Test for production of antimicrobial substances by bacteria - (Apr/28/2013 )

Hello,

I isolated bacteria from a natural lipid mixture which has shown to exert antimicrobial effects in the diffusion test. I am interested in whether the antimicrobial substances are of bacterial origin or if the native lipids themselves are antimicrobial.

The bacteria are lipophilic. When growing them in liquid medium supplemented with Twee-80 as lipid scource and afterwards performing a diffusion test with the liquid culture there is no antimicrobial effect.

But it might be that the bacteria use the lipids in the mixture, modify them and thereby conferring antimicrobial activity.
How can I test for that?

On the one hand, I need to include the lipid mixture in the growth medium, on the other hand I have get rid of them before conducting diffusion test to make sure that only bacterial metabolites will be included in the diffusion test.


My idea is preparing agar medium where I substitute Tween-80 for the natural lipid mixture. I inoculate the agar plates with the isloated bacteria and incubate. The colonies will be taken and suspended in water and a diffusion test will be conducted.

What do you think? I am wondering if the microbial metabolites will diffuse into the agar in that the diffusion test will lack any antimicrobial activity - even if the bacteria produced antimicrobial substances from the lipid mixture.

Thank you very much!

Describe the "natural lipid mixture."

Tween80 as lipid source? Do you add the lipid mixture too?

What bacteria do you use? usually they aren't that lipophilic, except a few that produce a hydrophobic coating, and these are susceptible to the presence of surfactants like Tween80

Tween80 is used as surfactant in media containing hydrophobic substances (e.g. hydrophobic C-sources like PAHs). Did you test the media with only Tween80 to say they can use it?

Tween 80 is used in alot of applications - it's a nonionic surfactant used in drug and cosmetic testing to neutralize preservatives like paraben. Others have used it as media additions to source unsaturated fatty acid (oleic) for mutants that required this in their membranes and I've also seen it reportedly used as substrate- both latter app'spresumed esterase activity to separate the fatty acid from the ethyoxylated sorbitan.

Think the original post reported no inhibition when grown with Tween 80 - only saw it in the bacteria/undescribed "natural lipid" combination. With only this, you really can't separate the two very effectively. You'd have to find some uncontaminated "natural lipid" to test -with this, one way to address this would be to incorporate the clean lipid mixture into an agar medium - inoculating a lawn of the indicator(of inhibition) culture and spot inoculating the bacterium in question.

Hello,
thanks for your suggestions!

@Xabi
The addition of Tween is essential since there is no detectable growth without. The isolated bacteria are Corynebacteria. This genus includes several lipophilic species which can be cultured by the addition of Tween-80. For C. jeikeium it has been shown that it lacks type I fatty acid synthase genes which is responsible for the synthesis of a large variety of fatty acids in C. ammoniagenes. Lipids in Corynebacteria are typically of straight chain saturated and monounsaturated and they are needed to form corynemycolic acids that are attached to the cell wall to establish a permeability barrier. Tween-80 represents a monounsaturated fatty acid and is widely used for the cultivation of Corynebacteria.

@Phil
It will be hard to find a bacteria free source but I could try including the lipid in the agar then autoclaving and test if my indicator strains will grow. If so, I could follow your recommendation.
One prolblem I will be likely to encounter is that my indicator strains will dominate and overgrow the isolated Corynebacteria. The latter are fastidious and slow growing and I normally incubate for 3 days for well isolated single colonies.
I have never done spot inoculation before, so it would be nice if you stated your opinion.

What maybe works is cutting and removing a small piece of the lipid mixture-containing agar and filling this hole with the same type of agar of ~45°C which has been inoculated with the Corynebacteria. Afterwards, I incubate for two days and the bacteria will hopefully grow inside the agar. Then, I inoculate the entire plate with the indicator strain and incubate for another one day checking for a zone of inhibition around the cut agar piece.

What do you think?

Yup - that's what I meant in offering the oleic acid as a source of unsaturated fatty acid - as you say monounsaturated.

i don't see much you can do that's defensible with the bacteria-lipid starting material. From your discussion, can we assume this natural lipid is obtained from skin? There's prob. enough data re. composition of skin lipids that you can assemble a reasonable mixture modeling the system that wouldn't include your isolate. The agar manipulations will be a little awkward but you can figure something out. Be aware that esterase-produced fatty acid can affect pH sufficiently to inhibit. Adding buffered media to test might help disclose if inhibition is seen and you could also include or add after growth Calcium salt to make the fatty acid soap.

Being a Corynebacteria you are right. To incorporate the lipids into agar you can check the protocols for lipase activity using oils such as olive oil. Basically you'd need to emulsify the oil into the agar after autoclaving. The best instrument is the Ultraturrax, but a common blender properly autoclaved works well too. Depending on the Tween and lipid concentration you can try to autoclave together and check if the lipids remain emulsified.

You should sterilise the lipids though it may be a tough issue. In some places dry heat is suggested but I'm aware that it may alter the lipid composition. Irradiation would be the best option but unavailable for almost everybody. If you wanna try sterile filtration, Millipore Fluoropore might be one of the few options. I know these filters can be used for almost anything: alkalis, acids, organic solvents,... but I'm not sure if they will be able to filter oily substances properly

Doubt filtration would work for emulsions. In any case, the natural lipid/bacterial mixture identified as having potential antimicrobial activity already has a bacterial presence so sterilization by any means would be largely after the fact. You could try other lipid mixtures If the question is just the capability of the microbe, but you really ought to get one of similar composition to the natural if that (assume in situ efficacy) is your primary concern.