Changes In Cell Membrane Permeability Research Articles

Membrane-Targeting Amphiphilic Honokiol Derivatives Containing an Oxazole Moiety as Potential Antibacterials against Methicillin-Resistant Staphylococcus aureus.

Infections with methicillin-resistant Staphylococcus aureus (MRSA) are becoming increasingly serious, making the development of novel antimicrobials urgent. Here, we synthesized some amphiphilic honokiol derivatives bearing an oxazole moiety and investigated their antibacterial and hemolytic activities. Bioactivity evaluation showed that E17 possessed significant in vitro antibacterial activity against S. aureus and MRSA, along with low hemolytic activity. Moreover, E17 exhibited rapid bactericidal properties and was not susceptible to resistance. Mechanistic studies indicated that E17 interacts with phosphatidylglycerol and cardiolipin of bacterial cell membranes, leading to changes in cell membrane permeability and polarization, increased intracellular ROS, and leakage of DNA and proteins, thus accelerating bacterial death. Transcriptome analysis further demonstrated that E17 has membrane-targeting effects, affecting the expression of genes related to cell membranes and ABC transporter proteins. Notably, in vivo activity showed that E17 has prominent anti-MRSA efficacy, comparable to vancomycin, and is expected to be a new anti-MRSA drug candidate.

Preliminary Exploration of the Biophysical Mechanisms of Pulsed Magnetic Field- Induced Cell Permeabilization.

Pulsed magnetic field treatment can enhance cell membrane permeability, allowing large molecular substances that normally cannot pass through the cell membrane to enter the cell. This research holds significant prospects for biomedical applications. However, the mechanism underlying pulsed magnetic field-induced cell permeabilization remains unclear, impeding further progress in research related to pulsed magnetic field. Currently, hypotheses about the mechanism are struggling to explain experimental results. Therefore, this study developed a parameter-adjustable pulsed magnetic field generator and designed experiments. Starting from the widely accepted hypothesis of "induced electric fields by pulsed magnetic field," we conducted a preliminary exploration of the biophysical mechanisms underlying pulsed magnetic field-induced cell permeabilization. Finally, we have arrived at an intriguing conclusion: under the current technical parameters, the impact of the pulsed magnetic field itself is the primary factor influencing changes in cell membrane permeability, rather than the induced electric field. This conclusion holds significant implications for understanding the biophysical mechanisms behind pulsed magnetic field therapy and its potential biomedical applications.

Comparative physiological and transcriptomic analysis of sono-biochemical control over post-acidification of Lactobacillus delbrueckii subsp. bulgaricus

Thermosonication (UT) prestress treatments combining with varied fermentation patterns has been revealed as an effective method to regulate post-acidification as exerted by Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii), but sono-biochemical controlling mechanisms remain elusive. This study employed physiological and transcriptomic analysis to explore the response mechanism of L. delbrueckii to UT-induced microstress (600 W, 33 kHz, 10 min). UT stress-induced inhibition of acidification of L. delbrueckii during (post)-fermentation was first confirmed, relying on the UT process parameters such as stress exposure duration and UT power. The significantly enhanced membrane permeability in cells treated by 600 W for 10 min than the microbes stressed by 420 W for 20 min suggested the higher dependence of UT-derived stresses on the treatment durations, relative to the ultrasonic powers. In addition, ultrasonication treatment-induced changes in cell membrane integrity enhanced and/or disrupted permeability of L. delbrueckii, resulting in an imbalance in intracellular conditions associated with corresponding alterations in metabolic behaviors and fermentation efficiencies. UT-prestressed inoculum exhibited a 21.46% decrease in the membrane potential during the lag phase compared to untreated samples, with an intracellular pH of 5.68 ± 0.12, attributed to the lower activities of H+-ATPase and lactate dehydrogenase due to UT stress pretreatments. Comparative transcriptomic analysis revealed that UT prestress influenced the genes related to glycolysis, pyruvate metabolism, fatty acid synthesis, and ABC transport. The genes encoding 3-oxoacyl-[acyl-carrier-protein] reductases I, II, and III, CoA carboxylase, lactate dehydrogenase, pyruvate oxidase, glucose-6-phosphate isomerase, and glycerol-3-phosphate dehydrogenase were downregulated, thus identifying the relevance of the UT microstresses-downregulated absorption and utilization of carbohydrates with the attenuated fatty acid production and energy metabolisms. These findings could contribute to provide a better understanding of the inactivated effects on the post-acidification of L. delbrueckii by ultrasonic pretreatments, thus providing theoretical basis for the targeted optimization of acidification inhibition efficiencies for yogurt products during chilled preservation processes.

Co-exposure to pentachlorophenol (PCP) and cadmium (Cd) triggers apoptosis-like cell death in Eschericia coli

Pentachlorophenol (PCP) - cadmium (Cd) complex pollution has been identified as a form of persistent soil pollution in south China, exerting detrimental impacts on the indigenous soil bacterial communities. Hence, it is worthwhile to investigate whether and how bacterial populations alter in response to these pollutants. In this study, Escherichia coli was used as a model bacterium. Results showed that PCP exposure caused bacterial cell membrane permeability changes, intracellular ROS elevation, and DNA fragmentation, and triggered apoptosis-like cell death at low exposure concentration and necrosis at high exposure concentration. Cd exposure caused severe oxidative damage and cell necrosis in the tested bacterial strain. The co-exposure to PCP and Cd elevated the ROS level, stimulated the bacterial caspase activity, and induced DNA fragmentation, thereby leading to an apoptosis-like cell death. In conclusion, PCP-Cd complex pollution can cause bacterial population to decrease through apoptosis-like cell death pathway. However, it is worth noting that the subpopulation survives under the complex pollution stress.

Antibacterial activity and action mode of crude bacteriocin C2-1 from Ligilactobacillus salivarius C2-1 against Listeria monocytogenes CICC 21633

The potential use of Ligilactobacillus salivarius-derived bacteriocin for the control of L. monocytogenes was investigated. The purpose of the study was to explore the antibacterial action mode of crude bacteriocin C2-1 from Ligilactobacillus salivarius C2-1 against Listeria monocytogenes CICC 21633. The inhibition zone diameter of crude bacteriocin C2-1 was 27.82 ± 0.3 mm against L. monocytogenes CICC 21633, and the minimum inhibitory concentration (MIC) of bacteriocin C2-1was 13.98 μg/mL. Bacteriocin C2-1 significantly inhibited the normal growth of L. monocytogenes CICC21633, is sensitive to proteinase K, trypsin, and pepsin, and has good thermal stability and acid tolerance. The antibacterial mode of bacteriocin C2-1 against L. monocytogenes CICC 21633 was discussed based on changes in cell membrane permeability, integrity, and morphological structure of L. monocytogenes CICC 21633 by measuring the conductivity, UV-absorbing substance content and the observation via scanning electron microscopy, respectively. These results demonstrated that bacteriocin C2-1 affected the cell membrane permeability and integrity of L. monocytogenes CICC 21633, resulting in intracellular material leakage. Furthermore, bacteriocin-producing genes were identified in the genome sequence using antiSMASH and BAGEL4 assays. Bacteriocin C2-1 might be a good biological food preservative to prevent L. monocytogenes infestation.

Investigating the Role of Extracellular Matrix Stiffness in Modulating the Ferroptosis Process in Hepatocellular Carcinoma Cells via Scanning Electrochemical Microscopy.

Extracellular matrix (ECM) stiffness modulates a variety of cellular processes, including ferroptosis, a process with significant potential implications for hepatocellular carcinoma (HCC) fibrosis and cirrhosis. However, the exact relationship between ECM stiffness and HCC ferroptosis is yet unclarified, partially due to the lack of in situ information on key parameters of the ferroptosis process of living HCC cells. This study pioneers the use of in vitro mechanical microenvironment models of HCC and the scanning electrochemical microscopy (SECM) technique for understanding this interplay. We first cultured HuH7 cells on 4.0, 18.0, and 44.0 kPa polyacrylamide (PA) gels to simulate early, intermediate, and advanced HCC ECM stiffness, respectively. Then, we used SECM to in situ monitor changes in cell membrane permeability, respiratory activity, and reactive oxygen species (ROS) levels of erastin-induced HuH7 cells on PA gels, finding that increasing ECM stiffness potentiates ferroptosis, including increased membrane permeabilization and H2O2 release as well as reduced respiratory activity. Through further transcriptome sequencing and molecular biology measurements, we identified a critical role for focal adhesion kinase (FAK)-mediated yes-associated protein (YAP) in regulating the ferroptosis process dependent on ECM stiffness, which provides novel insights into the mechanical regulation of ferroptosis in HCC cells and may pave the way for innovative therapeutic strategies.

Neher and Sakmann: Single Ion Channels.

Most biological functions have their basis in a rapid change in cell membrane permeability. Hodgkin and Huxley showed regulation of the flow of molecules and ions between the nerve cell and its environment (Nobel Prize 1963), by recording macroscopic currents. Two Germans Neher and Sakmann showed that specific ion channels actually exist, and specific membrane proteins act as gates or agents for active transport regulating in physiological and pathological processes.

A translocation fluorescent probe for analyzing cellular physiological parameters in neurological disease models.

Neurological disorders are closely linked to the alterations in cell membrane permeability (CMP) and mitochondrial membrane potential (MMP). Changes in CMP and MMP may lead to damage and death of nerve cells, thus triggering the onset and progression of neurological diseases. Therefore, monitoring the changes of these two physiological parameters not only benefits the accurate assessment of nerve cell health status, but also enables providing key information for the diagnosis and treatment of neurological diseases. However, the simultaneous monitoring of these two cellular physiological parameters is still challenging. Herein, we design and synthesize two quinolinium-carbazole-derivated fluorescent probes (OQ and PQ). As isomers, the only difference in their chemical structures is the linking position of the carbazole unit in quinoline rings. Strikingly, such a subtle difference endows OQ and PQ with significantly different organelle-staining behaviors. PQ mainly targets at the nucleus, OQ can simultaneously stain cell membranes and mitochondria in normal cells, and performs CMP and MMP-dependent translocation from the cell membrane to mitochondria then to the nucleus, thus holding great promise as an intracellular translocation probe to image the changes of CMP and MMP. After unraveling the intrinsic mechanism of their different translocation abilities by combining experiments with molecular dynamics simulations and density functional theory calculations, we successfully used OQ to monitor the continuous changes of CMP and MMP in three neurological disease-related cell models, including oxidative stress-damaged, Parkinson's disease, and virus-infected ones. Besides providing a validated imaging tool for monitoring cellular physiological parameters, this work paves a promising route for designing intracellular translocation probes to analyze cellular physiological parameters associated with various diseases.

Low-intensity pulsed ultrasound in obstetrics and gynecology: advances in clinical application and research progress.

In the past decade, research on ultrasound therapy in obstetrics and gynecology has rapidly developed. Currently, high-intensity ultrasound has been widely used in clinical practice, while low-intensity ultrasound has gradually emerged as a new trend of transitioning from pre-clinical research to clinical applications. Low-intensity pulsed ultrasound (LIPUS), characterized by a non-invasive low-intensity pulse wave stimulation method, employs its non-thermal effects to achieve safe, economical, and convenient therapeutic outcomes. LIPUS converts into biochemical signals within cells through pathways such as cavitation, acoustic flow, and mechanical stimulation, regulating molecular biological mechanisms and exerting various biological effects. The molecular biology mechanisms underlying the application of LIPUS in obstetrics and gynecology mainly include signaling pathways, key gene expression, angiogenesis, inflammation inhibition, and stem cell differentiation. LIPUS plays a positive role in promoting soft tissue regeneration, bone regeneration, nerve regulation, and changes in cell membrane permeability. LIPUS can improve the treatment benefit of premature ovarian failure, pelvic floor dysfunction, nerve damage caused by intrauterine growth restriction, ovariectomized osteoporosis, and incomplete uterine involution through the above biological effects, and it also has application value in the adjuvant treatment of malignant tumors such as ovarian cancer and cervical cancer. This study outlines the biological mechanisms and applications of LIPUS in treating various obstetric and gynecologic diseases, aiming to promote its precise application and provide a theoretical basis for its use in the field.

Lyososome Membrane Permeabilization Induces Plasma Membrane Macropores to Activate K +-Efflux-Dependent NLRP3 Inflammasomes in Murine Dendritic Cells

Abstract NLRP3 is a cytosolic sensor protein in myeloid leukocytes that recruits the adaptor protein ASC and activates caspase-1 in functional NLRP3 inflammasomes. Caspase-1 coordinately cleaves pro-IL-1β to bioactive IL-1β and gasdermin D (GSDMD) to induce N-GSDMD macropores in the plasma membrane (PM). The latter mediate efflux of mature IL-1β and initiate progression to pyroptosis, a lytic cell death. NLRP3 activation is triggered by perturbed cellular homeostasis with decreased cytosolic [K +] being the best-characterized signal. The required K +efflux is typically induced by the ionophore nigericin or extracellular ATP as an agonist for P2X7 receptor channels. K +efflux-dependent activation of NLRP3 is also triggered by soluble or particulate agents which accumulate in lysosomes to induce lysosomal membrane permeabilization (LMP) and release of hydrolytic enzymes into the cytosol; particulates include alum widely used as a vaccine adjuvant. The mechanisms by which LMP rapidly triggers changes in plasma membrane permeability remain undefined. We used a model of murine bone marrow derived dendritic cells (BMDC) treated with the soluble LMP agent Leu-Leu-O-methyl ester (LLME) to elicit K+ efflux and NLRP3 activation. Notably, this K+ efflux was coincident with a rapidly induced influx of propidiumiodide (PI), a 668 Da organic salt routinely used to track assembly of plasma membrane macropores such as GSDMD. Equivalent LLME-induced PI influx was observed in wildtype and Gsdmd−/−BMDC and was unaffected by inhibition of NLRP3 or caspase-1. These observations indicate that LMP triggers rapid activation of an endogenous pore-forming protein distinct from GSDMD. Current studies are evaluating gasdermin E or pannexin-1 as candidates. Supported by grants from the NIH (P01-AI141350 and R01-EY014362)

INTRAORAL APPLICATION OF LOW LEVEL LASER THERAPY -A REVIEW

Lowlevel laser therapy (LLLT) is a relatively new technique in the dental field. It is now used by many clinicians in dentistry. Among its most popular applications are the stimulation of the healing process and pain relief. This paper will discuss and review the most recent intraoral applications, its dosage, its applicability in dentistry, and the hazards connected with it are all discussed in this article so that it can be used to provide greater dental care.Low-level laser treatment (LLLT) elicits biological responses in the body by using light energy in the form of adenosine triphosphate (ATP). Pain relief, wound healing, muscle relaxation, immune system regulation, and neuron regeneration are all aided by increased cellular energy and changes in cell membrane permeability. The clinical consequences of LLLT are investigated in this article, as well as how it can be used in the dentistry area.

Research progress on components and mechanisms of neurotoxicity induced by traditional Chinese medicine.

Over the years, the safety of traditional Chinese medicine (TCM) has received widespread attention, especially the central nervous system-related adverse reactions. Indeed, the complexity of TCM has limited the widespread application of TCM. The article summarizes the main components associated with neurotoxicity, including alkaloids, terpenes, flavonoids, saponins, proteins, and heavy metals, by reviewing the literature on the neurotoxicity of TCM. It has been established that the neurotoxicity mechanisms mainly include mitochondrial damage, oxidative damage, inhibition of cell proliferation (including transcriptional and DNA damage), changes in cell membrane permeability, and apoptosis. By reviewing the latest literature, this paper provides the foothold for follow-up studies and can assist clinicians in preventing neurotoxicity via rational and safe TCM drug use.

Antimicrobial activities and mechanism of sturgeon spermary protein extracts against Escherichia coli.

This study aimed to evaluate the antimicrobial activities and mechanism of sturgeon spermary protein extracts (SSPE) against Escherichia coli. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. Cell structural change was analyzed using scanning electron microscopy-energy dispersive X-ray spectrometry and transmission electron microscope. Moreover, pH, zeta potential, membrane potential, intracellular ATP concentrations and the interaction of SSPE with genomic DNA were analyzed. Results showed that molecular weight of SSPE is 13.4 kDa, the content of basic amino acids is the highest, in which arginine accounts for 73.2%. The MIC and MBC of SSPE for E. coli were 0.05 and 5 mg/mL, respectively. After SSPE treatment, cell membrane permeability changes, zeta potential decrease and genomic DNA lysis occurred in E. coli, which indicated it exerted bacteriostatic effects either independently or simultaneously by destroying the cell membrane and genomic DNA. These findings indicated that SSPE has potential to be a natural antiseptic.

GC-MS Analysis of Volatile Constituents, Antibacterial Activity, and Mechanism of Action of the Essential Oil from Cinnamomum Cassia Bark Against Vancomycin-Resistant Enterococci

Background & Objectives: An alarming rise in multi-drug resistant bacterial pathogen like Vancomycin-resistant Enterococci (VRE) has culminated in a global health threat in today’s healthcare environment and has caused significant morbidity and mortality in patients. The field of medicinal herbs has developed at an exponential rate in subsequent decades. Acknowledging the antimicrobial mechanisms of essential oils and their components is vital for developing them as a potential new source of antibiotics that can work as an alternative treatment for multi-drug resistant bacterial infections. Methods: The current study investigates the physiochemical properties and a variety of analytical and biological activities to determine the activity of Cinnamomum cassia essential Oil against drug-resistant microorganisms by utilizing a Time kill curve and evaluating the Minimum Inhibitory Concentration (MIC) of oil. The antimicrobial properties were assessed using a growth curve and the cell membrane’s integrity. The essential oil was retrieved by hydro-distilling Cinnamon bark, and the volatile components of the essential oil were determined by Gas Chromatography-Mass Spectrometry (GC-MS). Result: GC-MS analysis detected major components such as Cinnamaldehyde-(E) (37.40%); 2-Propenal, 3-phenyl (11.70%) and 1,6-Octadien-3-ol,3,7- dimethyl- (5.97%). The minimum inhibitory concentration (MIC) and growth curves against VRE were also evaluated. The liberation of cellular contents, as well as changes in cell membrane permeability, have been studied. The MIC value of the essential oil against Vancomycin-resistant Enterococci was 15.625 μg/mL speculating that the essential oil seemed to be effective against the drug-resistant culture. When the essential oil of Cinnamomum cassia was administered to the bacterial culture, the constituents of the bacterial cell were expelled into the medium.

Inactivation efficacy and mechanisms of atmospheric cold plasma on Alicyclobacillus acidoterrestris: Insight into the influence of growth temperature on survival.

The bactericidal effect of dielectric barrier discharge-atmospheric cold plasma (DBD-ACP, 20, and 30 kV) against Alicyclobacillus acidoterrestris on the saline solution and apple juice was investigated. Results show that DBD-ACP is effective for the inactivation of A. acidoterrestris by causing significant changes in cell membrane permeability and bacterial morphology. The effect of culture temperatures on the resistance of A. acidoterrestris to DBD-ACP was also studied. A. acidoterrestris cells grown at 25°C had the lowest resistance but it was gradually increased as the culture temperature was increased (25–45°C) (p < 0.05). Moreover, results from Fourier transform infrared spectroscopy (FT-IR) and Gas Chromatography-Mass Spectrometer (GC-MS) analysis showed that the increase in the culture temperature can gradually cause the decreased level of cyclohexaneundecanoic acid in the cell membrane of A. acidoterrestris (p < 0.05). In contrast, cyclopentaneundecanoic acid, palmitic acid, and stearic acid showed an increasing trend in which the fluidity of the bacterial cell membrane decreased. This study shows a specific correlation between the resistance of A. acidoterrestris and the fatty acid composition of the cell membrane to DBD-ACP.

Effects of cinnamaldehyde against planktonic bacteria and biofilm formation of Shigella flexneri

Cinnamaldehyde (CA) has demonstrated anti-inflammatory, anti-tumor and anti-cancer activities; Its antimicrobial and antibiofilm actions against Shigella flexneri, on the other hand, have not been investigated. Sh. flexneri is a gram-negative foodborne pathogen that can be widely found in nature and some industrial production environments. In this current research, our aim was to examine the influences of CA on planktonic bacteria and biofilm formation. The minimum inhibitory concentration (MIC) of CA against Sh. flexneri strain was 100 μg/mL, while bacteria treated with CA showed a longer lag phase compared with the untreated control. CA effectively inactivated the Sh. flexneri in LB broth and fresh lettuce juice. CA treatment resulted in cell membrane permeability changes and dysfunction, as proven by cell membrane depolarization, decreased intracellular ATP concentration. In addition, CA was also discovered to increase the level of reactive oxygen species (ROS) in cells, and induce morphological changes in cells. Crystal violet staining showed that the biomass of biofilm was decreased significantly with CA in 24 h. Light microscopy and field emission scanning electron microscopy (FESEM) observations demonstrated decreased biofilm adhesion and destruction of biofilm architecture after treatment with CA. These findings indicated that CA acts as a natural bacteriostatic agent to control Sh. flexneri in food processing and production.

Improving MR cell size imaging by inclusion of transcytolemmal water exchange.

The goal of the current study is to include transcytolemmal water exchange in MR cell size imaging using the IMPULSED model for more accurate characterization of tissue cellular properties (e.g., apparent volume fraction of intracellular space ) and quantification of indicators of transcytolemmal water exchange. We propose a heuristic model that incorporates transcytolemmal water exchange into a multicompartment diffusion-based method (IMPULSED) that was developed previously to extract microstructural parameters (e.g., mean cell size and apparent volume fraction of intracellular space ) assuming no water exchange. For ≤ 5ms, the water exchange can be ignored, and the signal model is the same as the IMPULSED model. For ≥ 30 ms, we incorporated the modified Kärger model that includes both restricted diffusion and exchange between compartments. Using simulations and previously published in vitro cell data, we evaluated the accuracy and precision of model-derived parameters and determined how they are dependent on SNR and imaging parameters. The joint model provides more accurate values for cell sizes ranging from 10 to 12 microns when water exchange is fast (e.g., intracellular water pre-exchange lifetime ≤ 100 ms) than IMPULSED, and reduces the bias of IMPULSED-derived estimates of , especially when water exchange is relatively slow (e.g., &gt; 200 ms). Indicators of transcytolemmal water exchange derived from the proposed joint model are linearly correlated with ground truth values and can detect changes in cell membrane permeability induced by saponin treatment in murine erythroleukemia cancer cells. Our results suggest this joint model not only improves the accuracy of IMPULSED-derived microstructural parameters, but also provides indicators of water exchange that are usually ignored in diffusion models of tissues.

The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability.

Two-Round Treatment With Propidium Monoazide Completely Inhibits the Detection of Dead Campylobacter spp. Cells by Quantitative PCR.

Campylobacter spp. are known as important foodborne gastroenteric pathogens worldwide. Campylobacter spp. can exist in a viable but non-culturable (VBNC) state under unsuitable environmental conditions, which is undetectable by conventional culture methods. Quantitative polymerase chain reaction (qPCR) can be used to detect VBNC Campylobacter spp.; however, both viable and dead bacteria are detected during qPCR and are indistinguishable. Propidium monoazide (PMA), which can only enter dead bacterial cells through a damaged cell wall/cell membrane, binds to DNA and inhibits qPCR. PMA treatment has been performed along with qPCR (PMA-qPCR) to detect viable bacteria. However, the efficacy of detection inhibition differed among studies, and PMA can potentially enter living cells after changes in cell membrane permeability. In this study, we optimized the PMA treatment method by conducting it before qPCR. Two-round PMA treatment completely inhibited the qPCR signals from dead cells, whereas single-round PMA treatment failed to facilitate this. An optimized PMA-qPCR method was developed using commercial chicken meat, and VBNC Campylobacter spp., which are undetectable using conventional culture-based methods, were successfully detected. In conclusion, this study presents a novel, efficient PMA treatment method for the detection of viable Campylobacter spp., including VBNC Campylobacter spp., in chicken meat. We believe that this method will aid the reliable risk assessment of commercial chicken meat.

Transcriptomic Responses of Salmonella enterica Serovars Enteritidis in Sodium Hypochlorite.

Salmonella enterica serovars Enteritidis (S. Enteritidis) can survive extreme food processing environments including bactericidal sodium hypochlorite (NaClO) treatments generally recognized as safe. In order to reveal the molecular regulatory mechanisms underlying the phenotypes, the overall regulation of genes at the transcription level in S. Enteritidis after NaClO stimulation were investigated by RNA-sequencing. We identified 1399 differentially expressed genes (DEG) of S. Enteritidis strain CVCC 1806 following treatment in liquid culture with 100 mg/L NaClO for 20 min (915 upregulated and 484 downregulated). NaClO stress affects the transcription of genes related to a range of important biomolecular processes such as membrane damage, membrane transport function, energy metabolism, oxidative stress, DNA repair, and other important processes in Salmonella enterica. First, NaClO affects the structural stability of cell membranes, which induces the expression of a range of outer and inner membrane proteins. This may lead to changes in cell membrane permeability, accelerating the frequency of DNA conversion and contributing to the production of drug-resistant bacteria. In addition, the expression of exocytosis pump genes (emrB, yceE, ydhE, and ydhC) was able to expel NaClO from the cell, thereby increasing bacterial tolerance to NaClO. Secondly, downregulation of genes related to the Kdp-ATPase transporter system (kdpABC) and the amino acid transporter system (aroP, brnQ and livF) may to some extent reduce active transport by bacterial cells, thereby reducing their own metabolism and the entry of disinfectants. Downregulation of genes related to the tricarboxylic acid (TCA) cycle may drive bacterial cells into a viable but non-culturable (VBNC) state, resisting NaClO attack by reducing energy metabolism. In addition, significant upregulation of genes related to oxidative stress could mitigate damage caused by disinfectants by eliminating alkyl hydroperoxides, while upregulation of genes related to DNA repair could repair damage to bacterial cells caused by oxidative stress. Therefore, this study indicated that S. Enteritidis has genomic mechanisms to adapt to NaClO stress.