Eric Krause

An Examination of Antibiotic Resistance Genes in Pseudomonas chlororaphis in Response to Bacteriophage Therapy

April 3, 2021   /  

Name: Eric Kraus
Major: Biochemistry and Molecular Biology
Advisor: Dr. Stephanie Strand, Dr. Sara Martin (second reader)

Extensive antibiotic use over recent decades has led to increased levels of antibiotic resistance among bacteria. A current alternative treatment, phage therapy uses bacteriophages to infect and lyse bacteria while leaving the host organism unaffected. Evolution of phage resistance in bacteria can lead to the deletion, mutation, or expression level changes of certain genes. Increased antibiotic susceptibility is one of these benefits, providing an exciting new treatment option when phages are administered in conjunction with antibiotics. This study examined the effects of phage-resistance on the antibiotic susceptibility of Pseudomonas chlororaphis 14B11. The susceptibility profile of the 14B11 parent strain was determined against 10 different antibiotics. Then, the parent strain was grown with the PC1 bacteriophage to promote emergence of phage-resistant mutants. Mutant antibiotic susceptibility was tested and compared to the parent strain. A total of 27 mutants were isolated, where 26 mutants displayed increased susceptibility to at least one antibiotic. The following genes can confer antibiotic resistance and were selected for study in the mutants: MexE, MexF, OprN, MexG, MexH, MexI, OpmD, and CyoE. Ongoing efforts to amplify candidate genes via PCR and examine parent and mutant sequences for mutations have continued.

Eric will be online to field comments on April 16:
noon-2pm EDT (PST 9-11am, Africa/Europe: early evening) and 4-6 pm EDT (PST 1-3pm, Africa/Europe: late evening)

45 thoughts on “An Examination of Antibiotic Resistance Genes in Pseudomonas chlororaphis in Response to Bacteriophage Therapy”

  1. Thank you for your excellent presentation, and congratulations on the completion of your I.S.! Bacteriophage-antibiotic therapy is such an interesting area of research. What was the most intriguing or enjoyable aspect of pursuing this topic?

    1. Macy, I think that the most interesting aspect of pursuing my topic was learning how to perform different techniques in the lab and modifying them when certain procedures would not work (almost always PCR and gel electrophoresis in my case). While this was frustrating, it allowed me to learn even more skills that I think will be useful in the future.

  2. This was an amazingly well done study! The presentation of both bacteriophages and antibiotics was made easy to understand for someone who is not familiar in this field of research.
    One question I had about this study would be if you had considered focusing on the effects of differing bacteriophages on specific strains of bacteria that were found to be naturally susceptible / insusceptible to antibiotic treatment? (I.e. performing this experiment with bacteriophages then with antibiotics).

    1. Cameron, Thank you for the comment! Ideally, I would have liked to repeat these procedures using different kinds of phages each time (or even a combination of phages at once, known as a phage cocktail). This may lead to the evolution of differing levels of antibiotic susceptibilities compared to the ones found in the study. Although this would have consumed more time, this would have produced more phage-antibiotic combinations that display synergistic activity.

    1. I also had a few questions. Why was the susceptibility levels different between Mero. Control and Amp. Control?
      Has this experiment ever been done before in the lab by other scientists?

      1. Andrew, thanks for the question! Antibiotic susceptibility experiments are often performed on bacteria that are pathogenic towards humans, however the bacteria used in the study (P. chlororaphis) is non-pathogenic, therefore resulting in sparse information about its antibiotic susceptibility. Although some previous literature and IS projects have looked at a few of the antibiotics tested here, I did select certain antibiotics that I did not see in the literature.

        As for the meropenem and ampicillin tests, I think you may be referring to the differences between the experimental trials (the control trials exhibited similar growth for both). Although I did not expect to see this happen, I think it may be due to the fact that meropenem is a relatively newer antibiotic (use began in the 80’s/90’s) and still remains resistant to some bacterial enzymes that degrade B-lactams. Ampicillin has been used for much longer than meropenem, therefore providing more time for resistance to evolve. These factors may explain varying resistance between the two despite both being in the same class.

  3. What a clear and great presentation. Your topic was also very interesting and is definitely something to look out for in the coming years. A few questions I would like to ask is what made you choose this topic over your other ones, did you ever want to switch after you started, and what were your overall challenges?..

    Congratulations!

    1. Nadia, thank you for the question. As far as choosing this topic, I initially became interested in studying bacteria, viruses, and treatment methods for these infections from some powerful experiences I gained on a mission trip years ago. As I was searching for an opportunity to link my interest to an IS project, I stumbled upon the idea of using phages and antibiotics together in dual therapy. This instantly caught my attention, and I knew this was the project I wanted to pursue. Although many portions of the project were frustrating, I never thought about switching the topic. The main challenges for me began with finding literature on the specific bacteria I used (Pseudomonas chlororaphis). Other bacteria in the Pseudomonas genus dominate the literature due to their role in hospital-acquired infections. Therefore, I had to spend additional time searching for a few quality articles on P. chlororaphis. Another challenging aspect was the PCR/gel electrophoresis section. I attempted to isolate some candidate genes through this method, but the full length of the procedure reached 4-6 hours for one gene. With 8 genes and multiple mutants, the time commitment was intense. The PCR/gel procedure never fully worked, but I did learn a lot from the process.

  4. Great presentation and congratulations! One question I had pertains to Phase 1 of your project. Is there a reason why the resistance levels varied within specific antibiotic classes? I would assume that each class of antibiotics would have a similar effect since they target the same process in the cell, but this is not the case.

    1. Daphne, thank you for the question! I would assume that the resistance levels varied for a multitude of reasons. For the B-lactam class (meropenem and ampicillin in this case), meropenem is a newer, synthetic B-lactam that entered used in the 80s/90s. It is also resistant to some bacterial enzymes that degrade other B-lactams. Ampicillin has been in use for longer and is more susceptible to these bacterial enzymes. For these reasons, it may make sense that the parent strain was more susceptible to meropenem than ampicillin. As for the aminoglycosides (kanamycin, streptomycin, gentamicin), I am largely unsure. Literature has suggested that varied levels of resistance to these antibiotics arise due to how frequently they are used in clinical settings, but this idea would not apply here since P. chlororaphis is non-pathogenic to humans.

  5. Great job on your presentation! I have a question regarding how you selected your mutant colonies to be used for phase 2 of antibiotic testing? Were colonies chosen at different serial dilutions? And if so, was there any difference in viral or antibiotic resistance between the colonies grown with a lower dilution as opposed to higher dilution?

    Also, how did you decide on studying Pseudomonas chlororaphis 14B11 specifically as opposed to other bacteria?

    1. Brayden, thank you for the questions! Yes, the mutants were selected at different serial dilution concentrations. The choice to do this did not stem from any idea that mutants from different plates would behave differently. Rather, I attempted to select clearly isolated colonies wherever I saw them best. There was no difference in antibiotic or viral resistance between mutants selected at different dilutions: all mutants were fully resistant to the virus and antibiotic susceptibilities did not correlate with the dilution they were isolated from.

  6. Eric, this was such a good presentation. I especially liked the section on how resistance arises and I only had a shallow understanding of what causes that. The project presentation was terrific! Enjoy your success and congratulations!

  7. Thank you for such a interesting presentation! It was so kind of you to explain the background of the study. It helped me a lot while I followed along with the process of the research. Moreover, the topic was so interesting, that I so into your presentation!

    I do have three question to your presentation. I wonder why you have choses the Pseudomonas Chloroaphis as your bacteria to study, and am curious on its effect on the other viruses; I am curious why you have specifically chose certian antibiotics and created a mixture of it. Was there a specific standard? Moreover, I am curious what must have happened to one of the mutants that was not able to increase susceptibility.

    Thank you once again for a marvelous presentation!

    1. Minjin, thank you for the questions! I chose P. chlororaphis for my study due to its use in many IS projects similar to mine over the past few years. As for other viral effects on it, I am not entirely sure of all the viruses that can infect it. My research only used the PC1 virus, which can infect P. chlororaphis and cause cell lysis. However, this virus is not well characterized. I would like to expose the bacteria to different types of viruses, but unfortunately time did not allow for this to happen. As for antibiotics, I chose specific ones due to their association with some of the genes I was studying (changes in some genes may affect the bacteria’s susceptibility to that antibiotic class). Literature review allowed for this to happen. Also, I chose some antibiotics out of pure curiosity (sulfa, rifampicin, ciprofloxacin, etc.) just to see what the susceptibility levels of P. chlororaphis was like towards them.

  8. This was very interesting Eric, Congrats on the accomplishment. I was shocked at the susceptibility profile of the 14B11 parent strain. Very cool!

  9. Very nice read Erik. Not my field of study but you seem to have a great understanding of the topic at hand. Looking forward to whatever do next.

    – K. Doody

  10. Congratulations on completing your I.S! The study of bacteriophage therapy and the consequential antibiotic resistance is a very interesting topic! You did a good job of explaining the various aspects of your study and I found it very helpful for you to give a brief background to bacteriophage therapy and and antibiotic resistance. I am also interested in future work that can be studied such as successful synergistic combinations in the organism.

    I have one question regarding your study. How were you able to edit your procedure in order to work around the issues you faced using Gel electrophoresis and PCR?

    1. Vincent, thank you for the question! I attempted to change almost every aspect of PCR and/or the gel electrophoresis procedure one step at a time. After my first round of PCR did not produce consistent results, I attempted a gradient PCR that would find the proper annealing temperature for each candidate gene. I also began to increase the PCR cycles from 34 to 40 in hopes to exponentially increase my amplicon concentration. Re-isolation of my template DNA from each mutant also occurred to ensure its viability. Even different polymerases were used. In gel electrophoresis, I attempted to increase the volume of PCR product loaded per column from 12 to 75 micro-liters. This involved using different molds for the gel. I also switched out the TAE buffer, GelRed dye, and other reagents to ensure these were not the issue. Some of these changes did produce improved results in comparison to past attempts, but the process still requires modifications for successful completion.

  11. Hi Eric, congratulations! This was a super interesting project and I enjoyed your presentation.
    During Phase 2 you selected 27 mutants – were these just 27 colonies that you were able to clearly isolate? If so, how did you choose them? Wasn’t there a possibility that some may still be the same mutant strain?
    Again, congratulations on completing IS and an engaging presentation.

    1. Shankar, thank you for the questions! There were many more than 27 colonies that I was able to isolate. I simply chose 1-5 mutants every 2 days to continue with due to time constraints (handling hundreds of mutants would have been troublesome). I attempted to choose the same amount at the beginning and end of the study in order to keep my focus uniform. And yes, there is a possibility that some of the mutants may still be the same mutant strain. However, if two isolated mutants were the exact same strain (identical DNA), I would have expected the susceptibility profiles generated in phase 2 to match up exactly with another mutant, which was not observed. However, full genome sequencing could unambiguously identify if any mutants are the same. This would cost approximately $500 per mutant. If I had the funds, it would definitely be something I would investigate.

  12. This is such an interesting study. This semester I have been working on a group project in Cell Physiology that also looks at combined bacteriophage and antibiotic therapy to reduce resistance. You mentioned that some of the strains that were selected for bacteriophage resistance actually had decreased resistance to antibiotics. What implications do you think this has for using this as a treatment in humans and/or animals?

    1. Bailey, thank you for the question and the background on your research! That is incredibly interesting, and I would love to talk more about your findings. As for your question about mutants with decreased resistance to antibiotics, I believe that raises an important concept in developing dual therapy treatments. Each bacterial strain will have a unique antibiotic susceptibility profile, and the evolution of phage resistance may alter these profiles. Therefore, the development of a generic ‘one size fits all’ synergistic treatment may actually do more harm than good. Each strain, whether in a clinical or research setting, should be tested with an array of antibiotics and phages to ensure a specific treatment that will not produce adverse results.

  13. Great work Eric! Congrats on all of your accomplishments. Good luck with your future endeavors!

    1. Thank you Noah! I enjoyed your presentation as well. Good luck with your plans after Wooster!

  14. Congratulations on your accomplishments, Eric. Your presentation truly reflects how much time and effort you put into your I.S.

    1. Thank you Matt! Excited to see what you accomplish on and off the field in your remaining time at Wooster.

  15. Congratulations on this major achievement and the excellent presentation, Eric!

    Best of luck moving forward!

  16. Congrats on your study Eric. This is an amazing work. I have a few questions. What accounted for the different growths in all the controls in phase I? Since the controls were grown without any anti-biotic, should all of them be the same? In phase II, what is the difference between the color red and purple given that these strains all grew less than the parent strain?

      1. Thinh, thank you for the questions. The antibiotic susceptibility testing of the parent strain occurred by taking 3 control trials (bacteria without antibiotic) and 3 experimental trials (bacteria + one specific antibiotic) and then measuring the growth afterwards. Due to the time it took to allow for the bacteria to grow and the number of antibiotics used, testing had to occur over weeks, therefore leading to multiple control trials being grown on different days.

        As for the phase II experiment, the difference between the red and the purple originates from differences in mutant growth in that antibiotic. Red indicates that the mutant simply grew worse than the parent strain in the antibiotic (OD units/mL difference for the mutant > OD units/mL difference for the parent strain). The purple also indicates that the mutant grew less in the antibiotic compared to the parent strain, but by a greater difference. If the mutant grew twice as less in antibiotic compared to the control, then it was labelled purple instead of red. I hope this answers your questions!

  17. Congratulations Eric! You have done great work this semester, and there are a couple of students who are excited to continue the investigations!

    1. Thank you Dr. Strand! I would not have been able to do it without all of your help along the way. I’m excited to see what the new students find!

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