Source: UNIVERSITY OF DAYTON submitted to NRP
PARTNERSHIP: MAGNETIC CELLULOSE BIOCONJUGATES FOR L. MONOCYTOGENES DISINFECTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030676
Grant No.
2023-67017-40045
Cumulative Award Amt.
$751,996.00
Proposal No.
2022-09029
Multistate No.
(N/A)
Project Start Date
Aug 1, 2023
Project End Date
Jul 31, 2026
Grant Year
2023
Program Code
[A1332]- Food Safety and Defense
Recipient Organization
UNIVERSITY OF DAYTON
300 COLLEGE PARK
DAYTON,OH 45469
Performing Department
(N/A)
Non Technical Summary
The United States has a complex food system where our foods come from multiple production, processing, and packaging facilities. These create numerous potential entry points for foodborne pathogens and require stringent surveillance and disinfection protocols. However, some pathogens, such as Listeria monocytogenes (Listeria), survive even under extreme environmental conditions, including low pH and temperatures. Once entering high-risk individuals, Listeria can cause infections with high mortality rates and hospitalization costs. Despite our best disinfection efforts, Listeria outbreaks continue to take place. Costly food recalls as a result of potential Listeria contamination are also frequent. These urgent issues can all be attributed to the ability of Listeria to persist in the food production and processing facilities. In fact, Listeria can adhere to surfaces and grow into a biofilm structure resistant to many disinfectants and sanitizers. This project aims to target and destroy Listeria biofilm structures through nanotechnology by generating composite structures at the nanoscale responsive to external stimuli such as magnetic fields. More specifically, we plan to create and characterize innovative nanobioconjugates that contain cellulose-derived materials with magnetic nanoparticles linked to synthetic or natural disinfectants. We plan to investigate the effectiveness of these nanobioconjugates against Listeria biofilm under magnetic fields. This collaborative and interdisciplinary effort will occur in two different institutions, one mid-size University and one minority- and Hispanic-serving University. The project will involve undergraduate as well as graduate students. Ultimately, our findings will bring advanced nanotechnology to combat the presence and persistence of foodborne pathogens to promote food safety.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
72340102020100%
Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2020 - Engineering;
Goals / Objectives
The overarching goal of the project entitled: "Magnetic Cellulose Nanobioconjugates for Listeria monocytogenes Disinfection'' is to develop unique magnetic functionalized nanobioconjugates comprised of cellulose nanocrystals (CNCs), magnetic nanoparticles, and disinfectants, to eliminate Listeria monocytogenes biofilms under applied magnetic fields. This work will be achieved by following a series of technical objectives:1) Prepare multiple nanobioconjugates by testing two cellulose nanocrystal types and three different disinfectants (benzalkonium chloride, benzalkonium bromide, and chitosan),2) Assess the toxicity and antimicrobial performance of as-prepared nanobioconjugates against L. monocytogenes in vitro to identify candidates suitable for biofilm studies,3) Characterize biofilm formation in diverse L. monocytogenes strains under relevant environmental conditions (e.g., low temperature, reduced oxygen availability, varying relative humidity),4) Perform diffusion, distribution, and disinfection studies of optimized nanobioconjugates at different concentrations with and without applied magnetic fields in L. monocytogenes biofilms prepared under various conditions.
Project Methods
The project directors (PDs) propose to achieve the four goals over a period of three years. To achieve these goals, the PDs split the goals into two main tasks.The first task involves preparing nanobioconjugates and analyzing Listeria monocytogenes in-vitro with the different nanomaterials and biocides of interest.Nanobioconjugates preparation. Nanocomposites comprised of cellulose nanocrystals and iron oxide (CNC@Fe3O4) nanoparticles will form by coating the CNCs with Fe3O4 through an established co-precipitation method. Two cellulose nanocrystal types will be studied: sulfated CNC (S-CNC) and TEMPO-oxidized CNCs (OS-CNC), followed by magnetite growth onto CNCs through pH-controlled dispersion. Finally, the as-prepared nanobioconjugates will be surface-functionalized through electrostatic interactions with three proposed disinfectants: benzalkonium chloride (BC), benzalkonium bromide (BB), and chitosan.Nanobioconjugates characterization and evaluation. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) studies will be performed to analyze the morphological properties of the nanocomposites and the nanobioconjugates. These results will help assess the dispersion of the magnetic nanoparticles onto the cellulose nanocrystal substrates.Spectroscopic techniques (ATR-FTIR, Raman, XPS) and X-ray diffraction (XRD) will be used to confirm the chemical structure of the nanobioconjugates. The composition of the magnetic cellulose nanocrystals will be examined through thermogravimetric analysis (TGA) and inductively coupled plasma-mass spectrometry (ICP-MS). Vibrating sample magnetometry (VSM) measurements will help determine magnetization properties. Moreover, zeta potential measurements at varying pH will determine the isoelectric point and the most effective pH values to maintain appropriate colloidal stability and adequate transport properties.UV-Vis spectroscopy results will help determine and evaluate the adsorption and desorption kinetics as a function of concentration, time, and temperature. Ultimately, these results can be used to develop treatment methods for biofilm treatments.In vitro studies of L. monocytogenes against nanocomposites, disinfectants, and nanobioconjugates. The toxicity of the biocomposites will be determined by standard lactate dehydrogenase release assay with cultured mammalian cells. The antibacterial efficacy of nanobioconjugates will be evaluated using standard disc diffusion assays. In toxicity and antibacterial efficacy assays, we will compare the effects of disinfectant-only, nanocomposite-only, and nanobioconjugates. These comparisons will allow us to establish disinfectants' antibacterial activity and efficacy with or without nanocomposite conjugations. Moreover, the systemic characterizations will help sort candidate nanobioconjugates into high priority (low toxicity, high efficacy) and low priority (low/high toxicity, low efficacy) for subsequent studies in biofilms.The project's second task involves utilizing the proposed nanobioconjugates to attack and eliminateListeria Biofilms. A thorough understanding of biofilm formation will also be obtained under different environmental and substrate conditions. The methods to follow include:Listeria Biofilm formation will be analyzed in response to low oxygen availability, refrigeration temperatures (4-10°C), and humidity (10 - 90 %) on 24 strains L. monocytogenes on surface materials commonly found in food industries (stainless steel and polyethylene terephthalate). Such a full multifactorial analysis in replicates will help us determine the critical factors (strain, temperature, oxygen availability, humidity, and surface materials) that can influence biofilm formation. We will then examine the response of these biofilms to three different biocides (BC, BB, and chitosan) over different periods of time under the same condition where the neat biofilm was grown. This comparison will help establish a further understanding of the susceptibility kinetics of biofilms to biocides alone.Disinfection studies. Interactions of nanocomposites and bioconjugates with L. monocytogenes biofilms will be determined. Specifically, three disinfectant coatings, BC, BB, and chitosan, will be assessed on the CNC@Fe3O4 to identify the most colloidally stable combination and toxic to L. monocytogenes. 2D imaging will be done by conventional scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). To obtain 3D images, a series of techniques will be attempted, including focused ion beam (FIB)-SEM and LSCM. We will then evaluate and analyze the survival rate of L. monocytogenes against conjugated nanoparticles as a function of time using confocal microscopy and selective fluorescent labels to distinguish live versus dead bacterial populations accurately. L. monocytogenes disinfection studies will also use alternative current (AC) magnetic field(s) to demonstrate that applying a magnetic field will enhance L. monocytogenes disinfection and elimination rates. Demonstrating the efficacy of the nanobioconjugates under applied magnetic fields will be the project's main milestone and significant outcome.

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.

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