A Simplified Amplification-Free Strategy with Lyophilized CRISPR-CcrRNA System for Drug-Resistant Salmonella Detection.

Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drug-resistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for the detection of drug-resistant Salmonella has an ultra-sensitive detection limit of 84 CFU mL-1 within 30 min, with good linearity in the range of 102 -106 CFU mL-1 . In real-world applications, spiked recoveries range from 76.22% to 145.91%, with a coefficient of variation less than 10.59%. AFC-TRFIA offers a cost-effective, sensitive, and virtually equipment-independent platform for preventing foodborne illnesses, screening for drug-resistant Salmonella, and guiding clinical use.

Recent advances in biosynthesis of mycotoxin-degrading enzymes and their applications in food and feed.

Mycotoxins are secondary metabolites produced by fungi in food and feed, which can cause serious health problems. Bioenzymatic degradation is gaining increasing popularity due to its high specificity, gentle degradation conditions, and environmental friendliness. We reviewed recently reported biosynthetic mycotoxin-degrading enzymes, traditional and novel expression systems, enzyme optimization strategies, food and feed applications, safety evaluation of both degrading enzymes and degradation products, and commercialization potentials. Special emphasis is given to the novel expression systems, advanced optimization strategies, and safety considerations for industrial use. Over ten types of recombinases such as oxidoreductase and hydrolase have been studied in the enzymatic hydrolysis of mycotoxins. Besides traditional expression system of Escherichia coli and yeasts, these enzymes can also be expressed in novel systems such as Bacillus subtilis and lactic acid bacteria. To meet the requirements of industrial applications in terms of degradation efficacy and stability, genetic engineering and computational tools are used to optimize enzymatic expression. Currently, registration and technical difficulties have restricted commercial application of mycotoxin-degrading enzymes. To overcome these obstacles, systematic safety evaluation of both biosynthetic enzymes and their degradation products, in-depth understanding of degradation mechanisms and a comprehensive evaluation of their impact on food and feed quality are urgently needed.

Combined metabolomics and transcriptomics analysis reveals the mechanism of antibiotic resistance of Salmonella enterica serovar Typhimurium after acidic stress.

Drug-resistant Salmonella is widely distributed in the meat production chain, endangering food safety and public health. Acidification of meat products during processing can induce acid stress, which may alter antibiotic resistance. Our study investigated the effects of acid stress on the antibiotic resistance and metabolic profile of Salmonella Typhimurium, and explored the underlying mechanisms using metabolomic and transcriptomic analysis. We found that acid-stressed 14028s was more sensitive to small molecule hydrophobic antibiotics (SMHA) while more resistant to meropenem (MERO). Metabolomic analysis revealed that enhanced sensitivity to SMHA was correlated with increased purine metabolism and tricarboxylic acid cycle. Transcriptomic analysis revealed the downregulation of chemotaxis-related genes, which are also associated with SMHA sensitivity. We also found a significant downregulation of the ompF gene, which encodes a major outer membrane protein OmpF of Salmonella. The decreased expression of OmpF porin hindered the influx of MERO, leading to enhanced resistance of the bacteria to the drug. Our findings contribute to greatly improve the understanding of the relationship between Salmonella metabolism, gene expression, and changes in drug resistance after acid stress, while providing a structural framework for exploring the relationship between bacterial stress responses and antibiotic resistance.

Exploring the impact of fungal spores from agricultural environments on the mice lung microbiome and metabolic profile.

Exposure to particulate matter (PM) from agricultural environments has been extensively reported to cause respiratory health concerns in both animals and agricultural workers. Furthermore, PM from agricultural environments, containing fungal spores, has emerged as a significant threat to public health and the environment. Despite its potential toxicity, the impact of fungal spores present in PM from agricultural environments on the lung microbiome and metabolic profile is not well understood. To address this gap in knowledge, we developed a mice model of immunodeficiency using cyclophosphamide and subsequently exposed the mice to fungal spores via the trachea. By utilizing metabolomics techniques and 16 S rRNA sequencing, we conducted a comprehensive investigation into the alterations in the lung microbiome and metabolic profile of mice exposed to fungal spores. Our study uncovered significant modifications in both the lung microbiome and metabolic profile post-exposure to fungal spores. Additionally, fungal spore exposure elicited noticeable changes in α and ß diversity, with these microorganisms being closely associated with inflammatory factors. Employing non-targeted metabolomics analysis via GC-TOF-MS, a total of 215 metabolites were identified, among which 42 exhibited significant differences. These metabolites are linked to various metabolic pathways, with amino sugar and nucleotide sugar metabolism, as well as galactose metabolism, standing out as the most notable pathways. Cysteine and methionine metabolism, along with glycine, serine and threonine metabolism, emerged as particularly crucial pathways. Moreover, these metabolites demonstrated a strong correlation with inflammatory factors and exhibited significant associations with microbial production. Overall, our findings suggest that disruptions to the microbiome and metabolome may hold substantial relevance in the mechanism underlying fungal spore-induced lung damage in mice.

Current research progress of mammalian cell-based biosensors on the detection of foodborne pathogens and toxins.

Foodborne diseases caused by pathogens and toxins are a serious threat to food safety and human health; thus, they are major concern to society. Existing conventional foodborne pathogen or toxin detection methods, including microbiological assay, nucleic acid-based assays, immunological assays, and instrumental analytical method, are time-consuming, labor-intensive and expensive. Because of the fast response and high sensitivity, cell-based biosensors are promising novel tools for food safety risk assessment and monitoring. This review focuses on the properties of mammalian cell-based biosensors and applications in the detection of foodborne pathogens (bacteria and viruses) and toxins (bacterial toxins, mycotoxins and marine toxins). We discuss mammalian cell adhesion and how it is involved in the establishment of 3D cell culture models for mammalian cell-based biosensors, as well as evaluate their limitations for commercialization and further development prospects.

Effects of maternal supplementation with fully oxidised ß-carotene on the reproductive performance and immune response of sows, as well as the growth performance of nursing piglets.

The present study was conducted to evaluate the impact of dietary fully oxidised ß-carotene (OxBC, C40H60O15) supplementation during the perinatal period on immune status and productivity in a sow model. At day 85 of pregnancy, 150 sows were allocated to one of three dietary treatments with fifty sows per treatment. The three experimental diets were supplemented with 0, 4 or 8 mg/kg OxBC in the basal diet. The feeding trial was conducted from gestation day 85 until day 21 of lactation. Dietary OxBC supplementation greatly enhanced colostrum IgM, IgA and IgG levels, and the IgM and IgG content of 14-d milk. Dietary OxBC supplementation decreased the TNF-α and IL-8 levels in colostrum, as well as the TNF-α and IL-18 levels in 14-d milk. There was also a tendency towards an increase in the soluble CD14 level in 14-d milk. Although dietary treatments did not affect average daily feed intake nor backfat thickness loss during lactation, dietary OxBC supplementation tended to enhance litter weight and individual piglet weight at weaning. There was a trend towards increased lactose concentration in 14-d milk with increasing dietary OxBC. It is concluded that dietary supplementation with OxBC during the perinatal period enhances the lactose concentration of sow milk and the immune status of sows, which is reflected by improved cytokine status and immunoglobulin concentrations in colostrum and milk, and thus tending to increase litter weight and individual piglet weight at weaning. The results also provide a scientific nutritional reference for perinatal mothers due to the biological similarity between pigs and humans.

Opposite estrogen effects of estrone and 2-hydroxyestrone on MCF-7 sensitivity to the cytotoxic action of cell growth, oxidative stress and inflammation activity.

There are many kinds of estrogens, and endogenous estrogens produce a variety of estrogen metabolites with similar structure but with different physiological effects after metabolism in vivo. Studies have shown that estrone (E1) widely occurs in the environment and animal-derived food. Because of its estrogen effect, E1 can have adverse effects on the human body as an endocrine disruptor. In this study, we found that E1 and 2-hydroxyestrone (2-OH-E1), the hydroxylation metabolite of estrogen, have opposite proliferative effects on breast cancer cells (MCF-7) through cell proliferation experiments and comparison of their effects by molecular docking and detection of ROS, Ca2+, and cell pathway proteins. The effects of 2-methoxyestrone (2-MeO-E1) and 16α-hydroxyestrone (16α-OH-E1) on the biochemical and protein levels of MCF-7 were further studied to compare the effects of metabolic sites and modes on estrogen effects. Hydroxylation of E1 at the C2 site weakened the estrogen effect, down-regulated the expression of the mammalian target of rapamycin (mTOR) and protein kinase B (Akt) pathway proteins, inhibited the proliferation of cancer cells, and enhanced anti-oxidative stress and anti-inflammation. Methoxylation at the C2 position also inhibited the expression of inflammatory and oxidative stress pathway proteins but did not greatly affect the estrogen effects. However, hydroxylation on C16 had no significant effect on the biological effects of estrogen. Therefore, the structural changes of estrogen on C2 are important reasons for the different physiological effects of estrogen and its metabolites. Thus, by regulating the gene Cytochrome P450 1B1(CYP1B1), which affects the hydroxylation metabolism of estrogen, and promoting the hydroxylation of estrone at the C2 position, the estrogen effect of estrone can be effectively reduced, thus reducing the harm its poses in food and the environment.

A novel fluorescent molecularly imprinted polymer SiO2 @CdTe QDs@MIP for paraquat detection and adsorption.

Paraquat (PQ) residue is harmful for human health, agriculture, and the aquatic environment. This paper proposes a novel fluorescent molecularly imprinted polymer (MIP), SiO2 @CdTe QDs@MIP, for PQ detection and adsorption. The MIP was synthesized using 3-aminopropyltriethoxysilane as the functional monomer, 4,4'-bipyridyl as the template molecule, and tetraethoxysilane as the cross-linker. In addition, CdTe quantum dots featuring unique optical characteristics and excellent photochemical stability were combined as signal reporter. The synthesized MIP had a Brunauer-Emmett-Teller surface area of 68.2 m2 /g, pore volume of 0.42 cm3 /g and pore size of 6.9 nm, demonstrating the potential for both PQ detection and adsorption. For PQ detection, the MIP could achieve three orders of magnitude better than the limit of detection, and one order of magnitude wider detection range than existing methods. The PQ recovery values for real samples of water and corn were 96.4-102.1% and 93.9-97.3%, respectively. The amount of PQ detected by the MIP was within 98.05% on average of that using high-performance liquid chromatography. For PQ adsorption, the MIP had an adsorption capacity of 3.36 mg/g, and followed a pseudo-second-order kinetic model with excellent toxicological characteristics. Overall, the novel SiO2 @CdTe QDs@MIP proposed in this paper could facilitate an efficient and convenient method for PQ detection and adsorption.

Metabolic Transition of Milk Triacylglycerol Synthesis in Response to Varying Levels of Three 18-Carbon Fatty Acids in Porcine Mammary Epithelial Cells.

This study aimed to examine the effects of increasing levels of three 18-carbon fatty acids (stearate, oleate and linoleate) on mammary lipogenesis, and to evaluate their effects on the milk lipogenic pathway in porcine mammary epithelial cells (pMECs). We found that increasing the three of 18-carbon fatty acids enhanced the cellular lipid synthesis in a dose-dependent manner, as reflected by the increased (triacylglycerol) TAG content and cytosolic lipid droplets in pMECs. The increased lipid synthesis by the three 18-carbon fatty acids was probably caused by the up-regulated expression of major genes associated with milk fat biosynthesis, including CD36 (long chain fatty acid uptake); GPAM, AGPAT6, DGAT1 (TAG synthesis); PLIN2 (lipid droplet formation); and PPARγ (regulation of transcription). Western blot analysis of CD36, DGAT1 and PPARγ proteins confirmed this increase with the increasing incubation of 18-carbon fatty acids. Interestingly, the mRNA expressions of ACSL3 and FABP3 (fatty acids intracellular activation and transport) were differentially affected by the three 18-carbon fatty acids. The cellular mRNA expressions of ACSL3 and FABP3 were increased by stearate, but were decreased by oleate or linoleate. However, the genes involved in fatty acid de novo synthesis (ACACA and FASN) and the regulation of transcription (SREBP1) were decreased by incubation with increasing concentrations of 18-carbon fatty acids. In conclusion, our findings provided evidence that 18-carbon fatty acids (stearate, oleate and linoleate) significantly increased cytosolic TAG accumulation in a dose-dependent manner, probably by promoting lipogenic genes and proteins that regulate the channeling of fatty acids towards milk TAG synthesis in pMECs.

Loop-mediated isothermal amplification-based microfluidic chip for pathogen detection.

Due to the significant growth of food production, the potential likelihood of food contamination is increasing. Foodborne illness caused by bacterial pathogens has considerably increased over the past decades, while at the same time, the species of harmful microorganisms also varied. Conventional bacterial culturing methods have been unable to satisfy the growing requirement for food safety inspections and food quality assurance. Therefore, rapid and simple detection methods are urgently needed. The loop-mediated isothermal amplification (LAMP) technology is a highly promising approach for the rapid and sensitive detection of pathogens, which allows nucleic acid amplification under isothermal conditions. The integration of the LAMP assay onto a microfluidic chip is highly compatible with point-of-care or resource-limited settings, as it offers the capability to perform experiments in combination with high screening efficiency. Here, we provide an overview of recent advances in LAMP-based microfluidic chip technology for detecting pathogens, based on real-time or endpoint determination mechanisms. We also discuss the promoting aspects of using the LAMP technique in a microfluidic platform, to supply a guideline for further molecular diagnosis and genetic analysis.

Ultrasensitive Fluorometric Angling Determination of Staphylococcus aureus in Vitro and Fluorescence Imaging in Vivo Using Carbon Dots with Full-Color Emission.

Rapid, accurate, and safe screening of foodborne pathogenic bacteria is essential to effectively control and prevent outbreaks of foodborne illness. Fluorescent sensors constructed from carbon dots (CDs) and nanomaterial-based quenchers have provided an innovative method for screening of pathogenic bacteria. Herein, an ultrasensitive magnetic fluorescence aptasensor was designed for separation and detection of Staphylococcus aureus (S. aureus). Multicolor fluorescent CDs with a long fluorescent lifetime (6.73 ns) and high fluorescence stability were synthesized using a facile hydrothermal approach and modified cDNA as a highly sensitive fluorescent probe. CD fluorescence was quenched by Fe3O4 + aptamer via fluorescence resonance energy transfer (FRET). Under optimal conditions, the FRET-based aptasensor can detect S. aureus accompanied by a wide linear range of 50-107 CFU·mL-1 and a detection limit of 8 CFU·mL-1. Compared with other standard methods, this method was faster and more convenient, and the entire test was finished within 30 min. The capability of the aptasensor was simultaneously investigated on food samples. Additionally, the developed CDs exhibited excellent biocompatibility and were thus applied as fluorescent probes for bioimaging both in vitro and in vivo. This new platform provided an excellent application of the CDs for detecting and bioimaging pathogenic bacteria.

Ultrasensitive fluorometric determination of iron(iii) and inositol hexaphosphate in cancerous and bacterial cells by using carbon dots with bright yellow fluorescence.

An ON-OFF-ON dual-function fluorescent nanoprobe is described for the trace detection of ferric ions and inositol hexaphosphate (IP6) in living cells. It is based on the use of yellow-fluorescent nitrogen-doped carbon dots (YN-CDs). Highly fluorescent YN-CDs were synthesized by a hydrothermal process. They have an absolute quantum yield of 2.15% and excitation/emission peaks at 420/575 nm. Fluorescence is quenched by Fe3+via photo-induced electron transfer. The quenchometric assay has a 34 nM detection limit for Fe(iii). On addition of IP6 which has a high affinity for Fe3+ due to the formation of Fe-O-P bonds, fluorescence becomes gradually restored. The resulting ON-OFF-ON assays for Fe(iii) and IP6 are reliable and sensitive. IP6 can be detected at concentrations as low as 2 nM. The nanoprobe was then applied to the determination of Fe3+ and IP6 in living cells in a food matrix. Furthermore, YN-CDs exhibited excellent biocompatibility. Hence, the probe can be applied as a fluorescent ink for bioimaging, both in vitro (cancer cells and bacteria) and in vivo (nematodes and mice).

A novel magnetic fluorescent biosensor based on graphene quantum dots for rapid, efficient, and sensitive separation and detection of circulating tumor cells.

We describe a "turn-on" magnetic fluorescent biosensor based on graphene quantum dots (GQDs), Fe3O4, and molybdenum disulfide (MoS2) nanosheets. It is used for rapid, efficient, and sensitive separation and detection of circulating tumor cells (CTCs). A facile approach (electrochemical synthesis method) for the preparation of photoluminescent GQDs functionalized with an aptamer [epithelial cell adhesion molecule (EpCAM) receptors] and a magnetic agent for one-step bioimaging and enrichment of CTCs is described. MoS2 nanosheets, as a fluorescence quencher, and the aforementioned aptamer@Fe3O4@GQD complex were assembled to construct "turn-on" biosensing magnetic fluorescent nanocomposites (MFNs). This system exhibits low cytotoxicity and an average capture efficiency of 90%, which is higher than that of other magnetic nanoparticles on account of the one-step CTC separation method. In addition, the MFNs could quickly identify and label CTCs within 15 min, surpassing other one-step and two-step marker detection methods. Furthermore, because of the presence of aptamers, the MFNs have specific capability to capture CTCs (both low- and high-EpCAM-expressing cells). In addition, high-sensitivity detection of up to ten tumor cells in whole blood was achieved. Therefore, the MFNs have great potential to be used as universal biosensing nanocomposites for fluorescence-guided tumor cell enrichment and bioimaging. Graphical abstract ᅟ.

High-throughput sequencing analysis of bacterial community composition and quality characteristics in refrigerated pork during storage.

Spoilage bacteria seriously influence meat quality. In this study, the bacterial community, sensory scores, pH, and total volatile basic nitrogen (TVB-N) in refrigerated (4 °C) pork, the most commonly consumed meat in China, were investigated. In a high-throughput sequencing analysis of the V3-V4 region of the 16S rDNA gene, 259 bacterial genera were belonging to 21 phyla were identified. With the passage of time, the bacterial community diversity decreased. After 5 days, Pseudomonas, Acinetobacter and Photobacterium were dominant in refrigerated pork, especially Photobacterium, which rarely associated with meat spoilage. Our results suggest that these taxa contribute to refrigerated pork spoilage. During storage, pH and TVB-N showed similar trends. Additionally, total viable counts (TVC) increased steadily and sensory score decreased. On day 5, TVC, pH, TVB-N and sensory scores changed dramatically, and sensory scores indicating that the shelf life of refrigerated pork was less than 5 days. The predicted metabolic pathways, based o the data of 16S rDNA, indicated an abundant carbohydrate metabolism and amino metabolism in refrigerated pork. This study provides insight into the determinants of shelf life. Furthermore, it provides insight into the process involved in refrigerated pork spoilage.

Comet-like Heterodimers "Gold Nanoflower @Graphene Quantum Dots" Probe with FRET "Off" to DNA Circuit Signal "On" for Sensing and Imaging MicroRNA In Vitro and In Vivo.

Cardiovascular diseases have recently become the number one cause of death worldwide and the risk of getting cardiovascular diseases is doubled as the age increases. MicroRNA-34a (miRNA-34a) as an important potential sensor of aging and cellular senescence could be used in early diagnostics. Herein, a new ultrasensitive platform on the basis of the fluorescence resonance energy transfer (FRET) "off" to DNA circuit signal "on" principle was established, termed comet-like heterodimers gold nanoflower (AuNF) @ graphene quantum dots (GQDs) probe. We discussed that the distance of 4 nm between AuNF and GQDs would increase fluorescence quenching efficiency, and light up sensitivity after the probe combined with a target miRNA initiating DNA circuit strategy. The target miRNA-34a can be quantified down to 0.1 fM, which is about 2 orders of magnitude lower than the existing sensing protocols. Furthermore, we constructed the aging myocardial cell and animal model, and the nanoprobe presented low cytotoxicity and satisfied signal imaging in vitro and in vivo. Significantly, this platform herein is envisioned to provide a reliable guidance for early diagnosing cardiovascular diseases and proposing therapeutic protocols.

Ultrasensitive "FRET-SEF" Probe for Sensing and Imaging MicroRNAs in Living Cells Based on Gold Nanoconjugates.

MicroRNAs (miRNAs), a kind of single-stranded small RNA molecule, play significant roles in the physiological and pathological processes of human beings. Currently, miRNAs have been demonstrated as important biomarkers critically related to many diseases and life nature, including several cancers and cell senescence. It is valuable to establish sensitive assays for monitoring the levels of intracellular up-regulated/down-regulated miRNA expression, which would contribute to the early prediction of the tumor risk and cardiovascular disease. Here, an oriented gold nanocross (AuNC)-decorated gold nanorod (AuNR) probe with "OFF-enhanced ON" fluorescence switching was developed based on fluorescence resonance energy transfer and surface enhanced fluorescence (FRET-SEF) principle. The nanoprobe was used to specifically detect miRNA in vitro, which gave two linear responses represented by the equation F = 1830.32 log C + 6349.27, R2 = 0.9901, and F = 244.41 log C + 1916.10, R2 = 0.9984, respectively, along with a detection limit of 0.5 aM and 0.03 fM, respectively. Furthermore, our nanoprobe was used to dynamically monitor the expression of intracellular up-regulated miRNA-34a from the HepG2 and H9C2 cells stimulated by AFB1 and TGF-ß1, and the experimental results showed that the new probe not only could be used to quantitively evaluate miRNA oncogene in vitro, but also enabled tracking and imaging of miRNAs in living cells.

Using fluorescence immunochromatographic test strips based on quantum dots for the rapid and sensitive determination of microcystin-LR.

A novel immunosensor for the detection of microcystin-LR (MC-LR) was constructed with use of immunochromatographic test strips (ICTS). Quantum dots were chosen to be the fluorescent labels for the immune sensor in ICTS because of their excellent optical and electronic properties with a relatively narrow emission spectrum. The detection sensitivity of the ICTS was related to the concentration of the fluorescent probe and the amount of the MC-LR standards. Under optimal conditions, with MC-LR as the target, the ICTS sensor had a linear range from 0.25 to 5 µg/L, with a correlation coefficient of 0.9901 and a detection limit of 0.1 µg/L. Furthermore, the repeatability of the ICTS was good, and the coefficient of variation was 10%. The ICTS immunosensor allows the reliable detection of MC-LR in water, and has potential in simple, sensitive detection applications. Graphical Abstract A novel method was developed to detect MC-LR using QDs based immunochromatographic test strip.

Electrochemical sensor for determination of tulathromycin built with molecularly imprinted polymer film.

A novel tulathromycin (TLTMC) electrochemical sensor based on molecularly imprinted polymer (MIP) membranes was constructed. p-Aminothiophenol (p-ATP) and TLTMC were assembled on the surface of gold nanoparticles (AuNPs) modified on the gold electrode (GE) by the formation of Au-S bonds and hydrogen-bonding interactions. Besides, polymer membranes were formed by electropolymerization in a polymer solution containing p-ATP, tetrachloroaurate(III) acid (HAuCl4), tetrabutylammonium perchlorate (TBAP), and a template molecule TLTMC. A novel molecular imprinted sensor (MIS) in this experiment was achieved after the removal of TLTMC. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were used to illustrate the process of electropolymerization and its optimal conditions. The electrode with MIP obtained the linear of response range, which was between 3.0 × 10(-12) mol L(-1) and 7.0 × 10(-9) mol L(-1), and the limit of detection was 1.0 × 10(-12) mol L(-1). All the obtained results indicate that the MIS tends to be an effective electrochemical technique for the determination of TLTMC in real-time and in a complicated matrix.

Lysine and valine weaken antibiotic resistance in Salmonella Typhimurium induced by disinfectant stress.

Disinfectants are widely used in food production and environmental sanitation to prevent illness, but bacteria resistance to these disinfectants and co-resistance to antibiotics pose a threat to public health. This study investigated the impact of commonly used disinfectants on the resistance of Salmonella Typhimurium (ST) to disinfectants and antibiotics, and explored the metabolic mechanisms underlying the resistance changes. The results showed that subinhibitory concentrations of disinfectants had a minor impact on the resistance of ST to four disinfectants. However, chlorine-containing disinfectants stress enhanced bacteria resistance to ampicillin, while quaternary ammonium compounds stress increased resistance to tetracycline and gentamicin. Untargeted metabolomics analysis revealed significant changes in glutathione metabolism and lysine and valine degradation pathways after disinfectant exposure. Specifically, ST activated lysine decarboxylation, leading to a significant decrease in lysine levels after benzalkonium chloride exposure, while valine and leucine degradation pathways were activated by sodium hypochlorite stress. The addition of downregulated L-lysine and L-valine increased the sensitivity of ST to antibiotics, providing further evidence for the findings of metabolomics. This study provides guidance for the proper use of disinfectants in food processing and establishes a strategy based on metabolomics to control antibiotic-resistant bacteria.

Multispectral pathogens detection in food using multiplex hyperbranched saltatory rolling circle amplification.

Foodborne illnesses caused by Salmonella and Staphylococcus aureus are a significant public health concern, leading to societal and economic repercussions. It is important to develop a simple and straightforward bacteria detection and identification method. A triple-probe multiplex rolling circle amplification technique has been developed to simultaneously detect Salmonella Typhimurium and S. aureus. This method utilizes fluorophore-labeled long padlock probes targeting S. Typhimurium invA and S. aureus glnA specific genes, along with a pH-based detection approach for direct visual identification. The multiplex hyperbranched saltatory rolling circle amplification assay at 30 °C has showed promising results with synthetic targets within 30 min and real bacteria within 2 h after establishing the detection settings. The assay is specific for S. aureus and S. Typhimurium, with a limit of detection of 39 µM for fluorescence and 78 µM for colorimetric. In the simulative test of this method for the detection of S. Typhimurium and S. aureus in milk, the limit of detection for the fluorescence signal after 2 h of amplification was 10 CFU/mL and 5 CFU/mL, respectively. The detection method was evaluated to be stable enough to detect pathogen for 3.29 months. Consequently, this triple-probe-multiplex rolling circle amplification method displays notable specificity, sensitivity, as well as ease of interpretation when testing food samples for harmful pathogens.