Enhancing gluten detection assay development through optimization of gliadin extraction conditions.

Gluten consumption can lead to severe health conditions in certain individuals, and following a strict gluten-free diet is often the only effective treatment option. Therefore, it is crucial to develop a gluten detection method that is accurate, sensitive, and specific to ensure the absence of gluten. An important aspect of developing effective gluten detection tests is the implementation of an efficient gluten extraction method. In this study, we conducted an evaluation of various buffer conditions for gliadin extraction from both heat-treated and non-heat-treated food samples. These buffer conditions included ethanol, 2-mercaptoethanol, guanidine hydrochloride, detergents, chelating agents, and deep eutectic solvents. Among the tested conditions, a combination of 2-mercaptoethanol and guanidine hydrochloride demonstrated significantly higher extraction efficacy compared to most other conditions. Furthermore, we explored the use of a less toxic extraction buffer, choline chloride, which exhibited a 1.4-fold higher extraction efficiency than the combination of 2-mercaptoethanol and guanidine hydrochloride (p < 0.05). Choline chloride showed great potential as a preferred buffer for commercial gliadin extraction kits, suitable for both heat-treated and non-heat-treated food samples. Overall, our findings highlight the importance of optimizing the gluten extraction process to improve the accuracy and reliability of gluten detection methods, ultimately contributing to the development of effective tools for individuals following a strict gluten-free diet.

Generation of a Beta-Cell Transplant Animal Model of Diabetes Using CRISPR Technology.

Since insulin deficiency results from pancreatic beta-cell destruction, all type 1 and most type 2 diabetes patients eventually require life-long insulin injections. Insulin gene synthesis could also be impaired due to insulin gene mutations as observed in diabetic patients with MODY 10. At this point, insulin gene therapy could be very effective to recompense insulin deficiency under these circumstances. For this reason, an HIV-based lentiviral vector carrying the insulin gene under the control of insulin promoter (LentiINS) was generated, and its therapeutic efficacy was tested in a beta-cell transplant model lacking insulin produced by CRISPR/Cas9-mediated genetically engineered pancreatic beta cells. To generate an insulin knockout beta-cell transplant animal model of diabetes, a dual gene knockout plasmid system involving CRISPR/Cas9 was transfected into a mouse pancreatic beta cell line (Min6). Fluorescence microscopy and antibiotic selection were utilized to select the insulin gene knockout clones. Transplantation of the genetically engineered pancreatic beta cells under the kidney capsule of STZ-induced diabetic rats revealed LentiINS- but not LentiLacZ-infected Ins2KO cells transiently reduced hyperglycemia similar to that of MIN6 in diabetic animals. These results suggest LentiINS has the potential to functionally restore insulin production in an insulin knockout beta-cell transplant animal model of diabetes.

Cell Death in Escherichia coli: Incomplete Base Excision Repair under Depletion of DapB and Dxr Proteins.

The generation of reactive oxygen species (ROS) within the cell is a significantly shared aspect of bacterial cell death against different stress conditions. The main cell death mechanism due to the generation of reactive oxygen species is then the incomplete base excision repair (BER) in response to oxidized nucleotides. In their recent article in mBio, C. C. Gruber, V. M. P. Babu, K. Livingston, H. Joisher, and G. C. Walker (mBio 13[1]:e03756-21, 2022) report two new stress conditions regarding the depletion of DapB and Dxr, which indeed cause similar mechanisms for cell death. These two stress conditions trigger highly distinctive stress response mechanisms within the cell, but the ultimate cell death mechanism is a result of a shared process. These findings prove that the disturbance in the homeostasis of cells under a variety of different stresses initiates cell death mechanisms through the production of ROS, generation of 8-oxo-dG and the incomplete BER.

The Recent Original Perspectives on Nonculture-Based Bacteria Detection Methods: A Comprehensive Review.

Foodborne diseases that are primarily caused by pathogenic bacteria are of major public health concern globally. One of the key strategies in minimizing and controlling the risk of contamination of food with such pathogens requires establishing effective detection and tracking methods of zoonotic bacteria. Although culture-based pathogen detection methods are developed and widely used by the industry traditionally, nonculture-based zoonotic bacteria detection methods are now more widely investigated and used owing to the recent developments in nucleic acid and immunological-based detection methods. These rapid detection methods provide the opportunity of acquiring real-time test results and high-throughput screening of a large number of samples at a time. One of the key aspects of rapid detection methods is the development of effective sample processing methods as food samples are heterogeneous and highly complex in composition. In this review, variety of sample processing methods, in terms of nonspecific and target-specific sample processing as well as thorough overview of recent developments in nonculture-based bacteria detection methods are presented.

Mutant Construction and Integration Vector-Mediated Genetic Complementation in Listeria monocytogenes.

Genes that play a role in stress response mechanisms and other phenotypes of Listeria monocytogenes can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of L. monocytogenes using the plasmid pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758-2761, 2007). Following screening of mutant libraries, putative mutants are identified and the transposon is localized, leading to identification of the genes responsible for the phenotype of interest. To confirm the role of the transposon-harboring gene in the relevant phenotype, transposon mutants are genetically complemented with the wild-type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177-4186, 2002).

Mutant construction and integration vector-mediated gene complementation in Listeria monocytogenes.

Genes that play role in stress response mechanisms and other phenotypes of bacteria can be identified by construction and screening of mutant libraries. In this chapter, we describe the construction and screening of mutant libraries of Listeria monocytogenes using a plasmid, pMC38, carrying a mariner-based transposon system (TC1/mariner) and constructed by Cao et al. (Appl Environ Microbiol 73:2758-2761, 2007). Following screening of the mutant library, putative mutants are identified and the transposon is localized, leading to identification of the genes that play possible roles in the phenotype of interest. To confirm the role of the gene in the relevant phenotype, transposon mutants are genetically complemented with the wild type gene using the site-specific temperature-sensitive integration vector pPL2, constructed by Lauer et al. (J Bacteriol 184:4177-4186, 2002).

Isolation of Listeria monocytogenes from Food and Water: Official and Experimental Protocols.

Listeria monocytogenes is frequently encountered in foods but often at low concentrations and typically in the presence of other microbiota, including nonpathogenic Listeria spp. The potential of L. monocytogenes to cause severe human disease mandates sensitive, accurate, and rapid detection in foods. Isolation of L. monocytogenes from foods is critical, not only for routine surveillance, but also for epidemiologic investigations. Isolation of the pathogen from water (especially surface water used for irrigation) is similarly important, as produce has been implicated in listeriosis outbreaks and contaminated water can be involved in contamination of produce. This unit provides basic protocols for the isolation of L. monocytogenes from foods and water.

Bovine Staphylococcus aureus: dose response to iodine and chlorhexidine and effect of iodine challenge on antibiotic susceptibility.

Staphylococcus aureus is a gram-positive organism that is frequently associated with clinical or subclinical mastitis. The use of germicidal teat dips is one of the measures taken by the dairy industry to control mastitis. Iodine and chlorhexidine compounds are commonly used disinfectants in teat dips. We determined the minimum inhibitory concentrations (MIC) of iodine for 37 isolates of Staph. aureus and observed variations in MIC. Seven of these Staph. aureus isolates were selected as genotype group representatives based on their pulsed-field gel electrophoresis patterns. Dose responses against iodine and chlorhexidine were determined for the 7 genotype group representatives. The response of these isolates to iodine varied significantly, whereas all isolates were susceptible to chlorhexidine, even at concentrations as low as 0.0002%. We also evaluated whether exposure of Staph. aureus to sublethal levels of iodine influenced subsequent antibiotic susceptibility. No differences in antibiotic susceptibility of Staph. aureus were observed among cultures grown in brain heart infusion broth with and without supplemental iodine. The observed variation in iodine dose responses of Staph. aureus may have implications for the occurrence of Staph. aureus mastitis on dairy farms.

Purine biosynthesis mutants (purA and purB) of serotype 4b Listeria monocytogenes are severely attenuated for systemic infection in intragastrically inoculated A/J Mice.

In this study, we demonstrate that purA and purB transposon mutants of serotype 4b Listeria monocytogenes were severely impaired in their ability to colonize the gastrointestinal tract and cause systemic infection of the spleen, liver, and gallbladder following intragastric inoculation of A/J mice. The mutant strains were also impaired in their ability to multiply within Caco-2 human intestinal epithelial cells. Neither mutant was affected in resistance to synthetic gastric fluid (pH 4.5). These findings indicate that purine biosynthesis is critical for gastrointestinal virulence of L. monocytogenes serotype 4b in mice.

Inactivation of a cold-induced putative rna helicase gene of Listeria monocytogenes is accompanied by failure to grow at low temperatures but does not affect freeze-thaw tolerance.

Freeze-thaw tolerance (cryotolerance) of Listeria monocytogenes is markedly influenced by temperature of growth of the bacteria, and may involve responses to low-temperature stresses encountered during freezing and thawing. A cold-sensitive mariner-based transposon mutant of L. monocytogenes F2365 was found to harbor a single insertion in LMOf2365_1746, encoding a putative RNA helicase, and earlier shown by other investigators to be induced during 4 degrees C growth of L. monocytogenes. The mutant had normal growth at 37 degrees C but completely failed to grow at either 4 or 10 degrees C, and had impaired growth and reduced swarming on soft agar at 25 degrees C. However, the mutation had no discernible influence on the ability of the bacteria to tolerate repeated freezing and thawing after growth at either 25 or 37 degrees C. The findings suggest that the transposon insertion in the putative helicase gene, in spite of the severely cold-sensitive phenotype that accompanies it, does not affect the ability of the bacteria to cope with cold-related stresses encountered during repeated freezing and thawing.

Temperature-dependent requirement for catalase in aerobic growth of Listeria monocytogenes F2365.

Listeria monocytogenes is a Gram-positive, psychrotrophic, facultative intracellular food-borne pathogen responsible for severe illness (listeriosis). The bacteria can grow in a wide range of temperatures (1 to 45°C), and low-temperature growth contributes to the food safety hazards associated with contamination of ready-to-eat foods with this pathogen. To assess the impact of oxidative stress responses on the ability of L. monocytogenes to grow at low temperatures and to tolerate repeated freeze-thaw stress (cryotolerance), we generated and characterized a catalase-deficient mutant of L. monocytogenes F2365 harboring a mariner-based transposon insertion in the catalase gene (kat). When grown aerobically on blood-free solid medium, the kat mutant exhibited impaired growth, with the extent of impairment increasing with decreasing temperature, and no growth was detected at 4°C. Aerobic growth in liquid was impaired at 4°C, especially under aeration, but not at higher temperatures (10, 25, or 37°C). Genetic complementation of the mutant with the intact kat restored normal growth, confirming that inactivation of this gene was responsible for the growth impairment. In spite of the expected impact of oxidative stress responses on cryotolerance, cryotolerance of the kat mutant was not affected.

Impact of growth temperature and agar versus liquid media on freeze-thaw tolerance of Yersinia enterocolitica.

Yersinia enterocolitica is a foodborne pathogen well known for its ability to grow at low temperatures. Recent studies with another psychrotrophic foodborne pathogen, Listeria monocytogenes, revealed that temperature of growth had pronounced impact on survival following repeated freezing and thawing (cryotolerance). Listerial cryotolerance was significantly more pronounced when bacteria were grown at 37 degrees C than following growth at either 4 degrees C or 25 degrees C. However, it is not known whether such impact of growth temperature is a general adaptation shared with other foodborne pathogens. In this study, we investigated the impact of growth temperature (4 degrees C, 25 degrees C, and 37 degrees C) on cryotolerance of Y. enterocolitica. In strong contrast to findings previously obtained with Listeria spp., cryotolerance of Y. enterocolitica was impaired following growth in liquid media at 37 degrees C, with cell concentration dropping to undetectable levels (<10(1) colony forming unit/mL) following as few as six freeze-thaw cycles. On the other hand, when the bacteria were grown at 4 degrees C, cryotolerance was significantly higher (p < 0.05), and substantial survival was maintained even after 18 cycles (2-5 log reduction, depending on strain). Enhanced cryotolerance was also observed with cultures grown at 25 degrees C. Bacteria grown at 37 degrees C on agar were significantly more cryotolerant than following growth at 37 degrees C in liquid media (p < 0.05). The data suggest species-specific impact of growth temperature and liquid versus agar medium on cryotolerance of cold-tolerant bacteria.

Role of growth temperature in freeze-thaw tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37 degrees C but are strongly repressed below 30 degrees C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37 degrees C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at -20 degrees C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37 degrees C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25 degrees C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37 degrees C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.

Impact of antibiotic stress on acid and heat tolerance and virulence factor expression of Escherichia coli O157:H7.

This study was conducted to determine the effect of antibiotic stress on the virulence factor expression, simulated gastric fluid (SGF; pH 1.5) survival, and heat tolerance (56 degrees C) of Escherichia coli O157:H7. The MIC for three antibiotics (trimethoprim, ampicillin, and ofloxacin) was determined for two E. coli O157:H7 strains (ATCC 43895 [raw hamburger isolate] and ATCC 43890 [fecal isolate]) by the dilution series method. Subsequently, cells were stressed at the MIC of each antibiotic for 4 h, and poststress tolerance and virulence factor production were evaluated. Heat tolerance (56 degrees C) was determined by the capillary tube method, and SGF (pH 1.5) survival was used to assess acid tolerance. Virulence factor expression (stx, hlyA, and eaeA) was evaluated by the creation of lacZ gene fusions and then use of the Miller assay (a beta-galactosidase assay). Stressed and control cells were evaluated in triplicate. The MIC for trimethoprim was 0.26 mg/liter for both strains; for ampicillin, it was 2.05 mg/liter for both strains; and for ofloxacin, it was 0.0256 and 0.045 mg/liter for each strain. Heat tolerance and SGF survival following antibiotic stress decreased when compared with control cells (P < 0.05). Exposure to ofloxacin increased stx and eaeA expression (P < 0.05). Exposure to ampicillin or trimethoprim increased eaeA expression (P < 0.05). hly expression increased following trimethoprim stress (P < 0.05). Antibiotics can increase E. coli O157:H7 virulence factor production, but they do not produce a cross-protective response to heat or decreased pH.