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Bacteriophages (phages) have gained considerable attention as effective antimicrobial agents that infect and kill pathogenic bacteria. Based on this feature, phages have been increasingly used to achieve food safety. They are stored in a medium or buffer to ensure stability; however, they cannot be directly applied to food under these conditions due to reasons such as regulatory considerations and concerns about marketability. This study developed a stabilizing solution that allowed the maintenance of phage activity for extended periods at room temperature while being directly applicable to food. The stability of phages stored in distilled water was relatively low. However, adding a stabilizer composed of sugars and salts improved the survival rates of phages significantly, resulting in stability for up to 48 weeks at room temperature. When Escherichia coli O157:H7-contaminated vegetables were washed with tap water containing phages, the phages reduced the pathogenic E. coli count by over 90% compared with washing with tap water alone. Additionally, when pathogenic E. coli-contaminated vegetables were placed in a phage-coated container and exposed to water, the coating of the container dissolved, releasing phages and lysing the pathogenic E. coli. This led to a significant 90% reduction in pathogenic E. coli contamination compared to that after water rinsing. These results suggest an effective and economical method for maintaining phage activity and establishing the potential for commercialization through application in the food industry. [ABSTRACT FROM AUTHOR]

An impedance sensing system is a family of biosensors that measure changes in electrical impedance to perform their functions. Physical and chemical changes in the impedance of the sensing element, such as changes in the concentration of a target analyte or changes in the physical properties of the sensing element, can result in changes in the impedance of the sensing element. Many impedance biosensors have been developed for the detection of pathogens in the past few decades. Several types of biosensors have been developed for the detection of infections, including transduction elements, biorecognition components, and electrochemical approaches. In this review, we discuss the characteristics and pathogenic factors associated with 2,3-butanediol-producing Klebsiella pneumoniae collected using impedance sensors. An impedance sensing system was introduced as a great method for monitoring the virulence factors of Klebsiella spp. in situ. Klebsiella pneumoniae produces virulence factors, including capsules, lipopolysaccharides, fimbriae, and siderophores, as part of its pathogenesis. It is possible to examine virulence factors' pathogenic characteristics in vitro and in vivo using real tissues or mouse models in order to conduct experiments. For the monitoring of virulence factors in situ, a novel alternative method has been developed to mimic the environment of real tissues. For the purpose of developing tissue-mimicking models, mucin and mannose were used to modify the surface of gold electrodes. These components are known to contribute to the adhesion of pathogens to epithelial cells in mammals. [ABSTRACT FROM AUTHOR]

In this study, we report an approach for the automated separation and concentration of pathogenic bacteria from food samples as a pretreatment step before detection. We developed an automated immunomagnetic separation (IMS) system, which included pre-enrichment of food samples, incubation with immunomagnetic beads, and separation and concentration of immunomagnetic beads along with bound target bacteria using an automated instrument based on IMS technology. Magnetic recovery bars covered with glass cylinders were utilized for separation of magnetic beads in food samples introduced into collection tubes. The separated magnetic beads with bound bacteria were dispersed in 1 mL phosphate-buffered saline (PBS) in concentration tubes. The efficiency of separation and concentration of the target pathogenic bacteria by the fabricated system were determined using plate counting with E. coli O157:H7 and whole milk as the model target bacteria and real food matrix, respectively. To achieve high concentration and recovery yield, the spiked milk samples were enriched with buffered peptone water before incubation with immunomagnetic beads and introduction into the collection tubes. The entire procedure from pre-enrichment through to concentration was completed within 2 h with the automated IMS system. The proposed approach achieved over 100-fold concentration of inoculated E. coli O157:H7 from milk samples with over 20% recovery of viable cells after pre-enrichment. These results illustrated the feasibility of the developed auto-IMS platform for rapid inspection of food pathogens. [ABSTRACT FROM AUTHOR]

We introduce a system for the efficient separation and concentration of pathogenic bacteria using biologically prepared immunomagnetic beads. Amylose magnetic beads (AMBs) were synthesized by an enzymatic reaction of amylosucrase from Deinococcus geothermalis (DGAS). The simple and rapid conjugation of AMBs and antibodies was achieved by the MBP-SPG fusion protein. MBP (maltose binding protein) binds to the surface of an AMB owing to its intrinsic affinity to the di-glucose in the AMB. SPG (streptococcal protein G) fused to the MBP has specific affinity to the Fc region of the antibody. Anti- Escherichia coli O157 antibodies were conjugated to the AMBs through a MBP-SPG linker without any physical and chemical treatments. The efficiency of separation and concentration of the target E. coli O157:H7 by the functionalized AMBs was revealed by plating counting, conventional polymerase chain reaction (PCR), and real-time RCR analysis. The immuno-AMBs effectively separated and concentrated the target bacteria from a commercial milk sample spiked with known number of bacteria, which was then analyzed by PCR to a detection limit of 10 CFU/mL. On the other hand, no PCR product was produced when milk was introduced directly to a PCR reaction. These results show that MBP-SPG is an effective linker and the resulting immuno-AMBs are capable of separating and concentrating the target bacteria from a food matrix. [ABSTRACT FROM AUTHOR]

The food industry requires rapid and simple detection methods for preventing harm from pathogenic bacteria. Until now, various technologies used to detect foodborne bacteria were time-consuming and laborious. Therefore, we have developed an automated immunomagnetic separation combined with a colorimetric assay for the rapid detection of E. coli O157:H7 in food samples. The colorimetric detection method using enzymatic reaction is fascinating because of its simplicity and rapidity and does not need sophisticated devices. Moreover, the proposed procedures for the detection of bacteria in food take less than 3 h including pre-enrichment, separation and detection steps. First, target-specific immunomagnetic beads were introduced to contaminated milk in a pre-enrichment step. Second, the pre-enriched sample solution containing target bacteria bound on immunomagnetic beads was injected into an automated pretreatment system. Subsequently, the immunomagnetic beads along with target bacteria were separated and concentrated into a recovery tube. Finally, released β-galactosidase from E. coli O157:H7 after lysis was reacted with chlorophenol red β-galactopyranoside (CPRG) used as a substrate and the colorimetric change of CPRG was determined by absorbance measuring or the naked eye. By the proposed approach in this study, we could detect 3 × 102 CFU/mL of E. coli O157:H7 from a milk sample within 3 h. [ABSTRACT FROM AUTHOR]

Abstract: We introduce a nanoporous membrane based impedimetric immunosensor for the label-free detection of bacterial pathogens in whole milk. A simple and rapid method to modify a commercially available alumina nanoporous membrane with hyaluronic acid (HA) effectively reduced the non-specific binding of biomolecules and other cells, and permitted successful immobilization of antibodies. Escherichia coli O157:H7, one of the most harmful food-borne pathogenic bacteria, was tested as a model pathogen in this study. The ionic impedance of electrolytes through nanopores, due to antibody–pathogen interactions, was monitored by impedance spectra and analyzed by normalized impedance change (NIC). The regression equation for the NIC at 1kHz versus concentration of E. coli O157:H7 (10–105 cfu/ml) was obtained, and the detection limit found to be as low as 10cfu/ml. In addition, the proposed immunosensor was successfully used for the detection of E. coli O157:H7 in whole milk samples with the detection limit as low as 83.7cfu/ml with 95% probability. The specificity of the immunosensor was also demonstrated using non-target bacteria, including Staphylococcus aureus, Bacillus cereus, and non pathogenic E. coli DH5α. This study shows that a HA-functionalized nanoporous membrane-based impedimetric sensor is capable of detecting pathogenic bacteria in whole milk without any pretreatment. This is a significant step for evaluating the safety of food and environmental samples and other medical diagnostics. [Copyright &y& Elsevier]