Bactericidal efficacy of plasma-activated water against Vibrio parahaemolyticus on Litopenaeus vannamei.

In this study, the antimicrobial mechanism of plasma-activated water (PAW) against Vibrio parahaemolyticus and the effectiveness of PAW in artificially contaminated Litopenaeus vannamei were investigated. The results demonstrated a significant reduction (p < 0.05) in viable counts of V. parahaemolyticus with increasing plasma discharge time (5, 10, 20, and 30 min) and PAW immersion time (3, 5, 10, 20, and 30 s). Specifically, the count of V. parahaemolyticus decreased by 2.1, 2.7, 3.3, and 4.4 log CFU/mL after exposed to PAW 5, PAW 10, PAW 20, and PAW 30 for 30 s, respectively. Significant cell surface wrinkling, accompanied by notable nucleic acid and protein leakage were observed after treatment with PAW. The permeability of the inner and outer cell membranes was significantly increased (p < 0.05), along with an increase in electrical conductivity (p < 0.05). The reactive oxygen species (ROS) within V. parahaemolyticus cells were significantly increased (p < 0.05), while superoxide dismutase (SOD) activity, and the relative expression of the ompW, emrD, and luxS genes were significantly decreased (p < 0.05). A reduction number of 1.3, 1.8, 2.1, and 2.2 log CFU/g of V. parahaemolyticus in artificially contaminated L. vannamei was obtained with PAW for 5 min. The study elucidated that PAW could destroy cell membranes, leading to cell death. The findings would strengthen strategies for V. parahaemolyticus control and provide a potential application of PAW for preserving aquatic products.

Determination of flurbiprofen in rat plasma using ultra-high performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was developed to determine flurbiprofen in rat plasma. A triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source was used in negative ion mode. Acetonitrile precipitation was selected to prepare samples. Flurbiprofen and internal standard flurbiprofen-d5 were analyzed on an Acquity UPLC BEH C18 column with the mobile phase consisting of acetonitrile and water, and a gradient procedure was used for separation. The retention time of flurbiprofen was 0.67 min, and the whole running time was only 1.2 min. The detection was performed on a triple quadrupole tandem mass spectrometer using multiple reaction monitoring mode via an ESI source with optimized mass spectrometry parameters. The calibration curve was linear in the range of 25.0-1.00 × 104 ng/mL (r ≥ 0.99). The within-run and between-run relative standard deviations were not more than 13.9%. The within-run and between-run relative errors were from -9.0% to 3.4%. There was no significant matrix effect, and recovery was high. This method was fully validated, including whole blood stability in rat plasma, and successfully applied to the pharmacokinetic study in which 100% incurred sample reanalysis met the criteria.

Regulation of the pigment production by changing Cell morphology and gene expression of Monascus ruber in high-sugar synergistic high-salt stress fermentation.

AIMS: Extreme environment of microbial fermentation is the focus of research, which provides new thinking for the production and application of Monascus pigments (MPs). In this work, the high-sugar synergistic high-salt stress fermentation (HSSF) of MPs was investigated. METHODS AND RESULTS: The Monascus fungus grew well under HSSF conditions with 35 g L-1 NaCl and 150 g L-1 glucose, and the extracellular yellow pigment and intracellular orange pigment yield in HSSF was 98% and 43% higher than that in conventional fermentation, respectively. Moreover, the mycelial morphology was maintained in a better status with more branches and complete surface structure, indicating good biocatalytic activity for pigment synthesis. Four extracellular yellow pigments (Y1, Y2, Y3, and Y4) were transformed into each other, and ratio of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (P < 0.05) changed. Moreover, the ratio of unsaturated fatty acids to saturated fatty acids (unsaturated/saturated) was significantly (P < 0.05) increased, indicating that the metabolism and secretion of intracellular and extracellular pigment might be regulated in HSSF. The pigment biosynthesis genes mppB, mppC, mppD, MpPKS5, and MpFasB2 were up-regulated, whereas the genes mppR1, mppR2, and mppE were down-regulated, suggesting that the gene expression to regulate pigment biosynthesis might be a dynamic change process in HSSF. CONCLUSIONS: The HSSF system of MPs is successfully performed to improve the pigment yields. Mycelial morphology is varied to enhanced pigment secretion, and gene expression is dynamically regulated to promote pigment accumulation in HSSF.

Transcription factors: switches for regulating growth and development in macrofungi.

Macrofungi (or mushrooms) act as an extraordinarily important part to human health due to their nutritional and/or medicinal value, but the detailed researches in growth and development mechanisms have yet to be explored further. Transcription factors (TFs) play indispensable roles in signal transduction and affect growth, development, and metabolism of macrofungi. In recent years, increasing research effort has been employed to probe the relationship between the development of macrofungi and TFs. Herein, the present review comprehensively summarized the functional TFs researched in macrofungi, including modulating mycelial growth, fructification, sclerotial formation, sexual reproduction, spore formation, and secondary metabolism. Meanwhile, the possible effect mechanisms of TFs on the growth and development of some macrofungi were also revealed. Specific examples of functional characterizations of TFs in macrofungi (such as Schizophyllum commune and Coprinopsis cinerea) were described to a better comprehension of regulatory effect. Future research prospects in the field of TFs of macrofungi are discussed. We illustrated the functional versatility of the TFs in macrofungi based on specific examples. A systematical realization of the interaction and possible mechanisms between TFs and macrofungi can supply possible solutions to regulate genetic characteristics, which supply novel insights into the regulation of growth, development and metabolism of macrofungi. KEY POINTS: • The functional TFs researched in macrofungi were summarized. • The possible effect mechanisms of TFs in macrofungal were described. • The multiple physiological functions of TFs in macrofungi were discussed.

Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selectively suppress the DNA amplification of dead cells. The results showed the optimum concentration of PMA is 5.0 µg/mL. The detection limit of this assay by mPCR was 103 CFU/mL in the culture broth, and by PMA-mPCR was 104 CFU/mL both in pure culture and a food matrix (milk and ground beef). In addition, the detection of mixed viable and dead cells was also explored in this study. The detection sensitivity ratio of viable and dead counts was less than 1:10. Therefore, the PMA-mPCR assay proposed here might provide an efficient detection tool for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella and also have great potential for the detection and concentration assessment of VBNC cells.

Biofilm Formation and Control of Foodborne Pathogenic Bacteria.

Biofilms are microbial aggregation membranes that are formed when microorganisms attach to the surfaces of living or nonliving things. Importantly, biofilm properties provide microorganisms with protection against environmental pressures and enhance their resistance to antimicrobial agents, contributing to microbial persistence and toxicity. Thus, bacterial biofilm formation is part of the bacterial survival mechanism. However, if foodborne pathogens form biofilms, the risk of foodborne disease infections can be greatly exacerbated, which can cause major public health risks and lead to adverse economic consequences. Therefore, research on biofilms and their removal strategies are very important in the food industry. Food waste due to spoilage within the food industry remains a global challenge to environmental sustainability and the security of food supplies. This review describes bacterial biofilm formation, elaborates on the problem associated with biofilms in the food industry, enumerates several kinds of common foodborne pathogens in biofilms, summarizes the current strategies used to eliminate or control harmful bacterial biofilm formation, introduces the current and emerging control strategies, and emphasizes future development prospects with respect to bacterial biofilms.

Cold-adaptive mechanism of psychrophilic bacteria in food and its application.

Psychrophilic bacteria are a type of microorganisms that normally grow in low-temperature environments. They are usually found in extremely cold environments. However, as people's demand for low-temperature storage of food becomes higher, psychrophilic bacteria have also begun to appear in cold storage and refrigerators, which has become a food safety hazard. In this paper, the optimal cooling strategies of psychrophilic bacteria are reviewed from the aspects of the cell membrane, psychrophilic enzymes, antifreeze proteins, cold shock proteins, gene regulation, metabolic levels and antifreeze agents, and the principle of psychrophilic mechanism is briefly described. The application of thermophilic bacteria and its products adapted to cold environments in food fields are analyzed. The purpose of this paper is to provide ideas for future research on psychrophilic bacteria based on the mechanism and application of psychrophilic bacteria.

[Management of immunocompromised renal transplant patients infected with coronavirus disease 2019].

OBJECTIVE: To analyze the treatment process of a renal transplant patient infected with coronavirus disease 2019 (COVID-19), and discuss the management strategy for the immunocompromised hosts. METHODS: The diagnosis and treatment of a case of transplant patients with COVID-19 admitted to Horgos designated hospital of Xinjiang Uygur Autonomous Region in October 2021 were reviewed. The medical history and laboratory and imaging examination treatment and outcome of this case were analyzed. RESULTS: The recipient was a middle-aged male with a time from renal transplantation of 3 years. The onset was moderate to low fever, accompanied by cough and fatigue. Chest CT showed multiple ground glass shadows under the pleura of both lungs, mainly in both lower lungs, gradually worsening until "white lung" appeared, with early renal and cardiac insufficiency. In the course of treatment, immunosuppressants were reduced and the dosage of glucocorticoid was increased. In the early stage, due to renal insufficiency and hyperkalemia, dialysis was conducted for 3 times. Oral abidol and Lianhua Qingwen capsule were given as antiviral and anti-infection treatment. Special immunoglobulin and convalescent plasma of COVID-19 were used to boost the immunity of patients. The patient was eventually clinically cured. CONCLUSIONS: The clinical manifestations and diagnosis of COVID-19 for the kidney transplantation recipient are not significantly different from other populations, but immunocompromised hosts are more likely to suffer from organ dysfunction. The adjustment of immunosuppressants and glucocorticoids, respiratory support, selection of antibiotics, organ protection, nutritional support and traditional Chinese medicine intervention in the treatment of renal transplant recipients with severe COVID-19 need further discussion.

The application and research progress of bacteriophages in food safety.

The abuse of antibiotics and the emergence of drug-resistant bacteria aggravate the problem of food safety. Finding safe and efficient antibiotic substitutes is an inevitable demand for ensuring the safety of animal-derived food. Bacteriophages are a kind of virus that can infect bacteria, fungi or actinomycetes. They have advantages of simple structure, strong specificity and nontoxic side effects for the human body. Bacteriophages can not only differentiate live cells from dead ones but also detect bacteria in a viable but nonculturable state. These characteristics make bacteriophages more and more widely used in the food industry. This paper describes the concept and characteristics of bacteriophages, and introduces the application of bacteriophages in preharvest production, food processing, storage and sales. Several methods of using bacteriophages to detect foodborne pathogens are listed. Finally, the advantages and limitations of bacteriophages in the food industry are summarized, and the application prospect of bacteriophages in the food industry is discussed.

Research on the drug resistance mechanism of foodborne pathogens.

Foodborne diseases caused by foodborne pathogens are one of the main problems threatening human health and safety. The emergence of multi-drug resistant strains due to the abuse of antibiotics has increased the difficulty of clinical treatment. Research on the drug resistance mechanism of foodborne pathogens has become an effective method to solve multi-drug resistant strains. In this paper, the four main drug resistance mechanisms, including reduced cell membrane permeability, efflux pump mechanism, target site mutation mechanism, and enzymatic hydrolysis, were used to systematically analyze the drug resistance of Salmonella, Listeria monocytogenes, and Escherichia coli. And the new methods were discussed that may be used to solve the drug resistance of foodborne pathogens such as CRISPR and bacteriophages. This review provided a certain theoretical basis for the treatment of foodborne diseases.

The diagnostic tools for viable but nonculturable pathogens in the food industry: Current status and future prospects.

Viable but nonculturable (VBNC) microorganisms have been recognized as pathogenic contaminants in foods and environments. The failure of VBNC cells to form the visible colonies hinders the ability to use conventional media for their detection. Efficient and rapid detection of pathogens in the VBNC state is a prerequisite to ensure the food safety and public health. Despite their nonculturability, VBNC cells have distinct characteristics, such as morphology, metabolism, chemical composition, and gene and protein expression, that have been used as the basis for the development of abundant diagnostic tools. This review covers the current status and advances in various approaches for examining microorganisms in the VBNC state, including but not limited to the methodological aspects, advantages, and drawbacks of each technique. Existing methods, such as direct viable count, SYTO/PI dual staining, and propidium monoazide quantitative polymerase chain reaction (PCR), as well as some techniques with potential to be applied in the future, such as digital PCR, enhanced-surface Raman spectroscopy, and impedance-based techniques, are summarized in depth. Finally, future prospects for the one-step detection of VBNC bacteria are proposed and discussed. We believe that this review can provide more optional methods for researchers and promote the development of rapid, accurate detecting methods, and for inspectors, the diagnostic tools can provide data to undertake risk analysis of VBNC cells.

Effects of quorum sensing on the biofilm formation and viable but non-culturable state.

Quorum sensing exists widely in all kinds of microorganisms and is a communication channel for microorganisms. Many bacterial processes, including virulence factor expression, biofilm formation, and viable but non-culturable (VBNC) cell resuscitation, are mediated by quorum sensing, and biofilm formation complicates the treatment of various infections. Foodborne pathogens can enter VBNC state in extreme environments, and pathogens in VBNC state can evade traditional detection and resuscitate under appropriate conditions, causing potential harm to human health. The disruption of quorum sensing may decisively help control biofilm formation and VBNC cell resuscitation. This review describes the quorum sensing systems of various bacteria and major fungi, and summarizes the role of bacterial quorum sensing system in biofilm formation and VBNC resuscitation. In addition, the relationship between quorum sensing inhibitors (QSI) with biofilms and VBNC is also discussed.

Organic Modifications of SBA-15 Improves the Enzymatic Properties of its Supported TLL.

In this study, lipase from Thermomyces lanuginosus (TLL) was immobilized onto the parent and organic groups modified SBA-15, and the enzymatic properties of the obtained immobilized TLL samples were investigated. 1) Activity of SBA-15-TLL at 2862.78 ± 293.24 U/g was obtained. 2) Most of the organic groups modification favored a great improvement in activity, and higher activity over 12000 U/g was observed for N-phenylaminomethyl and phenyl group modification. 3) Most of the supported TLL showed better thermostability in air while poor in phosphate buffer, with over 80% vers less than 20% of their initial activity retained after 4 h incubation at 70℃. 4) The n-dodecyl, phenyl and N-phenylaminomethyl group functionalization decreased the sensitivity of immobilized TLL in extreme pH values. 5) The n-octyl and 2-(propoxymethyl)oxirane group modification confered the supported TLL good reusability, and over 60% of their initial activity was retained after five successive cycles of reuse.

Quorum-Sensing Regulation of Antimicrobial Resistance in Bacteria.

Quorum sensing is a cell-to-cell communication system that exists widely in the microbiome and is related to cell density. The high-density colony population can generate a sufficient number of small molecule signals, activate a variety of downstream cellular processes including virulence and drug resistance mechanisms, tolerate antibiotics, and harm the host. This article gives a general introduction to the current research status of microbial quorum-sensing systems, focuses on the role of quorum-sensing systems in regulating microbial resistance mechanisms, such as drug efflux pump and microbial biofilm formation regulation, and discusses a new strategy for the treatment of drug-resistant bacteria proposed by using quorum quenching to prevent microbial resistance.

Investigation of the mycelial morphology of Monascus and the expression of pigment biosynthetic genes in high-salt-stress fermentation.

Extreme environments, for example high-salt-stress condition, that can induce secondary metabolite biosynthesis in fungi are a promising and effective strategy for producing natural Monascus pigments used as food colourants and nutraceutical supplements. In this study, the relationship between the mycelial morphology and expression of pigment biosynthetic genes in high-salt-stress fermentation (HSF) with Monascus ruber CGMCC 10910 was investigated. The Monascus fungus grew well under HSF conditions with 35 g/l NaCl, and the intracellular yellow pigment yield in HSF was 40% higher than that in conventional batch fermentation (CBF). Moreover, the mycelial morphology was maintained in a better state, with a hyphal diameter of 5-6 µm in HSF, indicating good biocatalytic activity for pigment synthesis. The rate of the relative content of intracellular orange pigments to yellow pigments (O/Y) significantly (p < 0.05) changed, and the extracellular yellow pigments were transformed into each other, indicating that the pigment biosynthesis pathway was changed to promote yellow pigment accumulation in HSF. The pigment biosynthesis genes MpPKS5, MpFasB2, mppE, mppD and mppB were significantly (p < 0.05) up-regulated by approximately 58.4-106.1%, whereas the regulatory genes mppR1 and mppR2 were significantly (p < 0.05) down-regulated by approximately 23.2% and 59.0% in HSF. Notably, the mppE gene was highly correlated with (r > 0.95, p < 0.05) hyphal diameter. These findings indicated that the cultivation of the Monascus fungus under high-salt-stress conditions was beneficial for pigment biosynthesis by controlling the mycelial morphology to regulate gene expression. This study first described the relationship between the mycelial morphology and expression of pigment biosynthetic genes in Monascus during fermentation. KEY POINTS: • High-salt-stress fermentation (HSF) was first performed to improve Monascus pigment yield. • Pigment biosynthesis was enhanced by maintaining the mycelial morphology in an improved state in HSF. • Gene expression was up-/downregulated to promote yellow pigment accumulation in HSF. • The mycelial morphology was highly related to the expression of pigment biosynthetic genes in HSF.

Production of diacylglycerols through glycerolysis with SBA-15 supported Thermomyces lanuginosus lipase as catalyst.

BACKGROUND: In this study, SBA-15 was functionalized by silane coupling reagents, then lipase from Thermomyces lanuginosus (TLL) was immobilized onto the parent and the organically modified SBA-15 for diacylglycerol (DAG) production through glycerolysis. RESULTS: Diacylglycerol content of 54.77 ± 0.63%, and triacylglycerol (TAG) conversion of 77.75 ± 1.24%, were obtained from the parent SBA-15 supported TLL-mediated glycerolysis reaction in a solvent-free system. However, poor performance was unexpectedly observed when co-solvents were introduced into the reaction system. After organic modification, the functionalized SBA-15 supported TLL samples all exhibited reasonable performance, producing DAG content over 40 wt% and TAG conversion over 70 wt%. Higher DAG content, up to 59.19 ± 1.10%, was observed from the phenyl group-modified SBA-15 supported TLL. The operational stability of the immobilized TLL samples in glycerolysis was also improved after organic functionalization. The phenyl group-modified SBA-15 supported TLL showed good reusability in the present glycerolysis reaction, and 95.21 ± 4.87% of the initial glycerolysis activity remained after five cycles of reuse. CONCLUSION: The organic modification of SBA-15 improved the catalytic performance of its supported TLL in glycerolysis, in terms of TAG conversion, DAG content, and reusability. © 2019 Society of Chemical Industry.

Transcriptomic Analysis of Viable but Non-Culturable Escherichia coli O157:H7 Formation Induced by Low Temperature.

Escherichia coli O157:H7 is one of the most common pathogenic bacteria that pose a threat to food safety. The aim of this study was to investigate the mechanisms of the formation of viable but non-culturable (VBNC) E. coli O157:H7 induced by low temperature (-20 °C) using RNA sequencing (RNA-Seq) transcriptomics analysis. The results of the present investigation revealed the presence of 2298 differentially expressed genes in VBNC cells, accounting for 46.03% of the total number of genes. Additionally, GO function and KEGG pathway enrichment analysis were performed to investigate the functional and related metabolic pathways of the differentially expressed genes. We found that the ion transport, protein synthesis, and protein transmembrane transport activities were significantly improved in the VBNC cells, indicating that E. coli O157:H7 cells synthesized a considerable amount of protein to maintain the levels of their functional metabolic processes and life activities in the VBNC state. In conclusion, we suggest that the increased synthesis of proteins such as SecY, FtsY, and Ffh might indicate that they are the key proteins involved in the improvement of the transmembrane transport activities in VBNC E. coli O157:H7 cells, maintaining their functional metabolism in the VBNC state and enhancing their survival ability under low temperatures.

Microfluidic-Based Approaches for Foodborne Pathogen Detection.

Food safety is of obvious importance, but there are frequent problems caused by foodborne pathogens that threaten the safety and health of human beings worldwide. Although the most classic method for detecting bacteria is the plate counting method, it takes almost three to seven days to get the bacterial results for the detection. Additionally, there are many existing technologies for accurate determination of pathogens, such as polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA), or loop-mediated isothermal amplification (LAMP), but they are not suitable for timely and rapid on-site detection due to time-consuming pretreatment, complex operations and false positive results. Therefore, an urgent goal remains to determine how to quickly and effectively prevent and control the occurrence of foodborne diseases that are harmful to humans. As an alternative, microfluidic devices with miniaturization, portability and low cost have been introduced for pathogen detection. In particular, the use of microfluidic technologies is a promising direction of research for this purpose. Herein, this article systematically reviews the use of microfluidic technology for the rapid and sensitive detection of foodborne pathogens. First, microfluidic technology is introduced, including the basic concepts, background, and the pros and cons of different starting materials for specific applications. Next, the applications and problems of microfluidics for the detection of pathogens are discussed. The current status and different applications of microfluidic-based technologies to distinguish and identify foodborne pathogens are described in detail. Finally, future trends of microfluidics in food safety are discussed to provide the necessary foundation for future research efforts.

Contrast of Real-Time Fluorescent PCR Methods for Detection of Escherichia coli O157:H7 and of Introducing an Internal Amplification Control.

Various constituents in food specimens can inhibit the PCR assay and lead to false-negative results. An internal amplification control was employed to monitor the presence of false-negative results in PCR amplification. In this study, the objectives were to compare the real-time PCR-based method by introducing a competitive internal amplification control (IAC) for the detection of Escherichia O157:H7 with respect to the specificity of the primers and probes, analytical sensitivity, and detection limits of contamination-simulated drinking water. Additionally, we optimized the real-time fluorescent PCR detection system for E. coli O157:H7. The specificity of primers and probes designed for the rfbE gene was evaluated using four kinds of bacterial strains, including E. coli O157:H7, Staphylococcus aureus, Salmonella and Listeria monocytogenes strains. The real time PCR assay unambiguously distinguished the E. coli O157:H7 strains after 16 cycles. Simultaneously, the lowest detection limit for E. coli O157:H7 in water samples introducing the IAC was 104 CFU/mL. The analytical sensitivity in water samples had no influence on the detection limit compared with that of pure cultures. The inclusion of an internal amplification control in the real-time PCR assay presented a positive IAC amplification signal in artificially simulated water samples. These results indicated that real-time fluorescent PCR combined with the IAC possessed good characteristics of stability, sensitivity, and specificity. Consequently, the adjusted methods have the potential to support the fast and sensitive detection of E. coli O157:H7, enabling accurate quantification and preventing false negative results in E. coli O157:H7 contaminated samples.