Progress 08/01/24 to 07/31/25
Outputs
Target Audience:This project provided practical instruction and experiences for undergraduate students in Biology and Chemical Engineering, a graduate student in Materials Engineering, and a Postdoctoral fellow in Chemical and Biomedical Engineering. We expect to provide novel ways to treat and eradicateListeria monocytogenes biofilm formation, which could be of interest for many food industries with persistent biofilms. Changes/Problems:There were delays in acquiring the alternating magnetic field (AMF) system and installing it. The original company that was going to supply the AMF system failed to provide a unit that could deliver enough magnetic field strenghts (originally, we proposed 1000 Gauss). AMF Lifesystems supplied an AMF system, but unfortunately, it broke down due to clogging in the recirculating distilled water lines of the buildingand had to be sent back to the manufacturer for repair. We had to purchase a high-capacity chiller (~ $9K), which was not initially planned in the proposal.This has delayed the project by approximately 6 months; however, there are no changes in the goals or approach to this project. We are exploringthe idea of growingbiofilms in test tubes so these can be easily put under the applied magnetic field. Note that the maximum we can reach is ~ 500 Gauss. What opportunities for training and professional development has the project provided?An undergraduate student from UTSA, Erin McNeill, traveled to the University of Dayton to get trained on listeria biofilm growth. She came back to UTSA to grow biofilms that will be used in tasks 4.1 and 4.2. Erin presented a poster in the UTSA BMES/AIChE Research Symposium in April 2025. She will also submit an abstract to present at the AIChE student conference in Fall 2025. A postdoctoral fellow continues to gain experience supervising one undergraduate student and helping the Co-PI manage his lab on all aspects related to objectives 1 and 4 of this project. Two undergraduate students from the University of Dayton, Sarah Khan (Biology) and Joseph Rempe (Chemical Engineering), wererecruited and trained on the project in June 2025 and will continue the project in the coming months. An undergraduate student from the Ohio State University, Anastasia Cocieru, was selected for the Summer Undergraduate Research Experience program in the School of Engineering at the University of Dayton and was co-mentored to work on the nanocomposite synthesis, susceptibility testing, as well as nanocomposite effects on biofilm. How have the results been disseminated to communities of interest?Initial results were presented in 8 poster presentations. Additionally, 2 posters were presented at IAFP 2025, and these will be reported next year. We also published a review paper on magnetic nanoparticles in life science applications (products). There are four peer-reviewed papers in preparation for submission in the next few months. What do you plan to do during the next reporting period to accomplish the goals?Objective 1 - This objective is complete. However, the synthesis of the nanoparticles will continue as needed to complete the remaining objectives of the project. Objective 2 - We will begin toxicity testing for the synthesized nanocomposites. Objective 3 - We will begin the testing under different humidity conditions. Objective 4 - A focused ion beam (FIB) scanning electron microscope (SEM) is available at UTSA in the Kleberg Advanced Microscope Center (KAMC) and will be used to image the nanoparticles inside listeria biofilms. The biofilm samples will be prepared by fixating in 0.4% paraformaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer. They will be stained with 1% osmium tetroxide and saturated thiocarbohydrazidin DI water to increase contrast. These samples will then be dehydrated in ethanol and finally dried with hexamethyldisilazane (HMDS). The prepared samples will be milled and imaged in layers inside the FIB/SEM. These images be used to determine diffusion and distribution of the nanobioconjugates in the films. We will begin testing one of the magnetic nanobioconjugates under alternating magnetic fields (AMF) and listeria biofilms.
Impacts
What was accomplished under these goals?
Objective 1 - At UTSA, we continued synthesizing magnetite (Fe3O4) coated cellulose nanocrystals (CNCs) using sulfated CNCs (CNC-S) and Tempo-oxidized CNCs (CNC-T). A thorough characterization of these nanoparticles is complete including vibrating sample magnetometry, high resolution TEM, X-Ray Diffraction (XRD), and X-ray photoelectron (XPS) spectroscopy measurements. Two successful user proposals were accepted by the Center for Nanoscale Materials at Argonne National Laboratories to combine experimental and computational modeling of the results. At the University of Dayton, we prepared the three nanobioconjugates (benzalkonium chloride, benzalkonium bromide, and chitosan) and the characterization has been done. These includevibrating sample magnetometry, high resolution TEM, X-Ray Diffraction (XRD), and X-ray photoelectron (XPS) spectroscopy measurements, FT-IR, zeta-potential, and thermogravimetric analysis. Planktonic testing of Listeria monocytogenes and E. Colihave been done with the as-synthesized particles. We expect the submission of four publications from objectives 1 and 2. Objective 2 - We made the discovery of how high levels of benzalkonium chloride significantly enhance Listeria monocytogenes biofilm formation and are continuing to clarify the potential risks involved in such a phenomenon. We prepared a manuscript and are now working on revisions. We also conducted antimicrobial testing using three different susceptibility assays to better compare activities between the three nanobioconjugates. Objective 3 - We have completed the aerobic versus anaerobic biofilm characterizations for strain 10403s using two different surface materials and three different temperatures. The phenomenon of benzalkonium chloride enhancing biofilm was observed under all the conditions tested. Objective 4 - An IBC protocol was approved to begin work with L. monocytogenes biofilms at UTSA. The UTSA team is now growing their first L. monocytogenes biofilms, which will be used to study the diffusion and distribution of the nanobioconjugates. At the University of Dayton, nanobioconjugates were tested for their effects on biofilm formation. An alternating magnetic field (AMF) equipment was procured and installed during year 2 to perform magnetic hyperthermia studies.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Chand, K. and Vasquez-Guardado, E.S. (2025), Magnetic Nanoparticles: Synthesis and Applications in Life Sciences. ChemistryOpen 2500214. https://doi.org/10.1002/open.202500214
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
McNeill, E., Hasan, M.J., Sun, Y., Vasquez-Guardado, E., and Ure�a-Benavides, E.E. Magnetite Coated Cellulose Nanocrystals: Self-Assembly Interactions BMES/AIChE Research Symposium, San Antonio, T.X., April, 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Joynt, E., Herzog, S., and Sun, Y. Benzalkonium chloride enhances Listeria monocytogenes biofilm formation under various conditions Ohio Branch American Society for Microbiology Annual Meeting, Westerville, OH, April, 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Herzog, S., Joynt, E., McNamara, K., Evans, M., Hawkins, S., Ure�a-Benavides, E.E., Vasquez-Guardado, E., and Sun, Y. Higher concentrations of benzalkonium chloride increase lysis, aggregation, and enhance biofilm formation of Listeria monocytogenes Ohio Branch American Society for Microbiology Annual Meeting, Westerville, OH, April, 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Herzog, S., Evans, M., Hawkins, S., Joynt, E., Lang, A., McNamara, K., Miller, C., Ure�a-Benavides, E.E., Vasquez-Guardado, E., and Sun, Y. Higher concentrations of benzalkonium chloride enhance biofilm formation of Listeria monocytogenes American Society for Microbiology Microbe, Los Angeles, CA, June, 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Herzog, S., McNamara, K., Ure�a-Benavides, E.E., Vasquez-Guardado, E., and Sun, Y. Enhancement of biofilm in Listeria monocytogenes by benzalkonium chloride depends on the amount of the inoculum University of Dayton Stander Symposium, Dayton, OH, April 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Joynt, E., Ure�a-Benavides, E.E., Vasquez-Guardado, E., and Sun, Y. Benzalkonium chloride enhances Listeria monocytogenes biofilm formation under various conditions University of Dayton Stander Symposium, Dayton, OH, April 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Herzog, S., Ure�a-Benavides, E.E., Vasquez-Guardado, E., and Sun, Y. Higher concentrations of benzalkonium chloride enhance biofilm formation of Listeria monocytogenes University of Dayton Stander Symposium, Dayton, OH, April 2025. Poster Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Herzog, S., Evans, M., Hawkins, S., Joynt, E., Lang, A., McNamara, K., Miller, C., Ure�a-Benavides, E.E., Vasquez-Guardado, E., and Sun, Y. Higher concentrations of benzalkonium chloride enhance biofilm formation of Listeria monocytogenes Sigma-Xi Annual Celebration of Research, Dayton, OH, March 2025. Poster Presentation
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Progress 08/01/23 to 07/31/24
Outputs
Target Audience:This project provided practical instruction and experiencesfor undergraduate students in Biology and Chemical Engineering, a graduate student in Materials Engineering, and a Postdoctoral fellow in Chemical and Biological Engineering. Changes/Problems:There have been some problems with the kinetic adsorption analysis of benzalkonium chloride, benzalkonium bromide and chitosan onto CNC@Fe3O4 nanomaterials. The technique proposed was UV-Vis, but the readings are misleading due to the potential presence of nanomaterials in the dispersion. We are exploring the potential use of other techniques to identify the amount of organic material grafted onto the CNC@Fe3O4 as a function of time to analyze the kinetic adsorption of the three molecules. Preliminary susceptibility tests, however, confirm differences among the three types of coatings, suggesting that the formation of the as-proposed nanobioconjugates has occurred. What opportunities for training and professional development has the project provided?At UTSA, one undergraduate student Erin McNeill has been trained on the chemical synthesis of Fe3O4 nanoparticles and how to coat CNCs with them. She was also trained on the TEMPO oxidation reaction of CNCs. In general, Erin learned to perform nanoparticle synthesis in inert atmospheres at elevated temperatures. She also obtained an REU opportunity at Worchester Polytechnic Institute (WPI) during summer 2024, where she is learning protocols in growing cell cultures as well as growing biofilms on hydrogels. At UTSA, a postdoctoral fellow has gained experience training an undergraduate student and has helped the Co-PI manage his lab on all aspects related to objectives 1 and 4 of this project. At the University of Dayton, this work allowed the training and development of 7 undergraduate students, 1 Biology PhD student, and 1 Materials Engineering PhD student. Two of the undergraduate students graduated in May 2024 and will continue to medical school and graduate school. How have the results been disseminated to communities of interest?The results have been disseminated at two University of Dayton research symposia and one regional conference (Ohio Branch American Society for Microbiology), where the student won the Allan A. and Jann M. Ichida Award for Undergraduate Research Excellence. What do you plan to do during the next reporting period to accomplish the goals?Goal 1 - The synthesis of nanocomposite particles will continue throughout the project as it is needed to complete the remaining tasks. The characterization of the Fe3O4 coated CNC is still inconclusive in terms of the size distribution of the magnetite particles and the uniformity of the coating on the CNCs. In some instances, the CNCs become invisible to the electron beam, which is due to the low atomic number of carbon, oxygen and hydrogen atoms, the main elemental components of CNCs. Future work will focus on staining the composite nanoparticles with uranyl acetate to obtain better TEM images. We will also attempt to characterize the nanoparticle morphology using atomic force microscopy (AFM). In addition, a thorough characterization of the crystalline structure and magnetic properties of the nanoparticles will be completed with X-Ray diffraction (XRD), and vibrating sample magnetometry (VSM). Goal 2 Toxicity and antimicrobial activity of nanocomposites - We will continue cell culture studies to investigate the toxicity of the nanomaterials and conduct susceptibility testing to characterize the antimicrobial activities of the synthesized nanobioconjugates comprised of CNC-S and CNC-T nanocrystals. Goal 3 We are now investigating the mechanisms underlying the enhancement effects of BAC on CV staining when the cell numbers and activities in the biofilm were reduced. We are also making progress on other antimicrobials, focusing mainly on ways to prepare aqueous solutions of benzalkonium bromide (BAB) and chitosan. BAC and Biofilm - We aim to identify the mechanisms of BAC enhancement of CV staining. As CV staining is a very popular method to quantify biofilm growth, our results highlight the potential mis-interpretation of the outcomes from CV staining alone. We plan to submit a research manuscript based on these findings. Listeria monocytogenes strains - We have been focusing on one well-characterized strain 10403s and will start characterizing the effects of disinfectants and nanocomposites on more L. monocytogenes strains. Goal 4 - An undergraduate student will travel to Dayton to get trained on listeria biofilm growth and go back to UTSA to grow biofilms that will be used in tasks 4.1 and 4.2. Select conditions of biofilms will be grown in the lab based on the results from objectives 2 and 3. The interactions of the nanobioconjugates with the biofilm will be studied by a mixture of scanning electron microscopy (SEM) and laser scanning confocal microscopy methods (LSCM). The SEM studies will include focused ion beam analysis to attempt to obtain 3D images of the biofilms.
Impacts
What was accomplished under these goals?
Goal 1 - We have procured cellulose nanocrystals with sulfate groups on their surface (CNC-S) and coated them with magnetite (Fe3O4) at 1:2 and 1:4 CNC:Fe3O4 mass ratios. We characterized their hydrodynamic diameters through DLS, obtained transmission electron microscopy (TEM) images and measured their zeta potential as a function of pH. Some aggregation of the magnetite primary particles was observed, yet they remain attached to the CNCs. Benzalkonium chloride, benzalkonium bromide and chitosan have been attached to these substrates and ongoing efforts are being performed to characterize the functionalization and stability of the nanobioconjugates. We have also synthesized TEMPO-oxidized cellulose nanocrystals (CNC-T) and coated them with Fe3O4 at 1:2 and 1:4 mass ratios. They were also characterized through TEM, DLS and zeta potential. It was observed that the CNC-T nanoparticles were more colloidally stable than the CNC-S over a 24 hour period. The CNC-T nanoparticles coated with Fe3O4 also remained highly negative throughout a pH range from 1 to 10; while the Fe3O4-coated CNC-S showed a zeta potential with a magnitude smaller than -30 mV at pHs below 4. Goal 2 Susceptibility tests have been performed on L. monocytogene 10403s, incubated for one day, using brain heart infusion (BHI) media at 37oC. Currently benzalkonium chloride modified substrates showed larger diameter zones of inhibition as compared to benzalkonium bromide and chitosan functionalized nanobioconjugates. Goal 3 We finalized protocols to grow biofilms and analyze biofilms through crystal violet (CV) staining, MTT reduction assay, and colony-forming unit (CFU) quantification. We characterized biofilm under aerobic and anaerobic conditions using different surface materials and media at different temperatures. We discovered that based on CV staining, oxygen levels in most cases did not influence the level of biofilm growth. However, the CV staining of biofilm was significantly higher (p<0.001) in biofilms grown in diluted BHI (brain heart infusion media) than those grown in BHI. There was also a linear relationship in the effects of temperatures where the higher the temperatures (up to 37 °C), the more biofilm growth. We then began testing for the effects of antimicrobials on Listeria monocytogenes biofilms. When we tested using benzalkonium chloride either during biofilm growth or to treat established biofilms, we discovered that while benzalkonium chloride (BAC) was very effective in eliminating planktonic bacteria, it caused a dramatic increase in CV staining. Another curious observation we noted was that the effects of BAC on L. monocytogenes were not linear. Compared to BAC at 5% (v/v in BHI), BAC at 1% (v/v in BHI) was more effective in inhibiting planktonic bacteria and exhibited an extended enhancement effects on CV staining over 7 days These dramatic increases in CV staining typically are interpreted as increased biofilm growth. However, when we investigated further, there were very low levels of metabolic activities in these BAC-treated biofilms and similarly very small numbers of CFUs in these biofilms as well. Similar enhancement effects of BAC on biofilm growth were also observed with 316 stainless steel even though planktonic grow were inhibited. Goal 4 This objective is expected to begin in year 2 according to the proposal timeline. An IBC protocol has been submitted to UTSA for this part of the project and is awaiting approval.
Publications
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