Development of Real-Time Polymerase Chain Reaction (q-PCR) Assays for the Detection and Identification of Drug-resistance Genes in Carbapenem-resistant Enterobacteriaceae (CRE)

by

Print Friendly, PDF & Email

Richard A. Robison, Ph.D., Microbiology and Molecular Biology

I. The Specific aims for the project were as follows:

  1. Develop q-PCR assays for the various carbapenem-resistance genes, such as KPC, IMP, VIM, and NDM-1, and then multiplex them into single assays that can be used to quickly characterize an isolate.

II. Accomplishments to date related to the specific aims that were proposed:

  1. We successfully created q-PCR assays that accomplished our objectives in specific aim 1 above. Four specific singleplex assays for these drug-resistance genes have been optimized and are working well.
  2. We were also able to create q-PCR assays for the 2 most common species CRE species that are isolated in the U.S.: Escherichia coli and Klebsiella pneumoniae.
  3. We have quadraplexed an assay for KPC and NDM (the 2 most common CRE genes seen in the U.S.) and the assays for the 2 most common species noted in #2 above.
  4. We have completed bioinformatics work on the known plasmid sequences involved in CREs.
  5. We have also done some novel work with bacteriophage that can infect CREs.
  6. Students are continuing work on these projects. We have submitted the bioinformatics plasmid work for publication. It is currently in review. We hope to be able to submit a manuscript describing the q-PCR quadraplexed assay for publication soon. References for the publications/presentations related to this work are listed below:

 

Presentations from my lab: (Graduate students are in blue; undergraduate students are in red):

  1. Guerrero, I., Hoj, T.R., Berges, B., and Robison, R. Effects and differences between human and mouse cell lines during chikungunya virus infection. Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #32, April 15, 2017. Ogden, UT. Note: This presentation won 2nd place in the student poster competition.
  2. Thiriot, J., Hoj, T.R., and Robison, R. Galleria mellonella as a virulence model for Burkholderia pseudomallei: does it really work? Intermountain Branch of the American Society for Microbiology Annual Meeting. Abstract #36, April 15, 2017. Ogden, UT.
  3. Brown, O., Grose, J.H., and Robison, R.A. Evaluation of bacteriophages against extended-spectrum β-lactamase producing Enterobacteriaceae. The American Society for Microbiology TriBranch Meeting. April 6-7, 2018. Durango, CO.
  4. Mitton, J., Ogilvie, B., Tucker, J., and Robison, R. Providone-iodine vapor kills MRSA. The American Society for Microbiology TriBranch Meeting. Oral Presentation. April 6-7, 2018. Durango, CO.

Publications from work done in my lab: (Graduate students are in blue; undergraduate students are in red):

  1. Alizadeh, M., Wood, R.L., Buchanan, C.M., Bledsoe, C.G., Wood, M.E., McClellan, D.S., Blanco, R., Ravsten, T.V., Husseini, G.A., Hickey, C.L., Robison, R.A., and Pitt, W.G. 2017. Rapid separation of bacteria from blood – chemical aspects. Colloids and Surfaces B: Biointerfaces. 154: 365-372.
  2. Buchanan, C.M., Wood, R.L., Hoj, T.R., Alizadeh, M., Bledsoe, C.G., Wood, M.E., McClellan, D.S., Blanco, R., Hickey, C.L., Ravsten, T.V., Husseini, G.A., Robison, R.A., and Pitt, W.G. 2017. Rapid separation of very low concentrations of bacteria from blood. Journal of Microbiological Methods. 139: 48-53.
  3. Knob, R., Hanson, R.L., Tateoka, O.B., Wood, R.L., Guerrero-Arguero, I., Robison, R.A., Pitt, W.G., and Woolley, A.T. 2018. Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices. Journal of Chromatography A. 1562: 12-18.
  4. Card, G.E., Pickett, B.D., Ridge, P.G., and Robison, R.A. Characterization of carbapenemase resistance plasmids. Submitted to 2018, Under review.

III. Accomplishments to date related to the Robison lab mentoring environment:

We successfully conducted the Pathogenesis Journal Club (MMBIO 528R) during Winter and Fall semesters each year. We also offered MMBIO 518 (Pathobiology of CDC Select agents) during each odd Fall semester. Over 50 undergraduate and graduate students participated in these courses. The undergraduate mentoring courses (MMBIO 494R) assigned to my laboratory enrolled an average of 8 students each semester. We have also continued to hold weekly lab meetings and have had six lab parties during the past two years.

VI. Summary.

We have made very good progress relative to the overall specific aims of the MEG proposed in 2017, and have also fostered and maintained an excellent mentoring environment in which other research projects have been supported. To date, we have spent a majority of the funds awarded with about 45% of this amount expended on undergraduate student wages. The number and quality of our academic deliverables have been excellent.

Undergraduate and graduate students working in the Robison laboratory have participated in the following academic deliverables since 2016:

-32 presentations at scientific meetings

-16 peer-reviewed publications

In addition, the following graduate students have finished their Masters of Science degrees in my laboratory 2018: Hyrum Shumway, Olivia Brown, Galen Card, Joseph Thiriot, and Shreena Mody. Hyrum is in medical school, Galen and Joe are in a Ph.D. program and Olivia and Shreena are working in industry.