Effect of Probiotics on Antibiotic Resistance

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ayobacteria
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Effect of Probiotics on Antibiotic Resistance

Post by ayobacteria »

I had this idea for a science fair, I was thinking I could grow E. Coli in the presence of different probiotic foods on an agar plate, and then expose those to another agar plate with antibiotics to test the effects.

My question is about the experimental design for this experiment. I was planning on putting the probiotic foods into the LB agar broth itself, and then plating the E. Coli on top of that. But what is the best way to put food in the broth? For example, if I put some yogurt in the LB broth, autoclaved it, and poured it into a plate, would there be no problems? And how much yogurt would I put in?

Or for cheese, would I have to grind the cheese up into a paste, put the cheese in the broth, autoclave it, and the pour it into the plate?

Another question I had was, after E. Coli colonies form in the presence of the antibiotic, how do I transfer the E. Coli to another agar plate with antibiotics?

Also, I have access to a university metabolic analysis and engineering lab.

Thank you so much, any help is extremely appreciated!!!
brandimiller610
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Re: Effect of Probiotics on Antibiotic Resistance

Post by brandimiller610 »

Hi ayobacteria,

I really like your ideas for your project! I think you might face some challenges with your proposed methods though -- for example, you would need to establish methods for isolating the probiotics from these foods. Since you want to focus on the antibiotic resistant properties of probiotic bacteria, why don't you start with the pure cultures (these are what would be used anyway for fermentation to make yogurt, cheese, etc.) You could decide on which bacteria strains you want to use (Lactobacillus, Bifidobacterium, Streptococcus, and even some strains of Enterococcus are being screened and tested in current studies for their probiotic properties), culture them/transfer them to sterile discs, and then use the disc diffusion assay to determine their antibiotic properties against E. coli. You might even be able to test the antibiotic properties of the strains individually and together in culture. Here is a paper that describes these methods: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942577/

For the E. coli (or any bacteria), you can transfer colonies using an inoculating loop or pipette tip. You would sterilize the inoculating loop using a Bunsen burner; if you use a pipette tip, just make sure it is sterile (from the box). You pick up a single colony and spread the bacteria using the inoculating loop or pipette tip (or a cell spreader if you have one of those). Here is video showing this process: https://www.youtube.com/watch?v=0heifCiMbfY

Hope this helps! Let me know what you think of this idea and if you need any clarification or have any follow-up questions.

--Brandi
ayobacteria
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Joined: Tue Dec 21, 2021 5:50 pm
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Re: Effect of Probiotics on Antibiotic Resistance

Post by ayobacteria »

Thank you so so so much for your response, it has helped me tons!!

One question I did have though, I think the idea of using pure cultures is totally awesome, and I'll definitely try it, but is there also a way I could isolate the probiotics from the food itself?
brandimiller610
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Re: Effect of Probiotics on Antibiotic Resistance

Post by brandimiller610 »

Hi again,

It is definitely possible to isolate probiotics from dairy foods. This paper does a really good job of isolating the strains and using different techniques (i.e. Gram staining and catalase activity testing) to characterize the strains: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723976/. You just need to homogenize your samples, dilute them in broth media, and streak them for isolation on agar plates to get single, pure colonies. From there, you can run different assays to characterize them and/or use them for your antibiotic resistance experiments.

Hope this helps! Let us know if you need anything else!

--Brandi
ayobacteria
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Joined: Tue Dec 21, 2021 5:50 pm
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Re: Effect of Probiotics on Antibiotic Resistance

Post by ayobacteria »

Hello again,

Your help has been so helpful for my experiment so far and I'd like to thank you so so so much.

I did have one question though. If I were to put food, such as yogurt, into my LB agar broth, would autoclaving it change the attributes of the yogurt? Would the probiotics and beneficial bacteria in yogurt survive the conditions of the autoclave?

Thank you so much!
brandimiller610
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Re: Effect of Probiotics on Antibiotic Resistance

Post by brandimiller610 »

Hi again,

I am glad to hear that your experiments are going well! :)

To answer your question, yes, autoclaving yogurt will essentially kill the probiotic cultures. The autoclave uses heat and pressure to sterilize laboratory equipment and reagents at a temperature of 121 degrees C. Many probiotic bacteria are denatured/destroyed at temperatures as low as 50 degrees C, so autoclaving them will kill them. I think your best method of action, as discussed in the previous post, would be isolating the probiotics from yogurt and other dairy products, spreading them on Lb agar, and performing the disc diffusion assay to determine their antibiotic resistant properties. You will have to do various microbiological techniques, such as Gram staining and catalase activity testing, to identify the bacteria after isolating them. These papers might give you some insight: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627144/ and https://asm.org/Articles/2020/February/ ... wth,-Stain

Hope this helps!

--Brandi
brandimiller610
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Re: Effect of Probiotics on Antibiotic Resistance

Post by brandimiller610 »

I wanted to make a small correction to my previous post. After isolating your probiotics, you will want to spread them on MRS agar, not Lb agar. MRS is the best selective medium for growing probiotics, including Lactobacillus and Bifidobacterium.

Sorry for any confusion!

--Brandi
ayobacteria
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Re: Effect of Probiotics on Antibiotic Resistance

Post by ayobacteria »

This has really helped me a lot, thank you so much!
ayobacteria
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Re: Effect of Probiotics on Antibiotic Resistance

Post by ayobacteria »

Hello,

So I was wondering, if I was interested in the effects of the metabolites produced by probiotics in different foods, it wouldn’t matter if the probiotics are alive or not right?

More specifically, going back to my original idea of putting the yogurt and probiotic food into the LB agar itself and autoclaving it, could the experiment still work considering the effect that metabolites from the probiotics would produce? Even if the probiotics have been killed by the autoclave?

Thank you so much!
brandimiller610
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Re: Effect of Probiotics on Antibiotic Resistance

Post by brandimiller610 »

Hi again,

This is a very interesting question! My lab actually works with probiotics and we are investigating the beneficial effects of heat-killed bacteria on gut and brain health. So it is absolutely possible to observe beneficial effects from heat-killed bacteria and analyze their metabolic profiles (instead of probiotics, we call these postbiotics). However, there are very specific protocols regarding heat killing bacteria and many of them use temperatures between 70 and 80 degrees C to efficiently kill the bacteria (our lab uses 70 degrees C for 2 hrs). Therefore, the conditions of the autoclave would not be conducive for heat killing the bacteria to be used for further experiments.

Hope this helps!

--Brandi
ayobacteria
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Joined: Tue Dec 21, 2021 5:50 pm
Occupation: Student

Re: Effect of Probiotics on Antibiotic Resistance

Post by ayobacteria »

Thank you so much!
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