Isotachophoresis detection of viable bacteria for monitoring the quality of food contact surfaces and meat
Creators: name: Kumarajith, Thisara
Publisher: name: University of Tasmania
Year: 2024

description: As one of the world's top 3 beef exporters, the Australian red meat industry plays a crucial role in the country's economy. Production of safe meat is essential to consumers’ well?being and to maintaining the worldwide reputation for exceptional meat production. Meat is rich in nutrients making it an ideal substrate for numerous spoilage microorganisms and food?borne disease-causing pathogens. Hence, continuous surveillance systems that detect microbial growth are crucial to the food industry. Microbial contamination can occur at any stage of meat production. This creates unique challenges when monitoring for possible microbial contamination, as checking the microbial quality only at one stage is not enough to ensure the safety of the final product. The most common and accurate detection methods are culture-based techniques. However, these techniques include incubation steps which take at least 24 to 48 hours to generate results. This may cause problems if a contaminated carcass is present in the early stages of the production line, as it might not be discovered until later stages, by which time it could have contaminated other carcasses, increasing the waste. This thesis focused on developing a rapid microbial contamination detection system for the meat processing environment using the electrophoresis technique.<br>The recovery of a maximum number of bacteria from surfaces is necessary to improve the detection. Swab sampling, which is used in this study, is one of the most common surface sampling techniques used in food industries. Several types of swabs are available in the market, each made for unique testing purposes. The overall efficiency of swabs depends on two steps: uptake efficiency and release efficiency. Therefore, the first part of the thesis documents the research results comparing the uptake and release efficiencies of four different swabs: CleanFoam® (Texwipes, USA), FLOQSwabs™ (Copan diagnostic Inc., USA), Hydraflock swabs (Puritan medical products, USA), and Cotton swabs (LIVINGSTONE, Australia). The uptake efficiencies of the swabs were 57.93% ± 20.25, 86.96% ± 6.39, 84.85% ± 4.91, and 96.52% ± 1.92, respectively. However, uptake efficiencies of flock swabs and cotton swabs are not significantly different (P&gt;0.05). The release efficiency of the swabs depends on the type of releasing buffer used. Eight different buffers were assessed; Puritan Hydra flock swab with Tris-MOPS buffer produced the highest releasing efficiency of 93.42% ± 3.12, and the lowest reported was 17. 52% ± 17.84 for cotton swabs with Tris-borate plus 0.25% tween 80 buffer. Combining the results from both studies, an estimate of the overall efficiency of the swabs was obtained; the Puritan Hydra flock with Tris-MOPS buffer had maximum recovery (79.27%). Collectively, the data of this study shows that Flock swabs and foam swabs perform better than cotton swabs in recovering bacteria from surfaces.<br>The steps of developing the rapid bacterial quantifying method are presented in the thesis's second section, which also details the data from applying the proposed approach to surfaces and meat carcasses. Isotachophoresis, a type of electrophoresis, was used to isolate and quantify the total viable count with the help of nucleic acid stains (SYTO 9 and Propidium Iodide). SYTO 9 is a cyanine dye that intercalates to nucleic acids in both dead and live cells. In contrast, propidium iodide only stains the nucleic acids of cells with an impaired cell membrane. Isotachophoresis conditions for the analysis; Before analysis steps – flush the capillary with 1.8 % w/v HEC in 50 mM Tris HCl (pH 8.0) (75 psi for 8 min). For analysis, the capillary needs flushing with LE (Tris HCl 50 mM) at 10 psi for 9 min). Then an electrokinetic injection of the spacer analyte (Tris MES 90mM) at -16kV for 0.5 min with counter pressure (1.3 psi), and injection of the stained bacterial cells in terminating electrolyte (TE) (Tris TAPS (10 mM) at -16 kV) at -16 kV for 2 min with counter pressure (1.3 psi), followed by a continuation of isotachophoresis in TE with -16 kV applied at reversed polarity. The LODs of this method for detecting the total number of bacteria (dead + live cells) and for total viable bacteria in culture samples were 14 cells/ mL and 184 CFU/mL, respectively, within 30 min. When used for quantifying bacteria from steel surfaces, the required safety standard levels for meat contacting surfaces according to European standards (&gt;10 CFU/ cm<sup>2</sup> ) can be achieved by swabbing a 100 cm<sup>2</sup> area using Copan FLOQSwab and Tris-TAPS as the releasing buffer. The method was validated by comparison with culturing techniques which showed that both approaches provide similar data values. Meat surfaces were sampled using a Puritan Hydra flock swab. The same isotachophoresis conditions used to detect the bacteria on steel surfaces were used again here with the addition of a simple filtration method to the sample preparation. Simple 100 times dilution of the sample allows detection of the required safety standards (total viable count &gt;3.1625 × 10<sup>4</sup> CFU/cm<sup>2</sup> ). This system can be introduced into meat processing facilities as an alternative test for the time-consuming plate count method.<br>The third part of the thesis explored the possibility of using quantum dots to detect specific target bacteria when combined with a bacteria-specific antibody. The initial microscopic studies showed that the antibody quantum dot conjugates prepared by mixing biotinylated <i>E. coli</i>-specific antibodies with streptavidin quantum dots (λ<sub>em</sub> 645 nm) could successfully stain <i>E. coli</i> bacteria. Sample preparation included staining the bacteria offline for 30 min and then a simple centrifuge step to separate free antibody-quantum dot conjugates from the stained bacteria. Isotachophoresis of stained bacteria was carried out using Tris HCl (50 mM, pH 8.00) as the leading electrolyte and Tris TAPS (10 mM, pH 8) as the terminating electrolyte. The detection of 12 injected <i>E. coli </i>cells was achieved with the injection period of 0.7 psi for 20 sec and isotachophoresis at -12kV combined with forward pressure of 0.8 psi. The specificity of the quantum dot-linked <i>E. coli</i> antibody was tested in the presence of another bacilli-shaped bacteria, Bacillus subtilis, and no non-specific binding was observed between the conjugates and B. subtilis, confirming that the antibody could specifically identify <i>E. coli</i> bacteria from samples containing other bacteria.The rapid bacterial detection method developed by a combination of the isotachophoresis technique with industry-practice swab sampling shows a promising future for application in food processing facilities for monitoring the quality of surfaces and products such as meat in real-time. The approach can detect and quantify live bacteria in samples making it equivalent to the plate count method, however, unlike the plate count method, the developed method takes only 30 min. The addition of quantum-linked antibody staining to the isotachophoresis method enabled the specific detection of a target bacterial species.