate. Discs are available commercially, or – if you are working with non-commercial compounds – may be prepared by yourself. Be sure to prepare the inoculum for the disc diffusion assay according to the published guidelines (EUCAST). If you need to test several strains/isolates/species against some antibiotics, you might thing about an agar dilution assay (basically, what you have done, except that you inoculate not the entire agar surface with your bacteria; rather, you inoculate them as spots). However, even if you don’t want to measure the MIC you nevertheless should cover a range of concentrations and not just the two you mentioned. None of the available methods are really rapid, but workload/hands-on time differ. I guess that’s what it is about.
Answering your second question: if your isolate growth at a concentration (way) above the breakpoint it may be resistant, yes. If you have sparse growth on the 16 ug/mL plate and no growth on 32, one interpretation is that the majority of the population was inhibited (i.e. susceptible) by the lower concentration while the minor part was not. In this case, 8 ug/mL and lower concentrations would be helpful.
However, prerequisite for correct interpretation of your results is that you have worked under standardized rules. Did you work with freshly grown colonies of your test strain? Have you adjusted the MacFarland before streaking the agar? How long did your adjusted suspension stayed at which temperature before being streaked on the plate? For how long did you incubate the plates? Which type of agar have you used? Do you have a control which allows you to figure out that your agar plates with antibiotics did contain indeed the expected concentration of active compound? These and other parameters are important to judge if your isolate is resistant or not.
I used fresh overnight grown cultures. I did not adjust the MacFarland before streaking (why is this important?) the suspension was taken from 37 degrees shaker (where they were grown overnight) and streaked on LB agar plates..
I think you should use broth dilution method for antimicrobial sensivity. If you want to work rapidly and see many of bacteria are resistant or not, you can use dilution of an antimicrobial with breakpoint range. For example, florfenicol mic breakpoint is 8 ug/ml (EUCAST or CLSI) for enterobacteriaceae, if you want to test your samples only susceptible or resistant, you can use 4, 8 and 16 ug/ml concentration. You can read your results for susceptible (for growth on 4 ug/ml) or resistant (for growth on 8 or 16 ug/ml). This is the easiest way for your bacteria antimicrobial susceptibility.
Your bacteria puririty, concentration, incubation temperature, agar quality and other factors effect your test.
“I did not adjust the MacFarland before streaking (why is this important?)”
Susceptibility testing is normalized to a certain amount of bacteria in the assay. For agar dilution and disc diffusion assays, MacFarland must be adjusted to 0.5 according to CLSI and/or EUCAST guidelines. An overnight grown culture is most likely much higher in MacFarland (i.e. turbidity) and thus you inoculate to many bacteria into your test (may lead to a wrong resistant outcome or at least minor growth were no growth would be observed with the correct amount of bacteria).
As outlined by Muhammed Duman, all the parameters may affect the outcome of your test. While the effects may be minor, when testing with high concentration of antibiotics, meaning far above the breakpoint, results may be influenced when you work with concentrations of antibiotics close to the breakpoint. From our routine work (which is highly standardized according to EUCAST guidelines) we know that MIC’s can easily vary at about +/- 1 dilution step [which in your case would mean (8)-16-(32)].
Interesting topic! The agar diffusion method is definately a more valuable tool than agar plates with integrated antibiotics due to the concentration gradient you get (high to low as you move away from the tablet).
The Youtube video published by Harvard Med. School titled "The Evolution of Bacteria on a “Mega-Plate” Petri Dish (Kishony Lab)" might answer the question on why you tend to see "carpet growth" on plates with low antibiotic concentration and single colonies on higher concentrations, as evolution is a continuous process and spontaneous mutations might lead to resistant mutants that are able to survive in the close to breakpoint concentrations.
I'm not saying this is the reason, but it could definitely be part of it.
I would probably call the bacteria (susceptible)-intermediate-(resistant), but in vitro =/= in vivo anyways, so there are many ways to look at it!
PS: Out of curiousity, what species of bacteria are you working with?
Good luck!
https://www.youtube.com/watch?v=plVk4NVIUh8