Science Buddies: "Ask an Expert"

Hi! I have done some basic research on bacteria and antibiotic resistance. Like, how bacteria grow and what is the cause of bacterial resistance. I have also done some research a biocide that has been particularly used in cosmetics and disinfectants. I would like to know how this connects to antibiotics? Also, how can I design my experiment. Thank you!

Hi,

You are researching a really interesting topic, and one that is of significant importance right now with the increase in antibiotic resistant bacteria. Are you interested in the difference between biocides and antibiotics or are you interested in how biocides affect the development of antibiotic resistance? Biocides and antibacterials are probably an overlapping group. Technically, biocides are compounds that directly kill bacteria, whereas antimicrobials/antibacterials slow microbial growth. Antibiotics probably do function as a biocide, but they are generally considered a separate class of molecule. Perhaps one of the other experts can come and clarify this further.

As for designing your experiment, you are going to have to give a little more guidance on what you are trying to ask before we can help you design an experiment. Try to come up with the basic question you want answered, or even better, formulate a hypothesis. Then, we would be happy to help you design an experiment.

I'm attaching an article on biocides and antimicrobial resistance that may help give you some ideas and give you further background on this area.

Please post again in this forum if you have more questions or with more information on your topic so we can help you refine your experiments and hypothesis further.

JMP

Why is it that only 99.9% of bacteria are getting killed? What happens to that 0.1%? Do they become resistant to drugs/antibiotics?

Hi!

Is this the topic you're interested in to research for your science fair project? If yes, I think it's a great one; as currently there is a medical crisis, since many antibiotics are becoming less and less effective. In response to your question, yes, resistance is a huge factor in keeping the bacterial culture alive; if we killed all the bacteria, we wouldn't be seeing a resurgence in their growth.

Please post back with more questions as you narrow down your project!

Hello there,

I agree with scibuddyAK that antibiotic resistance is a very interesting topic to pursue as it has been a huge problem in treatments of microbial infections in the past and still remains a significant concern today. Resistance of certain bacterial species to antibiotics can occur naturally or be driven by pressure from treatments with antibiotics. Many bacteria in the environment produce their own antimicrobial compounds (from which many antibiotics have been derived actually) to kill off their competitors--hence, these bacteria also encode for resistance against these compounds so that they will not be killed by the own compounds that they produce and also from the compounds that their competitors synthesize. In medicine, often incorrect prescription of antibiotics to treat an infection or the prolonged use of antibiotics (much after the infection had already been cleared) can give rise to drug-resistant variants that can survive in the presence of the drug.

Antibiotic resistance can be acquired in several ways. First of all, certain bacteria species can transfer genes between each other. If one antibiotic-resistant bug happens to come into contact with an antibiotic-sensitive bug and they can transfer genetic material between each other, the antibiotic-resistant bug can pass on resistance genes to the sensitive bug, which will now render the sensitive bug antibiotic-resistant. Another mechanism is that mutations arising in bacterial proteins that are targeted by antibiotics can alter the protein structure slightly so the drug can no longer bind to the protein and inhibit its activity. Finally, bacteria can also upregulate membrane transporters to pump out the antibiotics so that the drug will not accumulate inside the bacterial cell and kill them.

Once you come up with some concrete questions/ideas for your project, or if you are curious about learning more about antibiotic resistance in general, feel free to ask us more questions!

Connie

Based on my research, it says that viral infections such as a cold or a flu cannot be treated by antibiotics but how come we take in paracetamols for flu? Doesn't it only make the bacteria become resistant to paracetamols?

Paracetamols are not antibiotics. Paracetamols are basically tylenol, and can be used to treat mild aches and pains and fever. It is often given during the flu to help bring fevers down and alleviate symptoms so people don't feel so miserable while they are sick. Paracetamols will not treat the virus causing the cold or the flu, and they also would not have any effect on bacteria if you had a bacterial infection. All it can do it make you feel better.

You are correct though that an antibiotic would not be beneficial for a viral infection like the cold or flu. Sometimes doctors mistakenly prescribe antibiotics for viral infections and this has been hypothesized to lead to the growth of antibiotic resistant bacteria, so the concept you are discussing is exactly right.

Have decided on a science fair project in this area?

Hi again! So I am in a process of conducting my experiment. My plan is to grow bacteria. One that is susceptible to ampicillin and one that is resistant. Then afer I grow these bacteria. I will pull out the DNA from the bacteria then amplify it (Polymerase Chain Reaction) then I will send the DNA to a lab to see their DNA sequence because I am trying to compare the DNA sequence of a susceptible and resistant bacteria like what is in a resistant bacteria's DNA sequence that is not in a susceptible bacteria. My question is, how do I make a bacteria resistant to antibiotics?

Hi,

Bacteria get their resistance to antibiotics from plasmids. Plasmids are small, circular DNA molecules that are not part of the larger genome of bacteria. Plasmids are also replicated when the bacteria divide and so they get passed on through generations. They can also be transferred to bacteria that do not have plasmids, so bacteria that are antibiotic resistant can transfer their plasmid to other nearby bacteria, which will now also be resistant.

The way we make bacteria resistant is by "transforming" the bacteria with a plasmid containing an antibiotic resistance gene. You can google "plasmids" and "antibiotic resistant plasmids" to learn more about this.

If you are trying to compare the DNA sequence of a resistant bacterium with a non-resistant bacterium, the difference will be in the plasmid that the bacterium contains, and this will be an antibiotic resistance gene.

Hello there,

Like the expert above had mentioned, in the lab we typically make bacteria resistant to antibiotics by transforming them with a plasmid that contains an antibiotic resistance gene. If you are going to do that, you have to make sure that you are working in a lab space and are taking the proper procedures to dispose of these bacteria because you do not want this antibiotic resistant strain to get out into the environment (even if it's something harmless like E. coli, it's still not a good idea because the plasmid could potentially be spread to other bacteria in the environment). However, if you are going to artificially make your bacteria antibiotic resistant by transforming a plasmid, then there is essentially no point in sequencing the resistant and susceptible strains because you know that if you started from the same strain background, then the only difference would be in the plasmid encoding the antibiotic resistance marker.

One other way you can go about this is to look at different environmental or clinical isolates of a certain bug (i.e. E. coli) and look at the difference in susceptibilities between the different isolates to certain antibiotics. If you find that some isolates are susceptible to say, ampicillin, but some are resistant, then it would be interesting to see whether the resistant strains have acquired a gene that allows them to become resistant to ampicillin. However, this would be slightly more complicated and definitely would require a lab that has access to these types of resources, especially with getting environmental or clinical isolates.

Sometimes, changes in the genome that encode resistance can be a lot more subtle than the presence or absence of an antibiotic resistance gene. For example, when people with bacterial infections are treated with antibiotics, there are certain bugs that can evolve to become resistant to the antibiotic, but not necessarily by acquiring a plasmid that encodes for resistance. They can just make a very small change to the target of the antibiotic (for example, change a couple of amino acids in the protein target so that the structure of the protein changes to the extent that the antibiotic can no longer bind to it), and if that is the case, then you would need to do further analysis to see if changing certain amino acids could confer that resistance. Bacteria also encode for a lot of transporters that have been implicated in pumping out antibiotics. It could be that when the bacteria are in the presence of the antibiotics, they start making more of these transporters in order to pump out the antibiotic. Hence, the difference between a susceptible and resistant strain may not necessarily be the presence or absence of an antibiotic resistance gene, but rather is in the difference in the amount of transporters produced by the strains.

Hope that helped! If any part of my explanation were unclear, don't hesitate to contact us with more questions!

Best,
Connie

What are some list of good bacteria that is inside in our body?

There are a nearly infinite number of bacteria inside our bodies. In fact, there are more bacterial cells in your body than there are "your" cells. We still haven't identified all of the different kinds of commensal bacteria in our gut, but some of them include E. coli, Lactobacillus, Bifidobacterium, Clostridium, and a bunch of others. There is also a fair amount of variation from person to person, particularly in the ratios of the different strains of bacteria to each other. If you want to learn more about this, I recommend that you spend some time searching for "commensal bacteria" or "gut flora" on Google to get some more background and specifics on different varieties. Also, if you are looking to culture commensal bacteria, be aware that a lot of the bacteria in the gut are not readily culturable.

I have thought of several ideas on what I could do for my experiment but I also have to consider biosafety rules in the science fair. I have finally came down to the hypothesis that chickens that have been exposed to antibiotics would show resistant bacteria in their feces compared to chickens that are organic. My problem right now is how will I extract DNA from their feces. I know that it would be hard since it has been composed but it is possible.

Hello there,

Isolating DNA from feces is definitely feasible and is widely done in labs that study the commensal bacteria living in our gut as well as in labs that work with intestinal pathogens. I'm not sure whether the texture or content of chicken feces would affect the extraction process, but it might be worth it to contact some local labs that work on commensal bacteria and ask them for their opinion on how you should isolate DNA from the feces. They should be able to give you some tips on which kits to use and on the procedures as well. They may also refer you to certain manufacturers of DNA extraction kits so you can ask them in more detail about whether their kits would work with chicken feces.

Your project is a super interesting one! Let us know if you have anymore questions.

Best,
Connie

Dear Ms. Connie,

Thank you for that information. Gladly, I have found a supply kit that extracts DNA chicken feces. I am just waiting for it to come in and start with my experiment. After I have extracted DNA, I will be using PCR to amplify its DNA. I know that I will be needing nucleotides, DNA template, Taq polymerase, and the DNA primers. I need to look for the DNA primers for ampicillin resistant gene. I tried looking it up but I was not sure if I found the right one. Are there any good websites where I could look into for these primers? Thank you

Hi,

Sounds like you have designed a really advanced and interesting project. I've isolated DNA from a lot of things, but not chicken poo! The main problem I see is that you will have DNAs from many different sources in the sample--from chicken cells, from their feed, and from bugs if they are free to eat them--as well as from all the bacterial species that live in their gut. Designing primers to target the bacterial ampicillin-resistance gene without false signals from the other DNAs will be a real challenge. Are you planning on doing real-time PCR or the regular kind?

I don't know anything about the gut flora of chickens. They probably have some strain of E coli but probably also some Gram-positives. I did a search for chicken E coli PCR primers and found a paper that might give you some primer ideas: http://aac.asm.org/content/46/10/3156.full

There are a number of free primer design programs on the web provided you have the DNA sequence for an ampicillin-resistance gene that you think will be present in the bacteria in your chicken feces. We buy our primers from IDT and they have several design and testing tools on their website (http://www.idtdna.com/pages/scitools?gc ... 7AodexcAxg). Their tech team is very helpful and I would suggest calling them and discussing your plans. I'm sure they would have some helpful suggestions. You are probably already familiar with this company. We also use NCBI's Primer-BLAST program, http://www.ncbi.nlm.nih.gov/tools/primer-blast/

Hope this is helpful for your project. Keep us posted on your progress.

Sybee

Hello there,

If you are isolating bacterial DNA from chicken feces, and then subsequently trying to use PCR to amplify ampicillin resistance genes, that may be a little tricky. This is because resistance can arise in different manners for different bugs. Hence, one specific mutation in a certain gene may confer amp resistance in one bacterial strain, but another different mutation in a different gene can very well confer amp resistance for another bacterial strain. I think your idea of using PCR to find the presence of resistant bacteria is a great idea, but it might be difficult given the different routes of resistance a bacterium can take. I think you may perhaps try plating the chicken feces isolated from chickens that haven't been treated or treated with antibiotics onto ampicillin plates and see how many bacteria grow on these antibiotic plates. You can then quantify the number of amp resistant bacteria per gram of feces isolated to see if chickens treated with antibiotics will have higher numbers of amp resistant bacteria. You may also try plating these samples onto plates with other antibiotics too to see what other kinds of antibiotic resistance are present in the bugs isolated from these chickens!

Other experts, if you have any better ideas, please share!

Best,
Connie