When dealing with plant crude extracts, antimicrobial and/or antibiofilm compounds might be present in them. So, what we usually do is that we test for MIC of the crude extracts and then if the crude extracts have MIC values, the antibiofilm assays would then utilize sub-MIC concentrations to see the effect of the crude extract on the formation of biofilms.
In my lab, in summary, we mix together the bacteria and crude extracts (at sub-MIC concentration) and let the bacteria form biofilm. After 24 hours, the biofilms would be quantified using crystal violet method. My question is, would the use of sub-MIC affect the growth of biofilm at the end of the experiment? I raise this question because in theory, even though sub-MIC concentration does not kill the bacteria, the presence of that certain amount of the antimicrobials in the crude extract might inhibit the growth of a fraction of the primary inoculum. Let's say I have 500,000 bacteria inoculated, at MIC concentration, all of the bacteria would be inhibited. But at sub-MIC concentration, perhaps 100,000 are killed (hypothetically). So, because the primary inoculum now is less compared to the untreated bacteria, the growth rate might be altered (insignificantly or significantly).
After 24 hours of incubation, would this affect the final amount of biofilms formed? And in the end, would the conclusion stating that the crude extract has antibiofilm effect be true? I would argue that the effect of antimicrobial at sub-MIC level might had contributed to this observation. Feel free to comment.
which culture are u using? mixed culture with different species of biofilm producers or pure( one species of biofilm producer) well it depends upon the culture u have chosen since there is diversity in biofilms. if ur crude extract has antimicrobial activity it may also show antibiofilm activity at sub MIC levels,i dont think pretreated culture is required to prove the antibiofilm activity of ur extract i suggest u to test the antibiofilm activity of extract by taking only on one type of biofilm producing strains.
Not all bacteria are killed at the MIC value, you have to proceed to the MBC to get that concentration and generally this concentration is higher than the MIC. I work with biofilms in the oral environment and many of these are commensal species that contribute to the ecological balance in the oral ecosystem. Working at sub-MIC concentration ensures that the biofilm is still allowed to form but controlled at its minimum.
I agree with Nuzhath that you have to test the antibiofilm activity as well and this may include antiadherence study.
I fear pellicle assay would not be significant unless you set controls with the same concentrations of plant extracts but in a medium where bacteria do not form biofilms, to test bacterial growth separately. Not only because what you mentioned, but also because certain plant extract might contain nutrients that bacteria can use to grow at a higher rate and, therefore, form a thicker pellicle. It is a general problem of this particular assay, and I do not understand why some results lacking the mentioned controls are accepted by referees... Good luck!
Currently I'm working with Staphylococcus aureus biofilms in pure culture forms. I have five strains that produce biofilms and the tests were carried out separately for each strains.
The following is my research plan:
1) Determine MIC for the plant extracts (for antimicrobial study)
2) Use sub-MIC concentration against S. aureus biofilms (for antibiofilm study)
So, my concern is that whether the usage of sub-MIC concentration affects the growth of biofilm since there might be less viable bacteria due to the presence of antimicrobials at that low concentration. If this is true, then it affects my conclusion that the extract has antibiofilm property. How would I know that the extract purely has antibiofilm property rather than having the antimicrobial compounds acting by inhibiting the bacteria.
I'm currently performing cell viability curve and biofilm growth, with treatments and negative controls. Hopefully this will answer the question.
sub inhibitory concentration of crude may so effective in inhibition of biofilm because as you know biofilm cells are 1000 times more resistance than planktonic cells so I would like to suggest that you should increase the concentration.
Many time low concentration of antimicrobial can cause the dispersal of biofilm cells but it is depends on which type of antimicrobials you are taking
Besides Sadhana Sagar considerations, we know about transitory decreasing of some microorganisms under sub inhibitory concentration effect affecting caracteristics of biofilm, in a not static situation.
Just to update everyone here. I did the experiment and I found that the growth curve for sub-MIC treated bacteria resulted in a tremendous delay in start of exponential phase i.e. exponential phase for untreated cultures started at about 5 hours whereas the exponential phase for the sub-MIC treated cultures started at about 16 hours. The outcomes observed clearly suggest that there was indeed antimicrobial effect acting on the treated cultures, although they reached the same maximum growth as the untreated cultures at 24 hours. So, in my opinion, I would say that the reduction in biofilms formed for the sub-MIC treated cultures might be due to the antimicrobial effect. If the growth has been delayed significantly, how much biofilms can they grow, given the same amount of time as the untreated cultures? There are arguments in my research team regarding this question. Some say that since both untreated and treated cultures reached the same level of growth in the end of the experiment, they should form the same amount of biofilms provided that no antibiofilm compounds are present. On the contrary, some believe that since the treated cultures only start the exponential phase about 10 hours later than the untreated cultures, the amount of biofilms that they can form would be less. In spite of this argument, one suggestion that I heard was to increase the time allowed for the biofilms to grow i.e. 48 hours instead of 24 hours. My concern is that if the antibiofilm compounds work on principles such as one-compound-one-bacteria, then the act of allowing more bacteria to grow might conceal the antibiofilm effect. However, I'm not entirely sure on whether this theory is even plausible.
Thanks for the update. In my opinion, a 10 hours delay before log phase is clearly a signal of antimicrobial activity. The fact that treated cultures end up reaching the same level of growth than untreated ones might be due to the rise of resistant mutants. An alternative explanation is that the antimicrobials select for a particular bacterial sub-population (i. e. matrix producers, or persisters), killing the rest. The delay would be caused by the lower amount of bacterial cells that start growing after the killing.
In both cases (mutants or sub-population), you would be comparing very different things, since the composition of the bacterial populations in the untreated cultures would be very different.
A quick test you can do is to recover bacteria from the treated cultures at 24 hours and re-inoculate with these bacteria. If mutants arose during the initial experiment or if a special sub-population was selected, you would expect these bacteria to grow faster the second time.
By the way, the way the growth is measured is critical in these kind of studies, since optical density can be affected by bacterial shape, cell lysis, etc. Plating or live/dead staining followed by flow cytometry would be more informative.
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