Doctor of Philosophy in Food Science (PhD)
Investigation and detection of antimicrobial resistance in foodborne bacteria using advanced technologies
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Food safety Food microbiology Molecular microbiology Nanotechnology Microfluidic "lab-on-a-chip" Food authentication Fabrication of nano-biosensor Gut microbiome Food microbiome
My supervisor is very considerate and always thinks for his students! He is very patient in listening to our ideas and struggles, and always provides the greatest supports to us! I'm very honored to be his student.
No abstract available.
The hard-to-treat chronic bacterial infection is one of the most significant challenges to conventional antibiotic therapy. These chronic infections represent an elevated risk for the development of severe clinical consequences. Bacteria can form biofilms or persister cells to withstand harsh stresses and antibiotic treatment. In addition, both biofilm and persister cells can restore the bacterial population upon the removal of stresses and antibiotic treatment. Hence, biofilm and persister cells are proposed to be one of the major survival strategies that associate chronic bacterial infections. As one of the major causes of human gastroenteritis in the world, Campylobacter jejuni was frequently identified in food production as well as in the environment. However, how can this microaerophilic microbe survive in the aerobic environment and disseminate throughout the food chain to eventually cause campylobacteriosis is not fully understood yet. We argued that bacterial biofilm and persister cells be two particular survival state of C. jejuni that contribute to the prevalence of C. jejuni. In this dissertation, particular survival modes of C. jejuni, known as biofilm and persister cells, were characterized. We found that C. jejuni could form both mono- and multispecies biofilms and biofilm formation was significantly influenced by environmental stresses. The extracellular DNA was the factor that mediated this influence. In addition, we identified the presence of C. jejuni persister cells which accounted for ~ 0.01% of the total population. The transcriptome analysis of persister cells indicated that the low metabolic activity and bacterial dormancy could play an important role in the formation of persister cells. In the end, a synergistic treatment using ajoene and Al₂O₃/TiO₂ nanoparticles in a combined manner was applied to generate a significant antimicrobial effect against C. jejuni. In this study, we comprehensively investigated the two major bacterial survival strategies, namely biofilm and persister cells, and applied innovative antimicrobial treatment to inactivate C. jejuni. The knowledge from this study provides insight to understand the survival and distribution of C. jejuni and aids in the development of intervention strategies to reduce the prevalence of C. jejuni and other pathogens.
The research work focuses on developing novel methods for determining small molecules in food matrices using molecularly imprinted polymers (MIPs) and surface enhanced Raman spectroscopy (SERS). MIPs are synthesized as artificial antibodies towards target molecules utilizing interactions between templates and functional monomers to impress complementary binding sites on polymers. MIPs selectively isolate templates from food extracts. SERS technique provides rapid and sensitive detection of MIPs-separated molecules. Statistical analysis including unsupervised principal component analysis (PCA), supervised simple linear regression and partial least square regression (PLSR) is employed to analyze SERS spectra. Chloramphenicol in milk and honey was determined using MIPs-packed solid phase extraction cartridge to isolate chloramphenicol from food matrices and dendritic silver to acquire SERS spectra of the eluted chloramphenicol. These spectra obtained from different spiked contents (0, 0.1, 0.5, 1, 5 ppm) of chloramphenicol in milk and honey were analyzed by PCA and PLSR (R > 0.9). MIPs particles were spread onto a thin layer chromatography (TLC) plate to determine Sudan I in paprika powder. Separation of Sudan I from paprika extract by an MIPs-TLC plate takes 30-40 s. SERS spectra obtained from Sudan I spot on the plate can be acquired within 1 s with gold colloid serving as SERS active substrate. A PLSR (R² = 0.978) model was constructed based on spiking levels (5, 10, 40, 70 and 100 ppm) of Sudan I in paprika powder. Histamine level in canned tuna was investigated using MIPs-polyvinyl chloride (PVC)-SERS method. MIPs-PVC films (recognition element) selectively extracted histamine from tuna extract. A gold colloid solution served as an eluting solvent to extract histamine from MIPs-PVC film and conducted a SERS detection of histamine. A PLSR model (R² = 0.947, RMSECV = 3.526) was built on SERS spectra of histamine with different spiking levels (3, 30 and 90 ppm) in canned tuna. The spectral results suggest the powerful separation of MIPs and sensitive detection of SERS. With statistical analysis, we have confirmed that SERS signals obtained by this MIPs-SERS approach rapidly and accurately quantify chloramphenicol in milk and honey, Sudan I in paprika powder and histamine in canned tuna.
Biofilm is a bacterial community that is responsible for most clinical infections and shows increased resistance to the conventional antimicrobials. Biofilm formation is mediated by quorum sensing (QS), by which bacteria produce and recognize autoinducers (AIs) and thereby coordinate their behaviors in a cell-density dependent manner. The purpose of this thesis project was to design and apply molecularly imprinted polymers (MIPs) to capture AIs, interrupt QS, and subsequently inhibit the formation of bacterial biofilms. Pseudomonas aeruginosa and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C₁₂-AHL) were selected as the bacterial model and target AI molecule, respectively. Photo-initiated bulk polymerization method was conducted to synthesize MIPs using 3-oxo-C₁₂-AHL as the template, itaconic acid (IA) or 2-hydroxyethyl methacrylate (HEMA) as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, 2,2’-azobis(2-methylpropionitrile) as the initiator and N,N-dimethylformamide as the porogen. Different functional monomers and different molar ratios of template: functional monomer: crosslinker were examined to optimize the adsorption capacity and affinity of the synthesized MIPs.Equilibrium rebinding study was conducted to evaluate the adsorption performance of MIPs. MIPs captured 55.2%-61.2% of 3-oxo-C₁₂-AHL in 20% acetonitrile. However, none of them showed good adsorption affinity due to the dominant non-specific binding. In 50% acetonitrile, IA-based MIPs (i.e., 1:6:25 and 1:8:25) demonstrated good adsorption affinity with imprinting factor >1. In biofilm inhibitory studies, P. aeruginosa biofilm was incubated with or without the presence of MIPs for 24 h. Biofilm biomass and sessile cell viability were determined by crystal violet assay and 2,3,5-triphenyl-tetrazolium chloride assay, respectively. Selective HEMA-based polymers (i.e., 1:8:25, 1:6:48 and 1:8:48) significantly (P
Foodborne illness is a growing concern world-wide, and Campylobacter in particular has been reported to cause approximately 145,000 foodborne illness cases every year in Canada. A recent annual report from the Canadian Integrated Program for Antimicrobial Resistance Surveillance reveals an increasing trend of antibiotic-resistant Campylobacter isolated from poultry sources across Canada. The large number of foodborne illnesses and emergence of the resistant strains of Campylobacter pose a serious threat in the agri-food industry. Hence, there is increasing urgency to find alternatives to conventional antimicrobials to reduce the prevalence of Campylobacter in the food supply chain while reducing the likelihood of resistance. Combining antimicrobials is a potential intervention strategy to reduce the growth of pathogens by expanding the spectrum of antimicrobial activity. In this study, carvacrol and zinc oxide nanoparticles (ZnO NPs) were investigated in regards to their synergistic antimicrobial effect against C. jejuni. The combination of these two agents for treatment is based upon current evidence of their individual antimicrobial activity. The objectives of this thesis project were to (1) determine the synergistic antimicrobial effect of carvacrol and ZnO NPs against C. jejuni, (2) investigate the macromolecular fingerprints and gene expression profile of C. jejuni after the combinational treatment, and (3) explain the potential mechanism of the synergistic antimicrobial effect. In this work, a macrobroth dilution method was used to test the antimicrobial effect of the compounds against C. jejuni. The macromolecular fingerprints of C. jejuni cells treated with carvacrol and ZnO NPs were investigated using confocal micro-Raman spectroscopy, whereas Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) was employed to study the variation in bacterial gene expression after the antimicrobial treatment. The synergistic antimicrobial effect of carvacrol and ZnO NPs against C. jejuni was clearly demonstrated using the time-kill curve. The macromolecular fingerprints and gene expression profile revealed the role of carvacrol in the synergistic antimicrobial effect against C. jejuni. The results of this study provide fundamental knowledge about bacterial stress in response to the synergistic antimicrobial effects. This antimicrobial combination may be utilized as an intervention strategy to reduce the prevalence of C. jejuni in agri-foods.
Atrazine is a harmful herbicide that can disrupt the hormonal system in humans and animals. Contamination of atrazine in various agri-food products occurs due to illegal uses. Detection of atrazine in foods is time consuming and expensive when the official methods such as gas chromatography-mass spectrometry are applied. In this thesis project, we developed a dual biosensor integrating molecularly imprinted polymers (MIPs) with gold nanoparticles (AuNPs)-based colorimetric assay and surface enhanced Raman spectroscopy (SERS), which can be applied for rapid, high-throughput and sensitive determination of trace levels of atrazine in agri-food products (e.g., apple juice).This biosensor includes three functions: separation, screening, and quantification. For separation, MIPs were synthesized using molecular imprinting technology by employing atrazine as the template molecule. MIPs-based solid phase extraction (MIPs-SPE) could selectively separate atrazine from apple juice with high recoveries (~93%). AuNPs-based colorimetric assays were able to rapidly detect atrazine due to ligands between atrazine molecules and the surface of AuNPs. High-throughput screening of a large number of samples could be achieved with simple color variation with the limit of detection (LOD) as low as 0.01 mg L-¹. For quantification, SERS tests were conducted using AuNPs for Raman spectral collection. Raman spectra of apple juice samples with different concentrations of atrazine were rapidly collected and analyzed by chemometrics. The calculated LOD equals to 0.0012 mg L-¹ and limit of quantification (LOQ) was 0.0040 mg L-¹, both of which meet the guidelines set up by Health Canada (i.e., 0.005 mg L-¹). Three types of AuNPs with different diameters (i.e., 43 nm, 27 nm, 11 nm) were synthesized and compared for the use of this biosensor. The largest AuNPs worked best for colorimetric assays while the medium-size AuNPs were the most suitable candidate for SERS tests. The extremely low LOD and LOQ strongly validate the potential application of this innovative dual biosensor for accurate and high-throughput determination of atrazine in foods.
After discovering the potential detriment of melamine to humans, the society calls for novel techniques to accomplish accurate, rapid, high-throughput, and on-line or in-field detection of melamine in foods, as required by the food industry and government laboratories. The aim of this study was to investigate different innovative biosensors combining antibodies or molecularly imprinted polymers (MIPs) with surface enhanced Raman spectroscopy (SERS) to determine melamine in a representative dairy product (i.e. milk). A “two-step” antibody-SERS biosensor was developed to detect melamine in whole milk. The anti-melamine antibody, produced by immunizing New Zealand white rabbits with melamine hapten-ovalbumin immunogen, was used to extract melamine from whole milk exclusively. After releasing melamine from the antibody, the eluents were deposited onto silver dendrite SERS-active substrate for SERS spectral collection. The limit of detection (LOD) calculated by the principal component analysis (PCA) model was lower than 0.79×10-³ mmol/L. The overall analysis was completed in 20 min.The MIP for the “two-step” MIPs-SERS biosensor was synthesized by bulk polymerization of melamine, methacrylic acid, ethylene glycol dimethacrylate and 2,2’-azobisisobutyronitrile. After confirming the specific affinity of the MIP towards melamine by adsorption capacity tests, MIP was used as sorbent for solid phase extraction (SPE) to extract melamine from whole milk. SERS spectra were collected by depositing the eluents from MISPE onto silver dendrite. The LOD and limit of quantification (LOQ) calculated by the linear regression model correlating relative intensity of melamine SERS band at 703 cm-¹ and melamine concentration in whole milk were 0.012 mmol L-¹ and 0.039 mmol L-¹, respectively, and the full analysis was accomplished in 18 min.“One-step” MIPs-SERS biosensor incorporated silver nanoparticles (AgNPs) into MIPs synthesized by bulk polymerization. Adsorption capacity tests verified the specific affinity of MIPs-AgNPs to melamine, and PCA model resulted in the LOD between 0.01 and 0.017 mmol L-¹ melamine in skim milk. The time required to detect melamine in skim milk was 25 min. The low LOD and LOQ, as well as rapid detection confirm the potential of applying these three types of biosensors for accurate and high-throughput detection of melamine in dairy products.
Real-time position sensing has a wide range of applications in motion control systems, parts inspectionand general metrology. Vision-based position sensing systems have signiﬁcant advantages over othersensing methods, including large measurement volume, non-contact sensing, and simultaneous mea-surement in multiple degrees-of-freedom (DOF). Existing vision-based position sensing solutions arelimited by low sampling frequency and low position accuracy. This thesis presents the theory, design,implementation and calibration of a new high-speed stereo-vision camera system for real-time positionsensing based on CMOS image sensors.By reading small regions around each target image rather than the full frame data of the sensor,the frame rate and image processing speed are vastly increased. A high speed camera interface isdesigned based on Camera Link technology, which allows a maximum continuous data throughputof 2.3Gbps. In addition, this stereo-vision system also includes ﬁxed pattern noise (FPN) correction,threshold processing, and sub-pixel target position interpolation.In order to achieve high position accuracy, this system is calibrated to determine its model parame-ters. The primary error sources in this system include target image noise, mechanical installation errorand lens distortion. The image sensor is characterized, and its FPN data is extracted, by experiment.The mechanical installation error and lens distortion parameters are identiﬁed through camera cali-bration. The proposed camera calibration method uses the 3D position reconstruction error as its costfunction in the iterative optimization. The optimization of linear and nonlinear parameters is decoupled.By these means, better estimation of model parameters is achieved. To verify the performance of theproposed calibration method, it is compared with a traditional single camera calibration method in sim-ulation and experiment. The results show that the proposed calibration method gives better parameterestimation than the traditional single camera calibration method.The experimental results indicate that the prototype system is capable of measuring 8 targets in 3-DOF at a sampling frequency of 8kHz. Comparison with a coordinate measurement machine (CMM)shows that the prototype system achieves a 3D position accuracy of 18μm (RMS) over a range of400mm by 400mm by 15mm, with a resolution of 2μm.